approaching translation at atomic resolution

review

The ribosome is the ribonucleoprotein particle that performsprotein synthesis using a messenger RNA template. The ribo-some (a 70S particle in prokaryotes) is composed of two sub-units. The small subunit (30S) mediates proper pairing betweentransfer RNA (tRNA) adaptors and the messenger RNA, whereasthe large subunit (50S) orients the 3′ ends of the aminoacyl (A-site) and peptidyl (P-site) tRNAs and catalyzes peptide bond for-mation. The ribosome must translocate directionally alongmRNA in 3 nucleotide steps to read the sequential codons. Thus,the ribosome is both an enzyme and a molecular motor1 (Fig. 1).

The essential features of ribosome function have been dissect-ed during 40 years of genetic, biochemical, and biophysicalanalysis. The current view ascribes the primary functions in theribosome to its RNA components, the 16S and 23S rRNAs(reviewed in ref. 2), though definitive proof that the ribosome isa ribozyme has remained elusive. Electron microscopy (EM) hasdelineated much of the global information about ribosome mor-phology. Recent advances in cryoelectron microscopy and sin-gle-particle image analysis have resulted in substantialimprovements in resolution3. Electron mictroscopy has not pro-vided atomic-level information, yet it is a powerful tool forunraveling the various functional states of the ribosome4.

Since the discovery that well-diffracting crystals of the largeribosomal subunit could be formed5, crystallographers haveenvisioned obtaining the ribosome structure at atomic resolu-tion. A year ago, several groups reported structures of the 30Sand 50S subunits at ∼ 4.5–5.5 Å resolution6–8. A structure of the70S ribosome with tRNAs substrates bound was also publishedat 7.8 Å resolution9. Now, marking one of the most stunningachievements of structural biology, atomic resolution structuresof 30S and 50S subunits have been determined (∼ 3 Å)10–14. Thesemassive structures (~0.9 and 1.7 MDa, respectively) provide asumptuous feast of RNA and protein structures, more thanquintupling the database of known RNA–protein complexes,and open the door to atomic-level discussions of the mechanismof translation.

The development of high-intensity synchrotron radiationsources and improved computational ability were essential tosolving these large structures (discussed in detail in ref. 13).Harnessing these developments through traditional isomor-phous replacement and anomalous scattering methods, theinvestigators used good old-fashioned hard work to solve these

nature structural biology • volume 7 number 10 • october 2000 855

breakthrough structures. The structure of the Haloarcula maris-mortui 50S subunit was solved by Steitz, Moore and coworkers toa resolution of 2.4 Å10,11, and the structure of the Thermus ther-mophilus 30S subunit was solved by Ramakrishnan and cowork-ers13,14 and Yonath, Franceschi and coworkers12 to resolutions of3.0 Å and 3.3 Å, respectively. (Figs 2–5) Each of these structuresallows almost complete tracing of the RNA and peptide chains.

The large subunitThe 50S subunit of H. marismortui contains two RNA chains, the23S rRNA (2,923 nts) and 5S rRNA (122 nts), and 31 proteins. Asa testimony to the quality of the structural data, the investigatorshad to perform protein sequence analysis using their electrondensity, since only 28 proteins had previously been sequenced.Though H. marismortui is an archaebacterium and a halophile,the similarities between this ribosome and that of Escherichia coliare extensive, readily allowing functional and structural linkageto the vast E. coli literature. Steitz, Moore and coworkers identify

1Department of Structural Biology, Stanford University School of Medicine, Stanford, California 21205, 94305-5126, USA. 2Howard Hughes Medical Institute andDepartment of Molecular Biology and Genetics, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.

Correspondence should be addressed to J.D.P. email: [email protected] and R.G. email: [email protected]

Fig. 1 Schematic of the morphology of the 70S ribosomal particle asrevealed by electron microscopy55. The 30S subunit (front) and 50S sub-unit (back) are shown, as are gross features described in the text. Theribosome is depicted as bound to mRNA and tRNA ligands. States oftRNA on the ribosome, based on the hybrid states model of Noller andcolleagues56, are indicated (taken from ref. 56 and modified). The A/Tstate refers to tRNA bound to the A site on the 30S subunit and to EF-Tu,the A/A state is tRNA bound to the A site on both subunits, the P/P stateis tRNA bound to the P site on both subunits. Following peptide bondformation, deacylated P-site tRNA moves spontaneously to the P/E statewhere the anticodon interacts in the 30S subunit P site and the 3′ end ofthe tRNA in the 50S subunit E site.

Approaching translation at atomic resolutionJoseph D. Puglisi1, Scott C. Blanchard1 and Rachel Green2

Atomic resolution structures of 50S and 30S ribosomal particles have recently been solved by X-ray diffraction.These ribosomal structures show often unusual folds of ribosomal RNAs and proteins, and provide molecularexplanations for fundamental aspects of translation. In the 50S structure, the active site for peptide bondformation was localized and found to consist of RNA. The ribosome is thus a ribozyme. In the 30S structures,tRNA binding sites were located, and molecular mechanisms for ribosomal fidelity were proposed. The 30Ssubunit particle has three globular domains, and relative movements of these domains may be required fortranslocation of the ribosome during protein synthesis. The structures are consistent with and rationalizedecades of biochemical analysis of translation and usher in a molecular age in understanding the ribosome.

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overall architecture. Although secondary structure maps of 23SrRNA divide the rRNA chain into six domains (Domains I–VI)that emanate from a central loop, the structure of the subunit isremarkably uniform (Fig. 2). Ribosomal proteins are largelyfound on the edges of the subunit–subunit face, and on the back-side, which is solvent exposed in the 70S particle. Indeed, the pro-teins seem to distribute evenly along the periphery of the particle,forming a protein lattice on which the rRNA structure forms (Fig.2). With the exception of proteins L1, L10, L11 and L12 that formthe tips of the 50S subunit’s protuberances, the proteins do notextend significantly beyond the particle envelope defined byrRNA.

The surface that interacts with the 30S subunit and the activesite cleft where tRNAs bind are conspicuously devoid of proteins.The authors take note of this and point out that these regions ofthe 50S subunit also contain the most highly conservedsequences of rRNA. Thus, the structure of the 50S subunit lendsthe most substantial evidence to date that rRNA plays the domi-nant role in the function of the ribosome. Domain IV of 23SrRNA forms the ridge of the cleft (Figs 2, 3) and the bulk of thesubunit interface as previously predicted from biochemicalexperiments15–17. Domain V of 23S rRNA forms the active sitecleft (see below) below and behind the domain IV ridge, also inagreement with a sea of biochemical data (reviewed in ref. 2).Although RNA structure forms the core of the active site cleft,this region of the ribosome does not appear to fold independent-ly of ribosomal proteins. Throughout the particle, proteinsappear intimately intertwined with the rRNA. More than a third

Fig. 2 Three-dimensional structures ofthe H. marismortui 50S subunit (top) andthe T. thermophilus 30S subunits (bot-tom). The RNA chains are shown in tanfor both subunits; 50S subunit proteinsare depicted in orange and 30S subunitproteins in blue. Rendering of proteins inthe 50S subunit was achieved by back cal-culating the positions of amide and car-boxyl atoms based on the Cα coordinates(PDB code 1FFK). This procedure was per-formed by Michael Levitt. (Top) Twoviews of the 50S subunit showing theface that interacts with the small subunit(left) and the solvent-accessible face(right). The positions of the missing L1and L7/L12 stalks are indicated. On thesubunit face, the long RNA ridge indomain IV of 23S rRNA that forms the lipof the active site cleft is indicated.(Bottom) Two views of the 30S subunit,showing the face that interacts with thelarge subunit (left) and the solvent-acces-sible face (right). On the subunit face, thelong RNA helix 44 is indicated. In bothsubunits, the interface region is com-posed mainly of RNA, whereas proteinsuniformly distribute over the solventexposed surface of both subunits.

review

most of the rRNA (2,711 of 2,923 nucleotides in 23S RNA and allof 5S RNA) and 27 proteins in their electron density. No cleardensity was observed for proteins L1, L10, L11 and L12 at 2.4 Åresolution or for the rRNA regions that interact with these pro-teins. In the traditional EM-defined ‘crown’ view of the 50S par-ticle (Fig. 1), seen from the surface that interacts with the 30Ssubunit, it is known that protein L1 constitutes the left handpoint of the crown and L10, L11 and L12 form the majority ofthe right-hand point of the crown (Fig. 2). These proteins havebeen crystallized in isolation and were visible in lower resolutionstructures6,9. Either the loss of these proteins during purifica-tion/crystallization or residual dynamics of these domains hasleft these functionally interesting regions of the subunit disor-dered.

The 50S subunit is ~250 Å in diameter and is roughly isomet-ric in mass distribution. The face that interacts with the 30S sub-unit is somewhat flattened, except for a deep cleft that lies behindan rRNA ridge that runs longitudinally across the equator of the50S subunit. This cleft is of sufficient size to accommodate the 3′acceptor arms of three tRNA molecules (the aminoacyl, peptidyland exit) and contains the active site for peptide bond formation(Fig. 3). Exiting downward from the center of this cleft is a tun-nel roughly 100 Å in length and 15 Å in diameter through whichthe polypeptide products of translation are released.

A striking feature of the 50S subunit is the relative distributionof proteins and nucleic acid within the particle. The 23S rRNArepresents a majority (∼ 75%) of the 50S subunit mass and itsglobular fold within the particle forms the bulk of its shape and

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of the nucleotides in 23S rRNAmake van der Waals contact with aribosomal protein.

The 50S subunit represents atrue collaboration between RNAand proteins; the physical proper-ties of the components createsomething that neither couldachieve on its own. All large sub-unit proteins except L12 interactdirectly with RNA. Most (23/29)RNA-binding proteins interactwith more than one domain of 23S rRNA, helping to create thecompact structure of the 50S subunit. The protein structuresobserved in both the 50S and 30S subunits are perhaps the mostnovel feature of the ribosome structures. Nearly half (13/30) ofthe large subunit proteins have unusual, extended structuraldomains that snake through regions of compact rRNA structureto stabilize interhelical contacts between RNA domains. Buriedextensions of proteins L2 and L3 reach deep into the compactrRNA core (Fig. 4). These protein moieties are among those thatmost closely approach the active site and thus have the greatestpotential to stabilize its structure. These data are remarkablyconsistent with previous protein extraction and reconstitutionexperiments18–20. The extended protein structures beautifullyhighlight the interdependence of the rRNA and proteins in thisribonucleoprotein assembly.

The ribosome structure explains how 23S rRNA, whose sec-ondary structure had been predicted by phylogenetic compar-isons21, folds within the 50S subunit. Phylogenetically conservedresidues either stabilize tertiary folds of rRNA, mediate sequence-specific contacts to proteins or reside on surface regions of theribosome for direct interaction with tRNA ligands or the 30S sub-unit. The global architecture of the rRNA is mediated by the nowstandard array of tertiary interactions, including pseudoknots,base triples, tetraloop–receptor interactions, and ribose zippers.Adenosine-mediated interactions, first observed in the P4-P6domain crystal structure of the group I intron22, are essential forclose packing of the RNA elements. The disproportionate num-ber of adenosines in bulge and loop regions of rRNA likely reflectsthe unusual contribution of this nucleotide to helix packing.

The structure of the 50S subunit will also be valuable to pro-tein folding and protein secretion enthusiasts. Beyond just syn-thesizing proteins, the ribosome plays a role in maturingpeptides as they are made. The peptide exit tunnel of the 50Ssubunit is lined with both rRNA and protein (with fewhydrophobic patches), and as many as 50 of the newly synthe-sized amino acids of a polypeptide are contained in the tunnelbefore they become solvent and chaperone accessible. Thepolypeptide tunnel with conspicuous constrictions, bends, andirregularities may play an intimate role in the protein foldingprocess. The structure also provides a physical map of the specif-


ic protein and RNA components of the ribosome that may beinvolved in protein folding and secretion which may be com-pared with existing biochemical data23, 24.

In the end, the finding of Steitz, Moore and coworkers that willreverberate through all areas of molecular biology far into thefuture is the demonstration that the active site for peptide bondformation by the 50S subunit is composed solely of RNA (Figs 4,5). While the notion that the ribosome is a ribozyme has beenincreasing in popularity for many years, Steitz, Moore andcoworkers prove this point beyond reasonable doubt by present-ing two supporting crystal structures of the 50S subunit withbound tRNA substrate analogs. One analog consists of a shorthelix (minihelix) that mimics the acceptor stem of tRNA with aterminal puromycin residue instead of adenosine. Puromycin isan aminoacyl tRNA analog equivalent to the terminal adenosine(A76) of tRNA linked through a 3′ amide linkage to a methoxy-tyrosine residue. A second analog is a transition-state analog ofpeptide bond formation first developed by Yarus and coworkers25.This inhibitor contains 3′-terminal CCdA (representing P-sitetRNA) covalently attached to the amino group of the puromycin(mimicking A-site tRNA) amino acid through a tetravalent phos-phate linkage. The phosphoramide group mimics the tetrahedralgeometry and the developing negative charge on the carbonyl ofthe presumed transition state for peptidyl transfer. The inhibitorbinds with increased affinity to 50S subunits relative to the indi-vidual substrate components, consistent with it representing atleast certain chemical properties of the transition state, thoughthe affinity is lower than that predicted for the transition state,suggesting limitations to the mimicry25.

The substrate analogs soaked into the 50S crystal lattice inter-act with residues of domain V within the active site cleft, near theentrance to the peptide exit tunnel. Neither substrate dramati-cally perturbs the structure of the 50S particle, except thatnucleotide A2637 (A2602 in E. coli) acquires interpretable densi-ty as a result of binding the Yarus analog. The puromycin residueof the transition state analog superimposes on that of the mini-helix bound to the A site, allowing the authors to create a modeldepicting the interaction of the 3′ ends of both tRNAs with theribosome simultaneously. The active site cleft consists entirely ofdomain V of 23S rRNA. None of the 30 large subunit proteins

Fig. 3 Side views of the 50S and 30Ssubunits. The subunit interfaces ofboth particles are flat. A tRNA mole-cule, drawn to scale, is positioned withits 3′ CCA-end pointed towards theactive site cleft of the 50S subunit andits anticodon stem-loop directedtowards the decoding site of the 30Ssubunit. Landmark features of eachparticle are indicated.


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come within 18 Å of the phosphate group representing the tetra-hedral intermediate and the catalytic center of the 50S subunit.The structure clearly shows that the ribosome is an RNA enzymewith proteins stabilizing the RNA structure of the active site.

Both tRNA analogs interact with nucleotides biochemicallyidentified as P-site or A-site binding determinants26,27. The C74and C75 residues of P-site tRNA of the analog developed byYarus form Watson-Crick base pairs with two guanosines, G2285and G2284 (G2252 and G2251 in E. coli) in the P-loop whereasC75 of A-site tRNA forms a Watson-Crick pair with G2588(G2553 in E. coli) in the A-loop (Fig. 6). These interactions withthe P-loop and A-loop had been predicted based on biochemicaland in vitro complementation analysis28,29. Interaction with thetwo recognition loops positions the tetrahedral phosphoramidewithin a complex RNA structure formed by the central loop ofdomain V RNA and its peripheral helices. Thus, nucleotidesfrom the A-loop, the P-loop, the 2600 helix (E. coli) and the cen-tral ring of domain V are juxtaposed together above the openingof the peptide exit channel — these nucleotides define the activesite (Figs 4, 6).

The resulting structure places the phosphoramide oxygen of thetetravalent phosphate of the transition state analog within hydro-gen bond distance of the N3 of A2486 (A2451 in E. coli). To formthis hydrogen bond in the crystals at pH 5.8, one of these atomsmust be protonated. The authors propose that an extensive net-work of hydrogen bonding interactions of A2486 coupled to a sol-


vent inaccessible phosphate on A2485 (A2450 in E. coli) might besufficient to shift the pKa of the N3 of this residue from a normalpKa of about 1.5. Shifted pKa values for adenosine N1 and cytosineN3 have been observed in other RNA structures, and are thoughtto result from coupling of favorable electrostatic and other non-covalent interactions upon protonation30–32. Such a proposedmechanism for perturbing the pKa of A2486 has similarity to theburied carboxylate of Asp102 in chymotrypsin that enhances thenucleophilicity of the active site serine.

In an accompanying study, Strobel and coworkers indepen-dently demonstrate that the pKa of A2451 in E. coli (A2486 in H. marismortui) is substantially perturbed33. Using dimethylsul-fate (DMS) as a protonation-sensitive probe, A2451 was found tobe the only accessible adenosine in the 50S subunit with a per-turbed pKa. Their results indicate that some group on A2451 (N1or N3) titrates at pH 7.6, far from the measured pKa values of 3.5and 1.5.

How then might such a nucleotide with a perturbed pKa andthe other active site residues conspire in catalysis of peptide bondformation? The authors propose a mechanism whereby A2486

Fig. 4 Global and close-up views of the A and P sites on the 50S subunit.The four proteins of the large subunit L2, L3, L4 and L10e, which containlong extended regions that penetrate closest to the active site of the par-ticle, are indicated in red. Nucleotides of the large subunit present in thestructure that are protected from chemical probes by the terminalresidues of A- and P-site tRNA are indicated in green and pink, respec-tively26. In the upper panel, A and P loop structures are rendered withcolor as well for purpose of clarity28,29. A, P and E sites spatially distributefrom right to left as indicated; tRNAs move sequentially through A, P and E sites in the translation elongation cycle.

Fig. 5 Global and close-up views of the A and P sites on the 30S subunit13.Protein S12, functionally important to tRNA selection at the A site, is rendered in blue. Nucleotides of the small subunit present in thestructure that are protected from chemical probes by A and P site tRNAare indicated in green and pink, respectively57. In the global view, themRNA-like oligonucleotide seen in the crystal structure reported byRamakrishnan et al. is rendered in P-site color as well.


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(A2451 in E. coli) acts as a general base, abstracting a protonfrom the amino group that attacks the aminoacyl ester bond ofthe P-site tRNA. No bound divalent ions, critical for manyribozymes that perform phosphodiester chemistry34, areobserved in the active site. However, a potassium ion is foundnear the active site interacting with G2102 (G2061 in E.coli) andG2482 (G2447 in E.coli); both of these residues directly contactA2486 (A2451). Both groups assert that the collective environ-ment surrounding A2486 (A2451 in E. coli), which may includecharge relay mechanisms and tautomeric shifting of bases, ulti-mately gives rise to a nucleotide empowered to catalyze the pep-tide bond forming reaction in the 50S subunit.

The atomic resolution structure and biochemical experimentsare compelling. The interactions of the CCA-moieties of thetRNA substrate analogs with the P- and A-loops were predictedby biochemical and genetic studies28, 29 and thus serve to validatethe placement of the analog. These constraints in turn limitpotential error in positioning of the phosphoramide linkage andthe surrounding active site nucleotides. The phenomenal overlapof nucleotides identified by chemical modification analysis asbeing protected by bound tRNA26, 35 and the nucleotides found inthe active site by crystallography cannot be coincidental. Thestructure must be relevant.

On a more conservative note, several issues should be consi-dered. The crystals may not be found in a catalytically active con-formation and molecular movement (subtle or less subtle) maybe required for peptide bond formation to occur. As the reactionis considerably less energetically demanding than proteolysis,orientation of the activated substrates of this reaction may besufficient to account for the observed rates of translation. Or,deprotonation by A2486 (H. marismortui) of the protonatedform of the amino acid (NH3

+), as observed bound to EF-Tu36,may well be sufficient to promote nucleophilic attack.Determination of the precise mechanism of peptidyl transferaseand the relative contributions made by various active site com-ponents to the reaction energetics will require rigorous enzymol-ogy coupled with further structural analyses.

The small subunitThe genetic code is deciphered on the 30S subunit where tRNAanticodons pair with mRNA codons. Two groups, Yonath andcoworkers12 and Ramakrishnan and coworkers13,14 present thestructure of the Thermus thermophilus 30S subunit. Althoughthe Ramakrishnan group has data at a slightly better resolution,the structures are globally similar. The small ribosomal subunitcontains a single RNA chain of 1,518 nucleotides (16S rRNA)and 20 proteins. The 30S particle has a distinctly different globalstructure than the 50S subunit (Figs 2, 3). Whereas the 50S parti-cle is thick and monolithic, the 30S particle is thin and flexible.


The particle is divided into three domains which each containone of the principle domains of 16S rRNA observed in the sec-ondary structure. The head, body and platform of the 30S sub-unit, terms that originate from EM reconstructions of 30Sparticles, are composed of the 3′ major, 5′ and central domains of16S rRNA secondary structure, respectively. The 3′ minordomain of 16SrRNA forms an extended helix (H44 in the E. colihelix numbering system) that runs down the long axis of the 30Ssubunit surface that interacts with the 50S subunit. All fourdomains of the 30S particle join at a narrow neck region (alsoEM derived nomenclature).

The general organization of RNA and protein within the 30Ssubunit is similar to that observed in the 50S subunit. Small sub-unit proteins cluster on the solvent-exposed surface of the parti-cle and are largely absent from the surface that interacts with the50S particle (Fig. 2). The exception to the rule is protein S12,which is found at the subunit interface bound to several func-tionally important components of the 30S particle. In contrast tothe 50S structure, the proteins in the more globular, flexible 30Sstructure do not penetrate as deeply into 16S rRNA. While manyof the small subunit proteins do contain extended domains thatinteract intimately with the rRNA, the more flexible nature of the30S structure eliminates the need for proteins that would com-pact the rRNA domains into a monolithic sphere. The small sub-unit also contains a short 26 residue peptide called Thx, whichhelps to stabilize the structure of the head domain. Similar to theinteractions seen in the 50S structure, RNA tertiary structure isoften stabilized by protein interactions, as well as by the standardRNA–RNA interactions discussed above. Indeed, Ramakrishnanand coworkers13,14 take note of the prevalence of adenosine-mediated helix packing interactions within the 30S subunit andmake an effort to catalog them.

A major function of the 30S subunit is decoding of mRNA, aprocess by which a tRNA is properly matched to the codon withinthe A site of the ribosome. Although neither 30S structure con-tains tRNAs or mRNA ligands, they provide insights into howtRNA–mRNA complexes are recognized by the 30S subunit.Ramakrishnan’s group reports that intersubunit interactions areobserved in the crystal lattice that place the ‘spur’, an element that

Fig. 6 Schematics of the active sites for a, decoding on the 30S subunitand b, peptide bond formation on the 50S subunit showing molecularinteractions known to occur during these processes. a, A1492 and A1493in helix 44 of 16S rRNA read the shape of the codon–anticodon helix(blue) formed by the A-site tRNA–mRNA complex (taken from ref. 54). b, The peptidyl transferase center of the 50S subunit showing the mod-eled interactions with the Yarus transition state analog. P-site tRNAresidues C74 and C75 (red) form base pairs with G2251 and G2252 (E. coli)in the P-loop, and A-site tRNA residue C75 forms a base pair with G2553(E. coli) of the A-loop. The line traces the phosphodiester backbone ofthe modeled transition state analog that links P-site and A-site tRNA; thetetrahedral phosphoramide group is shown as a red sphere. A2451 (E.coli) is in closest proximity to this charged group and may directly partic-ipate in the catalysis of peptide bond formation. A2602 wedges betweenthe P and A site substrates.

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Fig. 7 The ribosome is a molecular machine. The hybridstates model for translation elongation56. A, P and E siteson the 30S and 50S subunits are indicated schematically.a, Initially the P site is filled with peptidyl tRNA and the Asite is unoccupied. b, Aminoacyl-tRNA binds to the A siteas a ternary complex with EF-Tu–GTP in a codon-indepen-dent manner. c, Subsequently, the anticodon of theternary complex tRNA interacts with the A-site mRNAcodon on the 30S subunit d, GTP is hydrolyzed and EF-Tudissociates from the 3′ CCA-end of tRNA and the ribo-some e, allowing the acceptor end to engage in the Asite. f, Peptide bond formation occurs rapidly, transfer-ring the peptide chain to the A-site tRNA and the accep-tor end of P-site tRNA spontaneously moves to the E-site(P/E state) and the A-site tRNA moves to the P-site (A/P*state). g, Elongation factor G (EF-G) binds to the ribosomeas a complex with GTP. h, GTP hydrolysis leads to a transi-tion state for translocation where the ribosome disen-gages from the tRNA-mRNA complexes to allowmovement of the anticodons of the P- and A-site tRNAs. i, This movement results in full tRNA occupation of the E and P-sites, and an empty A site, ready to translate thenext codon.

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juts out of the 30S particle (corresponding to Helix 6, nts 61–106in E. coli), into the P site of the neighboring subunit. There is alsoelectron density for mRNA in the P site. This density may derivefrom the very 3′ end of 16S rRNA which is not observed in thestructure. Based on this P-site position, the authors modeled thepositions of adjacent A-site and exit site (E-site) tRNAs. Yonathand coworkers use the prior crystal structure of functional com-plexes9 to arrive at similar tRNA positions. In contrast to the largesubunit active site, these tRNA binding sites on the 30S subunit arecomposed of elements from more than one structural domain.Movement of the domains, mediated by tRNA interactions, pro-tein factors, and GTP hydrolysis, is likely an essential feature of 30Sfunction during decoding and translocation. Yonath and cowork-ers propose that the interdomain closure of the head and body ofthe 30S subunit latches the mRNA onto the ribosome.

Using the spur interaction as a model, Ramakrishnan andcoworkers13,14 propose details of P-site tRNA interaction with the30S subunit (Fig. 5). The P-site tRNA interacts with the 30S sub-unit mainly along the minor groove of its anticodon helix. Theinteractions occur with nucleotides 1338–1341 and 1229–1230(E. coli numbering) in the 3′ major domain, as well as with por-tions of proteins S13 and S9. A loop residue of the spur, equiva-lent to position 34 of a P-site anticodon, stacks upon C1400, thesite of a high efficiency UV crosslink between a P-site tRNA anti-codon and 16S rRNA37. The regions observed in contact with theP site mimic agree with prior biochemical studies of the P site38,39.The extensive 30S subunit–P-site tRNA interaction is consistentwith the high affinity of the P site40 and with hydroxyl radical pro-tection experiments41. This makes sense intuitively since the P sitemust hold on tightly to the peptidyl-tRNA while decoding andpeptide bond formation are taking place. Dissociation of the P-site tRNA would have drastic consequences.

Fidelity of translation in the A site requires specificity of tRNAselection over simple binding affinity. The modeled A-site


tRNA–mRNA complex binds within a wider andshallower site on the 30S subunit than the P site14.The interactions with the ribosome are much lessextensive than in the P site (Fig. 5), consistent withbiochemical data, and the lower affinity of A-sitetRNA–mRNA complexes for the ribosome40.Ribosomal components of the A-site include por-tions of the 530 loop, Helix 34 in the head, andA1492/93 at the base of the long penultimate stem.All of these regions of 16S rRNA had been previous-

ly implicated in A site function by biochemical experiments39.Again, it is striking how well biochemical experiments identifiedthe critical components of the A site. Only protein S12, whichhas been extensively implicated in decoding by classic geneticexperiments42,43, may directly participate in contacting the A-sitetRNA–mRNA complex. In contrast, the E site is composed main-ly of proteins, including S7 and S11.

The 30S subunit participates actively in tRNA selection. Alarge body of evidence suggests that there is an active site fordecoding, in which the 30S subunit discriminates correct fromincorrect tRNAs in the A site. Once a tRNA is selected and pep-tide bond formation has occurred, the A-site tRNA must translo-cate into the P site. The structures of the 30S subunit providepositions for the A- P- and E-site tRNAs but do not immediatelyreveal how either tRNA selection or the translocation processoccurs. The tRNA anticodons bind within a cleft that formsbetween the individual domains, and relative movements ofthese domains are likely involved in both decoding and translo-cation. Antibiotics that target the small subunit interfere withthese processes, and provide a means to access functional infor-mation on the 30S subunit.

The ribosome is the target of many clinically important antibi-otics44 (Fig. 7). The small subunit is the binding site for amino-glycosides such as paromomycin, gentamicin, streptomycin andspectinomycin, as well as the tetracyclines45. These antibioticsinterfere with critical functions of the ribosome, and thus theirmode of action sheds light on ribosome function. Spectinomycinbinds near Helix 34 of 16S rRNA and interferes with EF-G func-tion during translocation. The aminoglycosides paromomycinand streptomycin bind to the 30S subunit near the decoding sitein distinct areas; in fact, their binding is cooperative46. In theirpresence, there is a decrease in fidelity of translation47,48. Theaffinity of tRNA for the A site is increased by aminoglycosidebinding49 and the rate constants for kinetic steps of tRNA selec-

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tion are affected50. NMR studies on the RNA binding site forparomomycin in the presence and absence of the antibioticrevealed how aminoglycosides bind to rRNA51. The structuresuggested that the drugs stabilize the local conformation ofA1492 and A1493, which are involved in decoding. However, theglobal implications of drug binding to the 30S subunit could notbe determined by NMR.

Ramakrishnan and coworkers have solved the structure of the30S subunit with spectinomycin, paromomycin and strepto-mycin simultaneously bound14. The binding sites found for allthree antibiotics agree with prior biochemical and genetic stud-ies. Spectinomycin binds to Helix 34, and is near protein S5 inwhich resistance mutations are located52. Binding of the drugmay stabilize the RNA conformation of the head, and preventmovement of the head domain that may occur during transloca-tion. Streptomycin binds near the decoding site, and links fourregions of rRNA involved in A-site tRNA binding: Helix 44,Helix 1, Helix 27, and the 530 loop. In addition, protein S12directly interacts with the antibiotic, consistent with the highlevel streptomycin resistance that can occur upon mutation ofS12. Streptomycin stabilizes the structure of RNA around thedecoding site. In particular, the base pairing of H27 is in a config-uration that leads to miscoding (ram mutations, for ribosomalambiguity mutations)53. In this conformation, H27 docks tightlyinto the long penultimate stem (H44), and this docking likelyaffects the conformation of the A site. Paromomycin in thethree-drug complex binds in the major groove of H44, asobserved by NMR51. Upon binding, A1492 and A1493 are exten-sively flipped out into the minor groove. Apparently, antibioticsthat act on the 30S subunit affect ribosome function by stabiliz-ing particular conformations of ribosomal RNA.

The ribosome must distinguish correct versus incorrectcodon–anticodon pairs by structure-specific, sequence indepen-dent interactions. Based on NMR and biochemical studies,Yoshizawa et al.54 proposed that the mRNA backbone was contact-ed by A1492 and A1493. Ramakrishnan and coworkers13,14 proposea similar molecular basis for ribosomal decoding. The complex ofthe 30S subunit with the antibiotic represents a high affinity formfor tRNA–mRNA interaction in the A site. The flipped-out bases ofA1492 and A1493 would be positioned within the minor groove ofthe A-site codon-anticodon helix. A reasonable hypothesis is thatA1492 and A1493 sense the width of the minor groove, whichwould be distorted in non-cognate codon–anticodon pairs (Fig. 6).As suggested by NMR, paromomycin induces miscoding by stabi-lizing the flipped-out conformation of A1492 and A1493. The 30Ssubunit structure suggests that these nucleotides may representsome of the moving parts of this molecular machine.

ConclusionsThe structures of the 30S and 50S subunits are solved. As antici-pated, the ribosome is composed of active site clefts, catalyticcenters and channels. Most importantly, the ribosome is aribozyme. This is an amazing and satisfying result.

Decades of biochemistry and genetics can now be interpretedin atomic detail. Remarkably, these approaches identified thevast majority of the players in the active sites. As a general rule inthe ribosome, and perhaps in other RNPs, nucleotides that areaccessible to base-specific chemical probes have a high probabil-ity of being involved in critical functions — indeed, the likeli-hood improves if highly accessible and highly conservednucleotides are considered.

The euphoria of these results yields to a more sober reality.There are still many unanswered questions about the mecha-

nism of translation. How do the large and small subunits com-municate between their ‘active sites’ and how do they interactwith factors to promote the directional movements of translo-cation? The visceral power of structure can be overwhelming.However, the ribosome is a dynamic engine (Fig. 7), and wemust now reconcile the static structures solved by crystallogra-phy with the multiple states that the ribosome must sampleduring the translational cycle. The answers to these questionswill come with further mechanistic and structural studies. Butthe field of translation is now permanently changed.Hypotheses about ribosome function must now be framed onthe molecular level.

AcknowledgmentsThe authors thank E. Viani Puglisi and C. Merryman for useful discussions, M. Levitt for assistance generating full backbone coordinates for the 50S subunitproteins, and V. Ramakrishnan for providing 30S subunit coordinates prior topublication.

Received 8 September, 2000; accepted 12 September, 2000.

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