Helix 69 Is Key for Uniformity during Substrate Selection onthe Ribosome*□S

Received for publication, May 2, 2011, and in revised form, May 26, 2011 Published, JBC Papers in Press, May 27, 2011, DOI 10.1074/jbc.M111.256255

Rodrigo F. Ortiz-Meoz and Rachel Green1

From the Howard Hughes Medical Institute, Department of Molecular Biology and Genetics, The Johns Hopkins University Schoolof Medicine, Baltimore, Maryland 21205

Structural studies of ribosome complexes with bound tRNAsand release factors show considerable contacts between thesefactors and helix 69 (H69) of 23 S rRNA. Although biochemicaland genetic studies have provided some general insights into therole of H69 in tRNA and RF selection, a detailed understandingof these contributions remains elusive. Here, we present a pre-steady-state kinetic analysis establishing that two distinctregions of H69 make critical contributions to substrate selec-tion. The loop of H69 (A1913) forms contacts necessary for theefficient accommodation of a subset of natural tRNA species,whereas the base of the stem (G1922) is specifically critical forUGA codon recognition by the class 1 release factor RF2. Thesedata define a broad and critical role for this centrally locatedintersubunit helix (H69) in accurate and efficient substrate rec-ognition by the ribosome.

The ribosome is the ribonucleoprotein machine responsiblefor the faithful translation of the genetic material into theencoded polypeptide. The core RNA components of the ribo-some (the 16 S and 23 S rRNA) that reflect its earliest evolutionplay a key role in interactions that specify the selection oftRNAs and release factors on recognition of sense and stopcodons, respectively. During translation elongation, the selec-tion of a cognate ternary complex (composed of aa-tRNA,EFTu, and GTP) from solution is specified primarily in thesmall ribosomal subunit where three nucleotides in 16 S rRNAdirectly evaluate the geometry of the codon-anticodon interac-tion (1). Though different in molecular detail, the selection ofclass 1 release factors takes place in the same decoding center ofthe small ribosomal subunit where conserved protein featuresof the RF engage the stop codon. Thus, for both decodingevents, molecular interactions in the small subunit “decodingcenter” are early and critical contributors to the selection ofcognate substrates during the translational cycle.In addition to these interactions in the decoding center, it is

believed that other molecular interactions between the ribo-some and these substrates contribute to binding and specificity.Structural studies of tRNA (2–5), release factors (6–8), and

ribosome recycling factor (9, 10) bound to the ribosome revealthat H692 of the large subunit rRNA makes extensive contactswith all of these factors. This universally conserved helix formsan intersubunit bridge that is proximal to the substrate bindingcleft and directly contacts the functionally critical h44 of 16 SrRNA which contains two of the three nucleotides known todirectly interact with the decoding helix (codon-anticodon)during tRNA selection (A1492 and A1493).What is the specific nature of these molecular interactions,

and what do they suggest about the role of H69 in ribosomefunction? H69 contacts incoming tRNA in both the pre- andpostaccommodation steps near positions 25/26 of the D stemand position 38, located near the anticodon stem; tRNA muta-tions at these positions are linked tomiscoding phenotypes (11,12). Likewise, the class 1 RFsmake extensive contacts with H69during stop-codon recognition to extend theGGQdomain intothe large ribosomal subunit to promote catalysis. A particularmetastable helical element referred to as the “switch helix”appears to occupy a cavity in the ribosome that is normallyoccupied by H69 but that is displaced during stop codon recog-nition. H69 is heavily modified by the synthase RluD, whichpseudouridylates positions 1911, 1915, and 1917 (13). Recentstudies have shown that ribosomes isolated from a strain lack-ing RluD show defects in the ability of RF2 (but not RF1) tocatalyze the release of a peptide on UAA-programmed ribo-some complexes (14). Finally, other studies observed large con-formational changes inH69 in ribosome structures where ribo-some recycling factor is bound, consistent with the idea thatmovement of the helix plays a critical role in the dissociationreaction that ribosome recycling factor catalyzes (9). Simplepositioningmakes H69 an excellent candidate for participationin substrate selection events and overall ribosome function.In an effort to define the contribution of H69 to ribosome

function, the properties of ribosomes from which the helix hadbeen deleted were characterized (15). Although the�H69 ribo-somes displayed substantial defects in subunit association (andeven exhibit ribosome recycling factor-independent recycling)and RF1-mediated catalysis, the particles were surprisinglyactive in poly-Phe synthesis and displayed only modest defectsin accuracy during tRNA selection (the variant ribosomesincorporated �2-fold less leucine on the poly-Uridine tem-plate). These results were somewhat at odds with the expecta-tion that H69 would be critical to both tRNA and RF selectionevents mediated at the subunit interface.

* This work was supported, in whole or in part, by National Institutes of HealthGrant GM059425 and the Howard Hughes Medical Institute (to R. G.).

□S The on-line version of this article (available at http://www.jbc.org) containssupplemental Table S1 and Figs. S1 and S2.

1 To whom correspondence should be addressed: Dept. of Molecular Biologyand Genetics, The Johns Hopkins University School of Medicine, 702 PCTB,725 N. Wolfe St., Baltimore, MD 21205. Tel.: 410-614-4922; Fax: 410-955-9124; E-mail: ragreen@jhmi.edu. 2 The abbreviations used are: H69, helix 69; RF, release factor.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 286, NO. 29, pp. 25604 –25610, July 22, 2011© 2011 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

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Genetic screens (16–18) using in vivo-based reporters forstop codon read-through and frame-shifting had also previ-ously identified several positions of interest in H69. In particu-lar, these studies identified “restrictive”mutations in the termi-nal loop of the helix (C1914, �1911) based on observeddecreases in UGA read-through, and other “ribosomal ambigu-ity”mutations in the stem (G1922) based on observed increasesin UGA read-through. Although these studies highlighted apotential role for H69 in substrate selection events, theirgenetic nature did not allow for direct evaluation of ribosomefunction, especially because the read-through assay cannot dis-tinguish between defects in the tRNA and RF selection steps.Here, we examine the role of previously identified key resi-

dues in H69 using transient kinetic approaches to isolate andquantify defects in specific steps in the tRNA and RF selectionprocesses. We conclude that although contacts between theloop of H69 (A1913) are not essential for tRNA selection for amajority of tRNA substrates, these contacts are neverthelesscritical for acceptance of a subset of natural tRNAs (specifically,tRNAk2CAU

Ile on the AUA codon and tRNAICGArg on the CGA

codon). These tRNAs depend on noncanonical pairings in thethird codon position, andwe argue that this inherent instabilitynecessitates additional contacts with H69. In other experi-ments, we find that although contacts between the stem of H69(G1922) and class 1 RFs are generally not critical to selection,these contacts are specifically essential for facilitating the rec-ognition of UGA codons by RF2. These data argue for a criticalrole for H69 in the overall fidelity of translation.

EXPERIMENTAL PROCEDURES

Buffers and Reagents—All experiments were performed inHiFi buffer (50 mM Tris-HCl (pH 7.5) with 70 mM NH4Cl, 30mM KCl, 3.5 mM MgCl2, 0.5 mM spermidine, 8 mM putrescine,and 2 mM DTT). Accommodation and GTPase activationmeasurements were performed at 20 °C and release factorassays at 37 °C. mRNA was prepared by PCR and T7 transcrip-tion and features an epsilon sequence, a Shine-Dalgarnosequence, and a seven-nucleotide spacer followed by the codingsequence AUGXXXUUU, whereXXX is the codon specified inindividual experiments. EFTu and aaRS were purified as

described previously (11). tRNATrp and tRNATrp24/27/59 wereexpressed, isolated, and purified from the MY87 strain asdescribed previously (11). Other WT tRNA species were iso-lated from Escherichia coli MRE 600 total tRNA (RocheApplied Science) by aminoacylation using the correspondingaaRS and incubation with EFTu prior tomixing with ribosomalcomplexes. To account for differences in tRNA abundance, theamount of total tRNA used to form ternary complexes wasadjusted to ensure that a sufficient ternary complex was pro-vided for the reaction to proceed to completion (with 1.0 �M

ribosome complexes); RF1 and RF2 were purified as describedpreviously (19). Ribosomes (WT and mutants) were purified asdescribed previously (20).Kinetic Analysis—Accommodation and GTPase activation

measurements were carried out essentially as described previ-ously (11, 12, 21). Peptide release experiments were carried outas described previously (19), where 0.25 �m initiation com-plexes were typically mixed with 5 �M purified release factorprotein in a rapid quench apparatus. To account for any subunitassociation defects of variant ribosomes, all initiation com-plexes were supplemented with purified WT 30 S ribosomalsubunits (21).

RESULTS

Variant Ribosomes and Experimental Background—Struc-tural studies of the ribosome and its substrates have made thetargeted study of individual regions of potential importanceeasy to accomplish, once themost interesting targets have beenidentified. As the ternary complex encounters the ribosomal Asite (2, 4), the incoming tRNA is distorted into an A/T state.This strained conformation is stabilized not only by interac-tions with the decoding center residues (A1492, A1493, andG530) but also by interactions with the 16 S rRNA shoulder,protein S12, the L11 region of 23 S rRNA, and the terminal loopof H69. After GTP hydrolysis and dissociation of EFTu, thetRNA accommodates and adopts the A/A state (Fig. 1B). Wenote that although there many interactions outside the decod-ing center that appear to stabilize the A/T state, the interactionbetween H69 and the tRNA core (between residue A1913 ofH69 and residues 25/26 and 38 of the tRNA) is one of a few that

FIGURE 1. Positioning of Helix 69 of 23 S rRNA. A, schematic representation of H69 residues including pseudouridines (�) at positions 1911, 1915, and 1917.Mutations introduced in this study are highlighted in red (A1913U) and blue (G1922A). B, structure of a post-accomodation tRNA in the A/A state. Regionscorresponding to ribosomal tRNA binding sites are indicated underneath the mRNA (gold). Decoding center residues 1492 and 1493 (green) evaluate thegeometry of the codon-anticodon interaction. H69 (teal) is shown with residues highlighted as in A. C, structure of RF2 in the ribosomal A site with the GGQdomain extended into the peptidyl transferase center. Decoding center residues 1492 and 1493 are seen to adopt a different conformation than in B, and astacking interaction is observed between 1493 of 16 S rRNA and A1913 of H69 of 23 S rRNA. Figures were made from Protein Data Bank codes 2WDK/2WDL and2WH1/2WH2 (8, 35) using PyMOL software.

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appears to be maintained as the tRNA enters the A/A state.When a stop codon is present in the A site and a class 1 releasefactor binds to the ribosome,A1913 also appears to be involved,forming a stacking interaction with the displaced decodinghelix residue A1493 (Fig. 1C). As such, A1913 does not directlycontact the release factor in the accommodated state but maystill be important for function during this stage or earlier in theprocess during codon recognition (though we have no struc-tural information on a potential “preaccommodation” releasefactor). Interestingly, the terminal loop of H69 has been impli-cated in substrate selection on the ribosome becauseC1914A/U mutant ribosomes yield phenotypes of increasedframe-shifting and decreased UGA read-through in reporterassays. Another potential site of interest found in H69 is G1922(Fig. 1A). This residue, though somewhat remote from the ribo-somal A site and involved in a canonicalWatson-Crick interac-tion in the H69 stem, has been singled out by genetic screensbecause mutation of this residue results in substantial UGAstop codon read-through (16).To study the contributions of H69 to tRNA selection and

release factor function, we mutated these two separate sites(A1913U and G1922A) on H69 and evaluated the biochemicalproperties of the resulting large subunit variants (Fig. 1A). Bothribosome variants were generated by site-directedmutagenesisand purified as described previously (20). Because we antici-pated that both might have defects in ribosome association, allassays were performed in the presence of additional WT 30 Ssubunits (see “Experimental Procedures”).Transient Kinetic Analysis of Effects of H69 Mutations on

Sense Codon Recognition—Todecipher the role ofH69 in tRNAselection, we began by evaluating kinetic parameters accordingto the previously established kinetic and thermodynamicframework (22). Based on earlier studies, we anticipated thattherewere two steps of primary interest, GTPase activation andaccommodation, which are key determinants of the fidelity oftRNA selection. The first of these steps, GTPase activation,occurs when the incoming ternary complex is poised in theA/Tstate and sampling the codon. Codon-anticodon interactionstrigger conformational rearrangements in EFTu that lead to thehydrolysis of GTP and subsequent dissociation of EFTu fromthe ribosome complex. Because GTPase activation (k3) limitsthe rate of GTP hydrolysis (kGTP), we measure kGTP as a proxyfor this process as reported previously (11, 22). For this assay,ribosomes (WT and variant) were programmedwith anmRNAtemplatewith coding regionAUGXXXUUU (whereXXX is theindicated mRNA codon) and were subjected to initiation andelongation reactions that yield a peptidyl-tRNA (fMet-AA-tRNAAA) in the P site. Using these ribosome complexes, wemeasured the rates of GTP hydrolysis by EFTu stimulated by achosen single concentration of WT, A1913U, and G1922Aribosome complexes. We found no substantial differencesbetween ribosomal species in the rates of kGTP triggered byinteractions between tRNAGAU

Ile or tRNAk2CAUIle ternary complex

and ribosomes (WT, A1913U, or G1922A) carrying either theAUUorAUAcodons in theA site (supplemental Fig. S1). Thesedata suggest thatH69 isminimally involved in this early stage oftRNA selection.

The second key step in the tRNA selection framework repre-sents the “accommodation” of the tRNA into the A/A stateonce it is released from EFTu. As this conformational change israte-limiting for the subsequent peptide bond forming reaction(kpep) (23), the rate of peptidyl transfer can be used as a proxyformeasuring accommodation (k5). For thismeasurement,WTand variant ribosome complexes were prepared with fMet-tRNAfMet in the P site, and several distinct codons in the A siteand the rate of dipeptide formation was measured on additionof ternary complexes carrying distinct tRNA isoacceptors cor-responding to those codons (24). When we followed tRNAPhe

interactions with UUU and UUC (both Phe codons) pro-grammed complexes, we found the rates of accommodation tobe indistinguishable on WT, A1913U, and G1922A ribosomes(Fig. 2A and Table 1). These results are broadly consistent withprevious reports indicating that deletion of H69 has little effecton poly-Phe synthesis (15). Similar results were obtained

FIGURE 2. Effects of H69 mutations on tRNA accommodation. Observedrates of peptide bond formation measured by following the formation offMet-AA dipeptide for WT and H69 variant ribosomes for tRNAPhe on UUU andUUC codons (A), tRNATrp and a miscoding mutant (tRNATrp-24/27/59) on UGG (B)and UGA (C) codons. Error bars represent S.E. Figures reported are from atleast three (n � 3) independent experiments.

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with tRNATyr deciphering UAU and UAC codons andtRNAcmo5UGC

Ala or tRNAGGCAla deciphering GCU, GCC, GCA, and

GCG codons (supplemental Table S1).In our previous work, we found that tRNATrp-miscoding

tRNAs exploit an interaction with residue A1913 of H69 toeffectively accommodate on certain near-cognate codon ribo-some complexes (21). These results are extended in Fig. 2wherea potentmiscoding tRNATrp variant (tRNATrp-24/27/59) exhibitsno defects in cognate codon recognition on any ribosome thatwe have characterized (Fig. 2B), but exhibits clear defects inaccommodation on the near-cognate codon UGA in theA1913U ribosome background (Fig. 2C). Notably, on the near-cognate UGA codon, none of the ribosome variants signifi-cantly stimulates accommodation by WT tRNATrp, ruling outmiscoding as causative for the previously described increases instop codon read-through associated with the G1922A variant(Fig. 2C) (16). Indeed, while the variant tRNATrp-24/27/59 sub-stantially promotes accommodation on the UGA codon (0.77s�1 versus 0.03 s�1, a 25-fold effect, Fig. 2c), if anything, theG1922A ribosomes slightly attenuate this elevated rate (0.77s�1 versus 0.45 s�1).So far, taken together, the A1913U ribosomes appear to

behave normally with cognate codon-anticodon pairing inter-actions, only exhibiting alterations in the tRNA selection pro-file whenmiscoding by a variant tRNATrp is specifically probed.These data suggest that A1913 plays some specialized role instabilizing more compromised interactions between tRNA andthe ribosome. To further explore the sensitivity (or hyperaccu-racy) of the A1913U ribosomes, we looked at the decodingparameters of several codons that specifically depend on non-canonical wobble position interactions. When comparing twodifferent isoleucine codons (AUUandAUA) (Fig. 3A) and threedifferent arginine codons (CGU, CGA, and CGC) (Fig. 3B), wenoticed that accommodation rates on several of these codons(AUU, CGU, and CGC) were unaffected by the different H69mutations, whereas they were sensitive to substitution ofA1913 for the AUA (2.4 s�1 versus 0.37 s�1) and CGA (1.3 s�1

versus 0.20 s�1) codons.Another feature of tRNA selection that is commonly evalu-

ated is the overall rate of rejection of aa-tRNAs post-GTPhydrolysis; increases in rejection at this late stage show up asend point defects in the single turnover assay utilized to evalu-

ate accommodation rates. Interestingly, although we generallysee no difference in the rates of rejection between the ribosomespecies tested on other cognate codons, there is a striking rejec-tion of tRNAICG

Arg on the CGA codon, both with theWT and theA1913U variant ribosomes (Fig. 3C); we do not see end pointdefects for the other potentially challenging decoding event on

TABLE 1Peptidyl transfer rates of rRNA mutantsS.E. are calculated from at least three independent determinations.

Codon (tRNA) WT (kPEP) A1913U (kPEP) G1922A (kPEP) % Codona

s�1b s�1b s�1b

UUU (Phe) 4.7 (0.86) 4.6 (0.71) 4.6 (1.0) 2.2UUC (Phe) 2.5 (0.53) 2.3 (0.36) 2.1 (0.14) 1.7UGG (WT-Trp) 2.5 (0.37) 2.2 (0.27) 2.4 (0.47) 1.5UGG (Mut-Trp) 1.6 (0.19) 1.5 (0.29) 1.3 (0.13) 1.5UGA (WT-Trp) .04 (0.01) 0.02 (0.01) 0.04 (0.02) 0.09UGA (Mut-Trp) .77 (0.11) 0.03 (0.02) 0.45 (0.11) 0.09CGU (Arg-ICG) 3.0 (0.50) 2.9 (0.35) 3.2 (0.48) 2.1CGA (Arg-ICG) 2.4 (0.23) 0.37 (0.04) 1.9 (0.16) 0.40CGC (Arg-ICG) 2.2 (0.31) 2.1 (0.31) 1.9 (0.38) 2.2AUU (Ile-GAU) 2.1 (0.47) 2.1 (0.42) 1.9 (0.41) 3.0AUA (Ile-k2CAU) 1.3 (0.28) 0.20 (0.04) 0.79 (0.23) 0.43

a % Codon values calculated for E. coli K12 from the Genomic tRNA Database.b kPEP is calculated at 1 �M ribosomes and 0.15 �M ternary complex. Values presented do not include a calculation for the rate of rejection (k7) seen in the CGAmeasurements.

FIGURE 3. A1913 is critical for tRNA accommodation. Observed rates (kpep)of peptide bond formation measured by following the formation of fMet-AAdipeptide for wild-type (WT) and H69 variant ribosomes for tRNAIle on AUU(decoded by tRNAGAU

Ile ) and AUA (decoded by tRNAk2CAUIle ) codons (A) and

tRNAIGCArg on CGU, CGA, and CGC codons (B). Shown is the time course of pep-

tide bond formation (C) for tRNAIGCArg on CGU (circles) and CGA (squares) codons

with wild-type (WT, black lines) and A1913U (1913, red lines) ribosomes. Frac-tion dipeptide values are normalized to the plateau of the CGU measure-ments. Error bars represent S.E.

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the AUA isoleucine codon. These increased rejection ratesmust be accounted for when calculating the accommodationrate (k5) specifically on the CGA codon (22).TransientKineticAnalysis of Effects ofH69Mutations on Stop

Codon Recognition—In light of the many similarities betweenrecognition of sense and stop codons by tRNAs and RFs,respectively, we set out to evaluate the properties of the sameribosome variants in RF recognition and function. To deter-mine the contributions of specific regions of H69 to termina-tion, we tested the ability of our ribosome variants to promotepeptide release by both RF1 and RF2 on all three stop codons.As for the tRNA selection assays, ribosome complexes wereprogrammed with an mRNA directing the initiator [35S]fMet-tRNAfMet to the P site and the appropriate codons to the A site(UAA, UAG, or UGA). These complexes where subsequentlymixed with saturating amounts of the appropriate release fac-tor, RF1 or RF2, and the rates of peptide release were deter-mined (19). The activity of RF1, which normally recognizesUAA and UAG codons, appears to be completely unaffected bythe introduced changes in H69 (Fig. 4A); the rates of peptiderelease on both codons are similar for all ribosomes tested(Table 2). The relatively subtle changes to H69 studied herelikely explain the apparent discrepancy with earlier studieswhere defects in RF1 function were detected when the entirehelix (H69) was deleted (15). Interestingly, the activity of RF2 isdifferentially affected on its two cognate codons, UAA andUGA (Fig. 4B); whereas there were no appreciable defects forthe variant ribosomes in recognition by RF2 of UAA codons,there was a notable defect (krel reduced by �10-fold) on UGArecognition for the G1922A ribosomes (Table 2). To test

whether this decrease in function is a result of diminished bind-ing by RF2 to the G1922A ribosome, we measured the samerates with 3-fold more RF2 and found the observed rates to beunaffected (supplemental Fig. S2), indicating that the valuesmeasured reflect kcat for the release reaction.The defects associated with stop codon recognition by H69

variant ribosomes agree broadly with genetic screens that iden-tified positions in H69 that affected stop codon read-through(16). Although earlier interpretation of the genetic results wasambiguous in defining the affected step, our results clearly indi-cate that stop codon read-through results from specific defectsin UGA decoding by RF2 on the G1922A variant ribosomes; wenote that read-through was only observed on UGA, but not ona UAA or UAG codon at the same position (16).

DISCUSSION

Early cross-linking studies, structural work on the ribosome,and genetic screens have implicated H69 as playing an impor-tant role in tRNA selection during elongation by the ribosome.However, the details of the molecular mechanisms by whichH69 influences this process remain poorly understood. Here,we establish a critical role for H69 in tRNA selection and ter-mination by measuring the presteady-state kinetic parametersof several variant ribosomes carrying point mutations in thishelix. In particular, the terminal loop of H69 appears to be crit-ical for the recognition of certain tRNA species that depend onnoncanonical third position pairing interactions, whereas thestem of H69 appears to be critical specifically for recognition ofUGA stop codons by RF2. We suggest that the sensitivity ofthese particular decoding events (on typically rare codons inE. coli) results from nonoptimal interactions in the decodingcenter that are supplemented by additional more remote inter-actions between the ribosome and the substrate.Despite an abundance of structural data placing H69 in a

functionally critical region of the ribosome and genetic studiesimplicating the helix in core ribosome function, the most thor-ough biochemical study to date found that although H69 con-tributed substantially to subunit association (and thus recy-cling) and to termination by RF1, it contributed only modestlyto the fidelity of tRNA selection. In this earlier study, using aprocessive poly-Phe synthesis assay, the authors found thatribosomes lacking H69 are only modestly restrictive in decod-ing with �2-fold decreases in leucine misincorporation.Because this steady-state assay does not isolate the critical stepsof tRNA selection, more substantial effects on individual stepsin tRNAselection could have beenmasked by rate-limiting pro-cesses that are not affected by the selected ribosomal muta-tions. Another potential problem with the assay is that it only

FIGURE 4. G1922 is critical for RF2 recognition of UGA. Observed rates ofpeptide release (krel) measured by following the hydrolysis of fMet from fMet-tRNAfMet present in the ribosomal P site. A, rates of peptide release for releasefactor 1 (RF1) on UAA and UAG codons for WT and H69 variant ribosomes.B, rates of peptide release for release factor 2 (RF2) on UAA and UGA codonsfor WT and H69 variant ribosomes. Error bars represent S.E.

TABLE 2Peptide release rates of rRNA mutantsS.E. are calculated from at least three independent determinations.

Codon (RF) WT (krel) A1913U (krel) G1922A (krel) % Codona

s�1b s�1b s�1b

UAA (RF1) 0.92 (0.21) 1.1 (0.20) 1.2 (0.16) 0.21UAG (RF1) 0.89 (0.23) 0.87 (0.19) 0.85 (0.22) 0.03UAA (RF2) 2.1 (0.46) 2.0 (0.28) 1.4 (0.35) 0.21UGA (RF2) 1.0 (0.10) 0.92 (0.19) 0.08 (0.05) 0.09

a % Codon values calculated for E. coli K12 from the Genomic tRNA Database.b krel is calculated at 0.25 �M ribosomes and 5 �M release factor.

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measures the encounter of the ribosome with two differenttRNA substrates (tRNAPhe and tRNALeu) on a single codon(UUU) and, as such, ignores other tRNA-codon interactionsthat may have different requirements.Other key studies implicated H69 in tRNA selection, largely

through genetic screens that identified mutations in the ribo-some that affected UGA read-through or frame-shifting onmRNA transcripts that contain a stop codon in the middle ofthe gene (16–18). One class of mutations discovered in thesestudies clustered in the terminal loop of H69 (C1914, �1911)and generally exhibited slightly decreased rates of UGA read-through; as a representative of this class of mutation, wemutated residue A1913 as it also appears to be located in afunctionally critical location. Another class of H69 mutationuncovered by genetic screens was located in the stem of H69.Mutations in this region (G1922, G1921) exhibit very strongUGA read-through phenotypes; as a representative of this classofmutation,wemutatedG1922 (Fig. 1). The importance ofH69as a critical component of ribosome function is hinted at inthese studies as they find that a number of point mutations inthe region exhibit lethal and severe slow growth phenotypes. Bymeasuring rates of tRNA and RF selection under varied condi-tions with multiple substrates, we define the underlying ribo-some defect that results in distinct differences in UGA read-through for the chosen simple base substitutions (A1913U,G1922A) in H69. In particular, we were interested in knowingwhether a strong UGA read-through phenotype in reporterassays in vivo arises because of increases in the rate of incorpo-ration of tRNATrp or because of decreases in the activity of RF2.Although our earlier studies had suggested that an intactH69

provided additional stabilization to certain near-cognate mis-coding events during tRNA selection (21), our results here indi-cate that H69 plays a key role in facilitating “normal” codon-anticodon interactions during translation. It is well known thatthe first twomRNAcodon positions are deciphered by the ribo-some through strict Watson-Crick RNA base pairs. Decodingof the third position is known to be less stringent, allowing acertain breadth of noncanonical interactions. These nonca-nonical interactions principally include so called “wobble” pair-ing with G-U and U-G base pairs being the most commonexample (exemplified in this study by tRNAPhe on theUUC andUUU codons, respectively). Other wobble interactions includemore heavily modified bases such as queosine (a modified G)(as in tRNATyr, which decodes UAU and UAC), or uridine-5-oxyacetic acid (as in tRNAcmo5UGC

Ala , which decodes GCA andGCG). The ribosome variants characterized in this study(A1913U and G1922A) did not distinguish between these par-ticular noncanonical third position pairing interactions andother more canonical WC pairing interactions (Table 1 andsupplemental Table S1).Other known third position pairing interactions, however,

appear to rely more heavily on the identity of A1913 of H69, asindicated by the defects reported on in Fig. 3,A and B. The wellcharacterized wobble interactions with the post-transcription-ally modified nucleotide inosine that decodes C, U, and A resi-dues at the third codon position is one such example (25).Although the third position C and U (CGC and CGU) aredecoded through the purine-pyrimidine interactions I-C and

I-U with tRNAICGArg , the third position A (CGA) is decoded

through a purine-purine interaction, I-A, which results in asignificantly wide base pair in the decoding helix (26). Anotherexample is the AUA codon in E. coli, decoded by tRNAk2CAU

Ile ,which contains a lysidine (k2C) residue in its anticodon (5�-k2CAU-3�). The addition of this lysine moiety to the cytidineresidue in the first position of the anticodon changes the spec-ificity of tRNAk2CAU

Ile from AUG to AUA (27). Though littlework has focused on this particular modification, a similarchange also appears to be critical for the proper decoding ofAUA in archaea (28). For both of these examples, A1913U var-iant ribosomes are compromised in their ability to select thesetRNA species for accommodation into the ribosome.The dependence of these two inherently weak codon-anti-

codon interactions on the terminal loop of H69 for efficientacceptance into the ribosome leads to interesting questionsabout themechanismbywhich the ribosome achieves uniform-ity of tRNA selection (29). When a rare AUA or CGA codon(Table 1) is encountered by a translating ribosome, interactionsother than those determined by decoding center residues(A1493, A1492, G530) are apparently necessary for efficientaccommodation of the incoming tRNA. Indeed, even when theH69 interactions are intact (with aWT ribosome), we measureincreased rates of rejection (k7) of tRNAICG

Arg on the CGA codon,an observation consistentwith recent reports in Saccharomycescerevisiae identifying the inherently weak nature of the CGAcodon (30). Overall, as the accommodation defects that weobserve in A1913U ribosomes are of a magnitude comparableto those observed when the small subunit decoding residues(1493, 1492, 530) are themselves mutated (31), we propose thatthe terminal loop of H69 functions as a critical core componentin tRNA selection on the ribosome.Another interesting observation emerging from our studies

is that the variant H69 ribosomes appear to have no defects inGTPase activation, though it is generally thought that increasedrates ofGTPase activation and accommodation are triggered bycommon structural signals in the decoding center (22).Although there are numerous examples in the literature whereboth GTPase activation and accommodation are similarlyaffected by a miscoding tRNA (11, 12), we have recently shownthat amutation in the elbow region of tRNATrp-G59A stimulatesaccommodation, but not GTPase activation, on a near-cognatecodon (21). Interestingly, this variant tRNA also depends onH69 residue A1913 to stimulate this accommodation step.Recent structural work on tRNATrp miscoding variants in theA/T state has argued that miscoding depends on the formationof hydrogen bonding interactions in the tRNA core and not onaltered contacts with the ribosome (32). These data argue for aH69-specific contribution to accommodation (but not toGTPase activation) with special sensitivity to the third positiondiscrimination.RF selection is in many ways similar to tRNA selection

wherein large scale conformational rearrangements triggeredby interactions in the decoding center of the small subunit leadto optimal catalysis in the large subunit (19, 33). Nevertheless,the signals that lead to these outcomes are clearly somewhatdifferent because they depend on protein-RNA rather thanRNA-RNA interactions in the decoding center (6, 8, 34). One

H69 Is Critical for Ribosome Function

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likely critical difference is the stacking interaction that formsbetween A1913 of H69 and A1493 of H44 when cognate inter-actions in the decoding center are sensed. Interestingly, weshow here that A1913U ribosomes have no apparent defect ininteractions with RF1 or RF2 on authentic stop codons, sug-gesting that the requisite stacking interaction may not dependon the identity of the nucleotide found at position 1913 but onlyon the presence of some nucleotide that can stack. Earlier stud-ies similarly showed thatmutation ofA1493 had no effect onRFrecognition and catalysis on authentic stop codons (19).How then do we rationalize the defects in RF recognition by

G1922A ribosomes where the mutation of interest appears tobemore structurally remote from presumed critical contacts atthe decoding center? We notice that for A1913 to stack withresidue A1493 of the decoding center, H69 must undergo arelatively large conformational change which results in the for-mation of a new pocket within the ribosome core that cradles(and undoubtedly stabilizes) the so-called “switch” loop thatforms in an extended conformation of the RF. Because of theproximity of G1922 to this switch loop pocket, we speculatethat G1922A ribosomes are compromised in their ability tostabilize the extended conformation of RF2 bound to the ribo-some (Fig. 4B). The specificity of the defects, observed onlywithRF2-UGA interactions, can perhaps be attributed to subtle dif-ferences observed on comparing the switch loop regions of RF1and RF2 (34). For example, RF2 is seen to make additional con-tacts with H69 residues 1914 and 1915 (8), and these might besomewhat destabilized on interaction with the UGA-pro-grammed ribosome complex. Whatever the cause, the specificdeficiencies that we observe in the G1922A ribosomes are con-sistent with multiple earlier reports of RF-related deficiencieswith H69 variant ribosomes (14–16).A thorough biochemical examination of specific ribosome

variants and the defects associated with recognition of certain“cognate” interactions by tRNAs and RFs has allowed us toidentify a role for H69 in both tRNA accommodation and RF2function. This study highlights how individual mRNA codonsare distinctly dealt with by the translational machinery and dis-covers a new mechanism that the ribosome employs to ensureuniformity in tRNA selection. Perturbation of this mechanismslows down the rates of incorporation of natural tRNA sub-strates on the AUA and CGA codons, at a minimum, and therate of peptide release on the UGA codon. These findings notonly shed light on the mechanism of ribosomal codon recogni-tion but may also prove useful in manipulating the ribosomalmachinery to accept foreign amino acids into codons that weredeemed previously inaccessible or inefficient.

Acknowledgments—We thank B. Rogers and S. He for providingreagents and technical expertise for ribosome tagging and purificationsteps and H. Zaher for comments and suggestions on the manuscript.

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H69 Is Critical for Ribosome Function

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Rodrigo F. Ortiz-Meoz and Rachel GreenHelix 69 Is Key for Uniformity during Substrate Selection on the Ribosome

doi: 10.1074/jbc.M111.256255 originally published online May 27, 20112011, 286:25604-25610.J. Biol. Chem. 

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