ribozymes

Cell & Molecular

Biology

Ribozymes

Chapter 7

RNA with catalytic activity

Self-Splicing RNA

Self-splicing intronof bacterial tRNA

http://www.pdb.org/pdb/101/motm.do?momID=65



Self-Cleaving RNA

A riboswitch that becomes a self-cleaving ribozyme in the presence of glucosamine.




Riboswitch

“...regulatory elementsbuilt directly into a messenger RNA.”




Guanine riboswitch




RNA-Catalyzed RNAPolymerization: Accurate andGeneral RNA-Templated Primer

ExtensionWendy K. Johnston, Peter J. Unrau,* Michael S. Lawrence,

Margaret E. Glasner, David P. Bartel†

The RNA world hypothesis regarding the early evolution of life relies on thepremise that some RNA sequences can catalyze RNA replication. In support ofthis conjecture, we describe here an RNA molecule that catalyzes the type ofpolymerization needed for RNA replication. The ribozyme uses nucleosidetriphosphates and the coding information of an RNA template to extend an RNAprimer by the successive addition of up to 14 nucleotides—more than a com-plete turn of an RNA helix. Its polymerization activity is general in terms of thesequence and the length of the primer and template RNAs, provided that the3! terminus of the primer pairs with the template. Its polymerization is alsoquite accurate: when primers extended by 11 nucleotides were cloned andsequenced, 1088 of 1100 sequenced nucleotides matched the template.

The RNA world hypothesis states that early lifeforms lacked protein enzymes and dependedinstead on enzymes composed of RNA (1).Much of the appeal of this hypothesis comesfrom the realization that ribozymes would havebeen far easier to duplicate than proteinaceousenzymes (2–5). Whereas coded protein replica-tion requires numerous macromolecular com-ponents [including mRNA, transfer RNAs(tRNAs), aminoacyl-tRNA synthetases, and theribosome], replication of a ribozyme requiresonly a single macromolecular activity: anRNA-dependent RNA polymerase that synthe-sizes first a complement, and then a copy of theribozyme. If this RNA polymerase were itself aribozyme, then a simple ensemble of moleculesmight be capable of self-replication and even-tually, in the course of evolution, give rise to theprotein-nucleic acid world of contemporary bi-ology. Finding a ribozyme that can efficientlycatalyze general RNA polymerization wouldsupport the idea of the RNA world (1, 6, 7) andwould provide a key component for the labora-tory synthesis of minimal life forms based onRNA (8, 9).

Although progress has been made in find-ing ribozymes capable of template-directedRNA synthesis, none of these ribozymes hasthe sophisticated substrate-binding propertiesneeded for general polymerization (7). De-rivatives of self-splicing introns are able to

join oligonucleotides assembled on a tem-plate (10–12). However, the templates thatcan be copied are limited to those that matchthe oligonucleotide substrates, and it has notbeen possible to include sufficient concentra-tions of all the oligonucleotide substratesneeded for a general copying reaction. Morerecently, efforts have shifted to derivatives ofan RNA-ligase ribozyme that was isolatedfrom a large pool of random RNA sequences(13–15). Some derivatives are capable oftemplate-directed primer extension using nu-cleoside triphosphate (NTP) substrates, buttheir reaction is also limited to a small subsetof possible template RNAs (15). These ligasederivatives recognize the primer-templatecomplex by hybridizing to a particular un-paired segment of the template (Fig. 1A). Inusing a short segment of a special template todirect primer extension, these ribozymes re-semble telomerases more than they resemblethe enzymes that replicate RNA and DNA bymeans of general polymerization.Polymerase isolation. We have used the

catalytic core of the ligase ribozyme (14, 16)as a starting point for the generation of aribozyme with general RNA polymerizationactivity. The new polymerase ribozyme wasisolated from a pool of over 1015 differentRNA sequences. Sequences in the startingpool contained a mutagenized version of theparental ligase (Fig. 1B). To sample a broaddistribution of mutagenesis levels, the start-ing pool comprised four subpools in whichthe core residues of the ligase domain weremutagenized at levels averaging either 0, 3,10, or 20% (17). Two loops within the ligasedomain, both unimportant for ligase function,were replaced with 8-nucleotide (nt) random-

sequence segments (Fig. 1B). The 5! termi-nus of the ligase domain was covalently at-tached to an RNA primer so that moleculesable to catalyze primer extension could beselected by virtue of their attachment to theprimer that they extended.

In contrast to the parental ribozyme,which hybridizes to the template, a ribozymecapable of general polymerization must rec-ognize the primer-template complex withoutrelying on sequence-specific interactions.Therefore, the template RNA was designed tobe too short for extensive hybridization withthe ribozyme (Fig. 1B). For the parental ri-bozyme (Fig. 1A), the pairing between theribozyme and the template also comprised astem known to be necessary for ligase func-tion (16). To restore this stem, a short RNA,GGCACCA (purple RNA in Fig. 1B), wasintroduced to hybridize to the segment of theligase domain that formerly paired with thetemplate. Finally, because a more generalmode of primer-template recognition mightrequire the participation of an additionalRNA domain, a 76-nt random-sequence seg-ment was appended to the 3! terminus of thedegenerate ligase domain (Fig. 1B).

Sequence variants able to recognize theprimer-template in this new configuration andthen extend the primer with tagged nucleotideswere enriched by repeated rounds of in vitroselection and amplification (Table 1). RNAsthat extended their primer by using 4-thioUTPwere isolated on APM gels (urea denaturinggels cast with a small amount of N-acryloyl-aminophenylmercuric acetate, which impedesmigration of RNA containing 4-thioU) (18). Tofavor variants that recognize generic rather thansequence-specific features of the primer-tem-plate, different primer-template sequences andlengths were used in different rounds of selec-tion (Table 1). To favor the more efficientribozyme variants, the stringency of the selec-tion was increased in later rounds by requiringaddition of two tagged nucleotides, such asbiotinylated A and 4-thioU (19), and by de-creasing the time of incubation with the taggedNTPs (Table 1).

After eight rounds of selection and amplifi-cation, desirable variants had increased in abun-dance to the point that a detectable fraction ofthe pool molecules could extend their primer byusing both 4-thioUTP and radiolabeled ATP ina template-dependent fashion (Fig. 2). Othervariants able to tag themselves were detected asearly as round four, but most of these ri-bozymes added tagged nucleotides in the ab-sence of the template oligonucleotide, or theydecorated themselves at sites other than the 3!terminus of the primer (20). Seventy-four vari-ants from rounds 8 through 10 were cloned andfound to represent 23 sequence families, eachfamily having descended from a different an-cestral sequence of the starting pool. Ri-bozymes from two families extended their

Whitehead Institute for Biomedical Research, and De-partment of Biology, Massachusetts Institute of Tech-nology, Cambridge, MA 02142, USA.

*Present address: Department of Molecular Biologyand Biochemistry, Simon Fraser University, Burnaby,BC, V5A 1S6, Canada.†To whom correspondence should be addressed. E-mail: [email protected]

R E S E A R C H A R T I C L E

www.sciencemag.org SCIENCE VOL 292 18 MAY 2001 1319

Artificial Ribozymes

The RNA World

ribozymes

Education

rna primer

rna template

rna replication

rna ggcacca purple rna

rnacatalyzed rna nus

rna sequences

rna molecule

rna helix