the rna world reader

Insights & Perspectives

The RNA dreamtime

Modern cells feature proteins that might have supported a prebiotic polypeptide world but

nothing indicates that RNA world ever was

Charles G. Kurland

Modern cells present no signs of a putative prebiotic RNA world. However,

RNA coding is not a sine qua non for the accumulation of catalytic polypep-

tides. Thus, cellular proteins spontaneously fold into active structures that

are resistant to proteolysis. The law of mass action suggests that binding

domains are stabilized by specific interactions with their substrates. Random

polypeptide synthesis in a prebiotic world has the potential to initially pro-

duce only a very small fraction of polypeptides that can fold spontaneously

into catalytic domains. However, that fraction can be enriched by proteolytic

activities that destroy the unfolded polypeptides and regenerate amino acids

that can be recycled into polypeptides. In this open system scenario the

stable domains that accumulate and the chemical environment in which they

are accumulated are linked through self coding of polypeptide structure.

Such open polypeptide systems may have been the precursors to the cellu-

lar ribonucleoprotein (RNP) world that evolved subsequently.

Keywords:.domain selection; non-ribosomal peptidyl transferase; polypeptides;

proteolysis; ribozymes

Introduction

The standard model for the origin of lifeimagines that the first replication, trans-lation, and transcription systems weresupported by RNA without the interven-tion of proteins [1]. The impulses for thisconjecture were first, the discoveries ofintrons and exons in eukaryote mRNAsand second, the self splicing of someintron sequences in ribosomal RNA [2].

Here, a core assertion is that primordialmini-RNAs corresponding to the originalexons encoded the first polypeptides [1].In this scenario ‘‘RNA molecules beganto synthesize proteins, first by develop-ing RNA adapter molecules that can bindactivated amino acids and then byarranging them according to an RNAtemplate using other molecules such asthe RNA core of the ribosome’’. Twenty-four years later this dazzling speculation

has been reduced by ritual abuse tosomething like a creationist mantra.Hence, the title, borrowed from Collinset al. [3], alludes to an oral tradition oforigins passed on by the first Australians.Finally, the support for a prebiotic RNAworld consists solely of ingenious piece-meal chemical simulations in vitro thatwere obtained by chemists at great costand effort over a 20-year period [4]. Suchchemical simulations are accepted ashard evidence by RNA worlders, but intruth, they do not constitute proper evol-utionary evidence.

It might have been useful earlier onto address questions such as: Does theroutine identification of ribonucleopro-teins (RNPs) as remnants of a prebioticRNA world [4] support Gilbert’s conjec-ture [1] or do they beg the question? Ispartial simulation in vitro by a ribozymerunning at one-millionth the rate nor-mally catalyzed by a protein [5, 6] to betaken as evidence for the prebiotic pre-cedence of the ribozyme activity?Though there are impressive syntheticribozymes with convincing performancecharacteristics [7], why are thereno examples of naturally occurring,protein-free ribozymes to link the postu-lated protein-free RNA world to themodern cellular world [8, 3]?

The secret of the cage

RNase P is an RNP that mediates thematuration of transfer RNAs [9].Normally, RNase P has both RNA as wellas protein components. But recently, anRNA-free variant was discovered inhuman mitochondria and shown to

DOI 10.1002/bies.201000058

Department of Microbial Ecology, University ofLund, Solvegatan, Lund, Sweden

Corresponding author:Charles G. KurlandE-mail: [email protected]

Abbreviations:FSF, fold superfamily; NPTase, non-ribosomalpeptidyl transferase; PTC, peptidyl transferasecenter; RNP, ribonucleoprotein.

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mature tRNA precursors normally [10].If RNase P function can be supported byprotein alone, it is conceivable that suchprotein functions have participated inprebiotic systems as well. So, whichcame first, a protein or an RNP versionof the enzyme? In fact, a recent phylo-genetic survey suggests that an ances-tral protein version may have precededthe RNP version of RNase P [11].

Another instructive RNP is the bac-terial ribosome. Studies of these RNPsreveal the tension between routine bio-chemical observations and the expec-tations of RNA worlders, who refuse toaccept the simplest interpretations oftheir experiments. For example, gentledisruption of 50S subunits yields a com-pacted 23S RNA associated with a verysmall core of ribosomal protein and thisRNP supports peptidyl transferaseactivity with model substrates [12, 13].Though these are straightforwardresults, the authors are reluctant to con-cede the potential involvement of ribo-somal protein in the peptidyl transferaseactivity. Instead, they speculate that thecore of proteins in their active 23S RNApreparations is trapped in an imaginaryRNA cage; that is to say, the proteins arepassive prisoners and not active partici-pants [12, 13]. Noller [13] further com-ments that ‘‘true ‘ribocentrics’ willsimply view the latest aspect of the ribo-somal puzzle as a worthy challenge,whose solution promises to reveal oneof nature’s most ancient biologicalsecrets’’.

An attempt to simulate the peptidyltransferase center (PTC) with the aid of asynthetic ribozyme was equally obtuse.Variants of tailored polynucleotides thatcould simulate peptidyl transferaseactivity were synthesized and selectedin vitro [6]. The fastest variant of thetailored ribozymes had a kcat corre-sponding to 0.05 minute1 per peptide,which was compared with an elongationrate of 15–20 seconds1 for Escherichiacoli ribosomes [6]. In this comparisonthe kcat was at least four orders of mag-nitude smaller for the ribozyme than forthe ribosome. However, the kcat of E. coliribosomes in the peptidyl transferasereaction is closer to 100 seconds1

[14], which indicates that the ribozymeis five orders of magnitude slower thanthe bacterial ribosome. Nevertheless,Zhang and Cech [6] suggest that the‘‘peptidyl transferase reaction of the

selected ribozymes is ‘fundamentally’similar to that carried out by the ribo-some’’. Fundamentally similar?

The first crystallographic reports ofthe structure of archaeal 50S ribosomalsubunits described a roughly 20 A-diameter protein-free RNA domain thatwas identified as the PTC [15, 16]. Theseobservations contrasted decades of bio-chemistry that had identified proteincontributions to the bacterial PTC [17].

So, a fatwa was issued to clarify theX-ray revelations: ‘‘From this structurethey deduced . . . that RNA componentsof the large subunit accomplish the keypeptidyl transferase reaction . . . Thus,ribosomal RNA (rRNA) does not exist asa framework to organize catalyticproteins. Instead, the proteins are thestructural units and they help toorganize key ribozyme (catalytic RNA). . . elements, an idea long championedby Harry Noller, Carl Woese . . ., andothers’’ [18].

In unambiguous contrast to thisclaim, Zimmerman and collaborators[17] showed that deletion of E. coli ribo-somal protein L27, long thought to bepart of the bacterial PTC, severelydepresses bacterial growth rates, whichare restored when the protein isexpressed from a plasmid. Likewise,deletion of one to three N-terminal aminoacids of L27 had a similar debilitatingeffect on growth rates as well as on pep-tidyl transferase reaction rates in vitro.Most informative was their finding thatdeletion of the three N-terminal aminoacids strongly inhibits the labeling of L27by photo-activated tRNA at the P site.The experiments of Maguire et al. [17]rather clearly confirm a substantial chainof biochemical experiments initiated in1973 that consistently implicated L27 as aclose neighbor and probable participantin the peptidyl transferase reaction ofbacterial ribosomes.

The identification by Maguire et al.[17] of L27 as an essential part of the PTCin E. coli ribosomes was confirmed by ahigh-resolution structure of 70S activeribosomes with tRNA-filled A site and Psite that revealed two proteins interact-ing directly with tRNA at the putativePTC [19]. One of these is L27 and theother is L16, which was also previouslyimplicated in peptidyl transferaseactivity [17]. Thus, the universalprotein-free ribozyme at the heart ofthe ribosome is history.

Does RNA replace protein?

Comparisons with the findings fromarchaeal subunits [15, 16] are sugges-tively complicated by the apparentabsence of an L27 homolog from thearchaeal 50S ribosomal subunit.According to Voorhees et al. [19] theprojected orientation of L16 in the arch-aeal 50S subunits suggests that, like itshomolog in the E. coli ribosome, it maybe interacting with the elbow of thetRNA in the archaeal A site. In fact itwould be highly instructive if, aftermore stringent structural determi-nations are made, archaeal ribosomeswere indeed found to exploit aprotein-free RNA peptidyl transferasein a high-resolution structure for active70S particles. In that case, the PTCwould be just another example of anRNP featuring an interchangeability ofRNA and protein functions as in theRNA-free RNase P [10]. Indeed a crypticevolutionary trade-off between RNA andprotein may account for the observeddifferences between some archaealand bacterial PTCs.

The growth efficiency of a cellularprocess such as translation or transcrip-tion can be measured by its rate normal-ized to the molecular mass that isrequired to carry out that process [20].No biosynthetic cycle is as expensive to aprokaryote cell as translation becausenothing else involves as large an invest-ment in macromolecular equipment.This means that the biosynthetic costof RNA in terms of growth efficiency isroughly one tenth that for a polypeptidechain. In this context of growth effi-ciency, the observed differences betweensome bacterial and archaeal 50S subu-nits are consistent with the notion thatthe evolution of the archaeal translationapparatus involved a trade-off betweenthe costs of making that equipment andthe efficiency with which it works.

For cells such as the bacteriumE. coli, the growth optimization favorshigh rates of function in relatively richmedia. That is to say, a fast, protein-richPTC is an acceptable ribosomal designfor optimal growth under relatively gen-erous conditions [20, 21]. However, forcells such as some archaea the optionsmay be different because they areadapted to growth under conditions ofenergy stress [22]. Here, ‘‘cheap’’ struc-tural solutions for ribosomes are at a

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premium even if they might come at thecost of a somewhat slower rate of func-tion, as expected for ribozymes. Thepoint is that archaea growing underconditions of energy stress are con-strained to produce amino acids rela-tively slowly with the consequencethat optimal translation rates might becorrespondingly slow. Here, substi-tution of costly protein by less costlyRNAwould be a favored design strategy.Indeed, comparative data suggest thatthe evolution of the archaea involves aselective loss of proteins from ribo-somes, a loss that is most striking atthe crown of the archaeal ribosometree [23].

Valentine’s [22] insight into theadaptive specializations of the archaeamay account for the putative protein-free PTC of some archaeal ribosomes,and more generally, it may explainwhy there are two prokaryote domainsthat are descendents of the eukaryoteancestor: one for rich environmentsand one for more metabolically chal-lenging circumstances. Since the eukar-yotes are the ancestral lineage fromwhich the divergence of archaea andbacteria are thought to have been drivenby reductive pressure [24–27], relaxedreductive pressure would allow eukar-yote ribosomes to remain more protein-aceous than prokaryote ribosomes.

Accordingly, one prediction of thisscenario is that the PTC of eukaryoteribosomes will turn out to be moreprotein-rich than those of prokaryotes.Another is that the rates of protein-depleted archaeal ribosomes undercomparable conditions may prove tobe slower than those of bacterial ribo-somes. Astonishingly, it is currentlyimpossible to locate in the literatureribosomal rates of translation alongwith growth rates with which to makemeaningful comparisons.

Ribosomes areribonucleoproteinparticles, period!

An evolutionary trade-off between RNAand protein for the adaptations of ribo-somes to the realities of growth con-straints is consistent with an earlierview of ribosome structure. Voorheeset al. [19] note that even in E. coli ribo-somes the PTC seems to be rich in RNA.

This was not too surprising since ‘‘every-where’’ in the bacterial ribosome is rich inRNA. Prior to the revelation of RNAworld, an emergent idea was that riboso-mal RNA and proteins are not segregatedbut are intermixed with cooperative clus-ters of proteins organized around specificRNA domains [28]. Thiswas a data-driveninterpretation based on biochemicalstudies of ribosomes modified bymutations, antibiotics, cross-linkingagents, site-specific chemical agents,and partial assembly in vitro. TheE. coli ribosome along with all other ribo-somes was seen as a RNP particle, not asan RNA particle in protein drag.

Here, the self assembly of ribosomesgenerates mixed neighborhoods of RNAand proteins that provide the workingsurfaces for the translating ribosome[28]. The fact that a protein and anRNA domain cooperate in the assemblyprocess would not preclude either oneas a potential ligand for intermediates intranslation. In effect, the strict divisionof labor for ribosomal RNA andprotein proclaimed by Cech [18] is notnecessarily respected by ribosomes.Amino acyl-tRNAs, protein factors,and mRNAs are all relatively huge sub-strates. Accordingly, the binding ofthese substrates to ribosomal sites andtheir movements during translationspan correspondingly large RNPdomains [28]. This data-driven, low-resolution model has been well substan-tiated by high-resolution structural datafor the components of bacterial ribo-somes. For example, all proteins withthe exception of the oligomeric proteinL7/L12 are directly bound to ribosomalRNA [29]. Likewise, participation of RNAand protein at functional sites is con-firmed by high-resolution structures for70S ribosomes complexed with A-siteand P-site tRNAs [19].

Polypeptide world

Gilbert’s ‘‘big bang’’ scenario [1] can bereplaced by a data-driven, gradualistscheme in which a prebiotic polypeptideworld evolved into amodern RNPworld.Intelligent discussions of a putative pol-ypeptide world are found in Cairns-Smith [30], Kaufman [31], and Egel[32]. Here, the principle novelty is achemical scheme in which randomlygenerated, catalytic polypeptides may

have been selected through a proteolyticmechanism that enriches the popu-lation of polypeptides with biologicallyrelevant activities without the interven-tion of coding by RNA.

Highly relevant to this enrichmentscheme are the workings of a ubiquitouscellular catabolic pathway that pre-serves the stability of high-density cel-lular proteomes by destroying proteinsthat present aberrant sequences [33–35].Modern proteins are made up of one toseveral compact or folded domains (e.g.fold superfamilies or FSFs) along withterminal as well as interspersed linkersequences. The provision of robust fold-ing pathways for polypeptides synthes-ized on ribosomes is part of normalsequence selection in evolution [36].The result is that linkers are bound atsurfaces of domains or to other macro-molecules so that they, together withthe self-organizing domains, are pro-tected from the depredations of ubiqui-tous proteolytic ‘‘machines’’ suchas proteasomes [33–35]. In general,degrading enzymes require a sequencethat is less than 10 A in diameter to beaccommodated within the proteolyticsite [37]. Clearly, an amino acidsequence that is organized into a com-pact domain or one that is stably boundto ligands such as other domains, lipidmembranes, or chemical substrates isnot likely to pass through the 10 A gate-way leading to proteolysis.

The presentation of proteolyticallyaccessible amino acid sequences eitheras unbound linkers or as unfoldeddomains can result frommutation, trans-lation errors, and chemical modification,induced conformational rearrange-ments, or failure to bind a ligand [33–36]. Systematic destruction of aberrantproteins by proteasomes and their homo-logs rids cellular proteomes of potentialseeds for aggregation and precipitation,which otherwise would be lethal to cells.Indeed, the maintenance of the highprotein densities characteristic of all cel-lular proteomes has had a profoundinfluence on the evolution of cells[38, 26]. Human degenerative diseasesthat arise from mutations affectingprotein folding or from defective protea-some function underscore the impact ofproteolytic surveillance systems [39–42].The metabolic consequence of suchproteolytic surveillance is that proteindegradation is a significant catabolic

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flow in healthy cells. As much as thirtypercent of newly synthesized proteinsare destroyed by proteolysis [43], whilea steady-state background of proteinturnover of about two percent per houris observed in growing cells [44].

This ubiquitous catabolic flow wasintroduced to illustrate a particularlyinformative dimension of protein evol-ution: this is that mutant variantspresenting unfolded domains or unpro-tected strings would tend to be culledfrom populations as lethal alleles [45].For example, even an allele that isexpressed with a fully active catalyticsite in an unstable fold might be a lethalallele if its substrate does not protect thedomain from destruction. Accordingly,protein sequence evolution is not a ran-dom walk through amino acid sequencespace. It is canalized through modularsequences that are expressed as self-organized compacted domains and pro-tected linker sequences, which are thesole survivors of selection by proteolyticmachines [45]. Indeed, such modulardomains may correspond to the prod-ucts of exons though it must be said thatprotein chemists insist that the bound-aries of exons and domains are not iden-tical [46]. Perhaps a measure ofsequence drift over time may accountfor these boundary discrepancies.

Numerous enzymes that synthesizeoligopeptides without the assistance ofan mRNA or the rest of the moderntranslation apparatus have beendescribed in all three superkingdoms[47–49]. Their products are short pepti-des with activities as diverse as anti-biotics as well as neural transmittersand that ubiquity itself speaks for theirancient origins. Though these non-ribo-somal peptidyl transferases (NPTases)produce oligopeptides with definedamino acid sequences, more primitiveancestral enzymatic activities lackingsuch amino acid specificity can beexpected to have arisen in stochasticpopulations of polypeptides assembledby geochemical mechanisms [30–32].So, the second tier of prebiotic polypep-tides may have been composed of ran-dom sequences produced by NPTases.In this second phase, NPTases would beable to autocatalytically increase therates with which random polypeptidesequences could accumulate.

Among sufficiently large popu-lations of random amino acid

sequences, some small fraction wouldbe expected to fold spontaneously intostable active domains with functionssuch as the NPTases and proteolyticenzymes as well as any number of‘‘metabolic’’ activities. This expectationis supported by the observations ofKeefe and Szostak [50], who recoveredbiological activities from relativelysmall populations of random amino acidsequences polymerized in vitro. Thus,stochastic populations of polypeptidescontaining NPTases and proteolyticactivities etc. could generate a dynamicsituation in which random polypeptidesare continuously synthesized, but mostof these would be recycled by proteasesthat regenerate the amino acids. Thesmall core of polypeptides that is resist-ant to proteolysis would be, by hypoth-esis, that which could spontaneouslyfold into stable compacted domains,which are resistant to enzymatic attack.These diverse domains, stable toproteolysis, would be enriched for acorresponding diversity of catalyticactivities. This proposal can be testedin systems such as those described byKeefe and Szostak [50] to determinewhether exposure of randomly synthes-ized polypeptides to proteolysisincreases the specific activities of thepolypeptides for assayable functions.

Finally, it is reasonable to expect thatfolding into active, proteolysis-resistantdomains is facilitated by the binding ofcofactors and/or substrates specificto individual catalytic polypeptides.Here, the specific binding of small mol-ecules from the geochemical system totheir cognate polypeptides would tendthrough the law of mass action to trapthe polypeptides in a folded state that isresistant to proteolysis. In this way, pre-biotic geochemistry may have selectedpolypeptides that mediate cycles ofmetabolic intermediates. The predictedeffect of substrates on the proteolyticstabilities of random polypeptides canalso be studied in vitro as above.

Three steps to cells

In fact, a prebiotic polypeptide scenariomight account for the origins of theribonucleotides that accumulated priorto the debut of RNA. There has been atortured history of attempts to generateribonucleotides in the laboratory as

precursors required for the RNAs ofa putative prebiotic world [51, 52].Recently, an ingenious approach hassucceeded to synthesize pyrimidineribonucleotides in a reaction that mightconceivably have been supported byvolcanic outgassing [52]. However, thereare still questions about the feasibility ofthis reaction scheme in a prebioticenvironment and it still remains toaccount for the purine ribonucleotides[51, 52]. Finally, the scheme of Powneret al. [52] is not the textbook pathway forcellular biosynthesis of ribonucleotidesfrom three amino acids. For thesereasons it is still worth considering thealternative in which the ribonucleotideswere accumulated prebiotically accord-ing to the textbook schemes that mod-ern polypeptides follow.

Several innovations are required toenable a transition from the prebioticworld of polypeptides to biological sys-tems. One is the introduction of anmRNA analog to encode proteins, andassociated with that an analog of theaminoacyl-tRNA adapter to translatemRNAs, as in Gilbert’s proposal [1].However, the adapter initially neednot have been a tRNA because the abil-ity to recognize and to bind nucleotidetriplets in mRNA is not unique to RNA.Proteins in the form of release factorscan bind and recognize triplet codonswith a discriminatory capacity muchgreater than that of aminoacyl-tRNAs[53]. Likewise, proteins in the form ofaminoacyl-tRNA synthases recognizesubtle side chain differences of aminoacids and match these with cognatetRNA structures. Without this polypep-tide function the adapter hypothesiswould be just another RNA worldfantasy.

So, whatever the disadvantages maybe, a primitive translation machine mayhave exploited proteins for all functionsexcept that of mRNA. That is to say, theevolution and tuning of the translationmachinery may have been driven by theintroduction and progressive expansionof RNA functions at the expense ofprotein. Such a tendency is consistentwith the notion that the large protein-rich eukaryote ribosome is ancestral tothe more economic archaeal ribosomesthat have evolved with reduced proteincomplements under stringent reductivepressures ([24–27], Wang et al., inpreparation).



Hypotheses

A second elementary innovation wouldentail the ability to copy mRNA fromcomplementary polynucleotides. Aconservative guess is that this inno-vation arose when polypeptide-depen-dent random synthesis of polyribo-nucleotides was transformed into anenzymatic copy mechanism. Initially,it is simplest to imagine that makingmRNA copies as transcripts and repli-cating them as genomes were one andthe same function. At some point thesetwo functions were separated as inmodern cells.

The third innovation, which isessential for the evolution of geneticallydetermined sequences, is the creation ofa link between individual genetic deter-minants and their products to enableselection of competitive characters.Evolution has chosen cellular bound-aries to provide this link. As a result,the composite features of a cell’s pro-teome can be selected by their inte-grated influence on, for example, acell’s growth phenotype. Without sucha link, selection is impossible.Obviously, selection was for this veryreason impossible in the original formu-lation of RNA world [1].

There are at least two ways to thinkabout the origins of cellular bound-aries. One is to imagine that short poly-peptides with amphiphilic characterthat could mimic lipid moleculesformed the first membranous bound-aries as for example in nanotubulesmade in laboratories [32, 54, 55]. Theother is to employ lipids synthesized bypolypeptides to spontaneously formvesicles. In appropriate solutions lip-ids associate and behave astonishinglylike cellular membranes in modelexperiments [56–58]. Of course somecombination of both lipids and amphi-philic peptides may be more relevant.Since the lipids as well as lipid-likepeptides are potentially products ofpolypeptide enzymes, their emergencein the prebiotic world would not beexceptional.

Since each of these three innovationsis in the present view a spontaneousexpression of protein chemistry, it isassumed that the order of their appear-ances was random. However, all threeinnovations would have to cometogether to enable the transition fromprebiotic to biological systems.

Conclusions

In the 1970s I attended an EMBOWorkshop at which a French philoso-pher of science asserted thatMolecular Biology was merely ‘‘roman-tic idealism’’. I of course indignantlyrejected that comment out of hand.But 30 years later while trying to under-stand the explanatory power of Gilbert’s‘‘big bang’’ theory [1] for the origin of lifeI drifted back to that comment aboutromantic idealism. And, I began tounderstand why RNA world did notneed to explain anything in order tobe attractive to nearly all molecularbiologists.

RNA world is an expression of theinfatuation of molecular biologists withbase pairing in nucleic acids played outin a one-dimensional space with noreference to time or energy: ‘‘DNAmakes RNA makes protein’’ [59]. Thisis not chemistry. It is genetics. And,when true believers apply their geneticdogma to studies of chemical mechan-ism, the result is ‘‘the secret of the cage’’and a five order of magnitude kineticdiscrepancy described as a ‘‘fundamen-tal’’ similarity [12, 13, 6].

The positive side of this infatuationhas been the development of robustgenomics, especially bioinformatics.But RNA worlders are not likely to findmuch comfort in genome sequences. Avery recent phylogenomic study byCaetano-Anolles et al. [60] based onhundreds of fully sequenced genomeshas revealed a time line for cellular evol-ution in which protein domains (FSFs)that support conventional metabolicpathways precede the debut of thenucleic acids as well as the proteindomains associated with nucleic acidsin gene expression. This cellular timeline for the gradual elaboration ofRNA functions [60] is not inconsistentwith the thesis that the prebiotic worldwas a polypeptide world.

Protein folding into compactdomains is a kind of self coding.However, the protein code is not asimple iterative code that lends itselfto a copy mechanism. That is to say, itwould be difficult to build geneticsaround polypeptide interactions alone.On the other hand, self folding andproteolytic editing might be just goodenough to create a prebiotic chemical

platform from which a cellular geneticsystem might take off.

If my own initial reactions are any-thing to go by, RNA world or ‘‘RNAmakes RNA makes protein’’ has imme-diacy for molecular geneticists that islacking in the present scheme for a pre-biotic polypeptide world. Thus, theappeal of a polypeptide fold editingscheme rests on some familiarity withthe contours of protein structure as wellas with translational editing in moderncells. Regardless of its spontaneousappeal, I suggest that RNAworld shouldnow take its place on the shelf of ‘‘niceideas’’ along with Aristotle’s identifi-cations of whales as fish and the workerbee as a male.

AcknowledgmentsFor stimulation, critique, unpublishedinformation, and guidance in the liter-ature I am greatly indebted to O. Berg,G. Caetano-Anolles, L. Collins, M.Ehrenberg, R. Garrett, A. Harish, A.Liljas, D. Penny, D. Valentine, and M.Wang. My gratitude also goes to IrmgardWinkler for help with the manuscriptand to the Royal PhysiographicSociety, Lund, as well as the NobelCommittee for Chemistry at the RoyalSwedish Academy of Sciences,Stockholm, for generous support.

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46. Doolittle RF. 1995. The multiplicity ofdomains in proteins. Annu Rev Biochem 64:287–314.

47. Hancock REW, Chapple DS. 1999. Peptideantibiotics. Antimicrob Agents Chemother 43:1317–23.

48. Finking R, Marahiel MA. 2004. Biosynthesisof non-ribosomal peptide. Annu RevMicrobiol58: 453–88.

49. Marahiel MA. 2009. Working outside theprotein-synthesis rules: insights into non-ribo-somal peptide synthesis. J Pept Sci 15: 607–799.

50. Keefe AD, Szostak JW. 2001. Functionalproteins from a random-sequence library.Nature 410: 715–8.

51. Szostak JW. 2009. Systems chemistry onearly earth. Nature 459: 171–2.

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Biol Direct. 2012; 7: 23.Published online Jul 13, 2012. doi: 10.1186/17456150723

PMCID: PMC3495036

The RNA world hypothesis: the worst theory of the early evolution of life (exceptfor all the others)Harold S Bernhardt

Department of Biochemistry, University of Otago, P.O. Box 56, Dunedin, New ZealandCorresponding author.

Harold S Bernhardt: [email protected]

Received May 9, 2012; Accepted July 11, 2012.

Copyright ©2012 Bernhardt; licensee BioMed Central Ltd.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( http://creativecommons.org/licenses/by/2.0),w hich permits unrestricted use, distribution, and reproduction in any medium, provided the original w ork is properly cited.

This article has been cited by other articles in PMC.

Abstract

The problems associated with the RNA world hypothesis are well known. In the following I discuss some of thesedifficulties, some of the alternative hypotheses that have been proposed, and some of the problems with these alternativemodels. From a biosynthetic – as well as, arguably, evolutionary – perspective, DNA is a modified RNA, and so thechickenandegg dilemma of “which came first?” boils down to a choice between RNA and protein. This is not just aquestion of cause and effect, but also one of statistical likelihood, as the chance of two such different types ofmacromolecule arising simultaneously would appear unlikely. The RNA world hypothesis is an example of a ‘top down’(or should it be ‘present back’?) approach to early evolution: how can we simplify modern biological systems to give aplausible evolutionary pathway that preserves continuity of function? The discovery that RNA possesses catalytic abilityprovides a potential solution: a single macromolecule could have originally carried out both replication and catalysis. RNA– which constitutes the genome of RNA viruses, and catalyzes peptide synthesis on the ribosome – could have been boththe chicken and the egg! However, the following objections have been raised to the RNA world hypothesis: (i) RNA istoo complex a molecule to have arisen prebiotically; (ii) RNA is inherently unstable; (iii) catalysis is a relatively rareproperty of long RNA sequences only; and (iv) the catalytic repertoire of RNA is too limited. I will offer some possibleresponses to these objections in the light of work by our and other labs. Finally, I will critically discuss an alternativetheory to the RNA world hypothesis known as ‘proteins first’, which holds that proteins either preceded RNA inevolution, or – at the very least – that proteins and RNA coevolved. I will argue that, while theoretically possible, such ahypothesis is probably unprovable, and that the RNA world hypothesis, although far from perfect or complete, is the bestwe currently have to help understand the backstory to contemporary biology.

Reviewers

This article was reviewed by Eugene Koonin, Anthony Poole and Michael Yarus (nominated by Laura Landweber).

Keywords: RNA world hypothesis, Proteins first, Acidic pH, tRNA introns, Small ribozymes

Background

The problems associated with the RNA world hypothesis are well known, not least to its proponents [1,2]. In the

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following, I discuss some of these difficulties, some of the alternative hypotheses that have been proposed (including the‘proteins first’ hypothesis), and some of the problems with these alternative models. As part of the discussion, I highlightthe support provided to the RNA world concept by the discovery of some extremely small ribozymes. The activities ofthese provide support for proposals we have made previously for the identity of the first tRNA [3], for the origin ofcoded ribosomal protein synthesis [4], and for the evolution of an RNA world at acidic pH [5] (see also [6]). I alsorevisit the proposal for a replicase origin of the ribosome, and what has become the most commonly held model for theorigin of tRNA.

In modern biological systems, the components of DNA are synthesized from RNA components [7], and it thereforemakes sense to view DNA as a modified RNA. Similarly, the ribosome – the universal cellular machine that makesproteins – is composed mainly of RNA, and RNA is its active component, although there are indications that proteinsmay be playing an increasing role in some instances e.g.[8,9] (even in the case of nonribosomal peptide synthesis[10,11], the protein enzyme complexes that synthesize other proteins are of course themselves synthesized on theribosome). RNA functions as both catalyst (e.g. in peptide synthesis and tRNA maturation) and genome (in RNA virusessuch as HIV and influenza viruses). In contrast to nucleic acids, which associate according to the rules of base paircomplementarity, the intricacies of protein structure do not – normally – allow for an easy mechanism of replication,which presumably explains the evolution of a coded system for their synthesis (for an interesting discussion of thecontrasting molecular requirements for replication and catalysis, see [12]). Parsimony at least would seem to favour ascenario in which functions carried out by two classes of macromolecules in the modern system were, at an earlier stage,carried out by only one (for an alternative view however, see [13]). So which came first, the chicken or the egg? Proteinor RNA? This is an underlying current in the debate surrounding the RNA world hypothesis, which I address when Idiscuss the ‘proteins first’ hypothesis.

Before beginning, it is important to clear up a common source of confusion. The RNA world hypothesis does notnecessarily imply that RNA was the first replicating molecule to appear on the Earth (although a new paper by Bennerand colleagues argues that this was, in fact, the case [14]). The more general claim is that the RNA world comprised astage of evolution preceding – perhaps immediately – the RNA/protein/DNA world we now inhabit. In this way, thehypothesis is not incompatible with models such as the ‘crystalsasgenes’ concept of CairnsSmith [15], which proposesthat the first replicators were imperfectioncontaining layers of clay that were able to pass on these imperfections toproceeding layers (unfortunately, one experimental test of CairnsSmith’s model suggests that replicated defects arequickly overrun by random defects or noise [16]). Similarly, it has been hypothesized that RNA was preceded inevolution by a nucleic acid analogue – for example, one in which glycerol replaces ribose in the phosphodiester backbone– though pathways for the prebiotic synthesis of many such analogues are even less plausible than for RNA itself [17].

Discussion

The following objections to the RNA world hypothesis have been raised:

RNA is too complex a molecule to have arisen prebiotically

RNA is an extremely complex molecule, with four different nitrogencontaining heterocycles hanging off a backbone ofalternating phosphate and Dribose groups joined by 3′,5′ linkages. Although there are a number of problems with itsprebiotic synthesis, there are a few indications that these may not be insurmountable. Following on from the earlier workof Sanchez and Orgel [18], Powner, Sutherland and colleagues [19] have published a pathway for the synthesis ofpyrimidine nucleotides utilizing plausibly prebiotic precursor molecules, albeit with the necessity of their timed delivery(this requirement for timed delivery has been criticized by Benner and colleagues [14], although most origin of life modelsinvoke a succession of changing conditions, dealing as they do with the evolution of chemical systems over time; what iscritical is the plausibility of the changes). A particularly interesting aspect of the pathway is the use of UV light as amethod of isolating the naturally occurring nucleotides [18,19], suggesting a possible means of nucleotide selection (seealso [20]).



Although RNA is constructed with uniform 3′,5linked backbones, recent work by Szostak and colleagues hasdemonstrated that ribozymes and RNA aptamers retain partial function when the standard 3′,5′linkages are replacedwith a mixture of 3′,5′ and 2′,5′ linkages, suggesting that a degree of heterogeneity may be compatible with (or evenbeneficial to) RNA function and synthesis (J. Szostak, pers. commun.; [21]). This complements an earlier study by Ertemand Ferris [22] that showed that poly C oligonucleotides with mixed 3′,5′ and 2′,5′linkages are able to serve astemplates for the synthesis of poly G oligonucleotides by nonenzymatic replication. Such work suggests that ancestralsystems may not have been as tightly constrained as they are today.

Due perhaps to the molecular complexity of nucleic acids, metabolismfirst models (as opposed to replicationfirstmodels such as the RNA world hypothesis) highlight the importance of the initial generation of small molecules throughchemical or metabolic cycles. Establishment of a plausible energy source is a critical aspect of these models, some ofwhich propose that life arose in the vicinity of hot alkaline (pH 9–11) undersea hydrothermal vents, with energyprovided by pH and temperature gradients between the vent and the cooler, more acidic ocean [2326]. In some ways,metabolismfirst models appear not to conflict with the RNA world hypothesis, as they potentially offer a solution to thedifficulty of ribonucleotide and RNA synthesis. A large point of difference, however, comes with the claim that suchnucleic acidfree systems are capable of Darwinian evolution. Addressing this claim, Vasas et al.[27] have reported alack of evolvability in such systems, while Benner and colleagues have noted the lack of experimental support fromspecific chemical models [14]. A more recent paper by Vasas et al.[28], while seemingly contradicting their earlierpaper, uses a computational modeling approach without reference to a realworld chemical system (something noted bytwo of the reviewers in their published reviews).

RNA is inherently unstable

RNA is often considered too unstable to have accumulated in the prebiotic environment. RNA is particularly labile atmoderate to high temperatures, and thus a number of groups have proposed the RNA world may have evolved on ice,possibly in the eutectic phase (a liquid phase within the ice solid) [2933]. Two of these studies [31,32] demonstratedmaximal ribozymic activity at −7 to −8°C, possibly due to the combined effects of increased RNA concentration andlowered water activity. A possible difficulty with this scenario is that RNA sequences have an increased tendency to basepair at such temperatures, leading in some cases to the formation of intermolecular complexes [34] that potentially couldreduce catalytic activity.

A further problem is the susceptibility of RNA to basecatalyzed hydrolysis at pH >6 [35]. The phosphodiester bonds ofthe RNA backbone and the ester bond between tRNAs and amino acids – something similar to which would have beencritical for the evolution of ribosomal protein synthesis – are both more stable at pH 4–5 [5,6]. With our proposal forRNA world evolution at acidic pH [5], we have suggested that the primordial ‘soup’ may have been more likevinaigrette, while Hanczyc [36] has drawn a comparison with mayonnaise, with its emulsified mixture of oil in water (inlight of these, could there be potential for food science to provide insights for origin of life studies?) While Mg isimportant for stabilizing RNA secondary and tertiary structure, high Mg concentrations also catalyze RNAdegradation, which has been identified as a particular problem in the case of RNA template copying [21]. Here too,acidic pH offers a possible solution, as the positive charge on protonated cytosine and adenosine residues in acidicconditions may reduce the requirement for divalent cations. For example, a selfcleaving ribozyme with maximum activityat pH 4 isolated by in vitro selection, is active in the absence of divalent ions (including Mg ) [37]. RNA secondary(and tertiary) structure would appear to be compatible with the presence of protonated nucleotides, as we have found anincreased number of potentially protonated AC base pair ‘mismatches’ in the tRNAs from acidophilic archaeal specieswith reported cytoplasmic pHs of 4.66.2 [5].

Catalysis is a relatively rare property of long RNA sequences only

The RNA world hypothesis has been criticized because of the belief that long RNA sequences are needed for catalyticactivity, and for the enormous numbers of randomized sequences required to isolate catalytic and binding functions using

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in vitro selection. For example, the best ribozyme replicase created so far – able to replicate an impressive 95nucleotide stretch of RNA – is ~190 nucleotides in length [38], far too long a sequence to have arisen through anyconceivable process of random assembly. And typically 10,000,000,000,0001,000,000,000,000,000 randomizedRNA molecules are required as a starting point for the isolation of ribozymic and/or binding activity in in vitro selectionexperiments, completely divorced from the probable prebiotic situation. As Charles Carter, in a published review of ourrecent paper in Biology Direct[5], puts it:

“I, for one, have never subscribed to this view of the origin of life, and I am by no means alone. The RNA worldhypothesis is driven almost entirely by the flow of data from very high technology combinatorial libraries, whoserelationship to the prebiotic world is anything but worthy of “unanimous support”. There are several serious problemsassociated with it, and I view it as little more than a popular fantasy” (reviewer's report in [5]).

10 10 is an awful lot of RNA molecules. However, the discovery of a number of extremely short ribozymes suggeststhat long sequences – and hence the huge numbers of RNA molecules required to sample the necessary sequence space– might not have been necessary. In a section titled ‘Miniribozymes: small is beautiful, Landweber and colleagues [31]discuss a number of such small ribozymes, including a minimal size active duplex of only 7 nucleotides that selfcleaves.Regarding the relatively modest rate enhancement of this miniribozyme – three orders of magnitude less than the parentribozyme from which it is derived – the authors conclude: “the smallest molecules are likely to arise first, and any rateenhancement would have been beneficial in a prebiotic setting” [31]. Another, closely related, miniribozyme can ligate asmall RNA to its 5′ end, requiring only a single(!) bulged nucleotide in the context of a larger basepaired structurecontaining a strand break. Interestingly, the selfcleaving 7nucleotide sequence forms a part of the ligase ribozyme,demonstrating the closeness in sequence space of the two, albeit related, functions [31]. Equally as interesting from anRNA world perspective, Yarus and colleagues have recently isolated by in vitro selection a ribozyme that is able to betruncated to just 5 nucleotides, while retaining its ability to catalyze the aminoacylation in trans of a 4nucleotide RNAsubstrate [39]. Remarkably, only 3 nucleotides are responsible for this activity: 2 in the ribozyme and 1 in the substrate.In fact, even this much is not required: a variant of the parent ribozyme with a mutation of 1 of the 3 conservednucleotides is able to aminoacylate a substrate variant with the sequence GCCA (similar to the universal aminoacylated 3′terminus of tRNA), albeit at a reduced rate [40] (we have previously proposed a possible sequence for anaminoacylating ribozyme based on this variant that could have basepaired with the universal 3′ CCA termini of tRNAs(and proposed RNA hairpin precursors [41,3] through a double helix interaction, while also forming specific triple helixinteractions – at acidic pH – with other nucleotides in the tRNA [5]). As with the small ribozymes discussed byLandweber and colleagues, the rates of aminoacylation of Yarus' ribozymes are somewhat underwhelming: that of theoriginal 5nucleotide ribozyme is only 25fold higher than the uncatalyzed rate [39], while that of the variant is only 6foldhigher than the uncatalyzed rate [40] (for further discussion of the implications of such tiny ribozymes see [42], and [31]and references therein).

Although not quite as small as the ribozymes discussed above, Gross and colleagues have demonstrated that 12nucleotide and 20nucleotide nuclear tRNA introns from Arabidopsis thaliana and Homo sapiens – understood tobe cleaved by protein enzymes in vivo – are able to selfcleave in the presence of 10 mMMg , 0.5 mM spermine and0.4% Triton X100 [4345]. Although the introns form part of a larger pretRNA sequence, the nucleotides responsiblefor selfexcision are possibly confined to a 3 or 4nucleotide bulge region. The discovery of this intrinsic activity (whichadmittedly requires the presence of a low concentration of surfactant) supports previous proposals for the origin of tRNA[41,3,4]. Although there exist a number of other models for the origin of tRNA (one of which is discussed in detail in thefollowing section), a hairpin duplicationligation origin stands as a credible hypothesis [41,3] that has received supportfrom a number of sources [4648]. Briefly, the idea first proposed by Di Giulio [41] is that two (either identical orvery similar) hairpins, approximately half the size of contemporary tRNA, formed a ligated duplex due to the symmetry ofbasepairing interactions, possibly by an intronmediated mechanism [49] (Figure 1). It has been proposed previouslythat contemporary proteinspliced nuclear tRNA introns are descended from an ancestral selfsplicing group Itype intronthat catalyzed the ancestral ligation [49] (as depicted in Figure 1, the ancestral tRNA intron may have derived from a 3′

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extension of one of the precursor hairpins by a transcriptional runoff error). The findings of Gross and colleagues [4345]indicate that some normally proteincleaved nuclear tRNA introns have partially retained the ability to selfcleave. Thisability to selfcleave implies the reverse reaction – selfligation – is also possible, which could have produced the ligatedintroncontaining hairpin intermediate; subsequent intron selfcleavage could have produced the first prototRNA [49](Figure 1).

Figure 1A proposal for the origin of tRNA through the ligation of a hairpin duplexcatalyzed by an ancestral selfsplicing group Itype intron based onproposals by Di Giulio [41], and Dick and Schamel [49]. In this depiction,the intron is shown as originating ...

The catalytic repertoire of RNA is too limited

It has been suggested that the probable metabolic requirements of an RNA world [50] would have exceeded thecatalytic capacity of RNA. The majority of naturally occurring ribozymes catalyze phosphoryl transfer reactions – themaking and breaking of RNA phosphodiester bonds [51]. Although the most efficient of these ribozymes catalyze thereaction at a comparable rate to protein enzymes – and in vitro selection has isolated ribozymes with a far wider rangeof catalytic abilities [9,51] – the estimate of proteins being one million times fitter than RNA as catalysts seemsreasonable, presumably due to proteins being composed of 22 chemically rather different amino acids as opposed to the4 very similar nucleotides of RNA [12].

It is frequently forgotten however that proteins too have their catalytic limitations: after all, many enzyme active sitescontain cofactors and/or coordinated metal ions, suggesting that some reactions are ‘too hard’ for proteins as well (it isestimated that ~50% of proteins are metalloproteins [52], although of course not all these metal ions are found at theactive site). RNA riboswitches bind a range of protein cofactors, such as flavin mononucleotide, thiamine pyrophosphate,tetrahydrofolate, Sadenosylmethionine and adenosylcobalamin (a form of vitamin B12) [53]. In the case of the glmSriboswitch/ribozyme, the metabolite glucosamine6phosphate binds in the active site and appears to participate incatalysis [54]. Because of the ability of these naturally occurring RNA riboswitches to bind protein enzyme cofactors,and because many of these cofactors possess nonfunctional fragments of RNA – one of the earliest pointers to apossible ancestral RNA world [55] – it is likely that at least some of the cofactors now used by proteins were handeddown directly from the RNA world, where they played a similar if not identical role in assisting catalytic function [53].

One of the arguments for the RNA world hypothesis comes from the observation that RNAs are, in most cases, worsecatalysts than proteins. This implies that their presence in modern biological systems can best be explained by their beingremnants of an earlier stage of evolution, which were too embedded in biological systems to allow replacement easily. Analternative explanation is that they were coopted by a protein world due to their superior properties for the particularfunctions they perform. While such an explanation seems intuitively less likely, surprisingly it is held by some proponentsof the ‘proteins first’ model [5660] (discussed in more detail below).

Proteins first

An increasingly strident view is that protein either preceded RNA in evolution or, at the very least, that RNA and proteincoevolved, in what is known as the ‘proteins (or peptides) first’ hypothesis [5660]. Take, for example, Charles Kurlandin his 2010 piece in Bioessays[57], which is utterly scathing of the RNA world hypothesis and its fellow travelers:

“[The RNA world hypothesis] has been reduced by ritual abuse to something like a creationist mantra”, and

“[The] RNA world is an expression of the infatuation of molecular biologists with base pairing in nucleic acids played outin a onedimensional space with no reference to time or energy” [57].

On a less emotional note, Harish and CaetanoAnollés [60] earlier this year published a phylogenetic analysis of






ribosomal RNA and ribosomal proteins, concluding that the oldest region of the ribosome is a helical stem of the smallribosomal subunit RNA and the ribosomal protein that binds to it. As this helical stem has the important roles in themodern ribosome of decoding the mRNA message and in the movement of the two subunits relative to each other(including translocation of the mRNA message and tRNAs), Harish and CaetanoAnollés conclude that the originalfunction of the ribosome was as an RNA replicase (this idea, which has been suggested previously, is discussed in detailin the following section). In addition, because RNA and protein components of the ribosome apparently have similarages, Harish and CaetanoAnollés surmise that peptide synthesis has always been carried out by RNA in association withproteins, as is the case with the modern ribosome.

Without debating the merits or otherwise of their phylogenetic techniques, the most serious objection to these conclusionsis that phylogenetic analysis has the limitation that it can only analyze the protein sequence record as it has been capturedin DNA (this is true even for a phylogenetic analysis based on protein fold structures, as the only record we possess ofthese folds is their primary amino acid sequence as captured in the DNA). Therefore, any information we can recovercan only date from the advent of coded protein synthesis, as that is the point at which protein sequence became coded innucleic acid. In an online report [61] on Harish and CaetanoAnollés’ paper, Russell Doolittle makes this same point:

“This is a very engaging and provocative article by one of the most innovative and productive researchers in the field ofprotein evolution,” said University of California at San Diego research professor Russell Doolittle, who was not involvedin the study. Doolittle remains puzzled, however, by “the notion that some early proteins were made before the evolutionof the ribosome as a proteinmanufacturing system.” He wondered how – if proteins were more ancient than theribosomal machinery that today produces most of them –“the amino acid sequences of those early proteins were‘remembered’ and incorporated into the new system.” [61].

To which, CaetanoAnollés’ reported response is slightly puzzling:

“It requires understanding the boundaries of emergent biological functions during the very early stages of proteinevolution. However, the proteins that catalyze nonribosomal protein synthesis – a complex and apparently universalassemblyline process of the cell that does not involve RNA molecules and can still retain high levels of specificity – aremore ancient than ribosomal proteins. It is therefore likely that the ribosomes were not the first biological machines tosynthesize proteins.” ( [61]; italics in original).

It is certainly possible that there were functional noncoded peptides prior to the advent of coded protein synthesis. Thesecould have been formed either through random processes, by noncoded ribosomal synthesis prior to the advent of coding[4], by nonribosomal peptide synthesis catalyzed by specific ribozymes (analogous to nonribosomal peptide synthesiscatalyzed by protein enzymes in modern systems [62]), or by some combination of the above. It seems highly unlikely,however, that proteins synthesized proteins prior to the advent of the ribosome, as this would appear to suggest an infiniteregression series. As Doolittle [61] suggests, the critical point is that once coding evolved, the sequences of thesenoncoded proteins would have needed to be recapitulated by coded proteins; therefore the phylogenetic signal wouldonly go back to the point of recapitulation. Put another way, the earliest proteins phylogenetically speaking will be the firstproteins that were coded for. Presumably, if these sequences can still be detected in modern genomes, they would tendto be relatively short and somewhat indistinct traces only, as one might expect for the first proteins produced by arudimentary ribosome. In a sense then, one can say that the advent of coded protein synthesis has drawn a veil over theprevious life of proteins. Although it seems unlikely, complex proteins may have existed prior to this, but – as all recordof them has been erased by the advent of coding – that is as much as we can say (for an indepth discussion of theimplications of nonribosomal peptide synthesis for the RNA world hypothesis, see [62]).

RNA replicase origin of the ribosome

As mentioned above, Harish and CaetanoAnollés are not the first to suggest an RNA replicase origin of the ribosome(or small ribosomal subunit). The idea, which was possibly first proposed by Weiss and Cherry [63], is that “the ancestorof small subunit RNA was an RNA replicase that used oligonucleotides as a substrate” [63]. The hypothesis has grown in



scope to include the use of excised tRNA anticodons as the source of oligonucleotides, with the energy required forligation provided by concomitant peptide bond formation [6466]. However, as pointed out by Wolf and Koonin [67],such a ligase would have required a molecular machinery at least as complex as the modern ribosome, which wouldmake it an unlikely evolutionary forerunner. This notwithstanding, Weiss and Cherry’s original, simpler, model may havesome merit. If, as has been recently suggested, early RNA replication was performed by the ligation of shortoligonucleotides [68,69], or by a combination of nucleotide polymerization and oligonucleotide ligation [21], a ‘decoding’RNA able to proofread triplet base pair interactions for accuracy – similar to its role in the modern ribosome ofmaintaining the fidelity of the triplet codonanticodon interaction – might have played an important role. Interestingly, a49nucleotide hairpin comprising part of the decoding site of the small ribosomal subunit RNA has been found to bindboth poly U oligonucleotide and the tRNA anticodon stemloop in a similar fashion to the entire small subunit [70].This hairpin contains the two mobile nucleotides A and A (numbered according to the Escherichia coli smallribosomal subunit RNA sequence) that proofread the anticodoncodon helix in the modern ribosome [71]. It would beinteresting to test whether this hairpin is able to enhance the rate and/or accuracy of nonenzymatic ligation using a singlestranded RNA ‘template’ and short complementary oligonucleotides. If an enhancement were indeed demonstrated, sucha mechanism would be analogous to that utilized by the large ribosomal subunit, for which substrate positioning of the twotRNAs may constitute one of its main roles in catalyzing peptide synthesis [72].

As part of their model of early RNA replication by oligonucleotide ligation, Manrubia and colleagues propose that anincrease in the catalytic rate of the replicase/ligase would have occurred with an increase in sequence length through aprocess of bootstrapping [68,69]. Furthermore, they suggest that the first RNA replication possibly had a high errorrate:

“Highly mutagenic replication processes could have produced relatively large repertoires of short, genetically differentmolecules, some of them folding into secondary/tertiary structures able to perform selectable functions” [68].

Similarly, we have proposed that, in an RNA world evolving at acidic pH, nonstandard base pairing interactions due tobase protonation could have provided a means of increasing RNA sequence variation through nonenzymatic replication[5].

The origin of tRNA

Wiener and Maizels’ genomic tag hypothesis proposes that the 3′ (or ‘top’) half of tRNA originally functioned as a tagdemarking the 3′end of genomic RNAs for replication, and thus was the first part of tRNA to evolve [73]. Sun andCaetanoAnollés [74,75] have published phylogenetic evidence that they believe supports the genomic tag hypothesis byconfirming, “that the ‘top half’ of tRNA is more ancient than the ‘bottom half’” [75]. Noller [76] has observed that thetRNA top half (comprising the T arm and the acceptor stem – including the amino acid binding site) interacts almostexclusively with the large ribosomal subunit, while the bottom half (comprising the D and anticodon arms) interacts almostexclusively with the small subunit. Because peptide synthesis (a function of the large subunit) is usually viewed as moreancestral than decoding (a function of the small subunit) – a view which has support from a structural analysis by Bokovand Steinberg [77] – the top half of tRNA (which interacts with the large subunit) has been viewed as being moreancestral than the bottom half [73,78]. However, this ‘standard model’ for the origin of tRNA, and the results of Sun andCaetanoAnollés that support this model [74,75], are apparently both in conflict with Harish and CaetanoAnollés’ [60]more recent findings on the relative ages of the ribosomal subunits. As described above, these findings suggest that thesmall ribosomal subunit was the first to evolve, which is difficult to reconcile with the fact that the bottom half of tRNA(with which the small subunit mainly interacts), is, by theirs [74,75] and others [73,78] estimation, the newer half oftRNA. Equally, their finding that the large ribosomal subunit evolved more recently [60] is difficult to reconcile with thefact that the top half of tRNA (with which the large subunit mainly interacts), is, by theirs and others estimation, the olderhalf of tRNA. Incidentally, CaetanoAnollés and colleagues’ finding [75,79,80] that the most ancient tRNAs coded forselenocysteine, tyrosine, serine and leucine not only runs counter to other work in the area (see e.g.[81]), but – as thesetRNAs all possess long variable arms – appears to contradict their own finding that the “variable region was the laststructural addition to the molecular repertoire of evolving tRNA substructures” [74].

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As discussed above, a plausible scenario for the origin of tRNA is the duplication and subsequent ligation of an RNAhairpin approximately half the length of modern tRNA (or alternatively the ligation of two very similar hairpins) [41,3],with ligation possibly catalyzed by an ancestral selfcleaving intron [49] (see Figure 1). An important implication of suchan origin is that both tRNA halves are of equal antiquity, as both would have to be present for ligation to occur!However, due to the symmetry of the tRNA molecule, the top half, which is considered to be the more ancient, is in factmore ancientlike, as it retains the basepaired 3′ and 5′ ends of the original hairpin from which it derives. In contrast, thebottom half, considered to be the more recently acquired, contains the ‘join’ between the two hairpins, which has alteredthe conformation of the original hairpin, giving this bottom half a new structure. If one accepts a hairpin duplicationligation origin of tRNA, this explains why the top half of tRNA interacts with the peptidyl transferase region of the largeribosomal subunit: it is because this half retains the same structure (and possibly nucleotide sequence) as the hairpin fromwhich it derives, which originally interacted with the peptidyl transferase region of the large subunit. In fact and this pointhas been made by others [49] – this retention of structure probably favoured (or even enabled) the duplication event, asit meant the resultant tRNA was able to be aminoacylated by the same ribozyme synthetase that aminoacylated thehairpin precursor, and therefore the tRNA was able to participate in ribosomal protein synthesis. At the same time, theappearance of a novel structure at the ligation point – the anticodon loop – allowed for the subsequent evolution ofgenetic coding [4,3].

One of the strongest arguments in favour of the hairpin ligation being catalyzed by an ancestral selfcleaving intron [49](as depicted in Figure 1) is the presence of the highly conserved ‘canonical intron insertion position’ between nucleotides37 and 38 in the anticodon loop [41], where almost all eukaryotic nuclear (and the majority of archaeal) tRNA intronsare found, even though introns are only found in a subset of tRNA isoacceptors [82]. It has been proposed previouslythat this conserved position constitutes a 'molecular memory’ of the position of the ancestral intron that was responsiblefor the ligation that created the first tRNA [83]. If the canonical intron insertion position is ancestral, it implies thateukaryotic nuclear tRNAs (and possibly archaeal tRNAs) have a more ancestral structure than eubacterial tRNAs, whichusually lack tRNA introns altogether or possess selfsplicing introns at a variety of different positions in the molecule.Such a finding is consistent with the intronsearly hypothesis, and the proposal that eubacteria have undergone a processof intron loss [84,85].

Conclusions

I have argued that the RNA world hypothesis, while certainly imperfect, is the best model we currently have for the earlyevolution of life. While the hypothesis does not exclude a number of possibilities for what – if anything – preceded RNA,unfortunately the evolution of coded protein synthesis has drawn a veil over the previous history of proteins. The situationis different in the case of noncoding RNAs such as ribosomal RNA and tRNA, as these were able to replicate prior tothe evolution of ribosomal protein synthesis.

As we have noted previously [5], the proposal that the RNA world evolved in acidic conditions [5,6] offers a plausiblesolution to Charles Kurland's criticism [57] that the RNA world hypothesis makes no reference to a possible energysource. As de Duve [87] has noted, "the widespread use of protonmotive force for energy transduction throughout theliving world today is explained as a legacy of a highly acidic prebiotic environment and may be viewed as a clue to theexistence of such an environment" [87]. Although Russell, Martin and others [2326] have argued that proton andthermal gradients between the outflow from hot alkaline (pH 911) undersea hydrothermal vents and the surroundingcooler more acidic ocean may have constituted the first sources of energy at the origin of life, the lack of RNA stability atalkaline pH ( [5] and references within) would appear to make such vents an unlikely location for RNA world evolution.

Although possible, it seems unlikely that the AC base pair 'mismatches' found in the tRNA genes of Ferroplasmaacidarmanus and Picrophilus torridus (two species of archaebacteria with a reportedly acidic internal pH) [5] arecorrected by C to U RNA editing that occurs, for example, with some but not other plant chloroplast tRNAs [88,89].Such editing of secondary structure AC base pair mismatches has so far not been found to occur in archaebacteria;however, in a single archaeal species (Methanopyrus kandleri) a tertiary structure AC base pair found in 30 of its 34





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tRNAs undergoes C to U editing catalyzed by a cytidine deaminase CDAT8 [90]. M. kandleri is a unique organism thatcontains many 'orphan' proteins. CDAT8, which contains a cytidine deaminase domain and putative RNAbindingdomain, has no homologues in other arachaeal species, including F. acidarmanus and P. torridus (L Randau, pers.commun.; [90]). Definitive proof, however, that the AC base pairs in these two species are not modified would ofcourse require e.g. cDNA sequencing of the tRNAs.

Abbreviations

mRNA: messenger RNA; tRNA: transfer RNA.

Competing interests

The author declares that he has no competing interests.

Reviewers’ comments

Referee 1: Eugene Koonin

I basically agree with Bernhardt. The RNA World scenario is bad as a scientific hypothesis: it is hardly falsifiable and isextremely difficult to verify due to a great number of holes in the most important parts. To wit, no one has achieved bonafide selfreplication of RNA which is the cornerstone of the RNA World. Nevertheless, there is a lot going for the RNAWorld (Bernhardt summarizes much of the evidence, and I add more below) whereas the other hypotheses on the originof life are outright helpless. Moreover, as argued in some detail elsewhere [91], the RNA World appears to be anoutright logical inevitability. ‘Something’ had to start efficiently replicating to kick off evolution, and proteins do not havethis ability. As Bernhardt rightly points out, it is not certain that RNA was the first replicator but it does seem certain thatit was the first ‘good’ replicator. To clarify, this does not imply that the primordial RNA World did not have peptides; onthe contrary, it is plausible that peptides played important roles but they were not initially encoded in RNA.

Moreover, straightforward observations on modern proteins indicate that the role of RNA in the ancient translationsystem was much greater that it is in the modern system. Indeed, Class I aminoacyltRNA synthetases (aaRS) representonly a small branch on the complex evolutionary tree of Rossmannlike domains, so the common ancestor of all 10 ClassI aaRS emerged after extensive diversification of this particular class of protein domains had already taken place.Accordingly, one is compelled to conclude that a highfidelity translation system that alone would enable extensive proteinevolution existed already at the late stages of the hypothetical RNA World [92].

All this discussion is not pointless play with hypotheses. Realization of the unique status of the RNA World among theorigin of life scenarios is critical for maintaining the focus of research on truly important directions such as experimentaland theoretical study of the evolution of ribozymes rather than futile attempts to debunk the RNA World.

Referee 2: Anthony Poole

Harold Bernhardt’s review of the RNA world hypothesis is readable and timely. He presents a very openminded reviewof recent results and how they impact on old ideas, and distills a large amount of material. Aside from the admirableattempt to synthesize a vast array of ideas, a valuable contribution hidden within is the critical assessment of the view thatthe RNA world hypothesis needs to be abandoned in favour of a peptidesfirst model.

Author’s response: I have revised the abstract and introduction to include reference to my critique of the ‘proteins(or peptides) first’ hypothesis.

While I doubt that anyone seriously excluded peptides as part of a prebiotic milieu, the primacy of peptides doesneed careful consideration. In this regard, the explicit explanation of why a pregenetic code origin of proteinswill not be detectable from comparative genomic analyses is an important contribution. Perhaps this is obviousto some, but in light of a growing view that nonribosomal peptide synthesis preceded ribosomal peptide



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synthesis, it would seem that the community needs a reminder, and Bernhardt spells it out in a very informativemanner. Another issue with arguing for nonribosomal peptide synthesis preceding the ribosome is that there isan enormous difference in information input versus output. As discussed in[[62]], megaenzymes like cyclosporinare ~15000 amino acids in length and produce products of 11 amino acids in length – a factor of 10 is nottrivial. While nonribosomal peptide synthetases are modular and could in principle be engineered into minimalentities, the challenge of equalizing information input and output is significant regardless of one’s favouredprebiotic starting point. It is clear from reading Bernhardt’s review that the RNA community is much closer tothis than those who seek to replace primordial RNAbased replication with peptidebased replication.

Referee 3: Michael Yarus (nominated by Laura Landweber)

Almost always, progress to new understanding is sporadic, with insights coming in separated locales. Difficultiestemporarily immobilize discussion, but then are surmounted by a successful theory. This sometimes inchoate staggertoward a broader, more selfconsistent argument is all that can be expected, even of an ultimately successful idea.Discussions of the RNA world sometimes forget this, and demand e.g., the ultimate replicase today! But this essay byHarold Bernhardt remembers what has happened for other successful evolutionary ideas, like the big tree. For all itssuccesses, the tree is still being questioned under extreme prejudice in certain quarters, as is the RNA world.

Contrariwise, here we have here a sympathetic review of the support for the RNA world, which specifically makes thepoint that it fits our descent better than other ideas (You look like the son of a montmorillonite to me, ya mangy mutant!).It will be useful to those who want an entry to the RNA world literature, and could easily serve as the crux of a universitycourse.

However, this is also its weakness; the text is polite and respectful, even to those whose ‘contribution’ has beenotherwise. It treats even loony ideas (‘we need proteins to evolve translation!’) with deference. Or to put it in otherwords, it is edgeless – some attitude would be welcome. Some choice between hypotheses should go with the territory;some consequent makeorbreak predictions are the responsibilities of a guide. But as a gentle introduction, you will notfind better.

Author’s response: In revising the manuscript, I have – to some degree inadvertently – added a bit more bite!

Acknowledgements

This paper is dedicated to my mentor and colleague Professor Warren Tate, who was instrumental in my setting off onthis life of adventure and discovery and who encouraged me to write this paper. Many thanks to Hans Gross, GeorgeFox and Steven Benner for critical reading of an early draft of this manuscript and for their helpful suggestions. Thanks toLennart Randau for helpful information regarding his work on CDAT8 from M. kandleri. Thanks to Diana Yates from theUniversity of Illinois News Service and Russell Doolittle for permission to use material which first appeared there. Theresearch was conducted during tenure of a Health Sciences Career Development Award at the University of Otago.

The title is an adaptation of Sir Winston Churchill’s famous comment on democracy made in a speech to the House ofCommons on 11 November 1947: No one pretends that democracy is perfect or allwise. Indeed, it has been saidthat democracy is the worst form of government except all those other forms that have been tried from time totime.

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Review ArticleOrigins & Design 17:1

Gordon C. MillsDepartment of Human Biological Chemistry and GeneticsUniversity of Texas Medical BranchGalveston, TX 77555

Dean KenyonDepartment of BiologySan Francisco State University1600 Holloway AvenueSan Francisco, CA 94132

Introduction

One of the earliest published suggestions that RNA-catalyzed RNA replication preceded and gave rise to thefirst DNA-based living cells was made by Carl Woese in 1967, in his book The Genetic Code1. Similarsuggestions were made by Crick and Orgel2, for reasons that are not difficult to grasp. Prior to the discoveryof catalytic RNAs, proteins were considered by many to be the only organic molecules in living matter thatcould function as catalysts. DNA carries the genetic information required for the synthesis of proteins. Thereplication and transcription of DNA require a complex set of enzymes and other proteins. How then couldthe first living cells with DNA-based molecular biology have originated by spontaneous chemical processeson the prebiotic Earth? Primordial DNA synthesis would have required the presence of specific enzymes, buthow could these enzymes be synthesized without the genetic information in DNA and without RNA fortranslating that information into the amino acid sequence of the protein enzymes? In other words, proteins arerequired for DNA synthesis and DNA is required for protein synthesis.

This classic "chicken-and-egg" problem made it immensely difficult to conceive of any plausible prebioticchemical pathway to the molecular biological system. Certainly no such chemical pathway had beendemonstrated experimentally by the early 1960s. So the suggestion that RNA molecules might have formedthe first self-replicating chemical systems on the primitive Earth seemed a natural one, given the uniqueproperties of these substances.

They carry genetic information and (unlike DNA) occur primarily as single-stranded molecules that canassume a great variety of tertiary structures, and might therefore be capable of catalysis, in a manner similarto that of proteins. The problem of which came first, DNA or proteins, would then be resolved.

Self-replicating RNA-based systems would have arisen first, and DNA and proteins would have been addedlater. But in the absence of any direct demonstration of RNA catalysis, this suggestion remained only aninteresting possibility.

Then, in the early 1980s3, the discovery of self-splicing, catalytic RNA molecules (in the ciliated protozoanTetrahymena thermophila), put molecular flesh on the speculative bones of the idea of an early evolutionarystage dominated by RNA. These catalytic RNA molecules have subsequently been termed "ribozymes." "Onecan contemplate an RNA World," wrote Walter Gilbert in 1986, "containing only RNA molecules that serve

The RNA World: A Critique - Origins & Design 17:1. Mills, Gordon and... http://www.arn.org/docs/odesign/od171/rnaworld171.htm

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to catalyze the synthesis of themselves."4

The phrase "RNA World" stuck to the general hypothesis, and has since come to denote the RNA-first,DNA-and-proteins-later scenario depicted in Figure 1. The long-standing "chicken-and egg" puzzle at theorigin of life indeed appeared amenable to a solution:

The primordial...conundrum -- which came first, informational polynucleotides or functionalpolypeptides -- was obviated by the simple but elegant compaction of both genetic informationand catalytic function into the same molecule.5

A second impetus to the RNA world hypothesis came from the cluster of technical innovations now knowngenerally as ribozyme engineering. Naturally occuring RNA catalytic activities are actually restricted to asmall set of highly specialized reactions, e.g., the processing of RNA transcripts primarily in eukaryotic cells.However, ribozyme engineering, made possible by techniques such as DNA sequencing, in vitro transcriptionand the polymerase chain reaction [PCR]6, allow molecular biologists to manipulate RNA to whatever extentthe molecule will allow. Thus, the catalytic repertoire of RNA can be expanded beyond the naturallyoccurring activities -- in the main, by two broad strategies of ribozyme engineering.

One strategy involves the direct modification of existing species of ribozymes, to produce better or evennovel catalysts. This has been called the "rational design" approach. The other strategy employs pools of short(often 50-100 nucleotide units) randomized RNA molecules, which are subjected repeatedly to a selectionprocess designed to enhance the concentration of RNA molecules with the desired functional activity. Thefew selected molecules are then multiplied a million-fold or more by using the polymerase chain reaction,which uses activated nucleotide precursors and enzymes. This has been termed the "irrational design"method.

Judging from the progress in ribozyme engineering in recent years, it seems likely that new and improvedtypes of RNA catalysts will be produced in years ahead. Moreover, molecular biologists may discoveradditional catalytic roles of RNA in living cells, although the variety of such roles is not expected to rival thatof the protein enzymes. Thus, one might expect that the RNA World hypothesis will continue to havesupporters.

Yet beyond the immediate foreground of RNA World excitement lies a disquieting landscape of chemicalproblems, largely ignored in the recent literature on ribozyme engineering. As researchers broaden their focusto include the chemical plausibility of the RNA World itself, however7, these difficulties cannot be avoided.

Furthermore, the relevance of ribozyme engineering to naturalistic theories of the origin of life is doubtful atbest, primarily because of the necessity for intelligent intervention in the synthesis of the randomized RNA;then again in the selection of a few functional RNA molecules out of that mixture; then, finally, in theamplification of those few functional RNA molecules [see box, "What Do Ribozyme EngineeringExperiments Really Tell Us About the Origin of Life?"].

Hubert Yockey, borrowing a metaphor from Jonathan Swift, suggests that current origin-of-life research,including the RNA World hypothesis, floats improbably in mid-air like the roof of a house built by anarchitect of the Grand Academy of Lagado. This savant had contrived a method of building houses bybeginning at the roof and working downwards. "The architect pointed out that among the advantages of thisprocedure," Yockey notes8, "was that once the roof was in place [before the walls or foundation] the rest ofthe construction could proceed quickly and without interruption by weather." That "roof" -- consisting in thisinstance of tiles which represent the catalytic activities of RNA -- may look solid to those believers in the


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existence of a prebiotic RNA World. But is the roof really solid? Is it supported by walls and a foundation?

Once one peers over the edge of the roof to look beneath, we shall argue, the implausibility of the theoreticalstructure as a whole is inescapable. In what follows, we present the key postulates or presuppositions onwhich the RNA World hypothesis must rest (see Figure 2). Each represents an unsolved chemical problem, inevery case well-known to origin-of-life researchers. Unfortunately, in many articles on the RNA World, theseproblems are often collapsed into the "prebiotic soup" and "self-assembly" phases of the scenario, and receiveno discussion. We suggest that new discoveries about the catalytic activities of RNA should be seen for whatthey really are: not elucidating prebiotic processes on the early Earth, but rather as extending our knowledgeof the molecular biology of the cell in important ways (see below).

The relevance of catalytic RNA to the problem of the naturalistic origin of life is, however, a different matterentirely.

We take heart in noting that, despite the frequent neglect in much of the popular literature of the chemicaldifficulties of the RNA World scenario, many of the scientists involved with that hypothesis are quite candidin their assessment of the problems associated with it. These are represented for instance by the numerouscontributors to The RNA World7. Since the RNA World hypotheses are so broad, we will attempt to breakthem down into somewhat narrower postulates. In this way one may see more clearly some of thepresuppositions that are involved.

Problematic Chemical Postulates of the RNA World Scenario

Postulate 1: There was a prebiotic pool of beta-D-ribonucleotides.

Beta-D-ribonucleotides (see Figure 2) are compounds made up of a purine (adenine or guanine) or apyrimidine (uracil or cytosine) linked to the 1'-position of ribose in the beta-configuration.

There is, in addition, a phosphate group attached to the 5'-position of the ribose. For the four differentribonucleotides in this prebiotic scenario, there would be hundreds of other possible isomers.

But each of these four ribonucleotides is built up of three components: a purine or pyrimidine, a sugar(ribose), and phosphate. It is highly unlikely that any of the necessary subunits would have accumulated inany more than trace amounts on the primitive Earth. Consider ribose. The proposed prebiotic pathway leadingto this sugar, the formose reaction, is especially problematic9. If various nitrogenous substances thought tohave been present in the primitive ocean are included in the reaction mixture, the reaction would not proceed.The nitrogenous substances react with formaldehyde, the intermediates in the pathways to sugars, and withsugars themselves to form non-biological materials10. Furthermore, as Stanley Miller and his colleaguesrecently reported, "ribose and other sugars have suprisingly short half-lives for decomposition at neutral pH,making it very unlikely that sugars were available as prebiotic reagents."11

Or consider adenine. Reaction pathways proposed for the prebiotic synthesis of this building block start withHCN in alkaline (pH 9.2) solutions of NH4OH.12 These reactions give small yields of adenine (e.g., 0.04%)and other nitrogenous bases provided the HCN concentration is greater than 0.01 M. However, the reactionmixtures contain a great variety of nitrogenous substances that would interfere with the formose reaction.Therefore, the conditions proposed for the prebiotic synthesis of purines and pyrimidines are clearlyincompatible with those proposed for the synthesis of ribose. Moreover, adenine is susceptible to deaminationand ring-opening reactions (with half-lives of about 80 years and 200 years respectively at 37º C and neutral


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pH), making its prebiotic accumulation highly improbable13. This makes it difficult to see how anyappreciable quantities of nucleosides and nucleotides could have accumulated on the primitive Earth. If thekey components of nucleotides (the correct purines and pyrimidines, ribose, and phosphate) were not present,the possibility of obtaining a pool of the four beta-D-ribonucleotides with correct linkages would be remoteindeed.

If this postulate, the first and most crucial assumption, is not valid, however, then the entire hypothesis of anRNA World formed by natural processes becomes meaningless.

Postulate 2: Beta-D ribonucleotides spontaneously form polymers linked together by 3',5'-phosphodiester linkages (i.e., they link to form molecules of RNA; see figure 2).

Joyce and Orgel discuss candidly the problems with this postulate14. They note that nucleotides do not linkunless there is some type of activation of the phosphate group. The only effective activating groups for thenucleotide phosphate group (imidazolides, etc.), however, are those that are totally implausible in anyprebiotic scenario. In living organisms today, adenosine-5'-triphosphate (ATP) is used for activation ofnucleoside phosphate groups, but ATP would not be available for prebiotic syntheses. Joyce and Orgel notethe possible use of minerals for polymerization reactions, but then express their doubts about thispossibility15:

Whenever a problem in prebiotic synthesis seems intractable, it is possible to postulate theexistence of a mineral that catalyzes the reaction...such claims cannot easily be refuted.

In other words, if one postulates an unknown mineral catalyst that is not readily testable, it is difficult torefute the hypothesis.

Joyce and Orgel then note that if there were activation of the phosphate group, the primary polymer productwould have 5', 5'-pyrophosphate linkages; secondarily 2', 5'-phosphodiester linkages -- while the desired 3',5'-phosphodiester linkages would be much less abundant. However, all RNA known today has only 3',5'-phosphodiester linkages, and any other linkages would alter the three-dimensional structure and possibilitiesfor function as a template or a catalyst.

Even waiving these obstacles, and allowing for minute amounts of oligoribonucleotides, these moleculeswould have been rendered ineffective at various stages in their growth by adding incorrect nucleotides, or byreacting with the myriads of other substances likely to have been present. Moreover, the RNA moleculeswould have been continuously degraded by spontaneous hydrolysis and other destructive processes operatingon the primitive Earth16.

In brief, any movement in the direction of an RNA World on a realistically-modeled early Earth would havebeen continuously suppressed by destructive cross-reactions.

Postulate 3: A polyribonucleotide (i.e. RNA molecule), once formed, would have the catalytic activity toreplicate itself, and a population of such self-replicating molecules could arise.

The difficulty with this postulate is evident in the following quotation from Joyce and Orgel:

...it is assumed...that a magic catalyst existed to convert the activated nucleotides to a randomensemble of polynucleotide sequences, a subset of which had the ability to replicate. It seems tobe implicit that such sequences replicate themselves but, for whatever reason, do not replicate


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unrelated neighbors.17

They refer to this as a component of "The Molecular Biologists Dream," and discuss the difficulties inherentin such a view. In order for a stable population of self-replicating RNA molecules to arise -- a prerequisite forfurther evolution -- the RNA molecules must be able to replicate themselves with high fidelity, or thesequence specificity which makes self-replication possible at all will be lost. While "it is difficult to state withcertainty the minimum possible size of an RNA replicase ribozyme," Joyce and Orgel note, it seems unlikelythat a structure with fewer than 40 nucleotides would be sufficient. Suppose, then, that "there is some 50-mer[RNA molecule of 50 nucleotides length]," Joyce and Orgel speculate, that "replicates with 90% fidelity. ...Would such a molecule be expected to occur within a population of random RNAs?"

Perhaps: but one such self-replicating molecule will not suffice.

"Unless the molecule can literally copy itself," Joyce and Orgel note, "that is, act simultaneously as bothtemplate and catalyst, it must encounter another copy of itself that it can use as a template." Copying anygiven RNA in its vicinity will lead to an error catastrophe, as the population of RNAs will decay into acollection of random sequences. But to find another copy of itself, the self-replicating RNA would need(Joyce and Orgel calculate) a library of RNA that "far exceeds the mass of the earth."18

In the face of these difficulties, they advise, one must reject

the myth of a self-replicating RNA molecule that arose de novo from a soup of randompolynucleotides. Not only is such a notion unrealistic in light of our current understanding ofprebiotic chemistry, but it should strain the credulity of even an optimist's view of RNA'scatalytic potential. If you doubt this, ask yourself whether you believe that a replicase ribozymewould arise in a solution containing nucleoside 5'-diphosphates and polynucleotidephosphorylase!19

Postulate 4: Self-replicating RNA molecules wouild have all of the catalytic activities necessary tosustain a ribo-organism.

S.A. Benner et al. note20:

...one is forced to conclude that the last ribo-organism had a relatively complex metabolism thatincluded oxidation and reduction reactions, aldol and Claison condensations, transmethylations,porphyrin biosynthesis, and an energy metabolism based on nucleoside phosphates, all catalyzedby riboenzymes...It should be noted that this reconstruction cannot be weakened without losingmuch of the logical and explanatory force of the RNA World model.

Although Benner et al. speak of the last "ribo-organism," surely the first ribo-organism would have requirednearly all of the same metabolic capabilities in order to survive. It is also apparent that the scenario of Benneret al. would surely include enclosing the ribozymes within a membrane with the ability to transport ions andorganic molecules across that membrane.

Anyone who is familiar with biochemistry would recognize that it would take hundreds of differentribozymes, each with a particular catalytic activity, to carry out the metabolic processes described above. Itshould also be apparent that most of these metabolic capabilities would have to be functional within a shortperiod of time (certainly not hundreds of years), in the same microscopic region, or the ribo-organism wouldnever survive.


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When one recognizes that catalytic activities of RNA are just as dependent upon specific sequences ofnucleotides in RNA21 as protein enzymes are of amino acid sequences, then the probability of postulate 4being valid is seen to be vanishingly small.

Benner et al. note that the diverse catalytic properties of enzymes often require coenzymes or prostheticgroups. They mention particularly the iron-porphyrin, heme, and pyridoxal, but have no suggestion how these(and other co-enzymes) could have functioned in the catalytic activities of early RNA molecules.

The other unproven assumption of postulate 4 is that RNA molecules initially had all of these suggestedcatalytic activities, but nearly all of these activities have been subsequently lost. RNA molecules withcatalytic activity that are known today predominantly have nuclease or nucleotidyl transferase activity withsome minimal esterase actitivy22. There is no solid evidence that RNA molecules ever had the broad range ofcatalytic activities suggested by Benner et al., even though a number of the authors of The RNA World speakof present-day RNA molecules as being vestiges of that early RNA World.

Conclusion

We have more to learn about RNA, both in vivo (as used by organisms) and in vitro, in terms of its chemistrygenerally and functional properties in particular. RNA is a remarkable molecule.

The RNA World hypothesis is another matter. We see no grounds for considering it established, or evenpromising, except perhaps on the objectionable philosophical grounds of philosophical naturalism (and itsoperational offspring, methodological naturalism), according to which the best naturalistic hypothesis isperforce the hypothesis to be accepted. We consider that historical biology should be open to all empiricalpossibilities, including design -- and see the molecular biological system of organisms, of which RNA is sostunning a part, as exemplars of design.

We find ourselves, however, distinctly in the minority of biologists. If design exists at all, it is a matter ofsubjective intuition, the majority of our colleagues would claim, asserting with science writer George Johnsonthat "the point of science is...to explain the world through natural law."23

We would put the point rather differently. The point of science is to explain the world, through natural lawsor whatever other causes best account for the phenomena at hand.

Philosopher of science Stephen Meyer captures the point well:

The (historical) question that must be asked about biological origins is not "Which materialisticscenario will prove adequate?" but "How did life as we know it actually arise on earth?" Sinceone of the logically appropriate answers to this latter question is that "Life was designed by anintelligent agent that existed before the advent of humans," I believe it is anti-intellectual toexclude the "design hypothesis" without consideration of all the evidence, including the mostcurrent evidence, that would support it.24

Detecting design is not a matter of subjective intuition.25 To see design as a real causal possibility, however,one must break free of the constraints of naturalism.

What do Ribozome Engineering Experiments Tell Us About the Origin of Life?


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Notes

1. Carl Woese, The Genetic Code (New York: Harper and Row, 1967).2. F.H.C. Crick, "The origin of the genetic code," J. Mol. Biol. 38 (1968): 367-379; L.E. Orgel,"Evolution of the genetic apparatus," J. Mol. Biol. 38 (1968): 381-393.3. K. Kruger, P.J. Grabowski, A.J. Zaug, J. Sands, D.E. Gottschling, and T.R. Cech, "Self-Splicing RNA:Autoexcision and Autocyclization of the Ribosomal RNA Intervening Sequence of Tetrahymena," Cell31 (1982): 147-157.4. Walter Gilbert, "The RNA World," Nature 319 (1986): 618.5. I. Hirao and A.D. Ellington, "Re-creating the RNA World," Current Biology 5 (1995): 1017-1022; p.1017.6. Mullis, K.B. and Faloona, "Specific synthesis of DNA in vitro via a polymerase catalyzed chainreaction," Methods Enzymol 155 (1987): 335-350.7. G. Joyce, "RNA evolution and the origins of life," Nature 338 (1989): 217-224; T.J. Gibson and A.I.Lamond, "Metabolic complexity in the RNA World and implications for the origin of proteinsynthesis," J. Mol. Evol. 30 (1990): 7-15; G.F. Joyce and L.E. Orgel, "Prospects for understanding theorigin of the RNA World," in The RNA World, eds. R.F. Gesteland and J.F. Atkins (Cold Spring Harbor,NY: Cold Spring Harbor Laboratory Press, 1993), pp. 1-25.8. H.P. Yockey, "Information in bits and bytes: reply to Lifson's Review of Information Theory andMolecular Biology," BioEssays 17 (1995): 85-88; p. 87.9. R. Shapiro, "The improbability of prebiotic nucleic acid synthesis," Origins of Life 14 (1984):565-570; R. Shapiro, "Prebiotic ribose synthesis: a critical analysis," Origins of Life 18 (1988): 71-85.10. Recently it has been shown that reaction mixtures containing dilute glycoaldehyde phosphate andformaldehyde or glyceraldehyde-2-phophate will generate reasonably high yields of ribose2,4-diphosphate and a few other sugar phosphates in less amounts. See D. Muller, S. Pitsch, A. Kittaka,E. Wagner, C.E. Wintner, and A. Eschenmoser, "Chemie von alpha-aminonitrilen. Aldomerisierung vonglykoaldehydphosphat zu racemischen hexose- 2,4,6-triphosphaten und (in gegenwart vonformaldehyd) racemischen pentose 2,4-diphophaten: rac.allose-2,4,6-triphosphat und rac.-ribose-2,4,-diphosphat sind die reaktionshauptproduckte. Helv. Chim. Acta 73 (1990): 1410-1468; Joyce andOrgel, ibid. However, if these reactions are not also run in the presence of amines and other nitrogenouscompounds (i.e., in chemical mixtures of the complexity proposed for the "prebiotic soup"), theirrelevancy to the origin of life is problematical.11. Rosa Larralde, Michael P. Robertson, and Stanley L. Miller, "Rates of decomposition of ribose andother sugars: Implications for chemical evolution," Proc. Natl. Acad. Sci. USA 92 (1995): 8158-8160.The ribose half-lives are very short, Larralde et al. report: 73 minutes at pH 7.0 and 100º C and 44years at pH 7.0 and Oº C.12. J.P. Ferris, P.C. Joshi, E.H. Edelson, and J.G. Lawless, "HCN: a plausible source of purines,pyrimidines and amino acids on the primitive Earth," J. Mol. Evol. 11 (1978): 293-311.13. R. Shapiro, "The prebiotic role of adenine: a critical analysis," Origins of Life and the Evolution ofthe Biosphere 25 (1995): 83-98.14. Joyce and Orgel, ibid.15. Ibid., p.416. C. Thaxton, W. Bradley, and R. Olsen, The Mystery of Life's Origin (New York: PhilosophicalLibrary, 1984).17. Joyce and Orgel, ibid., p.7.18. Ibid., p.11.19. Ibid, p.13.


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20. S.A. Benner, M.A. Cohen, G.H. Gonnet, D.B. Berkowitz, and K.P. Johnsson, "Reading thePalimpest: Contemporary Biochemical Data and the RNA World," in The RNA World, eds. R.F.Gesteland and J.F. Atkins (Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press, 1993), pp.27-70; p. 57.21. T.R. Cech, "Mechanism and Structure of a Catalytic RNA Molecule," in 40 Years of the DoubleHelix, The Robert A. Welch Foundation 37th Conference on Chemical Research, 1993, pp. 91-110; seealso T.R. Cech, "Structure and Mechanism of the Large Catalytic RNAs: Group I and Group II Intronsand Ribonuclease P," in The RNA World, eds. R.F. Gesteland and J.F. Atkins (Cold Spring Harbor, NY:Cold Spring Harbor Laboratory Press, 1993), pp. 239-269.22. Ibid.23. George Johnson, Fire in the Mind: Science, Faith, and the Search for Order (New York: Alfred A.Knopf, 1995), p. 314.24. Stephen C. Meyer, "Laws, Causes, and Facts," in Darwinism: Science or Philosophy, eds. J. Buelland V. Hearn (Richardson, Texas: Foundation for Thought and Ethics, 1994), p.34.25. See William A. Dembski, "The Design Inference: Eliminating Chance Through Small Probabilities,"unpublished Ph.D. dissertation, 1995, Department of Philosophy, University of Illinois-Chicago Circle.

Copyright © 1996 Gordon C. Mills and Dean Kenyon. All rights reserved. International copyright secured.File Date: 6.22.96


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14 The RNA World

By the mid-1980s many researchers concluded that both DNA-first and

protein-first origin-of-life models were beset with many difficulties. As

a result, they sought a third way to explain the mystery of life's origin.

Instead of proposing that the first informational molecules were pro

teins or DNA, these scientists argued that the earliest stages of abiogen

esis unfolded in a chemical environment dominated by RNA molecules.,

The first scientist to propose this idea was Carl Woese, a microbiologist

at the University of Illinois. Walter Gilbert, a Harvard biophysicist, later

developed the proposal and coined the term by which it is now popu

larly known, the "RNA world."1

The RNA world is now probably the most popular theory of how

life began. Scientists in some of the most prestigious labs around the

world have performed experiments on RNA molecules in an attempt to

demonstrate its plausibility, and in the opinion of many scientists, the

RNA-world hypothesis establishes a promising framework for explain

ing how life on earth might have originated.

I had an encounter with one such scientist in the spring of 2000. I

had just written an article about DNA and the origin of life in the April

issue of a prominent New York journal of opinion.2 When the letters

to the editor came in, I initially blanched when I saw one from a fierce

critic named Kenneth R. Miller, a biology professor at Brown Uni

versity and a skilled debater. Had I made a mistake in reporting some

biological detail in my argument? When I saw his objection, however, I

was relieved. Miller claimed that my critique of attempts to explain the

The RNA World

Figure 14.1. Walter Gilbert, photographed in front of a chalkboard in his office at Harvard. Courtesy of Peter Menzel/Science Photo Library.

297

origin of biological information had failed to address the "RNA first"

hypothesis. Miller asserted that I had ignored "nearly two decades of

research on this very subject" and failed to tell my "readers of experi

ments showing that very simple RNA sequences can serve as biological

catalysts and even self-replicate."3

Miller was half right. I hadn't told my readers about these experi

ments. But I knew that two decades of research on this topic had not

solved the problem of the origin of biological information. Because of

space constraints and the format of the journal, I had decided not to address this issue in my original article. But now Miller's letter gave me

a chance to do so.

At the time I had been studying research articles from origin-of-life

specialists who were highly critical of the RNA-world hypothesis, and

in my response to Miller I cited and summarized many of their argu

ments. I heard nothing more from Miller on the matter, but as I at

tended various conferences over the next several years, I discovered that

he was far from alone. Despite the pervasive skepticism about the RNA

298 SIGNATURE IN THE CELL

world among leading origin-of-life researchers, many practicing molec

ular biologists, including some very prominent scientists at famous labs,

continued to share Miller's enthusiasm. Moreover, I discovered that

many of these molecular biologists had recently initiated new experi

mental work inspired by their confidence in the viability of the RNA

world approach. Had they solved the information problem?

Second Things First

The RNA world is a world in which the chicken and egg no longer con

found each other. At least that has been the hope. Building proteins re

quires genetic information in DNA, but information in DNA cannot be

processed without many specific proteins and protein complexes. This

problem has dogged origin-of-life research for decades. The discovery

that certain molecules of RNA possess some of the catalytic properties

seen in proteins suggested a way to solve the problem. RNA-first advo

cates proposed an early stage in the development of life in which RNA

performed both the enzymatic functions of modern proteins and the

information-storage function of modern DNA, thus sidestepping the

need for an interdependent system of DNA and proteins in the earliest

living system.

Typically RNA-first models have combined chance events and a law

like process of necessity, in particular, the process of natural selection.

As Gilbert and others envision it, a molecule of RNA capable of copying

itself (or copying a copy of itself) first arose by the chance association of nucleotide bases, sugars, and phosphates in a pre biotic soup (see Fig.

14.2). Then because that RNA enzyme could self-replicate, natural se

lection ensued, making possible a gradual increase in the complexity of

the primitive self-replicating RNA system, eventually resulting in a cell

with the features we observe today. Along the way, a simple membrane,

itself capable of self-reproduction, enclosed the initial RNA enzymes

along with some amino acids from the prebiotic soup.4

According to this model, these RNA enzymes eventually were re

placed by the more efficient proteins that perform enzymatic functions

in modern cells. For that to occur, the RNA-replicating system first

had to begin producing a set of RNA enzymes that could synthesize

The RNA World 299

Figure 14.2. The RNA World Scenario in Seven Steps. Step 1: The building

blocks of RNA arise on the early earth. Step 2: RNA building blocks link

up to form RNA oligonucleotide chains. Step 3: An RNA replicase arises

by chance and selective pressures ensue favoring more complex forms of

molecular organization. Step 4: RNA enzymes begin to synthesize proteins

from RNA templates. Step 5: Protein-based protein synthesis replaces

RNA-based protein synthesis. Step 6: Reverse transcriptase transfers genetic

information from RNA molecules into DNA molecules. Step 7: The modern

gene expression system arises within a proto-membrane.


proteins. As Gilbert has explained, in this step RNA molecules began

"to synthesize proteins, first by developing RNA adapter molecules that

can bind activated amino acids and then by arranging them according

to an RNA template using other RNA molecules such as the RNA core

of the ribosome."5 Finally, DNA emerged for the first time by a process

called reverse transcription. In this process, DNA received the informa

tion stored in the original RNA molecules, and eventually these more

stable DNA molecules took over the information-storage role that RNA

had performed in the RNA world. At that point, RNA was, as Gilbert

put it, "relegated to the intermediate role it has today-no longer the

center of the stage, displaced by DNA and the more effective protein

enzymes."6

I knew that origin-of-life theories that sound plausible when stated

in a few sentences often conceal a host of practical problems. And so

it was with the RNA world. As I investigated this hypothesis, both

before and after my exchange with Professor Miller, I found that many

crucial problems lurked in the shadows, including the one I had seen

before: the theory did not solve the problem of biological informa

tion-it merely displaced it.

Because so many scientists assume that the RNA world has solved

the problem of the origin of life, this chapter will provide a detailed

and, in some places, technical critique of this hypothesis. My critique

details five crucial problems with the RNA world, culminating in a

discussion of the information problem. To assist nontechnical readers,

I have placed some of this critique in notes for the scientifically trained.

I would ask technically minded readers to read these notes in full, be

cause in some cases they provide important additional support for, or

qualifications to, my arguments.

Each element of this critique stands mostly on its own. So if you find

that the technical material under one subheading presupposes unfamil

iar scientific concepts or terminology, take note of the heading, which

summarizes the take-home message of the section, and skip ahead to

the next one, or even the final two, which address the theory's greatest

weakness: its inability to explain the origin of biological information.

The RNA World 301

Problem I: RNA Building Blocks Are Hard to Synthesize and Easy to Destroy

Before the first RNA molecule could have come together, smaller con

stituent molecules needed to arise on the primitive earth. These in

clude a sugar known as ribose, phosphate molecules, and the four RNA

nucleotide bases (adenine, cytosine, guanine, and uracil). It turns out,

however, that both synthesizing and maintaining these essential RNA

building blocks, particularly ribose (the sugar incorporated into nucleo

tides) and the nucleotide bases, has proven either extremely difficult or

impossible to do under realistic prebiotic conditions.7 (See Fig. 14.3.) Consider first the problems with synthesizing the nucleotide bases. In

the years since the RNA world was proposed, chemist Robert Shapiro

has made a careful study of the chemical properties of the four nucleotide

bases to assess whether they could have arisen on the early earth under

Figure 14.3. The chemical structure and constituents of RNA.


realistic conditions. He notes first that "no nucleotides of any kind have been reported as products of spark-discharge experiments or in studies of meteorites." Stanley Miller, who performed the original prebiotic simulation experiment, published a similar study in 1998. 8 Moreover, even if they did somehow form on the early earth, nucleotide bases are too chemically fragile to have allowed life enough time to evolve in the manner Gilbert and other RNA-first theorists envision. Shapiro and Miller have noted that the bases of RNA are unstable at temperatures required by currently popular high-temperature origin-of-life scenarios. The bases are subject to a chemical process known as "deamination," in which they lose their essential amine groups (NH2). At 100 degrees C, adenine and guanine have chemical half-lives of only about one year; uracil has a half-life of twelve years; and cytosine a half-life of just nineteen days. Because these half-lives are so short, and because the evolutionary process envisioned by Gilbert would take so long-especially for natural selection to find functional ribozymes (RNA molecules with catalytic activity) by trial and error-Stanley Miller concluded in 1998 that "a high temperature origin of life involving these compounds [the RNA bases] therefore is unlikely."9 Miller further noted that, of the four required bases, cytosine has a short half-life even at low temperatures, thus raising the possibility that "the GC pair" (and thus RNA) "may not have been used in the first genetic material." Shapiro concurred. He showed that it would have been especially difficult to synthesize adenine and cytosine at high temperatures and cytosine even at low temperatures. Thus he concluded that the presumption that "the bases, adenine, cytosine, guanine and uracil were readily available on the early earth" is "not supported by existing knowledge of the basic chemistry of these substances."10

Producing ribose under realistic conditions has proven even more problematic. Prebiotic chemists have proposed that ribose could have arisen on the early earth as the by-product of a chemical reaction called the formose reaction. The formose reaction is a multistep chemical reaction that begins as molecules of formaldehyde in water react with one another. Along the way, the formose reaction produces a host of different sugars, including ribose, as intermediate by-products in the sequence of reactions. But, as Shapiro has pointed out, the formose reaction · vill not produce sugars in the presence of nitrogenous sub-

The RNA World 303

stances.11 These include peptides, amino acids, and an;iines, a category of molecules that includes the nucleotide bases.

This obviously poses a couple of difficulties. First, it creates a dilemma for scenarios that envision proteins and nucleic acids arising out of a prebiotic soup rich in amino acids. Either the prebiotic environment contained amino acids, which would have prevented sugars (and thus DNA and RNA) from forming, or the prebiotic soup contained no amino acids, making protein synthesis impossible. Of course, RNAfirst advocates might try to circumvent this difficulty by proposing that proteins arose well after RNA. Yet since the RNA-world hypothesis envisions RNA molecules coming into contact with amino acids early on within the first protocellular membranes (see above), choreographing the origin of RNA and amino acids to ensure that the two events occur separately becomes a considerable problem.

The RNA-world hypothesis faces an even more acute, but related, obstacle-a kind of catch-22. The presence of the nitrogen-rich chemicals necessary for the production of nucleotide bases prevents the production of ribose sugars. Yet both ribose and the nucleotide bases are needed to build RNA. (See note for details).12 As Dean Kenyon explains, "The chemical conditions proposed for the prebiotic synthesis of purines and pyrimidines [the bases] are sharply incompatible with those proposed for the synthesis of ribose."13 Or as Shapiro concludes: "The evidence that is currently available does not support the availability of ribose on the pre biotic earth, except perhaps for brief periods of time, in low concentration as part of a complex mixture, and under conditions unsuitable for nucleoside synthesis."14

Beyond that, both the constituent building blocks of RNA and whole RNA molecules would have reacted readily with the other chemicals present in the prebiotic ocean or environment. These "interfering cross-reactions" would have inhibited the assembly of RNA from its constituent monomers and inhibited any movement from RNA molecules toward more complex biochemistry, since the products of these reactions typically produce biologically inert (or irrelevant) substances.

Furthermore, in many cases, reactions (such as the formose reaction) that produce desirable by-products such as ribose also produce many undesirable chemical by-products. Unless chemists actively intervene, undesirable and desirable chemical by-products of the same reaction


react with each other to alter the composition of the desired chemicals

in ways that would inhibit the origin of life. In sum, synthesizing the

building blocks of the RNA molecule under realistic prebiotic condi

tions has proven formidably difficult.

Problem 2: Ribozymes Are Poor Substitutes for Proteins

Another major problem with the RNA world is that naturally occur

ring RNA molecules possess very few of the specific enzymatic proper

ties of proteins. To date, scientists have shown that RNA catalysts or

"ribozymes" can perform a small handful of the thousands of func

tions performed by modern proteins. Scientists have shown that some

RNA molecules can cleave other RNA molecules (at the phosphodi

ester bond) in a process known as hydrolysis. Biochemists also have

found RNAs that can link (ligate) separate strands of RNA (by catalyz

ing the formation of phosphodiester bonds). Other studies have shown

that the RNA in ribosomes (rRNA) promotes peptide-bond formation

within the ribosome15 and can promote peptide bonding outside the

ribosome, though only in association with an additional chemical cata

lyst.16 Beyond that, RNA can perform only a few minor functional roles

and then usually as the result of scientists intentionally "engineering" or "directing" the RNA catalyst (or ribozyme) in question.17

For this reason, claiming that catalytic RNA could replace proteins

in the earliest stages of chemical evolution is extremely problematic. To say otherwise would be like asserting that a carpenter wouldn't need

any tools besides a hammer to build a house, because the hammer per

formed two or three carpentry functions. True, a hammer does per

form some carpentry functions, but building a house requires many

specialized tools that can perform a great variety of specific carpentry

functions. In the same way, RNA molecules can perform a few of the

thousands of different functions proteins perform in "simple" single

cells (e.g., in the E. coli bacterium), but that does not mean that RNA

molecules can perform all necessary cellular functions.

The RNA World

Problem 3: An RNA-Based Translation and Coding System Is Implausible

305

The inability of RNA molecules to perform many of the functions of

protein enzymes raises a third and related concern about the plausibility

of the RNA world. RNA-world advocates offer no plausible explanation

for how primitive self-re'plicating RNA molecules might have evolved

into modern cells that rely on a variety of proteins to process genetic

information and regulate metabolism.18

To evolve beyond the RNA world, an RNA-based replication system

eventually would have to begin to produce proteins, and not just any

proteins, but proteins capable of template-directed protein manufac

ture. But for that to occur, the RNA replicator first would need to

produce machinery for building proteins. In modern cells it takes many

proteins to build proteins. So, as a first step toward building proteins,

the primitive replicator would need to produce RNA molecules capable

of performing the functions of the modern proteins involved in trans

lation. (Recall from Chapter 5 that translation is the process of build

ing proteins from the instructions encoded on an mRNA transcript.)

Presumably, these RNA molecules would need to perform the func

tions of the twenty specific tRNA synthetases and the fifty ribosomal

proteins, among the many others involved in translation. At the same

time, the RNA replicator would need to produce tRNAs and the many

mRNAs carrying the information for building the first proteins. These

mRNAs would need to be able to direct protein synthesis using, at first,

the transitional ribozyme-based protein-synthesis machinery and then,

later, the permanent and predominantly protein-based protein-synthesis

machinery. In short, the evolving RNA world would need to develop a

coding and translation system based entirely on RNA and also gener

ate the information necessary to build the proteins that later would be

needed to replace it.

This is a tall order. The cell builds proteins from the information

stored on the mRNA transcript (i.e., the copy) of the original DNA

molecule. To do this, a bacterial cell depends upon a translation and

coding system consisting of 106 distinct but functionally integrated

proteins as well as several distinct types of RNA molecules ( tRNAs,

mRNAs, and rRNAs).19 This system includes the ribosome (consisting


of fifty distinct protein parts), the twenty distinct tRNA synthetases,

twenty distinct tRNA molecules with their specific anticodons (all of

which jointly embody the genetic code), various other proteins, free

floating amino acids, ATP molecules (for energy), and-last, but not

least-information-rich mRNA transcripts for directing protein syn

thesis. Furthermore, many of the proteins in the translation system per

form multiple functions and catalyze coordinated multistep chemical

transformations (see Fig. 14.4).

Is it possible that a similar translation and coding system capable of

producing genetically encoded proteins might first have arisen using

only RNA catalysts (ribozymes)? Advocates of the RNA-world hy

pothesis have defended the possibility because of the demonstrated

catalytic properties of some RNA molecules. Eugene Koonin and

Yuri Wolf, two prominent scientists at the National Center for Bio

technology Information, recently reviewed the results of research on

the capacities of RNA catalysts in an important article assessing the

plausibility of an RNA-based translation system.20 They note that in

the last twenty years, molecular biologists have documented, or engi

neered, ribozymes that can catalyze "all three elementary reactions"21

required for translation, including aminoacylation (the formation of

a bond between an amino acid and an RNA), the peptidyl-transferase

reaction (which forms the peptide bond between amino acids), and

amino-acid activation (in which adenosine monophosphate is attached

to an amino acid).

At first glance, these results may seem to support the feasibility of

an RNA-based translation system. Nevertheless, significant reasons

to doubt this aspect of the RNA-world hypothesis remain, as Koonin

and Wolf note. First, though ribozymes have demonstrated the ca

pacity to catalyze representative examples of the three main types of

chemical reactions involved in translation, they have not demonstrated

the ability to catalyze anywhere near all the necessary reactions that

fall within these general classifications. Moreover, the gap between

"some" and "all" necessary reactions of a given type remains signifi

cant. For example, ribozyme engineers have successfully designed an

RNA molecule that will catalyze the formation of an aminoacyl bond

between itself and the amino acids leucine and phenylalanine. 22 But

no one has yet demonstrated that RNA can catalyze aminoacyl bonds

oT#et 'PTEI

([r.. IIJITrATIC>N, el.OIJ[rATIC>N,

& TEtMIIJATIC>N 'FACTo)

The RNA World

AkitJO AlI

307

AkitJOAlYL-tttJA

YtJT#ETAe

Figure 14.4. The main molecular components of the translation system:

twenty specific transfer-RNA molecules, twenty specific aminoacyl tRNA

synthetases, the ribosome with its two main subunits composed of fifty

proteins and ribosomal RNA, the messenger-RNA transcript, and a supply

of amino acids.


with the other eighteen protein-forming amino acids, still less with

the specificity required to make the resulting molecules useful for

translation. Yet establishing a genetic code requires molecules that can

catalyze highly specific aminoacylation for each of the twenty protein

forming amino acids. To say that RNA can catalyze "aminoacylation"

is true, but it obscures the distinction between part of a group and the

whole group, where having the whole group of molecules is necessary

to the function in question. Again, it takes more than a hammer to

build a house.

Second, unlike RNA catalysts (ribozymes), the protein-based en

zymes involved in translation perform multiple functions, often in

closely integrated or choreographed ways. Ribozymes, however, are

the one-trick ponies of the molecular world. Typically, they can per

form one subfunction of the several coordinated functions that a cor

responding enzyme can perform. But they cannot perform the entire

range of necessary functions, nor can they do so with the specificity

needed to execute the many sequentially coordinated reactions that

occur during translation.

Consider what ribozymes must do to rival the capacities of the syn

thetases that catalyze aminoacylation, which occurs between tRNA

molecules and their "conjugate" amino acids during translation in

actual cells. Researchers have demonstrated that certain RNA molecules

can bind a protein-forming amino acid, phenylalanine, to itself, thus

performing the function of aminoacylation. They have even isolated

a version of the RNA catalyst that binds only phenylalanine, achiev

ing a specificity of sorts. But the synthetase enzymes responsible for

aminoacylation in life must catalyze a complex two-stage chemical reac

tion involving three kinds of molecules: amino acids, ATP (adenosine

triphosphate ), and tRNAs.

In the first stage of this reaction, synthetases couple ATP to a spe

cific amino acid, giving it the stored energy (in the form of adenosine

monophosphate, AMP) needed to establish a bond with a tRNA

molecule. Next, synthetases couple specific tRNA molecules to spe

cific activated (AMP-charged) amino acids. These tRNAs have spe

cific shapes and anticodon sites that enable them to bond to mRNA

at the ribosome. Thus, synthetases help form molecular complexes

with a specificity of fit and with specific binding sites that enable

The RNA World 309

translation to occur in the context of a whole system of associated

molecules.

The RNA catalyst proposed as a precursor to the synthetase cannot

do this. It does not couple ATP to amino acids as a precursor to catalyz

ing aminoacylation. Instead, the ribozyme engineer provides "preade

nylated" amino acids (amino acids already linked to AMP molecules).

Nor does the RNA catalyst couple an amino acid to a specific tRNA

with a specific anticodon. The more limited specificity it achieves only

ensures that the RNA catalyst will bind a particular amino acid to

itself, a molecule that does not possess the specific cloverleaf shape or

structure of a tRNA. Moreover, this RNA does not carry an anticodon

binding site corresponding to a specific codon on a separate mRNA

transcript. Thus, it has no functional significance within a system of mol

ecules for performing translation. Indeed, no other system of molecules

has even been proposed that could confer functional significance or

specificity on the amino acid-RNA complexes catalyzed by the amino

acyl ribozyme.

Thus, even in the one case where ribozyme engineers have produced

an RNA-aminoacyl catalyst, the ribozyme in question will not produce

a molecule with a functional specificity, or capacity to perform coordi

nated reactions, equivalent to that of the synthetases used in modern

cells. Yet without this specificity and capacity to coordinate reactions,

translation-the construction of a sequence-specific arrangement of

amino acids from the specific RNA transcript-will not occur. 23

Similar limitations affect the RNA catalysts that have been shown

to be capable of peptidyl-transferase activity (i.e., catalyzing peptide

bonds between amino acids). These ribozymes (made of free-standing

ribosomal RNA) compare quite unfavorably with the capacities of the

protein-dominated ribosomes that perform this function in extant cells.

For example, researchers have found that free-standing ribosomal RNA

can only catalyze peptide-bond formation in the presence of another

catalyst. More important, apart from the proteins of the ribosome, free

standing ribosomal RNA does not force amino acids to link together

into linear chains, which is essential to protein function. (For more

details, see the note.)24


Why RNA Catalysts Can)t Do What True Enzymes Can There is a fundamental chemical reason for the limited functionality of

RNA catalysts-one that casts still further doubt on the RNA-world hy

pothesis and specifically on its account of the origin of the translation

system. Because of the inherent limitations of RNA chemistry, 25 single

RNA molecules do not catalyze the coordinated multistep reactions that

enzymes, such as synthetases, catalyze. Even if separate RNA catalysts

can be found that catalyze each of the specific reactions involved in trans

lation (which is by no means certain), that would leave us very far short

of a translation system. Each pony of the RNA world does only its one

trick. And even if all the ponies were present together, each one would

do only its particular trick separately, decoupled from the others. That's a

problem, because producing the molecular complexes necessary for trans

lation requires coupling multiple tricks-multiple crucial reactions-in a

closely integrated (and virtually simultaneous) way. True enzyme cata

lysts do this. RNA and small-molecule catalysts do not.

Here's the chemical backstory. Enzymes couple energetically fa

vorable and unfavorable reactions together into a series of reactions

that are energetically favorable overall. As a result , they can drive

forward two reactions where ordinarily only one would occur with

any appreciable frequency. Water runs downhill because of favor

able energetics provided by gravitational force. Water does not run

uphill, however, unless there is so much of it that it accumulates

and slowly rises up the ban k. Whether chemical reactions will occur

readily depends upon whether there is enough energy to make them

occur. Molecules with enough stored energy to establish new chemi

cal bonds will react readily with one another. Molecules with insuf

ficient stored energy will not react readily with each other unless vast

amounts of the reactants are provided (the equivalent of the rising

water flooding the banks).

Enzymes use a reaction that liberates energy to drive forward a re

action that requires energy, coupling energetically favorable and unfa

vorable reactions together. Enzymes can do this because they have a

complex three-dimensional geometry that enables them to hold all the

molecules involved in each step of the reaction together and to coor

dinate their interactions. But two independent catalysts cannot accom

plish what a compound catalyst (i.e., an enzyme) can. And so far RNA

The RNA World 311

ribozymes have demonstrated the capacity to act only as independent

catalysts, not true enzyme catalysts. RNA catalysts might catalyze some

energetically favorable reactions, but without the sophisticated active

sites of enzymes, they can't couple those favorable reactions to energeti

cally unfavorable reactions.26 (See Fig. 14.5.)

€"1ZYMATrlALLY loU'PL€1> TWo-S'r€'P €AlTrOIJ (AMIIJO AlYLATr0"1)

AA + ATP -+ Al-\WOACYL-Ak'P + z.'P,:. Akii.ioACYL-Ak'P + ti.iA - Al-\WOAlYL-ti.iA + Ak'P

Figure 14.5. Enzymes couple energetically favorable and unfavorable

reactions together into a series of reactions that are energetically favorable

overall. Enzymes can accomplish this because they have a three-dimensional

specificity that allows them to sequester and correctly position all the

molecules involved in a series of such reactions. RNA catalysts cannot do

this. The figure above shows an enzymatically mediated reaction called

aminoacylation. The diagram shows the specificity of fit between a tRNA

synthetase and a tRNA molecule during this two-stage chemical reaction.

The synthetase links the tRNA to a specific amino acid (AA) using energy

from ATP, thus coupling energetically favorable and unfavorable reactions.

Amino acids and ATP molecules are not pictured. They would be enveloped

by the synthetase during the reactions represented by the chemical equations.


Thus, the demonstration that RNA can catalyze "all the elementary

reactions" of translation, but neither the suite of functions nor the co

ordinated functions performed by the necessary enzyme catalysts of

the extant translation syste1'1, does little to establish the plausibility of

ribozyme-based protein synthesis, let alone the transition to enzyme

based protein synthesis, that the RNA-world scenario requires. The

inability to account for the origin of the translation system and ge

netic code, therefore, remains a formidable barrier to the success of the

RNA-world hypothesis.

Problem 4: The RNA World Doesn't Explain the Origin of Genetic Information

As I sifted through the primary scientific literature on the RNA-world

hypothesis, it did not take me long to realize that the hypothesis faced

significant problems quite apart from the central sequencing problem

that most interested me. Yet I also realized that it did not resolve the

mystery of the origin of biological information-which I had, here

tofore, called the DNA enigma. Indeed, I now realized that I might

just as easily have called that mystery the "RNA enigma," because the

information problem looms just as large in a hypothetical RNA world

as it does in a DNA world. This is not actually surprising. The RNA

world was proposed not as an explanation for the origin of biological

information, but as an explanation for the origin of the interdepen

dence of nucleic acids and proteins in the cell's information-processing

system. And as I studied the hypothesis more carefully, I realized that it

presupposed or ignored, rather than explained, the origin of sequence

specificity-information-in various RNA molecules.

Consider the step in the RNA-world scenario that I just examined

getting from a primitive replicator to a system for building the first

proteins. Even if a system of ribozymes for building proteins had arisen

from an RNA replicator, that system of molecules would still need

information-rich templates for building specific proteins. RNA-world

advocates give no account of the origin of that information beyond

vague appeals to chance. But as I argued in Chapters 8-10, chance is

not a plausible explanation for the information necessary for building

The RNA World 313

even one protein of modest length, let alone a set of RNA templates for

building the proteins needed to establish a protein-based translation

system and genetic code.

The need to account for these templates of information stands as a

formidable challenge to the RNA world. Nevertheless, the hypothesis

faces an even more basic information problem: the first self-replicating

RNA molecules themselves would have needed to be sequence-specific

in order to perform the function of replication, which is a prerequi

site of both natural selection and any further evolution toward cellular

complexity.

Though the RNA world was originally proposed as an explanation

for the "chicken and egg" functional interdependence problem, not the

information problem, some RNA-world advocates nevertheless appear

to think that it can somehow leapfrog the sequence-specificity require

ment. They imagine short chains ( oligomers) of RNA arising by chance

on the prebiotic earth. Then, after a sufficiently large pool of these mol

ecules had arisen, some would have acquired the ability to self-replicate.

In such a scenario the capacity to self-replicate would then favor the

survival of those RNA molecules that could do so and thus would favor

the specific sequences that the first self-replicating molecules happened

to have. Thus, self-replication arose again as a kind of "accidental choice

remembered."27

But like Quastler's DNA-first model discussed in the last chapter,

this scenario merely shifts the specificity problem out of view. First,

for strands of RNA to perform catalytic functions (including self

replication), they, like proteins, must display specific arrangements of

their constituent building blocks (nucleotides in the RNA case). In

other words, not just any sequence of RNA bases will be capable of self

replication. Indeed, experimental studies indicate that RNA molecules

with the capacity to replicate themselves, if they exist at all, are ex

tremely rare among possible RNA base sequences. Although no one has

yet produced a fully self-replicating RNA molecule,28 some research

ers have engineered a molecule that can copy a part of itself-though

only about 10 percent of itself and then only if a complementary primer

strand is provided to the ribozyme by the investigator. Significantly,

the scientists selected this partial self-replicator out of an engineered

pool of 1,000 trillion (1015) other RNA molecules, almost all of which


lack even this limited capacity for self-replication.29 This suggests that

sequences with this capacity are extremely rare and would be especially

so within a random (nonengineered) sample.

Further, for an RNA molecule to self-replicate, the RNA strand

must be long enough to form a complex structure. Gerald Joyce and

the late Leslie Orgel are two prominent origin-of-life researchers who

have evaluated the RNA-world scenario in detail. They consider, for

the sake of argument, that a replicase could form in a SO-base RNA

strand, though they are clearly skeptical that an RNA sequence of this

length would really do the job. 30 Experimental results have confirmed

their skepticism. Jack Szostak, a prominent ribozyme engineer, and his

colleagues have found that it typically takes at least 100 bases to form

structures capable of catalyzing simple ligation (linking) reactions. He

estimates that getting a ligase capable of performing the other functions

that polymerases must perform-"proper template binding, fidelity and

strand separation"-may require between 200 and 300 nucleotides.31

The ribozyme mentioned above-the one that can partially copy itself

required 189 nucleotide bases.32 It is presently unclear how many bases

would be needed to generate enough structural complexity to allow

true polymerase function, since no molecule capable of both complete

and unassisted self-replication has yet been engineered. It may be as low

as 189 bases, but it may be much higher, or it may simply be impos

sible. 33 Moreover, the problem may be more basic than length. RNA,

with its limited alphabet of four bases, may not even have the capacity to

form the complex three-dimensional shapes and distributions of charge

necessary to perform polymerase or replicase function.

In any case, even if we suppose that RNA-based RNA polymerases

( replicases) are possible, experimental evidence indicates that they would

have to be information-rich-both complex and specified-just like

modern DNA and proteins. Yet explaining how the building blocks of

RNA might have arranged themselves into information-rich sequences

has proven no easier than explaining how the parts of DNA might have

done so, given the requisite length and specificity of these molecules. As

Christian de Duve has noted in critique of the RNA-world hypothesis,

"Hitching the components together in the right manner raises addi

tional problems of such magnitude that no one has yet attempted to do

so in a prebiotic context."34

Ihe RNA World 315

Certainly, appeals to chance alone have not solved the RNA information problem. A 100-base RNA molecule corresponds to a space of possibilities equal to 4100 (or 1060). A 200-base RNA molecule corresponds to 4200 (or 10120) possibilities. Given this and the experiments mentioned above showing the rarity of functional ribozymes (to say nothing of polymerases) within RNA sequence space, the odds of a functional, self-replicating RNA sequence arising by chance are exceedingly small. Moreover, the odds against such an event occurring are only compounded by the likely presence of destructive cross-reactions between desirable and undesirable molecules within any realistic prebiotic environment.

To make matters worse, as Gerald Joyce and Leslie Orgel note, for a single-stranded RNA catalyst to produce an RNA identical to itself (i.e., to "self-replicate"), it must find an appropriate RNA molecule nearby to function as a template, since a single-stranded RNA cannot function as both replicase and template. Moreover, as they observe, this RNA template would have to be the precise complement of the replicase. Once this chance encounter occurred, the replicase molecule could make a copy of itself by making a complement of its complement (i.e., by transcribing the template), using the physics of nucleotide base pairing. 35

This requirement, of course, compounds the informational problem facing this crucial step in the RNA-world scenario. Even if an RNA sequence could acquire the replicase function by chance, it could perform that function only if another RNA molecule-one with a highly specific sequence relative to the original-arose close by. (See Fig. 14.6.) Thus, in addition to the specificity required to give the first RNA molecule self-. replicating capability, a second RNA molecule with an extremely specific sequence-one with essentially the same specificity as the original-would also have to arise. RNA-world theorists do not explain the origin of the requisite specificity in either the original molecule or its complement. Orgel and Joyce have calculated that to have a reasonable chance of finding two such complementary RNA molecules of a length sufficient to perform catalytic functions would require an RNA library of some 1048 RNA molecules. 36 The mass of such a library vastly exceeds the mass of the earth, suggesting the extreme implausibility of the chance origin of a primitive replicator system. They no doubt vastly underestimate the necessary size of this library and the actual improbability of a self-replicating couplet of


RNAs arising, because, as noted, they assume that a 50-base RNA might be capable of self-replication. (See note for qualifying details. )37

Given these odds, the chance origin of even a primitive selfreplicating system-one involving a pair of sequence-specific (i.e., information-rich) replicases-seems extremely implausible. And, yet, invoking natural selection doesn't reduce the odds or help explain the origin of the necessary replicators since natural selection ensues only after self-replication has arisen. As Orgel and Joyce explain, "Without evolution [i.e., prebiotic natural selection] it appears unlikely that a self-replicating ribozyme could arise, but without some form of selfreplication there is no way to conduct an evolutionary search for the first primitive self-replicating ribozyme."38

Robert Shapiro has resorted to one of my old standbys-Scrabble letters-to illustrate why neither chance, nor chance and natural selection combined, can solve the sequencing problem in the RNA world. While speaking in 2007 at a private conference on the origin of life, he asked an elite scientific audience to imagine an enormous pile of Scrabble letters. Then he said, "If you scooped into that heap [of letters], and you flung them on the lawn there, and the letters fell into a line which contained the words, 'To be or not to be, that is the question,' that is roughly the odds of an RNA molecule, given no feedback [natural selection]-and there would be no feedback, because it [the RNA molecule] wouldn't be functional until it attained a certain length and could copy itselfappearing on earth."39

If neither chance, nor chance and selection, can solve the RNA sequencing problem, can self-organization do the trick? It can't. RNA bases, like DNA bases, do not manifest bonding affinities that can explain their specific arrangements. Thus, no one has even attempted to solve the RNA sequencing problem by proposing a "self-organizational RNA world scenario." Instead, the same kind of evidentiary and theoretical problems emerge whether one proposes that genetic information arose first in RNA or DNA molecules. And every attempt to leapfrog the sequencing problem by starting with supposedly "informationgenerating" RNA replicators has only shifted the problem to the specific sequences that would be needed to make such replicators functional.

In addition, not only does the origin of RNA self-replication depend upon sequence specificity (information), but the transition from the

The RNA World 317

RNA-based translation system to the current protein-based translation

system would have required at some point the production of more than

100 different proteins, each of which would have in turn required an

information-rich nucleic acid to guide its construction.

Once again, the pink stuff was spreading.

L

2..

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Figure 14.6. The minimal requirements for template-directed RNA self

replication as envisioned by Joyce and Orgel. They insist that any RNA replicase would need to come into close proximity to an exact complementary

strand, thus increasing the needed sequence specificity associated with

getting such self-replication (and natural selection) started.


Problem 5: Ribozyme Engineering Does Not Simulate Undirected Chemical Evolution

Because of the difficulties with the RNA-world hypothesis and the

limited number of enzymatic functions that naturally occurring

ribozymes can perform, a new cottage industry has sprung up in

molecular biology. Scientists sympathetic to the RNA world have

sought to design new RNA molecules with heretofore unobserved

functions. In doing so, these scientists have hoped not only to learn

more about RNA chemistry, but also to demonstrate the plausibil

ity of the RNA-world hypothesis and possibly even to synthesize an

artificial form of life.40

These ribozyme-engineering experiments typically deploy one of two

approaches: the "rational design" approach or the "directed evolution"

approach. In both approaches, biologists try to generate either more

efficient versions of existing ribozymes or altogether new ribozymes

capable of performing some of the other functions of proteins. In the

rational-design approach, the chemists do this by directly modifying

the sequences of naturally occurring RNA catalysts. In the directed

evolution (or "irrational design") approach, scientists seek to simu

late a form of prebiotic natural selection in the process of producing

ribozymes with enhanced functional capacities. To manage this they

screen pools of RNA molecules using chemical traps to isolate mol

ecules that perform particular functions. After they have selected these

molecules out of the pool, they generate variant versions of these mol

ecules by randomly altering (mutating) some part of the sequence of

the original molecule. Then they select the most functional molecules

in this new crop and repeat the process several times until a discernible

increase in the desired function has been produced.

Most ribozyme-engineering procedures have been performed on li

gases, ribozymes that can link together two RNA chains ( oligomers)

by forming a single (phosphodiester) bond between them. Ribozyme

engineers want to demonstrate that these ligases can be transformed

into true polymerases or "replicases." These polymerases would not

only link nucleotide bases together (by phosphodiester bonds), but also

would stabilize the exposed template strands, and use the exposed bases

as a template to make sequence-specific copies.

The RNA World 319

Polymerases are the holy grail of ribozyme engineering. According

to the RNA-world hypothesis, once a polymerase capable of template

directed self-replication arose, then natural selection could have become

a factor in the subsequent chemical evolution of life. Since ligases can

perform one, though only one, of the several functions performed by

true polymerases, RNA-world theorists have postulated ligases as the

ancestral molecular species from which the first self-replicating poly

merase arose. They have tried to demonstrate the plausibility of this

conjecture by using ribozyme engineering to build polymerases (or rep

licases) from simpler ligase ribozymes.

To date, no one has succeeded in engineering a fully functional

RNA-based RNA polymerase, from either a ligase or anything else.41

Ribozyme engineers have, however, used directed evolution to enhance

the function of some common types of ligases. As noted, they also have

produced a molecule that can copy a small portion of itself. Leading ri

bozyme engineers such as Jack Szostak and David Bartel have presented

these results as support for an undirected process of chemical evolu

tion starting in an RNA world.42 Popular scientific publications and

textbooks have often heralded these experiments as models for under

standing the origin of life on earth and as the leading edge of research

establishing the possibility of evolving an artificial form of life in a test

tube.

Yet these claims have an obvious flaw. Ribozyme engineers tend to

overlook the role that their own intelligence has played in enhancing

the functional capacities of their RNA catalysts. The way the engineers

use their intelligence to assist the process of directed evolution would

have no parallel in a prebiotic setting, at least one in which only undi

rected processes drove chemical evolution forward. Yet this is the very

setting that ribozyme experiments are supposed to simulate.

RNA-world advocates envision ligases evolving via undirected pro

cesses into RNA polymerases that can replicate themselves from free

standing bases, thereby establishing the conditions for the beginning of

natural selection. In other words, these experiments attempt to simulate

a transition that, according to the RNA-world hypothesis, would have

taken place before natural selection had begun to operate. Yet in order to

improve the function of the ligase molecules, the experiments actually

simulate what natural selection does. Starting from a pool of random


sequences, the investigators create a chemical trap to isolate only those sequences that evince ligase function. Then they select those sequences for further evolution. Next they use a mutagenesis technique to generate a set of variant versions of these original ligases. Then they isolate and select the best sequences-those manifesting evidence of enhanced ligase function or indications of future polymerase function-and repeat the process until some improvement in the desired function has been realized.

But what could have accomplished these tasks before the first replicator molecule had evolved? Szostak and his colleagues do not say. They certainly cannot say that natural selection played this role, since the origin of natural selection as a process depends on the prior origin of the self-replicating molecule that Szostak and his colleagues are working so hard to design. Instead, in their experiment, Szostak and his colleagues play a role that nature cannot play until a self-replicating system, or at least a self-replicating molecule, has arisen. Szostak and his colleagues function as the replicators. They generate the crop of variant sequences. They make the choices about which of these sequences will survive to undergo another round of directed evolution. Moreover, they make these choices with the benefit of a foresight that neither natural selection nor any other undirected or unintelligent process can-by definition-possess.43 Indeed, the features of the RNA molecules that Szostak and his colleagues isolate and select are not features that would, by themselves in a precellular context, confer any functional advantage.

Of course, ligase enzymes perform functions in the context of modern cells and in that setting might confer a selectable advantage on the cells that possess them. But prior to the origin of the first selfreplicating protocell, ligase ribozymes would not have any functional advantage over any other RNAs. At that stage in chemical evolution, no self-reproducing system yet existed upon which any advantage could be conferred.

The ability to link (ligate) nucleotide chains is, at best, a necessary but not a sufficient condition of polymerase or replicase function. Absent a molecule or, what is more likely, a system of molecules possessing all of the features required for self-replication, nature would not favor any RNA molecule over any other. Natural selection as a process selects only

The RNA World 321

functionally advantageous features and only in self-replicating systems.

It passes its blind eye over molecules possessing merely necessary con

ditions or possible indicators of future function. Moreover, "it" does

nothing at all when mechanisms for replication and selection do not yet

even exist. In ribozyme-engineering experiments, engineers perform a

role in simulating natural selection that undirected natural processes

cannot play prior to the commencement of natural selection. Thus, even

if ribozyme experiments succeed in significantly enhancing the capaci

ties of RNA catalysts, it does not follow they will have demonstrated

the plausibility of an undirected process of chemical evolution. Insofar

as ribozyme-engineering experiments using a rational-design approach

(as opposed to a directed-evolution approach) involve an even more

overt role for intelligence, they exemplify the same problem (for ex

ample, see note 28).

Conclusion

As I have investigated various models that combined chance and neces

sity, I have noted an increasing sense of futility and frustration arising

among the scientists who work on the origin of life. As I surveyed the

literature, it became clear that this frustration had been building for

many years. In 1980, Francis Crick lamented, "An honest man, armed

with all the knowledge available to us now, could only state that in some

sense, the origin of life appears at the moment to be almost a miracle,

so many are the conditions which would have had to have been satisfied

to get it going."44 In 1988, the German biochemist and origin-of-life

researcher Klaus Dose followed suit with an equally critical assessment

of the state of the field. Dose explained that research efforts to date

had "led to a better perception of the immensity of the problem of the

origin of life on earth rather than to its solution. At present, all discus

sions on principal theories and experiments in the field either end in a

stalemate or a confession of ignorance."45 After attending a scientific

conference on the origin of life in 1989, one of my Cambridge supervi

sors returned to report, "The field is becoming increasingly populated

with cranks. Everyone knows everybody else's theory doesn't work, but

no one is willing to admit it about his own."

12/18/2014 The Origins of the RNA World


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Cold Spring Harb Perspect Biol. May 2012; 4(5): a003608.doi: 10.1101/cshperspect.a003608

PMCID: PMC3331698

The Origins of the RNA WorldMichael P Robertson and Gerald F Joyce

Departments of Chemistry and Molecular Biology and The Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, California92037Correspondence:Email: [email protected]

Copyright © 2012 Cold Spring Harbor Laboratory Press; all rights reserved

This article has been cited by other articles in PMC.

Abstract

The general notion of an “RNA World” is that, in the early development of life on the Earth, genetic continuity wasassured by the replication of RNA and genetically encoded proteins were not involved as catalysts. There is now strongevidence indicating that an RNA World did indeed exist before DNA and proteinbased life. However, argumentsregarding whether life on Earth began with RNA are more tenuous. It might be imagined that all of the components ofRNA were available in some prebiotic pool, and that these components assembled into replicating, evolvingpolynucleotides without the prior existence of any evolved macromolecules. A thorough consideration of this “RNAfirst”view of the origin of life must reconcile concerns regarding the intractable mixtures that are obtained in experimentsdesigned to simulate the chemistry of the primitive Earth. Perhaps these concerns will eventually be resolved, and recentexperimental findings provide some reason for optimism. However, the problem of the origin of the RNA World is farfrom being solved, and it is fruitful to consider the alternative possibility that RNA was preceded by some otherreplicating, evolving molecule, just as DNA and proteins were preceded by RNA.

1. INTRODUCTION

The general idea that, in the development of life on the Earth, evolution based on RNA replication preceded theappearance of protein synthesis was first proposed over 40 yr ago (Woese 1967; Crick 1968; Orgel 1968). It wassuggested that catalysts made entirely of RNA are likely to have been important at this early stage in the evolution of life,but the possibility that RNA catalysts might still be present in contemporary organisms was overlooked. Theunanticipated discovery of ribozymes (Kruger et al. 1982; GuerrierTakada et al. 1983) initiated extensive discussion ofthe role of RNA in the origins of life (Sharp 1985; Pace and Marsh 1985; Lewin 1986) and led to the coining of thephrase “the RNA World” (Gilbert 1986).

“The RNA World” means different things to different investigators, so it would be futile to attempt a restrictive definition.All RNA World hypotheses include three basic assumptions: (1) At some time in the evolution of life, genetic continuitywas assured by the replication of RNA; (2) WatsonCrick basepairing was the key to replication; (3) geneticallyencoded proteins were not involved as catalysts. RNA World hypotheses differ in what they assume about life that mayhave preceded the RNA World, about the metabolic complexity of the RNA World, and about the role of smallmolecule cofactors, possibly including peptides, in the chemistry of the RNA World.

There is now strong evidence indicating that an RNA World did indeed exist on the early Earth. The smoking gun is seenin the structure of the contemporary ribosome (Ban et al. 2000; Wimberly et al. 2000; Yusupov et al. 2001). The activesite for peptidebond formation lies deep within a central core of RNA, whereas proteins decorate the outside of this

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RNA core and insert narrow fingers into it. No amino acid side chain comes within 18 Å of the active site (Nissen et al.2000). Clearly, the ribosome is a ribozyme (Steitz and Moore 2003), and it is hard to avoid the conclusion that, assuggested by Crick, “the primitive ribosome could have been made entirely of RNA” (1968).

A more tenuous argument can be made regarding whether life on Earth began with RNA. In what has been referred to as“The Molecular Biologist's Dream” (Joyce and Orgel 1993), one might imagine that all of the components of RNA wereavailable in some prebiotic pool, and that these components could have assembled into replicating, evolvingpolynucleotides without the prior existence of any evolved macromolecules. However, a thorough consideration of this“RNAfirst” view of the origin of life inevitably triggers “The Prebiotic Chemist's Nightmare”, with visions of theintractable mixtures that are obtained in experiments designed to simulate the chemistry of the primitive Earth. Perhapsthis continuing nightmare will eventually have a happy ending, and recent experimental findings provide some reason foroptimism. However, the problem of the origin of the RNA World is far from being solved, and it is fruitful to consider thealternative possibility that RNA was preceded by some other replicating, evolving molecule, just as DNA and proteinswere preceded by RNA.

2. AN “RNAFIRST” VIEW OF THE ORIGIN OF LIFE

2.1. Abiotic Synthesis of Polynucleotides

This section considers the synthesis of oligonucleotides from ßDnucleoside 5′phosphates, leaving aside for now thequestion of how the nucleotides became available on the primitive Earth. Two fundamentally different chemical reactionsare involved. First, the nucleotide must be converted to an activated derivative, for example, a nucleoside 5′polyphosphate. Next the 3′hydroxyl group of a nucleotide or oligonucleotide molecule must be made to react with theactivated phosphate group of a monomer. Synthesis of oligonucleotides from nucleoside 3′phosphates will not bediscussed because activated nucleoside 2′ or 3′phosphates in general react readily to form 2′,3′cyclic phosphates.These cyclic phosphates are unlikely to oligomerize efficiently because the equilibrium constant for dimer formation is onlyof the order of 1.0 L/mol (Erman and Hammes 1966; Mohr and Thach 1969). In the presence of a complementarytemplate somewhat larger oligomers might be formed because the free energy of hybridization would help to driveforward the chain extension reaction.

In enzymatic RNA and DNA synthesis, the nucleoside 5′triphosphates (NTPs) are the substrates of polymerization.Polynucleotide phosphorylase, although it is a degradative enzyme in nature, can be used to synthesize oligonucleotidesfrom nucleoside 5′diphosphates. Nucleoside 5′polyphosphates are, therefore, obvious candidates for the activatedforms of nucleotides. Although nucleoside 5′triphosphates are not formed readily, the synthesis of nucleoside 5′tetraphosphates from nucleotides and inorganic trimetaphosphate provides a reasonably plausible prebiotic route toactivated nucleotides (Lohrmann 1975). Other more or less plausible prebiotic syntheses of nucleoside 5′polyphosphates from nucleotides have also been reported (Handschuh et al. 1973; Osterberg et al. 1973; Reimann andZubay 1999). Less clear, however, is how the first phosphate would have been mobilized to convert the nucleosides to5′nucleotides. Nucleoside 5′polyphosphates are highenergy phosphate esters, but are relatively unreactive in aqueoussolution. This may be advantageous for enzymecatalyzed polymerization, but is a severe obstacle for the nonenzymaticpolymerization of nucleoside 5′polyphosphates, which would occur far more slowly than the hydrolysis of the resultingpolynucleotide.

In a different approach to the activation of nucleotides, the isolation of an activated intermediate is avoided by using acondensing agent such as a carbodiimide (Khorana 1961). This is a popular method in organic synthesis, but itsapplication to prebiotic chemistry is problematic. Potentially prebiotic molecules such as cyanamide and cyanoacetyleneactivate nucleotides in aqueous solution, but the subsequent condensation reactions are inefficient (Lohrmann and Orgel1973).

Most attempts to study nonenzymatic polymerization of nucleotides in the context of prebiotic chemistry have usednucleoside 5′phosphoramidates, particularly nucleoside 5′phosphorimidazolides. Although phosphorimidazolides can be



formed from imidazoles and nucleoside 5′polyphosphates (Lohrmann 1977), they are only marginally plausible asprebiotic molecules. They were chosen because they are prepared easily and react at a convenient rate in aqueoussolution.

Nucleotides contain three principal nucleophilic groups: the 5′phosphate, the 2′hydroxyl, and the 3′hydroxyl group, inorder of decreasing reactivity. The reaction of a nucleotide or oligonucleotide with an activated nucleotide, therefore,normally yields 5′,5′pyrophosphate, 2′,5′phosphodiester, and 3′,5′phosphodiesterlinked adducts (Fig. 1A), in orderof decreasing abundance (Sulston et al. 1968). Thus the condensation of several monomers would likely yield anoligomer containing one pyrophosphate and a preponderance of 2′,5′phosphodiester linkages (Fig. 1B). There is littlechance of producing entirely 3′,5′linked oligomers from activated nucleotides unless a catalyst can be found thatincreases the proportion of 3′,5′phosphodiester linkages. Several metal ions, particularly Pb and UO , catalyze theformation of oligomers from nucleoside 5′phosphorimidazolides (Sleeper and Orgel 1979; Sawai et al. 1988). ThePb catalyzed reaction is especially efficient when performed in eutectic solutions of the activated monomers (inconcentrated solutions obtained by partial freezing of more dilute solutions). Substantial amounts of long oligomers areformed under eutectic conditions, but the product oligomers always contain a large proportion of 2′,5′linkages(Kanavarioti et al. 2001; Monnard et al. 2003).

Figure 1.

Phosphodiester linkages resulting from chemical condensation of nucleotides.(A) Reaction of an activated mononucleotide (N ) with an oligonucleotide(N –N ) to form a 3′,5′phosphodiester (left), 2′,5′phosphodiester ...

What kinds of prebiotically plausible catalysts might lead to the production of 3′,5′linked oligonucleotides directly fromnucleoside 5′phosphorimidazolides or other activated nucleotides? It is unlikely, but not impossible, that a metal ion orsimple acidbase catalyst would provide sufficient regiospecificity. The most attractive of the other hypotheses is thatadsorption to a specific surface of a mineral might orient activated nucleotides rigidly and thus catalyze a highlyregiospecific reaction.

The work of Ferris and coworkers provides support for this hypothesis (Ferris et al. 2004; Ferris 2006). They havestudied the oligomerization of nucleoside 5′phosphorimidazolides and related activated nucleotides on the clay mineralmontmorillonite (Ferris and Ertem 1993; Kawamura and Ferris 1994; Miyakawa and Ferris 2003). Some samples of themineral are effective catalysts, promoting the formation of oligomers even from dilute solutions of activated nucleotidesubstrates. Furthermore, the mineral profoundly affects the regiospecificity of the reaction. The oligomerization ofadenosine 5′phosphorimidazolide, for example, gives predominantly 3′,5′linked products in the presence ofmontmorillonite, but predominantly 2′,5′linked products in aqueous solution (Ding et al. 1996; Kawamura and Ferris1999). Once short oligomers have been synthesized, they can be further extended by adsorbing them on eithermontmorillonite or hydroxylapatite and repeatedly adding activated monomers, resulting in the accumulation of mainly3′,5′linked oligoadenylates up to 40–50 subunits in length (Ferris et al. 1996; Ferris 2002). However, even whenadsorbed on montmorillonite, the phosphorimidazolides of the pyrimidine nucleosides yield oligomers that arepredominantly 2′,5′linked.

Long oligomers have also been obtained from monomers in a single step using a different activated nucleotide in whichimidazole is replaced by 1methyladenine (Prabahar and Ferris 1997; Huang and Ferris 2003). Using the 1methyladenine derivative of adenylate or uridylate, oligomers containing up to 40 subunits were produced, consisting of ∼75%3′,5′linkages for oligoadenylate and ∼60% 3′,5′linkages for oligouridylate (Huang and Ferris 2006). Oligomerization ofthe 1methyladenine derivative of guanylate or cytidylate was less efficient, but all four activated monomers could be coincorporated, to at least a modest extent, within abiotically synthesized oligonucleotides.

Detailed analysis of this work on catalysis by montmorillonite suggests that oligomerization occurs at a limited number of

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structurally specific active sites within the interlayers of the clay (Wang and Ferris 2001). These sites must not besaturated with sodium ions, which appear to block access of the activated nucleotides (Joshi et al. 2009). Severaldifferent samples of montmorillonite have proven to be good catalysts, in part depending on their proton versus sodiumion content. It will be important to determine if there are other types of minerals that are comparably efficient catalysts ofoligonucleotide synthesis, and if so, to study the regiospecificity and sequence generality of the reactions they catalyze.

2.2. Nonenzymatic Replication of RNA

If a mechanism existed on the primitive Earth for the polymerization of activated nucleotides, it would have generated acomplex mixture of product oligonucleotides that differed in both length and sequence. The next stage in the emergenceof an RNA World would have been the replication of some of these molecules, so that a process equivalent to naturalselection could begin. The reaction central to replication of nucleic acids is templatedirected synthesis, that is, thesynthesis of a complementary oligonucleotide under the direction of a preexisting oligonucleotide. A good deal of workhas already been performed on this aspect of nonenzymatic replication. This work has been reviewed elsewhere (Joyce1987; Orgel 2004a), so only a summary of the results will be given here.

The first major conclusion is that most activated nucleotides do not undergo efficient, regiospecific, templatedirectedreactions in the presence of an RNA or DNA template. In general, only a small proportion of template moleculessucceed in directing the synthesis of a complete complement, and the complement usually contains a mixture of 2′,5′ and3′,5′phosphodiester linkages. After a considerable search, a set of activated nucleotides was found that undergo efficientand highly regiospecific templatedirected reactions. Working with guanosine 5′phospho2methylimidazolide (2MeImpG), it was shown that poly(C) can direct the synthesis of long oligo(G)s in a reaction that is highly efficient andhighly regiospecific (Inoue and Orgel 1981). If poly(C) is incubated with an equimolar mixture of the four 2MeImpNs(N = G, A, C, or U), less than 1% of the product consists of noncomplementary nucleotides (Inoue and Orgel 1982).Subsequent experiments suggested that this and the related reactions discussed later occur preferentially within thecontext of double helices that have a structure resembling the A form of RNA (Kurz et al. 1997, 1998; Kozlov et al.1999, 2000).

Random copolymers containing an excess of C residues can be used to direct the synthesis of products containing G andthe complements of the other bases present in the template (Inoue and Orgel 1983). The reaction with a poly(C,G)template is especially interesting because the products, like the template, are composed entirely of C and G residues. Ifthese products in turn could be used as templates, it might allow the emergence of a selfreplicating sequence. Selfreplication, however, is unlikely, mainly because poly(C,G) molecules that do not contain an excess of C residues tend toform stable selfstructures that prevent them from acting as templates (Joyce and Orgel 1986). The selfstructures are oftwo types: (1) the standard WatsonCrick variety based on C•G pairs, and (2) a quadrahelix structure that results fromthe association of four Grich sequences. As a consequence, any Crich oligonucleotide that can serve as a goodtemplate will give rise to Grich complementary products that tend to be locked in selfstructure and so cannot act astemplates. Overcoming the selfstructure problem using the standard C and G nucleotides is very difficult because itrequires the discovery of conditions that favor the binding of mononucleotides to allow templatedirected synthesis tooccur, but suppress the formation of long duplex regions that would exclude activated monomers from the template.

Some progress has been made in discovering definedsequence templates that are copied faithfully to yieldcomplementary products (Inoue et al. 1984; Acevedo and Orgel 1987; Wu and Orgel 1992a). Successful templatestypically contain an excess of C residues, with A and U residues isolated from each other by at least three C residues.Runs of G residues are copied into runs of C residues, so long as the formation of selfstructures by G residues can beavoided (Wu and Orgel 1992b). In light of the available evidence, it seems unlikely that a pair of complementarysequences can be found, each of which facilitates the synthesis of the other using nucleoside 5′phospho2methylimidazolides as substrates. Some of the obstacles to selfreplication may be attributable to the choice of reagentsand reaction conditions, but others seem to be intrinsic to the templatedirected condensation of activatedmononucleotides.



A related nonenzymatic replication scheme involves synthesis by the ligation of short 3′,5′linked oligomers (James andEllington 1999). This is certainly an attractive possibility, made more plausible by the discovery of analogous ribozymecatalyzed reactions (Bartel and Szostak 1993), but it faces two major obstacles. The first is the difficulty of obtaining thesubstrates in the first place. The second is concerned with fidelity. Pairs of oligonucleotides containing a single basemismatch, particularly if the mismatch forms a G•U wobble pair, still hybridize as efficiently as fully complementaryoligomers, except in a temperature range very close to the melting point of the perfectly paired structure. Maintainingfidelity would therefore be difficult under any plausible temperature regime.

Despite these problems, templatedirected ligation of short oligonucleotides may be a viable alternative to theoligomerization of activated monomers. Ferris' work discussed above suggests that predominantly 3′,5′linkedoligonucleotides might form spontaneously from activated nucleotides on some variety of montmorillonite (Ferris et al.1996) or on some other mineral. Oligonucleotide 5′triphosphates undergo slow but remarkably 3′,5′regiospecificligation in the presence of a complementary template (Rohatgi et al. 1996a,b). The combination of some such pair ofreactions might provide a replication scheme for polynucleotides starting with an input of activated monomers.

There also are efforts in what is sometimes termed “synthetic biology” to achieve nonenzymatic replication with moleculesthat resemble biological nucleic acids, but are not constrained by considerations of plausible prebiotic chemistry. Forexample, the 2′ and 3′hydroxyl groups of activated mononucleotides can be replaced by an amino group at eitherposition, providing enhanced nucleophilicity and resulting in more rapid templatedependent (and templateindependent)oligomerization (Lohrmann and Orgel 1976; Zielinski and Orgel 1985). Dinucleotide building blocks, consisting of 3′amino, 3′deoxynucleotide analogs can also be oligomerized in the presence of a suitable condensing agent (Zielinski andOrgel 1987). With additional modification of the nucleotide bases, it has been possible to carry out the templatedirectedcopying of nucleic acid sequences that contain of all four bases (Schrum et al. 2009). These efforts, although notexplaining the origin of the RNA World, contribute to understanding the chemical challenges that must be overcome inachieving the nonenzymatic replication of RNA.

2.3. The First RNA Replicase

The notion of the RNA World places emphasis on an RNA molecule that catalyzes its own replication. Such a moleculemust function as an RNAdependent RNA polymerase, acting on itself (or copies of itself) to produce complementaryRNAs, and acting on the complementary RNAs to produce additional copies of itself. The efficiency and fidelity of thisprocess must be sufficient to produce viable “progeny” RNA molecules at a rate that exceeds the rate of decompositionof the “parents.” Beyond these requirements, the details of the replication process are not highly constrained.

The RNAfirst view of the origin of life assumes that a supply of activated ßDnucleotides was available by some as yetunrecognized abiotic process. Furthermore, it assumes that a means existed to convert the activated nucleotides to anensemble of randomsequence polynucleotides, a subset of which had the ability to replicate. It seems to be implicit in themodel that such polynucleotides replicate themselves but, for whatever reason, do not replicate unrelated neighbors. It isnot clear whether replication involves one molecule copying itself (and its complement) or a family of molecules thattogether copy each other. These questions are set aside for the moment in order to first consider the question of whetheran RNA molecule of reasonably short length can catalyze its own replication with sufficiently high fidelity.

Accuracy and Survival

The concept of an error threshold, that is, an upper limit to the frequency of copying errors that can be tolerated by areplicating macromolecule, was first introduced by Eigen (1971). This important idea has been extended in a series ofmathematically sophisticated papers by McCaskill, Schuster, and others (McCaskill 1984a; Eigen et al. 1988; Schusterand Swetina 1988). Here only a brief summary of the subject is provided.

Eigen's model (1971) envisages a population of replicating polynucleotides that draw on a limited supply of activatedmononucleotides to produce additional copies of themselves. In this model, the rate of synthesis of new copies of a



particular replicating RNA is proportional to its concentration, resulting in autocatalytic growth. The net rate ofproduction is the difference between the rate of formation of errorfree copies and the rate of decomposition of existingcopies of the RNA. For an advantageous RNA to outgrow its competitors, its net rate of production must exceed themean rate of production of all other RNAs in the population. Only the errorfree copies of the advantageous RNAcontribute to its net rate of production, but all the copies of the other RNAs contribute to their collective production.Thus the relative advantage enjoyed by the advantageous individual compared with the rest of the population (oftenreferred to as the “superiority” of the advantageous individual) must exceed the probability of producing an error copy ofthat advantageous individual.

The proportion of copies of an RNA that are error free is determined by the fidelity of the component condensationreactions that are required to produce a complete copy. For simplicity, consider a selfreplicating RNA that is formed byn condensation reactions, each having mean fidelity q. The probability of obtaining a completely errorfree copy is givenby q , which is the product of the fidelity of the component condensation reactions. If an advantageous individual is tooutgrow its competitors, q must exceed the superiority, s, of that individual. Expressed in terms of the number ofreactions required to produce the advantageous individual,

For s > 1 and q > 0.9, this equation simplifies to

This is the “error threshold,” which describes the inverse relationship between the fidelity of replication, q, and themaximum allowable number of component condensation reactions, n. The maximum number of component reactions ishighly sensitive to the fidelity of replication, but depends only weakly on the superiority of the advantageous individual.For a selfreplicating RNA that is formed by the templatedirected condensation of activated mononucleotides, a total of2n – 2 condensation reactions are required to produce a complete copy. This takes into account the synthesis of both acomplementary strand and a complement of the complement.

It should be recognized that a marked superiority of one sequence over all other sequences could not be maintained overevolutionary time because novel variants would soon arise to challenge the dominant species. However, a marked initialsuperiority may be important in allowing an efficient selfreplicating RNA to emerge from a pool of less efficientreplicators. In the absence of other efficient replicators, a primitive selfreplicating RNA that operates with low fidelitymay gain a foothold by taking advantage of a somewhat less stringent error threshold. Whether or not this can occurdepends on its superiority. For example, an RNA that replicates 10fold more efficiently than its competitors and does sowith 90% fidelity could be no longer than 12 nucleotides, and a similarly advantageous RNA that replicates with 70%fidelity could be no longer than four nucleotides. It seems highly unlikely than any of the 17 million possible RNAdodecamers is able to catalyze its own replication with 90% fidelity, and even less likely that a tetranucleotide couldcatalyze its own replication with 70% fidelity. However, an RNA that replicates 10 fold more efficiently than itscompetitors and does so with 90% fidelity could be as long as 67 nucleotides, and one that replicates with 70% fidelitycould be as long as 20 nucleotides.

When selfreplication is first established, fidelity is likely to be poor and there is strong selection pressure favoringimprovement of the fidelity. As fidelity improves, a larger genome can be maintained. This allows exploration of a largernumber of possible sequences, some of which may lead to further improvement in fidelity, which in turn allows a stilllarger genome size, and so on. Once the evolving population has achieved a fidelity of about 99%, a genome length of

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about 100 nucleotides can be maintained, even for modest superiority values. This would allow RNAbased life tobecome firmly established. Until that time, it is a race between evolutionary improvement in the context of a sloppy selfreplicating system and the risk of delocalization of the genetic information because of overstepping the error threshold. Ifthe time required to bootstrap to high fidelity and large genomes is too long, there is a risk that the population willsuccumb to an environmental catastrophe before it has had the chance to develop appropriate countermeasures.

It is difficult to state with certainty the minimum possible size of an RNA replicase ribozyme. An RNA consisting of asingle secondary structural element, that is, a small stemloop containing 12–17 nucleotides, would not be expected tohave replicase activity, whereas a double stemloop, perhaps forming a “dumbbell” structure or a pseudoknot, might justbe capable of a low level of activity. A triple stemloop structure, containing 40–60 nucleotides, offers a reasonable hopeof functioning as a replicase ribozyme. One could, for example, imagine a molecule consisting of a pseudoknot and apendant stemloop that forms a cleft for templatedependent replication.

Suppose there is some 40mer that enjoys a superiority of 10 fold and replicates with 90% fidelity. This should beregarded as a highly optimistic but not outrageous view of what is possible for a minimum replicase ribozyme. Wouldsuch a molecule be expected to occur within a population of randomsequence RNAs? A complete library consisting ofone copy each of all 10 possible 40mers would weigh about 1 kg. There may be many such 40mers, encompassingboth distinct structural motifs and, more importantly, a large number of equivalent representations of each motif. As aresult, even a small fraction of the total library, consisting of perhaps 10 sequences and weighing about 1 g, might beexpected to contain at least one selfreplicating RNA with the requisite properties. It is not sufficient, however, that therebe just one copy of a selfreplicating RNA. The above calculations assume that a selfreplicating RNA can copy itself (orthat a fully complementary sequence is automatically available, as will be discussed later). If two or more copies of thesame 40mer RNA are needed, then a much larger library, consisting of 10 RNAs and weighing 10 g would berequired. This amount is comparable to the mass of the Earth.

At first sight, it might seem that one way to ease the error threshold would be for the replicase ribozyme to acceptdinucleotide or trinucleotide substrates, so that copies of the RNA could be formed by fewer condensation reactions.Calculations show that, over a broad range of superiority values, RNAs that are required to replicate with 90% fidelitywhen using mononucleotide substrates would be required to replicate with roughly 80% fidelity when using dinucleotidesubstrates or roughly 70% fidelity when using trinucleotide substrates. Thus the use of short oligomers offers only amodest advantage because of lessening of the error threshold, which likely would be outweighed by the greater difficultyof achieving high fidelity when discriminating among the 16 possible dinucleotide or 64 possible trinucleotide substrates,rather than among the four mononucleotides.

If one accepts the RNAfirst view that there was a prebiotic pool of randomsequence RNAs, and if one assumes thatthe pool included a replicase ribozyme containing, say, 40 nucleotides and replicating itself with about 90% fidelity, then itis not difficult to imagine how RNAbased evolution might have started. During the initial period a successful clone wouldhave expanded in the absence of competition. As competition for substrates intensified there would have followed asuccession of increasingly more advantageous individuals, each replicating within its error threshold. After a period ofintensifying competition, the single most advantageous species would have been replaced by a “quasispecies,” that is, amixture of the most advantageous individual and substantial amounts of closely related individuals that replicate almost asfast and almost as faithfully as the most advantageous one (Eigen and Schuster 1977; Eigen et al. 1988). Under theseconditions the persistence of a particular advantageous individual is no longer the problem, but one must understand theevolution of the composition of the quasispecies and the conditions for its persistence. This difficult problem has beenpartially solved by McCaskill (1984b). The general form of the solution is very similar to the error threshold described byEigen (1971), but with different values for the constant in the inequality. Thus concerns about the error threshold apply tothe quasispecies as well as to the succession of individuals. Practically speaking, however, once a quasispeciesdistribution of sophisticated replicators had emerged, the RNA World would have been on solid footing and, barring anenvironmental catastrophe, unlikely to lose the ability to maintain genetic information over time.

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Another ChickenandEgg Paradox

The previous discussion has tried mightily to present the most optimistic view possible for the emergence of an RNAreplicase ribozyme from a soup of randomsequence polynucleotides. It must be admitted, however, that this model doesnot appear to be very plausible. The discussion has focused on a straw man: The myth of a small RNA molecule thatarises de novo and can replicate efficiently and with high fidelity under plausible prebiotic conditions. Not only is such anotion unrealistic in light of current understanding of prebiotic chemistry (Joyce 2002), but it should strain the credulity ofeven an optimist's view of RNA's catalytic potential. If you doubt this, ask yourself whether you believe that a replicaseribozyme would arise in a solution containing nucleoside 5′diphosphates and polynucleotide phosphorylase!

If one accepts the notion of an RNA World, one is faced with the dilemma of how such a genetic system came intoexistence. To say that the RNA World hypothesis “solves the paradox of the chickenandtheegg” is correct if onemeans that RNA can function both as a genetic molecule and as a catalyst that promotes its own replication. RNAcatalyzed RNA replication provides a chemical basis for Darwinian evolution based on natural selection. Darwinianevolution is a powerful way to search among vast numbers of potential solutions for those that best address a particularproblem. Selection based on inefficient RNA replication, for example, could be used to search among a population ofRNA molecules for those individuals that promote improved RNA replication. But here one encounters another chickenandegg paradox: Without evolution it appears unlikely that a selfreplicating ribozyme could arise, but without some formof selfreplication there is no way to conduct an evolutionary search for the first, primitive selfreplicating ribozyme.

One way that RNA evolution may have gotten started without the aid of an evolved catalyst might be by usingnonenzymatic templatedirected synthesis to permit some copying of RNA before the appearance of the first replicaseribozyme. Suppose that the initial ensemble of monomers was not produced by random copolymerization, but rather by asequence of untemplated and templated reactions (Fig. 2), and further suppose that members of the initial ensemble ofmultiple stemloop structures could be replicated, albeit inefficiently, by the templatedirected process. This would havetwo important consequences. First, any molecule with replicase function that appeared in the mixture would likely find inits neighborhood similar molecules and their complements, related by descent, thus eliminating the requirement for twounrelated replicases to meet. Second, a majority of molecules in the mixture would contain stemloop structures. If it istrue that ribozyme function is favored by stable selfstructure, and if the basesequences of the stems in stemloopstructures are relatively unimportant for function, this model might provide an economical way of generating a relativelysmall ensemble of sequences that is enriched with catalytic sequences.

Figure 2.Nonenzymatic synthesis of multistemloop structures as a result of untemplated(open arrowhead) and templated (filled arrowhead) reactions. Templatedirected synthesis is assumed to occur rapidly whenever a template, activatedmonomers, and a suitable ...

How plausible is the assumption that replicases could act on sequences similar to themselves, while ignoring unrelatedsequences? This selectivity could be ensured by segregating individual molecules (or clonal lines) on the surface of mineralgrains, on the surface of micelles, or within membranes. Closely related molecules might be segregated as a groupthrough specific hydrogenbonding interactions (the family that sticks together, replicates together). For any segregationmechanism, weak selection would result if the replicating molecules are sufficiently dispersed that diffusion over theirintermolecular distance is slow compared with replication. Computer simulations have shown that under such conditionsof segregation, evolutionary bootstrapping can occur, resulting in progressively larger genomes that are copied withprogressively greater fidelity (Szabó et al. 2002). Alternatively, the requirement for replication of related, but notunrelated, sequences might be met through the use of “genomic tags” (Weiner and Maizels 1987). Among selfreplicatingsequences, it is plausible that some are restricted to copying molecules with a particular 3′terminal subsequence. Areplicator that happened by chance to carry a terminal sequence that matched the preference of its active site would






replicate itself while ignoring its neighbors.

Another resolution of the paradox of how RNA evolution was initiated without the aid of an evolved ribozyme is toabandon the RNAfirst view of the origin of life and suppose that RNA was not the first genetic molecule (CairnsSmith1982; Shapiro 1984; Joyce et al. 1987; Joyce 1989, 2002; Orgel 1989, 2004a). Perhaps RNA replication arose in thecontext of an evolving system based on something other than RNA (see the section “Alternative Genetic Systems”). Evenif this is true, all of the arguments concerning the relationship between the fidelity of replication and the maximumallowable genome length would still apply to this earlier genetic system. Of course, the challenge to those who advocatethe RNAlater approach is to show that there is an informational entity that is prebiotically plausible and is capable ofinitiating its own replication without the aid of a sophisticated catalyst.

2.4. Replicase Function in the Evolved RNA World

Although it is difficult to say how the first RNA replicase ribozyme arose, it is not difficult to imagine how such amolecule, once developed, would function. The chemistry of RNA replication would involve the templatedirectedpolymerization of mono or short oligonucleotides, using chemistry in many ways similar to that used by contemporarygroup I ribozymes (Cech 1986; Been and Cech 1988; Doudna and Szostak 1989). One important difference is that,unlike group I ribozymes, which rely on a nucleoside or oligonucleotide leaving group, an RNA replicase would morelikely make use of a different leaving group that provides a substantial driving force for polymerization and that, after itsrelease, does not become involved in some competing phosphoester transfer reaction.

From Ligases to Polymerases

The polymerization of activated nucleotides proceeds via nucleophilic attack by the 3′hydroxyl of a templateboundoligonucleotide at the αphosphorus of an adjacent templatebound nucleotide derivative (Fig. 3). The nucleotide is“activated” for attack by the presence of a phosphoryl substituent, for example a phosphate, polyphosphate, alkoxide, orimidazole group. As discussed previously, polyphosphates, such as inorganic pyrophosphate, are the most obviouscandidates for the leaving group. The condensation reaction could be assisted by favorable orientation of the reactivegroups, deprotonation of the nucleophilic 3′hydroxyl, stabilization of the trigonalbipyramidal transition state, and chargeneutralization of the leaving group. All of these tasks might be performed by RNA (Narlikar and Herschlag 1997;Emilsson et al. 2003), acting either alone (OrtolevaDonnelly and Strobel 1999) or with the help of a suitably positionedmetal cation or other cofactor (Shan et al. 1999; Shan et al. 2001).

Figure 3.Nucleophilic attack by the 3′hydroxyl of a templatebound oligonucleotide(N –N ) on the αphosphorus of an adjacent templatebound mononucleotide(N ). Dotted lines indicate base pairing to a complementary template. R is the...

The possibility that an RNA replicase ribozyme could have existed has been made abundantly clear by work involvingribozymes that have been developed in the laboratory through in vitro evolution (Bartel and Szostak 1993; Ekland et al.1995; Ekland and Bartel 1996; Robertson and Ellington 1999; Jaeger et al. 1999; Rogers and Joyce 2001; Johnston etal. 2001; McGinness and Joyce 2002; Ikawa et al. 2004; Fujita et al. 2009). Bartel and Szostak (1993), for example,began with a large population of randomsequence RNAs and evolved the “class I” RNA ligase ribozyme, an optimizedversion of which is about 100 nucleotides in length and catalyzes the joining of two templatebound oligonucleotides.Condensation occurs between the 3′hydroxyl of one oligonucleotide and the 5′triphosphate of another, forming a 3′,5′phosphodiester linkage and releasing inorganic pyrophosphate. This reaction is classified as ligation because of the natureof the oligonucleotide substrates, but involves the same chemical transformation as is catalyzed by modern RNApolymerase enzymes.

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Xray crystal structures of two RNA ligase ribozymes, the L1 and abovementioned class I ligases, have beendetermined, providing a glimpse into the mechanistic strategies that these two structurally and evolutionarily distinctribozymes use to catalyze the same reaction (Robertson and Scott 2007; Shechner et al. 2009) (Fig. 4). Both crystalstructures capture the product of the ligation reaction, and consequently offer an incomplete view of the reactionpathway. For example, the pyrophosphate leaving group is absent from the structures, so no conclusions can be drawnregarding potential ribozymeassisted orientation of the reactive triphosphate or charge neutralization of thepyrophosphate leaving group. There is, however, information regarding other aspects of the reaction mechanism that canbe inferred from the product structures.

Figure 4.Xray crystal structure of the (A) L1 ligase and (B) class I ligase ribozymes.Insets show the putative magnesium ion binding sites at the respective ligationjunctions. The structures are rendered in rainbow continuum, with the 5′triphosphatebearing ...

Both the L1 and class I ligases are dependent on the presence of magnesium ions for their activity. A prominent feature ofthe L1 structure (Fig. 4A) involves a bound metal ion in the active site, coordinated by three nonbridging phosphateoxygens, one of which belongs to the newly formed phosphodiester linking what originally were the two substrates. Thismagnesium ion is favorably positioned to help neutralize the increased negative charge of the transition state and,potentially, to activate the 3′hydroxyl nucleophile and to help orient the αphosphate for a more optimal inline alignment.In the case of the class I structure (Fig. 4B), no catalytic metal ions appear to have been retained in the vicinity of theactive site, although two magnesium ions are observed to participate in crucial structural interactions that help shape theactive site architecture. Despite the lack of direct observation of a catalytic metal at the active site, there is what appearsto be an empty metal binding site formed by two nonbridging phosphate oxygens, positioned directly opposite the ligationjunction in a manner similar to that observed for the magnesium binding site of the L1 ligase and reminiscent of thearrangement seen in protein polymerases. The lack of a metal in the crystal structure may simply be an artifact of thecrystallization process or may imply a local conformational change in the product that disfavors retention of the boundmetal. These structures show that, despite some remaining gaps in the detailed understanding of how these ribozymesfunction, the available information points to a universal catalytic strategy, very similar to that used by modern proteinbased RNA polymerases.

Subsequent to its isolation as a ligase, the class I ribozyme was shown to catalyze a polymerization reaction in which the5′triphosphatebearing oligonucleotide is replaced by one or more NTPs (Ekland and Bartel 1996). This reactionproceeds with high fidelity (q = 0.92), but the reaction rate drops sharply with successive nucleotide additions.

Bartel and colleagues performed further in vitro evolution experiments to convert the class I ligase to a bona fide RNApolymerase that operates on a separate RNA template (Johnston et al. 2001). To the 3′ end of the class I ligase theyadded 76 randomsequence nucleotides that were evolved to form an accessory domain that assists in the polymerizationof templatebound NTPs. The polymerization reaction is applicable to a variety of template sequences, and for wellbehaved sequences proceeds with an average fidelity of 0.967. This would be sufficient to support a genome length ofabout 30 nucleotides, although the ribozyme itself contains about 190 nucleotides. The ribozyme has a catalytic rate forNTP addition of at least 1.5 min , but its K is so high that, even in the presence of micromolar concentrations ofoligonucleotides and millimolar concentrations of NTPs, it requires about 2 h to complete each NTP addition (Lawrenceand Bartel 2003). The ribozyme operates best under conditions of high Mg concentration, but becomes degradedunder those conditions over 24 h, by which time it has added no more than 14 NTPs (Johnston et al. 2001).

Further optimization of the polymerase ribozyme using highly sophisticated in vitro evolution techniques has led toadditional improvements in its biochemical properties. By directly selecting for extension of an external primer on aseparate template, Zaher and Unrau (2007) were able to improve the maximum length of templatedependent

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polymerization to >20 nucleotides, with a rate that is ∼threefold faster than that of the parent for the first nine monomeradditions and up to 75fold faster for additions beyond 10 nucleotides. In addition, although not rigorously quantitated,the new ribozyme displays significantly improved fidelity, particularly with respect to discrimination against G•U wobblepairs. It is this improved fidelity that appears to be the underlying source for the observed improvements in the maximumlength of extension and the rate of polymerization.

A different RNA ligase ribozyme can operate on a separate RNA template in a largely sequencegeneral manner, anddoes so with a K that is at least 100fold lower than that of the classIderived polymerase (McGinness and Joyce2002). However, its catalytic rate is much lower as well, and it is unable to add more than a single NTP. Yet anotherRNA ligase ribozyme can operate on a separate template with the help of designed tertiary interactions that “clamp” thetemplate–substrate complex to the ribozyme (Ikawa et al. 2004). But it too is a relatively slow catalyst that cannot addmore than a single NTP.

A highly pessimistic view is that because there is no known polymerase ribozyme that combines all of the propertiesnecessary to sustain its own replication, no such ribozyme is possible. A more balanced view is that RNA clearly iscapable of greatly accelerating the templatedependent polymerization of nucleoside 5′polyphosphates. Such catalyticRNAs can operate in a sequencegeneral manner and with reasonable fidelity. It seems only a matter of time (and likelyconsiderable effort) before more robust polymerase ribozymes will be obtained. Nature did not have the opportunity toconduct carefully arranged evolution experiments using highlypurified reagents, but did have the luxury of much greaterreaction volumes and much more time.

RNA Replication

Despite falling short of the ultimate goal of a generalpurpose RNA polymerase ribozyme, a robust reaction system forRNAcatalyzed RNA replication has recently been shown. The system uses a pair of crossreplicating ligase ribozymesthat each catalyze the formation of the other, using a mixture of four different substrate oligonucleotides (Lincoln andJoyce 2009). In reaction mixtures containing only these RNA substrates, MgCl , and buffer, a small starting amount ofribozymes gives rise to many additional ribozymes through a process of RNAcatalyzed exponential amplification.Whenever the substrates become depleted, the replication process can be restarted and sustained indefinitely byreplenishing the supply of substrates.

Because the substrates are recognized by the ribozymes through specific WatsonCrick pairing interactions, evolutionexperiments can be performed by providing a variety of substrates that have different sequences in these recognitionregions and different corresponding sequences in the catalytic domain of the ribozyme. RNA replication was performedwith a library of 144 possible substrate combinations, resulting in the emergence of a set of highly advantageousreplicators that included recombinants which were not present at the start of the experiment. Until the advent of ageneralpurpose RNA polymerase ribozyme, the system of crossreplicating ligases offers the best platform to study thebiochemical properties and evolutionary behavior of an allRNA replicative system.

Nucleotide Biosynthesis

RNA replicase activity is probably not the only catalytic behavior that was essential for the existence of the RNA World.Maintaining an adequate supply of the four activated nucleotides would have been a top priority. Even if the prebioticenvironment contained a large reservoir of these compounds, the reservoir would eventually become depleted, and somecapacity for nucleotide biosynthesis would have been required.

Ribozymes have been obtained, through in vitro evolution, that catalyze some of the steps of nucleotide biosynthesis.Unrau and Bartel (1998), for example, developed a ribozyme that catalyzes a reaction between 4thiouracil and 5phosphoribosyl1pyrophosphate (PRPP) to form 4thiouridylate (Fig. 5A). The 4thiouracil is provided free in solutionand the PRPP is tethered to the 3′ end of the ribozyme. An optimized form of this ribozyme, containing 124 nucleotides,has an observed rate of 0.2 min in the presence of 4 mM 4thiouracil (Chapple et al. 2003). This is at least 10 fold

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faster than the uncatalyzed rate of reaction, which is too slow to measure. Unrau and colleagues used a similar approachto develop two different ribozymes that catalyze the formation of 6thioguanylate from 6thioguanine and tethered PRPP(Lau et al. 2004), as well as a third guanylate synthase ribozyme that arose as an unanticipated consequence of a relatedin vitro evolution experiment (Lau and Unrau 2009). The first two guanylate synthase ribozymes are slightly larger andhave about twofold higher catalytic efficiency compared with the uridylate synthase ribozyme, although guanylatesynthesis is expected to have a much higher uncatalyzed rate of reaction.

Figure 5.Known RNAcatalyzed reactions that are relevant to nucleotide biosynthesis.(A) Formation of 4thiouridylate from free 4thiouracil and ribozymetethered 5phosphoribosyl1pyrophosphate. (B) 5′phosphorylation of an oligonucleotideusing γthioATP ...

RNAcatalyzed synthesis of PRPP has not been shown, but a ribozyme has been obtained that catalyzes the 5′phosphorylation of oligonucleotides using γthioATP as the phosphate donor (Lorsch and Szostak 1994) (Fig. 5B). Theribozyme shows a rate enhancement of about 10 fold compared with the uncatalyzed rate of reaction. Once anucleoside 5′phosphate has been formed, it canphosphoryl be activated by another ribozyme that catalyzes thecondensation of a nucleoside 5′phosphate and a ribozymetethered nucleoside 5′triphosphate (Huang and Yarus 1997)(Fig. 5C). This results in the formation of a 5′,5′pyrophosphate linkage, which provides an activated nucleotide leavinggroup that can drive subsequent RNAcatalyzed, templatedirected ligation of RNA (Hager and Szostak 1997) (Fig. 5D).

None of these four RNAcatalyzed reactions has precisely the right format for the corresponding reaction in ahypothetical nucleotide biosynthesis pathway in the RNA World. However, they show that RNA is capable ofperforming the relevant chemistry with substantial catalytic rate enhancement. It remains to be seen whether ribozymescan be developed that catalyze the formation of the fundamental building blocks of RNA, Dribose and the fournucleotide bases, using starting materials that would have been abundant on the primitive Earth.

3. An “RNALATER” VIEW OF THE ORIGIN OF LIFE

3.1. Abiotic Synthesis of Nucleotides

The RNAfirst view of the origin of life proceeds from the assumption that pure ßDnucleotides were available in someprebiotic pool. How close to such a pool could one hope to get without magic (or evolved enzymes) on the primitiveEarth? Could one hope to achieve replication in a pool containing a more realistic mixture of organic molecules, including,of course, ßDribonucleotides? The synthesis of a nucleotide could occur in a number of ways. The simplest,conceptually, would be to synthesize a nucleoside base, couple it to ribose, and finally to phosphorylate the resultingnucleoside. However, a number of other routes are feasible, for example the assembly of the base on a preformed riboseor ribose phosphate, or the coassembly of the base and sugarphosphate.

The classical prebiotic synthesis of sugars is by the polymerization of formaldehyde (the “formose” reaction). It yields avery complex mixture of products including only a small proportion of ribose (Mizuno and Weiss 1974). This reactiondoes not provide a reasonable route to the ribonucleotides. However, a number of more recent experimental findings, tosome extent, address this deficiency.

The basecatalyzed aldomerization of glycoaldehyde phosphate in the presence of a halfequivalent of formaldehydeunder strongly alkaline conditions gives a relatively simple mixture of tetrose and pentosediphosphates and hexosetriphosphates, of which ribose 2,4diphosphate is the major component (Müller et al. 1990). Reactions of this kindproceed efficiently when 2 mM solutions of substrates are incubated at room temperature and pH 9.5 in the presence oflayered hydroxides such as hydrotalcite (magnesium aluminum hydroxide) (Pitsch 1992; Pitsch et al. 1995a). The

9








phosphates are absorbed between the positively charged layers of the mineral. The reaction proceeds under these milderconditions presumably because of the high concentration of substrates in the interlayer and because the positive charge onthe metal hydroxide layers favors enolization of glycoaldehyde phosphate. A reaction between glycoaldehyde orglyceraldehyde and the amidotriphosphate ion provides an ingenious and prebiotically plausible route to glycoaldehydephosphate and glyceraldehyde2phosphate, respectively, the two substrates in the above reactions (Krishnamurthy et al.2000).

A number of other studies have addressed the problems presented by the lack of specificity of the formose reaction andby the instability of ribose. The Pb ion is an excellent catalyst for the formose reaction and enables yields of thepentose sugars as high as 30% to be achieved (Zubay 1998). Furthermore, it seems likely that ribose is almostexclusively the first pentose product of the reaction and that the other pentoses are formed from it by isomerization. Otherrecent studies have addressed the problem presented by the instability of ribose. The four pentose sugars, includingribose, are all strongly stabilized in the presence of borate ions or calcium borate minerals (Ricardo et al. 2004).However, the effect of borate on the progress of the formose reaction has not been reported.

Many sugars, including the four pentoses, react readily with cyanamide to form stable bicyclic aminooxazolines (Sanchezand Orgel 1970) (Fig. 6A). Strikingly, the ribose derivative crystallizes readily from aqueous solution even when complexmixtures of related molecules, including a mixture of the aminooxazoline derivatives of the other three pentose sugars,are present (Springsteen and Joyce 2004). The crystals are multiply twinned, each crystal containing many small domainsof each of the two enantiomorphs. Thus the reaction of a mixture of racemic sugars with cyanamide followed bycrystallization might stabilize ribose, segregate it from other sugars, and present it in enantiospecific microdomains. Muchremains to be shown, but the reactions described above suggest that ribose synthesis, although still problematic, may notbe the intractable problem it once seemed.

Figure 6.

Potential prebiotic synthesis of pyrimidine nucleosides. (A) Reaction of ribosewith cyanamide to form a bicyclic product, with cyanamide joined at both theanomeric carbon and 2hydroxyl. (B) Analogous reaction of arabinose3phosphate to form a bicyclic ...

The synthesis of the nucleoside bases is one of the success stories of prebiotic chemistry. Adenine is formed withremarkable ease from ammonia and hydrogen cyanide (Orò 1961). This synthesis has been described as “the rock of thefaith” by Stanley Miller. Reasonably plausible syntheses of the other purine bases and of the pyrimidines have also beendescribed (Sanchez et al. 1967; Ferris et al. 1968; Robertson and Miller 1995; Orgel 2004b; Saladino et al. 2004). Thecoupling of the purine bases with ribose or ribosephosphate has been achieved under mild conditions, but in relativelylow yield (Fuller et al. 1972). The corresponding reaction with pyrimidines does not occur.

There is a different potential route for the prebiotic synthesis of pyrimidine nucleotides via arabinose aminooxazoline thatfirst was explored nearly 40 yr ago (Tapiero and Nagyvary 1971) and in recent years has begun to look very persuasive(Ingar et al. 2003; Anastasi et al. 2007; Powner et al. 2009). The earlier studies began with arabinose 3phosphate,which, like arabinose and other sugars, reacts with cyanamide to give the corresponding aminooxazoline (Fig. 6B). Thisin turn reacts with cyanoacetylene to form a tricyclic intermediate that hydrolyzes to produce a mixture of cytosinearabinoside3′phosphate and cytosine 2′,3′cyclic phosphate.

Sutherland and colleagues (Powner et al. 2009) have taken this approach further by starting simply with glycoaldehydeand cyanamide, which in the presence of 1 M phosphate at neutral pH gives 2aminooxazole in excellent yield (Fig. 6C).The phosphate both buffers and catalyzes the reaction, directing glycoaldehyde toward 2aminooxazole, rather than acomplex mixture of aldomerization products. Glyceraldehyde is then added, resulting in formation of the various pentoseaminooxazolines, including the arabinose compound. Arabinose aminooxazoline, in turn, can react with cyanoacetylene,

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also in phosphate buffer, to form cytosine 2′,3′cyclic phosphate as the major product. Perhaps equally intriguing,although given less emphasis in these studies, is that reaction of arabinose aminooxazoline with cyanoacetylene also givesa substantial yield of cytosine 2′,3′cyclic5′bisphosphate, which is more amenable to being converted to an activatedmonomer that would be suitable for polymerization.

There has been significant progress, especially recently, concerning the synthesis of the nucleosides and nucleotides fromprebiotic precursors in reasonable yield. However, the story remains incomplete because these syntheses still requiretemporally separated reactions using high concentrations of just the right reactants, and would be disrupted by thepresence of other closely related compounds. The reactions channel material toward the desired products, but otherfractionation processes must be discovered that provide the correct starting materials at the requisite time and place. This“preprebiotic” chemistry likely would involve a series of reactions catalyzed by minerals or metal ions, coupled with aseries of subtle fractionations of nucleotidelike materials based on adsorption on minerals, selective complex formation,crystallization, etc.

Even minerals could not achieve on a macroscopic scale one desirable separation, the resolution of Dribonucleotidesfrom their Lenantiomers. This is a serious problem because experiments on templatedirected synthesis using poly(C)and the imidazolides of G suggest that the polymerization of the Denantiomer is strongly inhibited by the Lenantiomer(Joyce et al. 1984). This difficulty may not be insuperable; perhaps with a different mode of phosphate activation, theinhibition would be less severe. However, enantiomeric crossinhibition is certainly a serious problem if life arose in aracemic environment.

It is possible that the locale for life's origins was not racemic, even though the global chemical environment containednearly equal amounts of each pair of stereoisomers. There likely were biases in the inventory of compounds delivered tothe Earth by comets and meteorites. For example, some carbonaceous chondrite meteorites contain a significantenantiomeric excess of Lamino acids that are known to be indigenous to the meteorite (Engel and Macko 1997; Croninand Pizzarello 1997; Pizzarello et al. 2003; Glavin and Dworkin 2009). These in turn could bias terrestrial syntheses,although the level of enantiomeric enrichment generally declines with successive chemical reactions. A special exceptionare a remarkable set of reactions and fractionation processes that amplify a slight chiral imbalance, even to the level oflocal homochirality (Kondepudi et al. 1990; Soai et al. 1995; Viedma 2005; Klussmann et al. 2006; Noorduin et al.2008; Viedma et al. 2008). These systems have in common both a catalytic process for amplification of samehandedmolecules and an inhibition process for suppression of oppositehanded molecules.

Some of the most appealing examples of chiral symmetrybreaking reactions involve saturating solutions of various aminoacids that form an equilibrium between the liquid phase and solid phase. The solid phase consists of either racemic orenantiopure crystals, and the liquid phase reflects whatever enantiomeric excess exists at the eutectic point for themixture. For some amino acids, such as serine and histidine, the enantiomeric excess at the eutectic is >90% (Klussmannet al. 2006). This means that, starting from a small concentration imbalance of D and Lisomers, the imbalance isamplified as both isomers enter the solid phase and the solution phase approaches the eutectic equilibrium. This andrelated nearequilibrium mechanisms (Noorduin et al. 2008; Viedma et al. 2008) could provide a means to achieve highenantiomeric enrichment in a local environment. This in turn could bias the production of ribose and the derivednucleotides.

Scientists interested in the origins of life seem to divide neatly into two classes. The first, usually but not always molecularbiologists, believe that RNA must have been the first replicating molecule and that chemists are exaggerating thedifficulties of nucleotide synthesis. They believe that a few more striking chemical “surprises” will establish that a pool ofracemic mononucleotides could have formed on the primitive Earth, and that further experiments with different activatinggroups, minerals, and chiral amplification processes will solve the enantiomeric crossinhibition problem. The secondgroup of scientists are much more pessimistic. They believe that the de novo appearance of oligonucleotides on theabiotic Earth would have been a near miracle. Time will tell which is correct.

3.2. Alternative Genetic Systems



The problems that arise when one tries to understand how an RNA World could have arisen de novo on the primitiveEarth are sufficiently severe that one must explore other possibilities. What kind of alternative genetic systems might havepreceded the RNA World? How could they have “invented” the RNA World? These topics have generated a good dealof speculative interest and some relevant experimental data.

Eschenmoser and colleagues have undertaken a systematic study of the properties of analogs of nucleic acids in whichribose is replaced by some other sugar, or in which the furanose form of ribose is replaced by the pyranose form(Eschenmoser 1999) (Fig. 7B). Strikingly, polynucleotides based on the pyranosyl analog of ribose (pRNA) formWatsonCrick paired double helices that are more stable than RNA, and pRNAs are less likely than the correspondingRNAs to form multiplestrand competing structures (Pitsch et al. 1993, 1995b, 2003). Furthermore, the helices twistmuch more gradually than those of standard nucleic acids, which should make it easier to separate strands of pRNAduring replication. Pyranosyl RNA appears to be an excellent choice as a genetic system; in some ways it seems animprovement compared with the standard nucleic acids. However, pRNA does not interact with normal RNA to formbasepaired double helices.

Figure 7.

The structures of (A) RNA; (B) pRNA; (C) TNA; (D) GNA; (E) PNA; (F)ANA; (G) diaminotriazinetagged (left) and dioxo5aminopyrimidinetagged(right) oligodipeptides; and (H) tPNA. ANA contains a backbone of alternatingD and Lalanine subunits. The ...

Most doublehelical structures reported in the literature are characterized by a backbone with a sixatom repeat.Eschenmoser and colleagues made the surprising discovery that an RNAlike structure based on threose nucleotideanalogs (TNA) (Fig. 7C), although it involves a fiveatom repeat, can still form a stable doublehelical structure withstandard RNA (Schöning et al. 2000). This provides an example of a pairing system based on a sugar that could beformed more readily than ribose: Tetroses are the unique products of the dimerization of glycoaldehyde, whereaspentoses are formed along with tetroses and hexoses from glycoaldehyde and glyceraldehyde. A structural simplificationof Eschenmoser's threose nucleic acid has been achieved by Meggers and colleagues (Zhang et al. 2005). They replacedthreose by its open chain analogue, glycol, in the backbone of TNA, resulting in glycol nucleic acid (GNA) (Fig. 7D).Complementary oligomers of GNA form antiparallel, doublehelices with surprisingly high duplex stabilities.

Peptide nucleic acid (PNA) is another nucleic acid analog that has been studied extensively (Fig. 7E). It was discoveredby Nielsen and colleagues in the context of research on antisense oligonucleotides (Egholm et al. 1992, 1993; Wittung etal. 1994). PNA is an uncharged, achiral analog of RNA or DNA in which the ribosephosphate backbone of the nucleicacid is replaced by a backbone held together by amide bonds. PNA forms very stable double helices withcomplementary RNA or DNA. Work in the Orgel laboratory has shown that information can be transferred from PNAto RNA, or from RNA to PNA, in templatedirected reactions, and that PNA/DNA chimeras are readily formed oneither DNA or PNA templates (Schmidt et al. 1997a,b; Koppitz et al. 1998). Thus it seems that a transition from a PNAWorld to an RNA World is possible.

The alanyl nucleic acids (ANA) are interesting for a different reason. They are polypeptides formed from nucleo aminoacids (Fig. 7F), but pairing structures can be formed only if the two enantiomers of their constituent αamino acids occurin a regular alternating sequence (Diederichsen 1996, 1997). Because abiotic syntheses of potentially chiral moleculeswould under almost all circumstances yield racemic products, pairing structures that can be formed from racemic mixturesare particularly attractive. The ANAtype backbone of alternating D and Lamino acids could, in principle, supportpaired, doublestranded structures based on a variety of sidechain interactions.

Eschenmoser and colleagues have examined repeating homochiral dipeptide backbones that have either triazines oraminopyrimidines attached at alternating positions (Mittapalli et al. 2007a,b) (Fig. 7G). In this case, not only is the











backbone a potential precursor to that of RNA, but also the bases have been replaced by potential precursors, such as2,4diaminotriazine ( NN), 2,4dioxotriazine ( OO), 2,4diamino5aminopyrimidine ( NN), and 2,4dioxo5aminopyrimidine ( OO). Oligomers containing either NN or OO subunits were found to pair strongly withcomplementary RNA, whereas oligomers containing either OO or NN subunits did not. Not surprisingly, therefore,pairing between complementary substituted oligodipeptides of the same type (either oligo[ NN]•oligo[ OO] oroligo[ NN]•oligo[ OO]) also was weak. However, crosspairing between oligo( NN) and oligo( OO) was robust(Mittapalli et al. 2007a). This raises the intriguing possibility that an informational polymer could have a mixedcomposition of NN and OO subunits, which would direct the synthesis of an opposing strand that has acomplementary sequence as well as a “complementary” backbone composition.

Even more radical are thioester peptide nucleic acids (tPNA) (Fig. 7H), containing a repeating dipeptide backbone withcysteine residues at alternating positions, which are transiently linked via a thioester to a nucleic acid base (Ura et al.2009). The bases are in dynamic equilibrium between the solution and cysteine positions along the backbone. Occupancyof a base at a particular position is enhanced by the presence of the complementary base on the opposing strand. In thisway the informational polymer can selfassemble in a templatedirected manner, with mismatched bases exchangingrapidly and matched bases remaining thioesterified for an extended period of time. Perhaps genetic information could bepropagated in such a system, although the fidelity of replication, and therefore the maximum number of informationalsubunits, is likely to be modest.

The studies described previously suggest that there are many ways of linking together the nucleotide bases into chains thatare capable of forming basepaired double helices. It is not clear that it is much easier to synthesize the monomers of pRNA, TNA, GNA, PNA, ANA, or tPNA than to synthesize the standard nucleotides. However, it is possible that abasepaired structure of this kind will be discovered that can be synthesized readily under prebiotic conditions. Theproperties of the NN and OOtagged oligodipeptides suggest that it may be fruitful to explore a broader range ofpotential precursors to RNA, changing the recognition elements as well as the backbone. A strong candidate for the firstgenetic material would be any informational macromolecule that is replicable in a sequencegeneral manner and derivesfrom compounds that would have been abundant on the primitive Earth, and preferably has the ability to crosspair withRNA.

The transition from an RNAlike World to the RNA World could take place in two ways. The transition might becontinuous if the preRNA template could direct the synthesis of an RNA product with a complementary sequence. Sucha transition, for example, from PNA to RNA, would preserve information. RNA could then act as a genetic material in aformerly PNA World. However, even if chimeras were involved in the transition, it is unlikely that the original function ofa PNA catalyst could be retained throughout the transition because PNA and RNA have such different backbonestructures. A direct and continuous transition from pRNA to RNA would not be possible because pRNA does notform complementary double helices with RNA, but this limitation does not apply to TNA and GNA.

The second type of transition can be described as a genetic takeover. A preexisting selfreplicating system evolves, forits own selective advantage, a mechanism for synthesizing and polymerizing the components of a completely differentgenetic system, and is taken over by it. CairnsSmith (1982) has proposed that the first genetic system was inorganic,perhaps a clay, and that it “invented” a selfreplicating system based on organic monomers. However, he clearlyrecognized the possibility of one organic genetic material replacing another (CairnsSmith and Davies 1977). Genetictakeover does not require any structural relationship between the polymers of the two genetic systems. It suggests thepossibility that the original genetic system may have been unrelated to nucleic acids.

The hypothesis of a genetic material completely different from nucleic acids has one enormous advantage—it opens upthe possibility of using very simple, easily synthesized prebiotic monomers in place of nucleotides. However, it also raisestwo new and difficult questions. Which prebiotic monomers are plausible candidates as the components of a replicatingsystem? Why would an initial genetic system invent nucleic acids once it had evolved sufficient synthetic knowhow togenerate molecules as complex as nucleotides?

T T APAP T AP

T APT T

AP AP T AP

T AP

T AP




A number of prebiotic monomers that might have made up a simple genetic material have already been suggested. Theyinclude hydroxy acids (Weber 1987), amino acids (Orgel 1968; Zhang et al. 1994), phosphomonoesters of polyhydricalcohols (Weber 1989), aminoaldehydes (Nelsestuen 1980), and molecules containing two sulfhydryl groups (Schwartzand Orgel 1985). The list could be expanded almost indefinitely. The discussion here concerns a small class of thesemonomers that appear to be particularly attractive in the light of recent work on enzyme mechanisms.

There is accumulating evidence that several enzymes that make or break phosphodiester bonds have two or three metalions at their active sites (Cooperman et al. 1992; Sträter et al. 1996). In the case of the editing site for phosphodiesterhydrolysis in the Klenow fragment of Escherichia coli DNA polymerase I, no other functional groups of the enzymecome close to the phosphodiester bond that is cleaved. This has led to the suggestion that the major role of the enzyme isto act as scaffolding on which to hang metal ions in precisely determined positions (Beese and Steitz 1991; Steitz 1998).A similar suggestion has been made for ribozymes on the basis of both indirect and direct evidence (Freemont et al.1988; Yarus 1993; Steitz and Steitz 1993; Shan et al. 1999, 2001; Stahley and Strobel 2005).

Perhaps these observations can be extended to suggest that, if informational polymers preceded RNA, they may alsohave been dependent on metal ions for their catalytic activity. If so, the range of prebiotic monomers that needs to beconsidered is greatly reduced. In addition to the functional groups that react to form the backbone, the monomers musthave carried metalbinding functional groups. If the metal ions involved were divalent ions such as Mg and Ca , theside groups are likely to have been carboxylate or phosphate groups. If transition metal ions were involved, sulfhydrylgroups and possibly imidazole derivatives are likely to have been important.

Prebiotic monomers suitable for building polymers that bind Mg or Ca include aspartic acid, glutamic acid, andserine phosphate among biologically important amino acids. ßamino acids, such as isoglutamic acid, hydroxydicarboxylicacids, such as αhydroxysuccinic acid, and hydroxytricarboxylic acids, such as citric acid, are other possible candidates.A polymer containing Daspartic acid, Laspartic acid, and glycine as its subunits is typical of potentially informational copolymers that might, in the presence of divalent metal ions, both replicate and function as a catalyst. Transitionmetal ionsmight play a corresponding role for polymers containing cysteine or homocysteine. The present challenge is to showreplication, or at least information transfer in templatedirected synthesis, in some such system.

What selective advantage could a simpler, metabolically competent system derive from the synthesis of oligonucleotides?This is a baffling question. Most arguments that come to mind do not stand up to detailed analysis. If, for example, onepostulates that nucleotides were first synthesized as parts of cofactors such as DPN, one must explain why the particularheterocyclic bases and sugars were chosen. Even if one supposes that among the many “experiments” in secondarymetabolism performed by early organisms one happened by accident on a pair of complementary nucleotides that couldform a replicating polymer, one must still explain how polymerization subsequently contributed to the success of the“inventor.” Could oligonucleotides, by hybridization, have functioned at first as selective “glues” for tying pairs ofmacromolecules together? Could RNA have been invented by one organism as “antisense” against the genome ofanother?

The discussion so far, even though highly speculative, is still conservative in overall outlook. It supposes that the originalinformationaccumulating system that led to the evolution of life on Earth was either RNA or some linear copolymer thatreplicated in an aqueous environment in much the same way as RNA. There remains a lingering doubt that the discussionis not on the right track at all; maybe the original system was not an organic copolymer (CairnsSmith 1982), or maybe itreplicated in a nonaqueous environment and RNA is an adaptation that permitted invasion of the oceans. Perhapssystems of high complexity can develop without any need for a genome in the usual sense (Dyson 1982; Kauffman 1986;Wächtershäuser 1988; De Duve 1991; Eschenmoser 2007). Perhaps …

Laboratory simulations of prebiotic chemistry are dependent on organic chemistry and can only explore the kinds ofreactions understood by organic chemists. A good deal is known about reactions in aqueous solution, but less aboutreactions at the interface between water and inorganic solids. Very little is known about reactions in systems in whichinorganic solids are depositing from aqueous solutions containing organic material. It is hard to see how speculative

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schemes involving heterogeneous aqueous systems can be tested until much more is known about the underlying branchesof chemistry.

4. CONCLUDING REMARKS

After contemplating the possibility of selfreplicating ribozymes emerging from pools of random polynucleotides andrecognizing the difficulties that must have been overcome for RNA replication to occur in a realistic prebiotic soup, thechallenge must now be faced of constructing a realistic picture of the origin of the RNA World. The constraints that musthave been met in order to originate a selfsustained evolving system are reasonably well understood. One can sketch outa logical order of events, beginning with prebiotic chemistry and ending with DNA/proteinbased life. However, it mustbe said that the details of this process remain obscure and are not likely to be known in the near future.

The presumed RNA World should be viewed as a milestone, a plateau in the early history of life on Earth. So too, theconcept of an RNA World has been a milestone in the scientific study of life's origins. While this concept does not explainhow life originated, it has helped to guide scientific thinking and has served to focus experimental efforts. Further progresswill depend primarily on new experimental results, as chemists, biochemists, and molecular biologists work together toaddress problems concerning molecular replication, ribozyme enzymology, and RNAbased cellular processes.

ACKNOWLEDGMENTS

This work was supported by research grant NNX07AJ23G from the National Aeronautics and Space Administration.Previous versions of this article, which were published in the First (1993), Second (1999), and Third (2006) Editions ofThe RNA World, were coauthored by Leslie Orgel, who died on October 27, 2007. Many portions of the text have notbeen changed in the current edition because they remain an accurate reflection of current scientific understanding. Thecontributions of Leslie Orgel to this work and to the scientific literature of the origins of life are gratefully acknowledged.

Footnotes

Editors: John F. Atkins, Raymond F. Gesteland, and Thomas R. Cech

Additional Perspectives on RNA Worlds available at www.cshperspectives.org

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