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Helix 69 Is Key for Uniformity during Substrate Selection on the Ribosome

2011; Elsevier BV; Volume: 286; Issue: 29 Linguagem: Inglês

10.1074/jbc.m111.256255

1083-351X

Rodrigo F. Ortiz‐Meoz, Rachel Green,

RNA Research and Splicing

Structural studies of ribosome complexes with bound tRNAs and release factors show considerable contacts between these factors and helix 69 (H69) of 23 S rRNA. Although biochemical and genetic studies have provided some general insights into the role of H69 in tRNA and RF selection, a detailed understanding of these contributions remains elusive. Here, we present a pre- steady-state kinetic analysis establishing that two distinct regions of H69 make critical contributions to substrate selection. The loop of H69 (A1913) forms contacts necessary for the efficient accommodation of a subset of natural tRNA species, whereas the base of the stem (G1922) is specifically critical for UGA codon recognition by the class 1 release factor RF2. These data define a broad and critical role for this centrally located intersubunit helix (H69) in accurate and efficient substrate recognition by the ribosome. Structural studies of ribosome complexes with bound tRNAs and release factors show considerable contacts between these factors and helix 69 (H69) of 23 S rRNA. Although biochemical and genetic studies have provided some general insights into the role of H69 in tRNA and RF selection, a detailed understanding of these contributions remains elusive. Here, we present a pre- steady-state kinetic analysis establishing that two distinct regions of H69 make critical contributions to substrate selection. The loop of H69 (A1913) forms contacts necessary for the efficient accommodation of a subset of natural tRNA species, whereas the base of the stem (G1922) is specifically critical for UGA codon recognition by the class 1 release factor RF2. These data define a broad and critical role for this centrally located intersubunit helix (H69) in accurate and efficient substrate recognition by the ribosome. IntroductionThe ribosome is the ribonucleoprotein machine responsible for the faithful translation of the genetic material into the encoded polypeptide. The core RNA components of the ribosome (the 16 S and 23 S rRNA) that reflect its earliest evolution play a key role in interactions that specify the selection of tRNAs and release factors on recognition of sense and stop codons, respectively. During translation elongation, the selection of a cognate ternary complex (composed of aa-tRNA, EFTu, and GTP) from solution is specified primarily in the small ribosomal subunit where three nucleotides in 16 S rRNA directly evaluate the geometry of the codon-anticodon interaction (1Ogle J.M. Brodersen D.E. Clemons Jr., W.M. Tarry M.J. Carter A.P. Ramakrishnan V. Science. 2001; 292: 897-902Crossref PubMed Scopus (960) Google Scholar). Though different in molecular detail, the selection of class 1 release factors takes place in the same decoding center of the small ribosomal subunit where conserved protein features of the RF engage the stop codon. Thus, for both decoding events, molecular interactions in the small subunit "decoding center" are early and critical contributors to the selection of cognate substrates during the translational cycle.In addition to these interactions in the decoding center, it is believed that other molecular interactions between the ribosome and these substrates contribute to binding and specificity. Structural studies of tRNA (2Schmeing T.M. Voorhees R.M. Kelley A.C. Gao Y.G. Murphy 4th, F.V. Weir J.R. Ramakrishnan V. Science. 2009; 326: 688-694Crossref PubMed Scopus (396) Google Scholar, 3Korostelev A. Trakhanov S. Laurberg M. Noller H.F. Cell. 2006; 126: 1065-1077Abstract Full Text Full Text PDF PubMed Scopus (412) Google Scholar, 4Li W. Agirrezabala X. Lei J. Bouakaz L. Brunelle J.L. Ortiz-Meoz R.F. Green R. Sanyal S. Ehrenberg M. Frank J. EMBO J. 2008; 27: 3322-3331Crossref PubMed Scopus (43) Google Scholar, 5Valle M. Sengupta J. Swami N.K. Grassucci R.A. Burkhardt N. Nierhaus K.H. Agrawal R.K. Frank J. EMBO J. 2002; 21: 3557-3567Crossref PubMed Scopus (264) Google Scholar), release factors (6Korostelev A. Zhu J. Asahara H. Noller H.F. EMBO J. 2010; 29: 2577-2585Crossref PubMed Scopus (83) Google Scholar, 7Laurberg M. Asahara H. Korostelev A. Zhu J. Trakhanov S. Noller H.F. Nature. 2008; 454: 852-857Crossref PubMed Scopus (271) Google Scholar, 8Weixlbaumer A. Jin H. Neubauer C. Voorhees R.M. Petry S. Kelley A.C. Ramakrishnan V. Science. 2008; 322: 953-956Crossref PubMed Scopus (232) Google Scholar), and ribosome recycling factor (9Pai R.D. Zhang W. Schuwirth B.S. Hirokawa G. Kaji H. Kaji A. Cate J.H. J. Mol. Biol. 2008; 376: 1334-1347Crossref PubMed Scopus (44) Google Scholar, 10Weixlbaumer A. Petry S. Dunham C.M. Selmer M. Kelley A.C. Ramakrishnan V. Nat. Struct. Mol. Biol. 2007; 14: 733-737Crossref PubMed Scopus (85) Google Scholar) bound to the ribosome reveal that H69 2The abbreviations used are: H69helix 69RFrelease factor. of the large subunit rRNA makes extensive contacts with all of these factors. This universally conserved helix forms an intersubunit bridge that is proximal to the substrate binding cleft and directly contacts the functionally critical h44 of 16 S rRNA which contains two of the three nucleotides known to directly 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 ribosome function? H69 contacts incoming tRNA in both the pre- and postaccommodation steps near positions 25/26 of the D stem and position 38, located near the anticodon stem; tRNA mutations at these positions are linked to miscoding phenotypes (11Cochella L. Green R. Science. 2005; 308: 1178-1180Crossref PubMed Scopus (176) Google Scholar, 12Ledoux S. Olejniczak M. Uhlenbeck O.C. Nat. Struct. Mol. Biol. 2009; 16: 359-364Crossref PubMed Scopus (62) Google Scholar). Likewise, the class 1 RFs make extensive contacts with H69 during stop-codon recognition to extend the GGQ domain into the large ribosomal subunit to promote catalysis. A particular metastable helical element referred to as the "switch helix" appears to occupy a cavity in the ribosome that is normally occupied by H69 but that is displaced during stop codon recognition. H69 is heavily modified by the synthase RluD, which pseudouridylates positions 1911, 1915, and 1917 (13Ejby M. Sørensen M.A. Pedersen S. Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 19410-19415Crossref PubMed Scopus (48) Google Scholar). Recent studies have shown that ribosomes isolated from a strain lacking RluD show defects in the ability of RF2 (but not RF1) to catalyze the release of a peptide on UAA-programmed ribosome complexes (14Kipper K. Sild S. Hetényi C. Remme J. Liiv A. Biochimie. 2011; 93: 834-844Crossref PubMed Scopus (11) Google Scholar). Finally, other studies observed large conformational changes in H69 in ribosome structures where ribosome recycling factor is bound, consistent with the idea that movement of the helix plays a critical role in the dissociation reaction that ribosome recycling factor catalyzes (9Pai R.D. Zhang W. Schuwirth B.S. Hirokawa G. Kaji H. Kaji A. Cate J.H. J. Mol. Biol. 2008; 376: 1334-1347Crossref PubMed Scopus (44) Google Scholar). Simple positioning makes H69 an excellent candidate for participation in 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 had been deleted were characterized (15Ali I.K. Lancaster L. Feinberg J. Joseph S. Noller H.F. Mol. Cell. 2006; 23: 865-874Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). Although the ΔH69 ribosomes displayed substantial defects in subunit association (and even exhibit ribosome recycling factor-independent recycling) and RF1-mediated catalysis, the particles were surprisingly active in poly-Phe synthesis and displayed only modest defects in accuracy during tRNA selection (the variant ribosomes incorporated ∼2-fold less leucine on the poly-Uridine template). These results were somewhat at odds with the expectation that H69 would be critical to both tRNA and RF selection events mediated at the subunit interface.Genetic screens (16O'Connor M. Mol. Genet. Genomics. 2009; 282: 371-380Crossref PubMed Scopus (20) Google Scholar, 17O'Connor M. Dahlberg A.E. J. Mol. Biol. 1995; 254: 838-847Crossref PubMed Scopus (108) Google Scholar, 18Hirabayashi N. Sato N.S. Suzuki T. J. Biol. Chem. 2006; 281: 17203-17211Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar) using in vivo-based reporters for stop codon read-through and frame-shifting had also previously identified several positions of interest in H69. In particular, these studies identified "restrictive" mutations in the terminal loop of the helix (C1914, Ψ1911) based on observed decreases in UGA read-through, and other "ribosomal ambiguity" mutations in the stem (G1922) based on observed increases in UGA read-through. Although these studies highlighted a potential role for H69 in substrate selection events, their genetic nature did not allow for direct evaluation of ribosome function, especially because the read-through assay cannot distinguish between defects in the tRNA and RF selection steps.Here, we examine the role of previously identified key residues in H69 using transient kinetic approaches to isolate and quantify defects in specific steps in the tRNA and RF selection processes. We conclude that although contacts between the loop of H69 (A1913) are not essential for tRNA selection for a majority of tRNA substrates, these contacts are nevertheless critical for acceptance of a subset of natural tRNAs (specifically, tRNAk2CAUIle on the AUA codon and tRNAICGArg on the CGA codon). These tRNAs depend on noncanonical pairings in the third codon position, and we argue that this inherent instability necessitates additional contacts with H69. In other experiments, 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 recognition of UGA codons by RF2. These data argue for a critical role for H69 in the overall fidelity of translation.DISCUSSIONEarly cross-linking studies, structural work on the ribosome, and genetic screens have implicated H69 as playing an important role in tRNA selection during elongation by the ribosome. However, the details of the molecular mechanisms by which H69 influences this process remain poorly understood. Here, we establish a critical role for H69 in tRNA selection and termination by measuring the presteady-state kinetic parameters of several variant ribosomes carrying point mutations in this helix. In particular, the terminal loop of H69 appears to be critical for the recognition of certain tRNA species that depend on noncanonical third position pairing interactions, whereas the stem of H69 appears to be critical specifically for recognition of UGA stop codons by RF2. We suggest that the sensitivity of these particular decoding events (on typically rare codons in E. coli) results from nonoptimal interactions in the decoding center that are supplemented by additional more remote interactions between the ribosome and the substrate.Despite an abundance of structural data placing H69 in a functionally critical region of the ribosome and genetic studies implicating the helix in core ribosome function, the most thorough biochemical study to date found that although H69 contributed substantially to subunit association (and thus recycling) and to termination by RF1, it contributed only modestly to the fidelity of tRNA selection. In this earlier study, using a processive poly-Phe synthesis assay, the authors found that ribosomes lacking H69 are only modestly restrictive in decoding with ∼2-fold decreases in leucine misincorporation. Because this steady-state assay does not isolate the critical steps of tRNA selection, more substantial effects on individual steps in tRNA selection could have been masked by rate-limiting processes that are not affected by the selected ribosomal mutations. Another potential problem with the assay is that it only measures the encounter of the ribosome with two different tRNA substrates (tRNAPhe and tRNALeu) on a single codon (UUU) and, as such, ignores other tRNA-codon interactions that may have different requirements.Other key studies implicated H69 in tRNA selection, largely through genetic screens that identified mutations in the ribosome that affected UGA read-through or frame-shifting on mRNA transcripts that contain a stop codon in the middle of the gene (16O'Connor M. Mol. Genet. Genomics. 2009; 282: 371-380Crossref PubMed Scopus (20) Google Scholar, 17O'Connor M. Dahlberg A.E. J. Mol. Biol. 1995; 254: 838-847Crossref PubMed Scopus (108) Google Scholar, 18Hirabayashi N. Sato N.S. Suzuki T. J. Biol. Chem. 2006; 281: 17203-17211Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). One class of mutations discovered in these studies clustered in the terminal loop of H69 (C1914, Ψ1911) and generally exhibited slightly decreased rates of UGA read-through; as a representative of this class of mutation, we mutated residue A1913 as it also appears to be located in a functionally critical location. Another class of H69 mutation uncovered by genetic screens was located in the stem of H69. Mutations in this region (G1922, G1921) exhibit very strong UGA read-through phenotypes; as a representative of this class of mutation, we mutated G1922 (Fig. 1). The importance of H69 as a critical component of ribosome function is hinted at in these studies as they find that a number of point mutations in the region exhibit lethal and severe slow growth phenotypes. By measuring rates of tRNA and RF selection under varied conditions with multiple substrates, we define the underlying ribosome 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 knowing whether a strong UGA read-through phenotype in reporter assays in vivo arises because of increases in the rate of incorporation of tRNATrp or because of decreases in the activity of RF2.Although our earlier studies had suggested that an intact H69 provided additional stabilization to certain near-cognate miscoding events during tRNA selection (21Ortiz-Meoz R.F. Green R. RNA. 2010; 16: 2002-2013Crossref PubMed Scopus (12) Google Scholar), our results here indicate that H69 plays a key role in facilitating "normal" codon-anticodon interactions during translation. It is well known that the first two mRNA codon positions are deciphered by the ribosome through strict Watson-Crick RNA base pairs. Decoding of the third position is known to be less stringent, allowing a certain breadth of noncanonical interactions. These noncanonical interactions principally include so called "wobble" pairing with G-U and U-G base pairs being the most common example (exemplified in this study by tRNAPhe on the UUC and UUU codons, respectively). Other wobble interactions include more heavily modified bases such as queosine (a modified G) (as in tRNATyr, which decodes UAU and UAC), or uridine-5-oxyacetic acid (as in tRNAcmo5UGCAla, which decodes GCA and GCG). The ribosome variants characterized in this study (A1913U and G1922A) did not distinguish between these particular noncanonical third position pairing interactions and other more canonical WC pairing interactions (Table 1 and supplemental Table S1).Other known third position pairing interactions, however, appear to rely more heavily on the identity of A1913 of H69, as indicated by the defects reported on in Fig. 3, A and B. The well characterized wobble interactions with the post-transcriptionally modified nucleotide inosine that decodes C, U, and A residues at the third codon position is one such example (25Curran J.F. Nucleic Acids Res. 1995; 23: 683-688Crossref PubMed Scopus (98) Google Scholar). Although the third position C and U (CGC and CGU) are decoded 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 a significantly wide base pair in the decoding helix (26Murphy 4th, F.V. Ramakrishnan V. Nat. Struct. Mol. Biol. 2004; 11: 1251-1252Crossref PubMed Scopus (115) Google Scholar). Another example is the AUA codon in E. coli, decoded by tRNAk2CAUIle, which contains a lysidine (k2C) residue in its anticodon (5′-k2CAU-3′). The addition of this lysine moiety to the cytidine residue in the first position of the anticodon changes the specificity of tRNAk2CAUIle from AUG to AUA (27Muramatsu T. Nishikawa K. Nemoto F. Kuchino Y. Nishimura S. Miyazawa T. Yokoyama S. Nature. 1988; 336: 179-181Crossref PubMed Scopus (360) Google Scholar). Though little work has focused on this particular modification, a similar change also appears to be critical for the proper decoding of AUA in archaea (28Ikeuchi Y. Kimura S. Numata T. Nakamura D. Yokogawa T. Ogata T. Wada T. Suzuki T. Suzuki T. Nat. Chem. Biol. 2010; 6: 277-282Crossref PubMed Scopus (99) Google Scholar). For both of these examples, A1913U variant ribosomes are compromised in their ability to select these tRNA species for accommodation into the ribosome.The dependence of these two inherently weak codon-anticodon interactions on the terminal loop of H69 for efficient acceptance into the ribosome leads to interesting questions about the mechanism by which the ribosome achieves uniformity of tRNA selection (29Ledoux S. Uhlenbeck O.C. Mol. Cell. 2008; 31: 114-123Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). When a rare AUA or CGA codon (Table 1) is encountered by a translating ribosome, interactions other than those determined by decoding center residues (A1493, A1492, G530) are apparently necessary for efficient accommodation of the incoming tRNA. Indeed, even when the H69 interactions are intact (with a WT ribosome), we measure increased rates of rejection (k7) of tRNAICGArg on the CGA codon, an observation consistent with recent reports in Saccharomyces cerevisiae identifying the inherently weak nature of the CGA codon (30Letzring D.P. Dean K.M. Grayhack E.J. RNA. 2010; 16: 2516-2528Crossref PubMed Scopus (100) Google Scholar). Overall, as the accommodation defects that we observe in A1913U ribosomes are of a magnitude comparable to those observed when the small subunit decoding residues (1493, 1492, 530) are themselves mutated (31Cochella L. Brunelle J.L. Green R. Nat. Struct. Mol. Biol. 2007; 14: 30-36Crossref PubMed Scopus (46) Google Scholar), we propose that the terminal loop of H69 functions as a critical core component in tRNA selection on the ribosome.Another interesting observation emerging from our studies is that the variant H69 ribosomes appear to have no defects in GTPase activation, though it is generally thought that increased rates of GTPase activation and accommodation are triggered by common structural signals in the decoding center (22Gromadski K.B. Rodnina M.V. Mol. Cell. 2004; 13: 191-200Abstract Full Text Full Text PDF PubMed Scopus (276) Google Scholar). Although there are numerous examples in the literature where both GTPase activation and accommodation are similarly affected by a miscoding tRNA (11Cochella L. Green R. Science. 2005; 308: 1178-1180Crossref PubMed Scopus (176) Google Scholar, 12Ledoux S. Olejniczak M. Uhlenbeck O.C. Nat. Struct. Mol. Biol. 2009; 16: 359-364Crossref PubMed Scopus (62) Google Scholar), we have recently shown that a mutation in the elbow region of tRNATrp-G59A stimulates accommodation, but not GTPase activation, on a near-cognate codon (21Ortiz-Meoz R.F. Green R. RNA. 2010; 16: 2002-2013Crossref PubMed Scopus (12) Google Scholar). Interestingly, this variant tRNA also depends on H69 residue A1913 to stimulate this accommodation step. Recent structural work on tRNATrp miscoding variants in the A/T state has argued that miscoding depends on the formation of hydrogen bonding interactions in the tRNA core and not on altered contacts with the ribosome (32Schmeing T.M. Voorhees R.M. Kelley A.C. Ramakrishnan V. Nat. Struct. Mol. Biol. 2011; 18: 432-436Crossref PubMed Scopus (94) Google Scholar). These data argue for a H69-specific contribution to accommodation (but not to GTPase activation) with special sensitivity to the third position discrimination.RF selection is in many ways similar to tRNA selection wherein large scale conformational rearrangements triggered by interactions in the decoding center of the small subunit lead to optimal catalysis in the large subunit (19Youngman E.M. He S.L. Nikstad L.J. Green R. Mol. Cell. 2007; 28: 533-543Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 33He S.L. Green R. Nat. Struct. Mol. Biol. 2010; 17: 465-470Crossref PubMed Scopus (21) Google Scholar). Nevertheless, the signals that lead to these outcomes are clearly somewhat different because they depend on protein-RNA rather than RNA-RNA interactions in the decoding center (6Korostelev A. Zhu J. Asahara H. Noller H.F. EMBO J. 2010; 29: 2577-2585Crossref PubMed Scopus (83) Google Scholar, 8Weixlbaumer A. Jin H. Neubauer C. Voorhees R.M. Petry S. Kelley A.C. Ramakrishnan V. Science. 2008; 322: 953-956Crossref PubMed Scopus (232) Google Scholar, 34Korostelev A. Asahara H. Lancaster L. Laurberg M. Hirschi A. Zhu J. Trakhanov S. Scott W.G. Noller H.F. Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 19684-19689Crossref PubMed Scopus (186) Google Scholar). One likely critical difference is the stacking interaction that forms between A1913 of H69 and A1493 of H44 when cognate interactions in the decoding center are sensed. Interestingly, we show here that A1913U ribosomes have no apparent defect in interactions with RF1 or RF2 on authentic stop codons, suggesting that the requisite stacking interaction may not depend on the identity of the nucleotide found at position 1913 but only on the presence of some nucleotide that can stack. Earlier studies similarly showed that mutation of A1493 had no effect on RF recognition and catalysis on authentic stop codons (19Youngman E.M. He S.L. Nikstad L.J. Green R. Mol. Cell. 2007; 28: 533-543Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar).How then do we rationalize the defects in RF recognition by G1922A ribosomes where the mutation of interest appears to be more structurally remote from presumed critical contacts at the decoding center? We notice that for A1913 to stack with residue A1493 of the decoding center, H69 must undergo a relatively large conformational change which results in the formation of a new pocket within the ribosome core that cradles (and undoubtedly stabilizes) the so-called "switch" loop that forms in an extended conformation of the RF. Because of the proximity of G1922 to this switch loop pocket, we speculate that G1922A ribosomes are compromised in their ability to stabilize the extended conformation of RF2 bound to the ribosome (Fig. 4B). The specificity of the defects, observed only with RF2-UGA interactions, can perhaps be attributed to subtle differences observed on comparing the switch loop regions of RF1 and RF2 (34Korostelev A. Asahara H. Lancaster L. Laurberg M. Hirschi A. Zhu J. Trakhanov S. Scott W.G. Noller H.F. Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 19684-19689Crossref PubMed Scopus (186) Google Scholar). For example, RF2 is seen to make additional contacts with H69 residues 1914 and 1915 (8Weixlbaumer A. Jin H. Neubauer C. Voorhees R.M. Petry S. Kelley A.C. Ramakrishnan V. Science. 2008; 322: 953-956Crossref PubMed Scopus (232) Google Scholar), and these might be somewhat destabilized on interaction with the UGA-programmed ribosome complex. Whatever the cause, the specific deficiencies that we observe in the G1922A ribosomes are consistent with multiple earlier reports of RF-related deficiencies with H69 variant ribosomes (14Kipper K. Sild S. Hetényi C. Remme J. Liiv A. Biochimie. 2011; 93: 834-844Crossref PubMed Scopus (11) Google Scholar, 15Ali I.K. Lancaster L. Feinberg J. Joseph S. Noller H.F. Mol. Cell. 2006; 23: 865-874Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 16O'Connor M. Mol. Genet. Genomics. 2009; 282: 371-380Crossref PubMed Scopus (20) Google Scholar).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 to identify a role for H69 in both tRNA accommodation and RF2 function. This study highlights how individual mRNA codons are distinctly dealt with by the translational machinery and discovers a new mechanism that the ribosome employs to ensure uniformity in tRNA selection. Perturbation of this mechanism slows down the rates of incorporation of natural tRNA substrates on the AUA and CGA codons, at a minimum, and the rate of peptide release on the UGA codon. These findings not only shed light on the mechanism of ribosomal codon recognition but may also prove useful in manipulating the ribosomal machinery to accept foreign amino acids into codons that were deemed previously inaccessible or inefficient. IntroductionThe ribosome is the ribonucleoprotein machine responsible for the faithful translation of the genetic material into the encoded polypeptide. The core RNA components of the ribosome (the 16 S and 23 S rRNA) that reflect its earliest evolution play a key role in interactions that specify the selection of tRNAs and release factors on recognition of sense and stop codons, respectively. During translation elongation, the selection of a cognate ternary complex (composed of aa-tRNA, EFTu, and GTP) from solution is specified primarily in the small ribosomal subunit where three nucleotides in 16 S rRNA directly evaluate the geometry of the codon-anticodon interaction (1Ogle J.M. Brodersen D.E. Clemons Jr., W.M. Tarry M.J. Carter A.P. Ramakrishnan V. Science. 2001; 292: 897-902Crossref PubMed Scopus (960) Google Scholar). Though different in molecular detail, the selection of class 1 release factors takes place in the same decoding center of the small ribosomal subunit where conserved protein features of the RF engage the stop codon. Thus, for both decoding events, molecular interactions in the small subunit "decoding center" are early and critical contributors to the selection of cognate substrates during the translational cycle.In addition to these interactions in the decoding center, it is believed that other molecular interactions between the ribosome and these substrates contribute to binding and specificity. Structural studies of tRNA (2Schmeing T.M. Voorhees R.M. Kelley A.C. Gao Y.G. Murphy 4th, F.V. Weir J.R. Ramakrishnan V. Science. 2009; 326: 688-694Crossref PubMed Scopus (396) Google Scholar, 3Korostelev A. Trakhanov S. Laurberg M. Noller H.F. Cell. 2006; 126: 1065-1077Abstract Full Text Full Text PDF PubMed Scopus (412) Google Scholar, 4Li W. Agirrezabala X. Lei J. Bouakaz L. Brunelle J.L. Ortiz-Meoz R.F. Green R. Sanyal S. Ehrenberg M. Frank J. EMBO J. 2008; 27: 3322-3331Crossref PubMed Scopus (43) Google Scholar, 5Valle M. Sengupta J. Swami N.K. Grassucci R.A. Burkhardt N. Nierhaus K.H. Agrawal R.K. Frank J. EMBO J. 2002; 21: 3557-3567Crossref PubMed Scopus (264) Google Scholar), release factors (6Korostelev A. Zhu J. Asahara H. Noller H.F. EMBO J. 2010; 29: 2577-2585Crossref PubMed Scopus (83) Google Scholar, 7Laurberg M. Asahara H. Korostelev A. Zhu J. Trakhanov S. Noller H.F. Nature. 2008; 454: 852-857Crossref PubMed Scopus (271) Google Scholar, 8Weixlbaumer A. Jin H. Neubauer C. Voorhees R.M. Petry S. Kelley A.C. Ramakrishnan V. Science. 2008; 322: 953-956Crossref PubMed Scopus (232) Google Scholar), and ribosome recycling factor (9Pai R.D. Zhang W. Schuwirth B.S. Hirokawa G. Kaji H. Kaji A. Cate J.H. J. Mol. Biol. 2008; 376: 1334-1347Crossref PubMed Scopus (44) Google Scholar, 10Weixlbaumer A. Petry S. Dunham C.M. Selmer M. Kelley A.C. Ramakrishnan V. Nat. Struct. Mol. Biol. 2007; 14: 733-737Crossref PubMed Scopus (85) Google Scholar) bound to the ribosome reveal that H69 2The abbreviations used are: H69helix 69RFrelease factor. of the large subunit rRNA makes extensive contacts with all of these factors. This universally conserved helix forms an intersubunit bridge that is proximal to the substrate binding cleft and directly contacts the functionally critical h44 of 16 S rRNA which contains two of the three nucleotides known to directly 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 ribosome function? H69 contacts incoming tRNA in both the pre- and postaccommodation steps near positions 25/26 of the D stem and position 38, located near the anticodon stem; tRNA mutations at these positions are linked to miscoding phenotypes (11Cochella L. Green R. Science. 2005; 308: 1178-1180Crossref PubMed Scopus (176) Google Scholar, 12Ledoux S. Olejniczak M. Uhlenbeck O.C. Nat. Struct. Mol. Biol. 2009; 16: 359-364Crossref PubMed Scopus (62) Google Scholar). Likewise, the class 1 RFs make extensive contacts with H69 during stop-codon recognition to extend the GGQ domain into the large ribosomal subunit to promote catalysis. A particular metastable helical element referred to as the "switch helix" appears to occupy a cavity in the ribosome that is normally occupied by H69 but that is displaced during stop codon recognition. H69 is heavily modified by the synthase RluD, which pseudouridylates positions 1911, 1915, and 1917 (13Ejby M. Sørensen M.A. Pedersen S. Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 19410-19415Crossref PubMed Scopus (48) Google Scholar). Recent studies have shown that ribosomes isolated from a strain lacking RluD show defects in the ability of RF2 (but not RF1) to catalyze the release of a peptide on UAA-programmed ribosome complexes (14Kipper K. Sild S. Hetényi C. Remme J. Liiv A. Biochimie. 2011; 93: 834-844Crossref PubMed Scopus (11) Google Scholar). Finally, other studies observed large conformational changes in H69 in ribosome structures where ribosome recycling factor is bound, consistent with the idea that movement of the helix plays a critical role in the dissociation reaction that ribosome recycling factor catalyzes (9Pai R.D. Zhang W. Schuwirth B.S. Hirokawa G. Kaji H. Kaji A. Cate J.H. J. Mol. Biol. 2008; 376: 1334-1347Crossref PubMed Scopus (44) Google Scholar). Simple positioning makes H69 an excellent candidate for participation in 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 had been deleted were characterized (15Ali I.K. Lancaster L. Feinberg J. Joseph S. Noller H.F. Mol. Cell. 2006; 23: 865-874Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). Although the ΔH69 ribosomes displayed substantial defects in subunit association (and even exhibit ribosome recycling factor-independent recycling) and RF1-mediated catalysis, the particles were surprisingly active in poly-Phe synthesis and displayed only modest defects in accuracy during tRNA selection (the variant ribosomes incorporated ∼2-fold less leucine on the poly-Uridine template). These results were somewhat at odds with the expectation that H69 would be critical to both tRNA and RF selection events mediated at the subunit interface.Genetic screens (16O'Connor M. Mol. Genet. Genomics. 2009; 282: 371-380Crossref PubMed Scopus (20) Google Scholar, 17O'Connor M. Dahlberg A.E. J. Mol. Biol. 1995; 254: 838-847Crossref PubMed Scopus (108) Google Scholar, 18Hirabayashi N. Sato N.S. Suzuki T. J. Biol. Chem. 2006; 281: 17203-17211Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar) using in vivo-based reporters for stop codon read-through and frame-shifting had also previously identified several positions of interest in H69. In particular, these studies identified "restrictive" mutations in the terminal loop of the helix (C1914, Ψ1911) based on observed decreases in UGA read-through, and other "ribosomal ambiguity" mutations in the stem (G1922) based on observed increases in UGA read-through. Although these studies highlighted a potential role for H69 in substrate selection events, their genetic nature did not allow for direct evaluation of ribosome function, especially because the read-through assay cannot distinguish between defects in the tRNA and RF selection steps.Here, we examine the role of previously identified key residues in H69 using transient kinetic approaches to isolate and quantify defects in specific steps in the tRNA and RF selection processes. We conclude that although contacts between the loop of H69 (A1913) are not essential for tRNA selection for a majority of tRNA substrates, these contacts are nevertheless critical for acceptance of a subset of natural tRNAs (specifically, tRNAk2CAUIle on the AUA codon and tRNAICGArg on the CGA codon). These tRNAs depend on noncanonical pairings in the third codon position, and we argue that this inherent instability necessitates additional contacts with H69. In other experiments, 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 recognition of UGA codons by RF2. These data argue for a critical role for H69 in the overall fidelity of translation.