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• W2549271860 abstract "•Cryo-EM structures of elongating, terminating, and stalled mammalian ribosomes•Eukaryotic-specific elements contribute to stringent sense and stop codon decoding•Pelota engages stalled ribosomes by destabilizing mRNA in the mRNA channel•Decoding complexes communicate recognition to GTPase activation in different ways In eukaryotes, accurate protein synthesis relies on a family of translational GTPases that pair with specific decoding factors to decipher the mRNA code on ribosomes. We present structures of the mammalian ribosome engaged with decoding factor⋅GTPase complexes representing intermediates of translation elongation (aminoacyl-tRNA⋅eEF1A), termination (eRF1⋅eRF3), and ribosome rescue (Pelota⋅Hbs1l). Comparative analyses reveal that each decoding factor exploits the plasticity of the ribosomal decoding center to differentially remodel ribosomal proteins and rRNA. This leads to varying degrees of large-scale ribosome movements and implies distinct mechanisms for communicating information from the decoding center to each GTPase. Additional structural snapshots of the translation termination pathway reveal the conformational changes that choreograph the accommodation of decoding factors into the peptidyl transferase center. Our results provide a structural framework for how different states of the mammalian ribosome are selectively recognized by the appropriate decoding factor⋅GTPase complex to ensure translational fidelity. In eukaryotes, accurate protein synthesis relies on a family of translational GTPases that pair with specific decoding factors to decipher the mRNA code on ribosomes. We present structures of the mammalian ribosome engaged with decoding factor⋅GTPase complexes representing intermediates of translation elongation (aminoacyl-tRNA⋅eEF1A), termination (eRF1⋅eRF3), and ribosome rescue (Pelota⋅Hbs1l). Comparative analyses reveal that each decoding factor exploits the plasticity of the ribosomal decoding center to differentially remodel ribosomal proteins and rRNA. This leads to varying degrees of large-scale ribosome movements and implies distinct mechanisms for communicating information from the decoding center to each GTPase. Additional structural snapshots of the translation termination pathway reveal the conformational changes that choreograph the accommodation of decoding factors into the peptidyl transferase center. Our results provide a structural framework for how different states of the mammalian ribosome are selectively recognized by the appropriate decoding factor⋅GTPase complex to ensure translational fidelity. Successful protein synthesis by ribosomes requires amino acids to be incorporated correctly during polypeptide elongation, translation to terminate at precise points, and quality control pathways to be engaged when translation is interrupted (Dever and Green, 2012Dever T.E. Green R. The elongation, termination, and recycling phases of translation in eukaryotes.Cold Spring Harb. Perspect. Biol. 2012; 4: a013706Crossref PubMed Scopus (266) Google Scholar). In eukaryotes, each of these events is mediated by specific factors (collectively termed as decoding factors in this study) that are delivered to the A site of the ribosome by a specialized member of a subfamily of translational GTPases. Members of this GTPase subfamily are structurally homologous but have non-redundant functions (Dever and Green, 2012Dever T.E. Green R. The elongation, termination, and recycling phases of translation in eukaryotes.Cold Spring Harb. Perspect. Biol. 2012; 4: a013706Crossref PubMed Scopus (266) Google Scholar): eEF1A delivers aminoacyl (aa)-tRNAs to sense codons; eRF3 delivers eRF1 to stop codons; and Hbs1l delivers Pelota (Dom34 in yeast) to stalled ribosomes. After delivery, the specificity of each decoding factor is inspected at the ribosomal decoding center before being accepted into the catalytic peptidyl transferase center (PTC) of the ribosome. Acceptance of each decoding factor by the ribosome has distinct and irreversible consequences: amino acid addition by aa-tRNA, translation termination by eRF1, and the initiation of mRNA and protein quality-control pathways by Pelota. Therefore, accurate decoding of the transcriptome and maintenance of protein homeostasis relies on decoding factor⋅GTPase complexes recognizing the appropriate ribosome-mRNA complex. Our mechanistic understanding of decoding derives primarily from functional and structural studies of sense codon recognition by aa-tRNAs and the bacterial eEF1A homolog, EF-Tu (Voorhees and Ramakrishnan, 2013Voorhees R.M. Ramakrishnan V. Structural basis of the translational elongation cycle.Annu. Rev. Biochem. 2013; 82: 203-236Crossref PubMed Scopus (186) Google Scholar). The accuracy of accepting the correct aa-tRNA is enhanced by a two-step mechanism that exploits the interactions at the decoding center twice. GTP hydrolysis by EF-Tu irreversibly separates an initial selection step from a secondary kinetic proofreading step (Blanchard et al., 2004Blanchard S.C. Gonzalez R.L. Kim H.D. Chu S. Puglisi J.D. tRNA selection and kinetic proofreading in translation.Nat. Struct. Mol. Biol. 2004; 11: 1008-1014Crossref PubMed Scopus (376) Google Scholar). During initial selection, aa-tRNA in complex with EF-Tu⋅GTP samples ribosomes in a configuration in which the aminoacyl group of the aa-tRNA is held by EF-Tu to prevent premature engagement with the PTC (Schmeing et al., 2009Schmeing T.M. Voorhees R.M. Kelley A.C. Gao Y.-G.G. Murphy 4th, F.V. Weir J.R. Ramakrishnan V. The crystal structure of the ribosome bound to EF-Tu and aminoacyl-tRNA.Science. 2009; 326: 688-694Crossref PubMed Scopus (399) Google Scholar). Cognate interactions between aa-tRNA and mRNA at the ribosomal decoding center are communicated to EF-Tu to activate GTP hydrolysis (Pape et al., 1998Pape T. Wintermeyer W. Rodnina M.V. Complete kinetic mechanism of elongation factor Tu-dependent binding of aminoacyl-tRNA to the A site of the E. coli ribosome.EMBO J. 1998; 17: 7490-7497Crossref PubMed Scopus (311) Google Scholar, Ogle et al., 2001Ogle J.M. Brodersen D.E. Clemons Jr., W.M. Tarry M.J. Carter A.P. Ramakrishnan V. Recognition of cognate transfer RNA by the 30S ribosomal subunit.Science. 2001; 292: 897-902Crossref PubMed Scopus (966) Google Scholar, Ogle et al., 2002Ogle J.M. Murphy F.V. Tarry M.J. Ramakrishnan V. Selection of tRNA by the ribosome requires a transition from an open to a closed form.Cell. 2002; 111: 721-732Abstract Full Text Full Text PDF PubMed Scopus (521) Google Scholar), which ultimately leads to the dissociation of EF-Tu⋅GDP from the ribosomal complex (Schmeing et al., 2009Schmeing T.M. Voorhees R.M. Kelley A.C. Gao Y.-G.G. Murphy 4th, F.V. Weir J.R. Ramakrishnan V. The crystal structure of the ribosome bound to EF-Tu and aminoacyl-tRNA.Science. 2009; 326: 688-694Crossref PubMed Scopus (399) Google Scholar). This frees the aa-tRNA to “accommodate” into the ribosomal PTC, a rate-limiting step that relies on the stability of the codon-anticodon interactions at the ribosomal decoding center (Pape et al., 1998Pape T. Wintermeyer W. Rodnina M.V. Complete kinetic mechanism of elongation factor Tu-dependent binding of aminoacyl-tRNA to the A site of the E. coli ribosome.EMBO J. 1998; 17: 7490-7497Crossref PubMed Scopus (311) Google Scholar). Important differences from the paradigm established by aa-tRNA⋅EF-Tu probably exist for eukaryotic decoding factor⋅translational GTPase complexes to account for higher translation accuracy (Kramer et al., 2010Kramer E.B. Vallabhaneni H. Mayer L.M. Farabaugh P.J. A comprehensive analysis of translational missense errors in the yeast Saccharomyces cerevisiae.RNA. 2010; 16: 1797-1808Crossref PubMed Scopus (82) Google Scholar), the evolutionary divergence of the mammalian ribosome, and the eukaryotic expansion of the translational GTPase family to deliver non-tRNA factors to the ribosomal A site (Atkinson et al., 2008Atkinson G.C. Baldauf S.L. Hauryliuk V. Evolution of nonstop, no-go and nonsense-mediated mRNA decay and their termination factor-derived components.BMC Evol. Biol. 2008; 8: 290Crossref PubMed Scopus (77) Google Scholar). Biochemical studies and moderate-resolution structures of several eukaryotic decoding complexes have revealed insights into conserved and distinct features of eukaryotic decoding complexes (Becker et al., 2011Becker T. Armache J.-P. Jarasch A. Anger A.M. Villa E. Sieber H. Motaal B.A. Mielke T. Berninghausen O. Beckmann R. Structure of the no-go mRNA decay complex Dom34-Hbs1 bound to a stalled 80S ribosome.Nat. Struct. Mol. Biol. 2011; 18: 715-720Crossref PubMed Scopus (126) Google Scholar, Dever and Green, 2012Dever T.E. Green R. The elongation, termination, and recycling phases of translation in eukaryotes.Cold Spring Harb. Perspect. Biol. 2012; 4: a013706Crossref PubMed Scopus (266) Google Scholar, Shoemaker and Green, 2012Shoemaker C.J. Green R. Translation drives mRNA quality control.Nat. Struct. Mol. Biol. 2012; 19: 594-601Crossref PubMed Scopus (263) Google Scholar, Taylor et al., 2012Taylor D. Unbehaun A. Li W. Das S. Lei J. Liao H.Y. Grassucci R.A. Pestova T.V. Frank J. Cryo-EM structure of the mammalian eukaryotic release factor eRF1-eRF3-associated termination complex.Proc. Natl. Acad. Sci. USA. 2012; 109: 18413-18418Crossref PubMed Scopus (51) Google Scholar, des Georges et al., 2014des Georges A. Hashem Y. Unbehaun A. Grassucci R.A. Taylor D. Hellen C.U.T. Pestova T.V. Frank J. Structure of the mammalian ribosomal pre-termination complex associated with eRF1∗eRF3∗GDPNP.Nucleic Acids Res. 2014; 42: 3409-3418Crossref PubMed Scopus (56) Google Scholar, Preis et al., 2014Preis A. Heuer A. Barrio-Garcia C. Hauser A. Eyler D.E. Berninghausen O. Green R. Becker T. Beckmann R. Cryoelectron microscopic structures of eukaryotic translation termination complexes containing eRF1-eRF3 or eRF1-ABCE1.Cell Rep. 2014; 8: 59-65Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). However, the molecular interactions that accompany initial selection, communicate information from the decoding center to each GTPase, and mediate decoding factor accommodation in each case remain incompletely understood. Using high-resolution electron cryomicroscopy (cryo-EM), we analyze the molecular basis of specificity at the decoding center for each mammalian decoding factor⋅translational GTPase complex, compare potential GTPase activation mechanisms, and describe the conformational changes governing the accommodation of decoding factors. These results provide new insights into how these related complexes are able to make discriminatory interactions to recognize the appropriate ribosome-mRNA substrates to maintain overall translational fidelity. Translational decoding complexes (here defined as the elongation complex, 80S⋅aa-tRNA⋅eEF1A; the termination complex, 80S⋅eRF1⋅eRF3; and the rescue complex, 80S⋅Pelota⋅Hbs1l) are transient states that either rapidly dissociate or progress to an accommodated state upon codon recognition. We therefore developed methods to trap or assemble these complexes (Figure S1 and STAR Methods). To prepare the elongation complex, ongoing in vitro translation reactions in rabbit reticulocyte lysate of an N-terminally tagged protein were inhibited by the elongation inhibitor didemnin B (Rinehart et al., 1981Rinehart Jr., K.L. Gloer J.B. Hughes Jr., R.G. Renis H.E. McGovren J.P. Swynenberg E.B. Stringfellow D.A. Kuentzel S.L. Li L.H. Didemnins: Antiviral and antitumor depsipeptides from a caribbean tunicate.Science. 1981; 212: 933-935Crossref PubMed Scopus (294) Google Scholar), and the ribosome-nascent chains (RNCs) were affinity purified via the partially synthesized nascent polypeptide. To generate the termination complex, we programmed and affinity purified RNCs with a UGA stop codon in the A site that were reconstituted with eRF1, eRF3, and the nonhydrolyzable GTP analog GMPPCP. Rescue complexes were prepared similarly to produce RNCs containing an empty A site (generated with a truncated mRNA), or an A site occupied by either a stop codon or an AAA codon within a polyadenylated (poly(A)) tail, that were reconstituted with Pelota, Hbs1l, and GMPPCP. The structure of each complex was solved by cryo-EM to between 3.3 and 3.8 Å resolution (Figure S2; Tables S1 and S2).Figure S2Quality of Cryo-EM Maps and Models, Related to Figure 1Show full captionThe EM map for each isolated RNC complex is shown colored according to individual factors (top row) or by local resolution (second row). Below each local resolution map are Fourier shell correlation (FSC) curves calculated between independent half maps (black), and calculated between the refined model and final map (purple), and with the self (blue) and cross-validated (magenta) correlations for each complex. The nominal resolution estimated from the map-to-map correlation at FSC = 0.143 is reported and agrees well with the model-to-map correlation at FSC = 0.5. The 80S⋅eRF1(AAQ)⋅ABCE1 map was generated by combining all of the datasets from (Brown et al., 2015bBrown A. Shao S. Murray J. Hegde R.S. Ramakrishnan V. Structural basis for stop codon recognition in eukaryotes.Nature. 2015; 524: 493-496Crossref PubMed Scopus (175) Google Scholar) to analyze eRF1 conformational changes during the termination pathway (see Figures 7 and S7).View Large Image Figure ViewerDownload Hi-res image Download (PPT) The EM map for each isolated RNC complex is shown colored according to individual factors (top row) or by local resolution (second row). Below each local resolution map are Fourier shell correlation (FSC) curves calculated between independent half maps (black), and calculated between the refined model and final map (purple), and with the self (blue) and cross-validated (magenta) correlations for each complex. The nominal resolution estimated from the map-to-map correlation at FSC = 0.143 is reported and agrees well with the model-to-map correlation at FSC = 0.5. The 80S⋅eRF1(AAQ)⋅ABCE1 map was generated by combining all of the datasets from (Brown et al., 2015bBrown A. Shao S. Murray J. Hegde R.S. Ramakrishnan V. Structural basis for stop codon recognition in eukaryotes.Nature. 2015; 524: 493-496Crossref PubMed Scopus (175) Google Scholar) to analyze eRF1 conformational changes during the termination pathway (see Figures 7 and S7). Each complex represents an unrotated ribosome containing canonical P- and E-site tRNAs (Figures 1, 2, 3, and S2). The GTPase (G) domain and domains 2 and 3 of each GTPase (Figure S3A) were well resolved, while the highly divergent N-terminal extensions of Hbs1l and eRF3 were not visualized, presumably due to their flexibility. Each decoding factor (Figure S3B) assumes a pre-accommodated conformation: the tRNA acceptor arm or the homologous M-C domains of eRF1 or Pelota interacts with the GTPase, and the tRNA anticodon stem loop or structurally distinct N domain of eRF1 or Pelota occupies the decoding center (Figures 1, 2, and 3).Figure 2Structure of the Mammalian Termination ComplexShow full caption(A) Overview of the termination complex assembled with eRF1 (purple) and eRF3 (orange).(B) Decoding center of the termination complex.(C) EM map density (contoured at 6σ) and model showing interactions of the mRNA containing the UGA stop codon (slate) with rRNA elements of the decoding center (yellow).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 3Structure of the Mammalian Rescue ComplexShow full caption(A) Overview of the rescue complex assembled with Pelota (pink) and Hbs1l (brown).(B) Decoding center of the rescue complex.(C) Hydrogen-bonding interactions between the β3′-β4′ loop of Pelota (pink) and 18S rRNA nucleotides (yellow).(D and E) Density corresponding to mRNA in the (D) termination or (E) rescue complexes both assembled on the same (NC-stop) mRNA stalled with the UGA stop codon in the A site. The ribosomal small subunit, P- and E-site tRNAs, and eRF1 or Pelota are indicated.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure S3Secondary Structure Topology Diagrams of Translational GTPases and Decoding Proteins, Related to Figure 1Show full caption(A) Topology diagram of the homologous regions of translational GTPases (e.g., eEF1A, eRF3, and Hbs1l), showing the G domain (red) and the two β-barrel domains (orange and yellow). The motifs important for GTP hydrolysis (Switch 1, Switch 2 (Sw2), and P loop) are highlighted.(B) Topology diagrams of eRF1 and Pelota, showing the divergent N domains and homologous M and C domains. The locations of the loop harboring the catalytic GGQ motif (blue) and the minidomain (mini) in eRF1 are indicated.View Large Image Figure ViewerDownload Hi-res image Download (PPT) (A) Overview of the termination complex assembled with eRF1 (purple) and eRF3 (orange). (B) Decoding center of the termination complex. (C) EM map density (contoured at 6σ) and model showing interactions of the mRNA containing the UGA stop codon (slate) with rRNA elements of the decoding center (yellow). (A) Overview of the rescue complex assembled with Pelota (pink) and Hbs1l (brown). (B) Decoding center of the rescue complex. (C) Hydrogen-bonding interactions between the β3′-β4′ loop of Pelota (pink) and 18S rRNA nucleotides (yellow). (D and E) Density corresponding to mRNA in the (D) termination or (E) rescue complexes both assembled on the same (NC-stop) mRNA stalled with the UGA stop codon in the A site. The ribosomal small subunit, P- and E-site tRNAs, and eRF1 or Pelota are indicated. (A) Topology diagram of the homologous regions of translational GTPases (e.g., eEF1A, eRF3, and Hbs1l), showing the G domain (red) and the two β-barrel domains (orange and yellow). The motifs important for GTP hydrolysis (Switch 1, Switch 2 (Sw2), and P loop) are highlighted. (B) Topology diagrams of eRF1 and Pelota, showing the divergent N domains and homologous M and C domains. The locations of the loop harboring the catalytic GGQ motif (blue) and the minidomain (mini) in eRF1 are indicated. As the ribosomes in the elongation complex (Figure 1A) are stalled at different codons by didemnin B, the density for the mRNA, aa-tRNAs, and the nascent chain are averages of the species captured. Despite this, the density at the decoding center is well defined, revealing that decoding in eukaryotes shares many features with that in bacteria (Ogle et al., 2001Ogle J.M. Brodersen D.E. Clemons Jr., W.M. Tarry M.J. Carter A.P. Ramakrishnan V. Recognition of cognate transfer RNA by the 30S ribosomal subunit.Science. 2001; 292: 897-902Crossref PubMed Scopus (966) Google Scholar). In particular, the decoding nucleotides A1824 and A1825 (A1492 and A1493 in bacteria) are flipped out of helix 44 (h44) of 18S rRNA. Together with G626 (G530 in bacteria) in the anti-conformation, these bases inspect the geometry of the minor groove of the codon-anticodon helix (Figure 1B) and help stabilize the A-site tRNA via hydrogen bonding. These interactions monitor Watson-Crick base-pairing at the first two codon positions (+1 and +2) while providing tolerance at the +3 wobble position. As in bacteria (Ogle et al., 2001Ogle J.M. Brodersen D.E. Clemons Jr., W.M. Tarry M.J. Carter A.P. Ramakrishnan V. Recognition of cognate transfer RNA by the 30S ribosomal subunit.Science. 2001; 292: 897-902Crossref PubMed Scopus (966) Google Scholar), the ribosomal protein uS12 projects a loop into the decoding center (Figures 1C and S4A). Gln61 (Lys44 in E. coli) at the apex of the loop indirectly hydrogen bonds with A1824 in its flipped-out position and with the +2 nucleotide. Pro62 adopts a conserved cis-peptide conformation (Noeske et al., 2015Noeske J. Wasserman M.R. Terry D.S. Altman R.B. Blanchard S.C. Cate J.H.D. High-resolution structure of the Escherichia coli ribosome.Nat. Struct. Mol. Biol. 2015; 22: 336-341Crossref PubMed Scopus (148) Google Scholar) that allows its backbone carbonyl to form a water- or metal-mediated hydrogen bond with the +3 nucleotide (Figures 1C and S4A). Additional hydrogen bonds may be introduced by environmental condition-dependent hydroxylation of Pro62 (Loenarz et al., 2014Loenarz C. Sekirnik R. Thalhammer A. Ge W. Spivakovsky E. Mackeen M.M. McDonough M.A. Cockman M.E. Kessler B.M. Ratcliffe P.J. et al.Hydroxylation of the eukaryotic ribosomal decoding center affects translational accuracy.Proc. Natl. Acad. Sci. USA. 2014; 111: 4019-4024Crossref PubMed Scopus (87) Google Scholar, Noeske et al., 2015Noeske J. Wasserman M.R. Terry D.S. Altman R.B. Blanchard S.C. Cate J.H.D. High-resolution structure of the Escherichia coli ribosome.Nat. Struct. Mol. Biol. 2015; 22: 336-341Crossref PubMed Scopus (148) Google Scholar). Notably, these hydrogen bonds are only with the mRNA backbone, allowing for wobble base-pairing at the +3 position. Relative to bacterial decoding, the eukaryotic-specific ribosomal protein eS30 may enhance the stability of a correct codon-anticodon interaction. In the presence of a cognate aa-tRNA, the N terminus of eS30 becomes ordered, allowing a conserved histidine (His76) to reach into a groove between the phosphate backbone of the anticodon +1 position and the two flipped-out decoding bases to form potentially stabilizing contacts (Figures 1B and 1D). Because this groove depends on the flipped nucleotides that accompany canonical codon-anticodon base-pairing, this interaction may preferentially stabilize cognate tRNAs to enhance discrimination. The A- and P-site tRNAs also appear to stabilize 15 residues at the C terminus of uS19 that interacts with the phosphate backbone of the P-site tRNA and may make electrostatic interactions with the A-site tRNA (Figure 1E). Similar tRNA-dependent transitions in ribosomal proteins are observed in bacteria, with the C terminus of uS13 instead of uS19 threading between the anticodon stem loops of the A- and P-site tRNAs in bacteria (Jenner et al., 2010Jenner L.B. Demeshkina N. Yusupova G. Yusupov M. Structural aspects of messenger RNA reading frame maintenance by the ribosome.Nat. Struct. Mol. Biol. 2010; 17: 555-560Crossref PubMed Scopus (218) Google Scholar). Deletion of the uS13 C terminus in bacteria is associated with a reduced rate of translation and less efficient tRNA selection (Faxén et al., 1994Faxén M. Walles-Granberg A. Isaksson L.A. Antisuppression by a mutation in rpsM(S13) giving a shortened ribosomal protein S13.Biochim. Biophys. Acta. 1994; 1218: 27-34Crossref PubMed Scopus (6) Google Scholar). Thus, the contacts formed by uS19, and especially by eS30, which is dependent on a cognate aa-tRNA, could increase the stability of aa-tRNAs during initial selection and accommodation, thereby reducing erroneous ejection of cognate aa-tRNAs during kinetic proofreading. Unlike translation elongation, the factors and mechanisms mediating translation termination are not conserved between prokaryotes and eukaryotes (Dever and Green, 2012Dever T.E. Green R. The elongation, termination, and recycling phases of translation in eukaryotes.Cold Spring Harb. Perspect. Biol. 2012; 4: a013706Crossref PubMed Scopus (266) Google Scholar). This includes the mechanism of stop codon recognition, as well as the role of termination-associated GTPases. Recent cryo-EM structures have revealed how accommodated eRF1 interacts with stop codons (Brown et al., 2015bBrown A. Shao S. Murray J. Hegde R.S. Ramakrishnan V. Structural basis for stop codon recognition in eukaryotes.Nature. 2015; 524: 493-496Crossref PubMed Scopus (175) Google Scholar, Matheisl et al., 2015Matheisl S. Berninghausen O. Becker T. Beckmann R. Structure of a human translation termination complex.Nucleic Acids Res. 2015; 43: 8615-8626Crossref PubMed Scopus (76) Google Scholar). However, the mechanism of stop codon recognition during the initial eRF1⋅eRF3 interaction with 80S ribosomes was unclear, as earlier structures had only visualized this complex at moderate resolution (Taylor et al., 2012Taylor D. Unbehaun A. Li W. Das S. Lei J. Liao H.Y. Grassucci R.A. Pestova T.V. Frank J. Cryo-EM structure of the mammalian eukaryotic release factor eRF1-eRF3-associated termination complex.Proc. Natl. Acad. Sci. USA. 2012; 109: 18413-18418Crossref PubMed Scopus (51) Google Scholar, des Georges et al., 2014des Georges A. Hashem Y. Unbehaun A. Grassucci R.A. Taylor D. Hellen C.U.T. Pestova T.V. Frank J. Structure of the mammalian ribosomal pre-termination complex associated with eRF1∗eRF3∗GDPNP.Nucleic Acids Res. 2014; 42: 3409-3418Crossref PubMed Scopus (56) Google Scholar, Preis et al., 2014Preis A. Heuer A. Barrio-Garcia C. Hauser A. Eyler D.E. Berninghausen O. Green R. Becker T. Beckmann R. Cryoelectron microscopic structures of eukaryotic translation termination complexes containing eRF1-eRF3 or eRF1-ABCE1.Cell Rep. 2014; 8: 59-65Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar, Muhs et al., 2015Muhs M. Hilal T. Mielke T. Skabkin M.A. Sanbonmatsu K.Y. Pestova T.V. Spahn C.M.T. Cryo-EM of ribosomal 80S complexes with termination factors reveals the translocated cricket paralysis virus IRES.Mol. Cell. 2015; 57: 422-432Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). To address this problem, programmed RNCs with a UGA stop codon in the A site were used to isolate three intermediate states along the canonical termination pathway: (1) delivery of eRF1 to the stop codon by eRF3; (2) accommodated eRF1; and (3) accommodated eRF1 after ABCE1 recruitment (Figures 2A, S1, S2, and S4B–S4D) (Brown et al., 2015bBrown A. Shao S. Murray J. Hegde R.S. Ramakrishnan V. Structural basis for stop codon recognition in eukaryotes.Nature. 2015; 524: 493-496Crossref PubMed Scopus (175) Google Scholar). The structures show that the stop codon maintains the same compacted geometry and interactions with the eRF1 N domain (Brown et al., 2015bBrown A. Shao S. Murray J. Hegde R.S. Ramakrishnan V. Structural basis for stop codon recognition in eukaryotes.Nature. 2015; 524: 493-496Crossref PubMed Scopus (175) Google Scholar, Matheisl et al., 2015Matheisl S. Berninghausen O. Becker T. Beckmann R. Structure of a human translation termination complex.Nucleic Acids Res. 2015; 43: 8615-8626Crossref PubMed Scopus (76) Google Scholar) throughout the termination pathway (Figures 2B and S4B–S4D), despite large rearrangements of the M and C domains of eRF1 (see below). In this configuration, the +2 and +3 stop codon bases stack with a flipped-out A1825, and the base following the stop codon (+4) stacks with G626 in the anti-conformation (Figures 2B, 2C, and S4E) (Brown et al., 2015bBrown A. Shao S. Murray J. Hegde R.S. Ramakrishnan V. Structural basis for stop codon recognition in eukaryotes.Nature. 2015; 524: 493-496Crossref PubMed Scopus (175) Google Scholar, Matheisl et al., 2015Matheisl S. Berninghausen O. Becker T. Beckmann R. Structure of a human translation termination complex.Nucleic Acids Res. 2015; 43: 8615-8626Crossref PubMed Scopus (76) Google Scholar). Improved density for the mRNA further reveals that the +5 base can stack with nucleotide C1698 of 18S rRNA, which protrudes into the mRNA channel (Figures 2B and 2C). The increased stability imparted by this additional stacking interaction explains why a +5 purine can increase the effectiveness of a “weak” stop codon with a +4 pyrimidine (McCaughan et al., 1995McCaughan K.K. Brown C.M. Dalphin M.E. Berry M.J. Tate W.P. Translational termination efficiency in mammals is influenced by the base following the stop codon.Proc. Natl. Acad. Sci. USA. 1995; 92: 5431-5435Crossref PubMed Scopus (225) Google Scholar). Pelota has been reported to bind stalled ribosomes with an empty A site as well as those with an mRNA-occupied A site without sequence preference (Shoemaker et al., 2010Shoemaker C.J. Eyler D.E. Green R. Dom34:Hbs1 promotes subunit dissociation and peptidyl-tRNA drop-off to initiate no-go decay.Science. 2010; 330: 369-372Crossref PubMed Scopus (233) Google Scholar). To determine the basis for this sequence-independent engagement by the rescue complex, we utilized our reconstitution method to assemble 80S⋅Pelota⋅Hbs1l complexes with an A site that lacked mRNA (assembled on a truncated mRNA), or that contained either the UGA stop codon or the AAA sense codon (due to translation stalling within a poly(A) tail) (Figures 3A, S1, and S2). The complex assembled on a truncated mRNA shows that the β3′-β4′ loop of Pelota extends from the N domain to protrude into the empty mRNA channel, following the path normally taken by mRNA (Figures 3B and S4F). A similar path is taken by the shorter β3′-β4′ loop of yeast Dom34 as observed at moderate resolution (Becker et al., 2011Becker T. Armache J.-P. Jarasch A. Anger A.M. Villa E. Sieber H. Motaal B.A. Mielke T. Berninghausen O. Beckmann R. Structure of the no-go mRNA decay complex Dom34-Hbs1 bound to a stalled 80S ribosome.Nat. Struct. Mol. Biol. 2011; 18: 715-720Crossref PubMed Scopus (126) Google Scholar). However, the higher-resolution information in our map allows the details of this interaction to be analyzed. The highly conserved residue (Arg45) at the top of the β3′-β4′ loop appears to play an anchoring role in the complex. Arg45 can hydrogen bond with His100, which is part of a conserved (Y/F/H)HT sequence on β6′ that int" @default.
• W2549271860 created "2016-11-30" @default.
• W2549271860 creator A5007776918 @default.
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• W2549271860 date "2016-11-01" @default.
• W2549271860 modified "2023-10-12" @default.
• W2549271860 title "Decoding Mammalian Ribosome-mRNA States by Translational GTPase Complexes" @default.
• W2549271860 cites W1195318580 @default.
• W2549271860 cites W1514008151 @default.
• W2549271860 cites W1891202747 @default.
• W2549271860 cites W1968010838 @default.
• W2549271860 cites W1978402156 @default.
• W2549271860 cites W1979098238 @default.
• W2549271860 cites W1981447303 @default.
• W2549271860 cites W1981930726 @default.
• W2549271860 cites W1982295411 @default.
• W2549271860 cites W1983167449 @default.
• W2549271860 cites W1983435302 @default.
• W2549271860 cites W1990139062 @default.
• W2549271860 cites W2003521251 @default.
• W2549271860 cites W2017411951 @default.
• W2549271860 cites W2019696945 @default.
• W2549271860 cites W2020663470 @default.
• W2549271860 cites W2027255663 @default.
• W2549271860 cites W2031880123 @default.
• W2549271860 cites W2034246870 @default.
• W2549271860 cites W2037521932 @default.
• W2549271860 cites W2038276231 @default.
• W2549271860 cites W2039162233 @default.
• W2549271860 cites W2041504837 @default.
• W2549271860 cites W2047745452 @default.
• W2549271860 cites W2050782453 @default.
• W2549271860 cites W2054770020 @default.
• W2549271860 cites W2055381555 @default.
• W2549271860 cites W2056372900 @default.
• W2549271860 cites W2062717825 @default.
• W2549271860 cites W2063325492 @default.
• W2549271860 cites W2063742123 @default.
• W2549271860 cites W2072696446 @default.
• W2549271860 cites W2073504319 @default.
• W2549271860 cites W2076052277 @default.
• W2549271860 cites W2085063254 @default.
• W2549271860 cites W2086812659 @default.
• W2549271860 cites W2091807611 @default.
• W2549271860 cites W2092964977 @default.
• W2549271860 cites W2095197488 @default.
• W2549271860 cites W2097604487 @default.
• W2549271860 cites W2100455255 @default.
• W2549271860 cites W2103903051 @default.
• W2549271860 cites W2104825395 @default.
• W2549271860 cites W2111776302 @default.
• W2549271860 cites W2114323152 @default.
• W2549271860 cites W2117430673 @default.
• W2549271860 cites W2118391959 @default.
• W2549271860 cites W21200892 @default.
• W2549271860 cites W2120396539 @default.
• W2549271860 cites W2123360206 @default.
• W2549271860 cites W2123716335 @default.
• W2549271860 cites W2124026197 @default.
• W2549271860 cites W2124081058 @default.
• W2549271860 cites W2126780661 @default.
• W2549271860 cites W2132307548 @default.
• W2549271860 cites W2132629607 @default.
• W2549271860 cites W2132650893 @default.
• W2549271860 cites W2133576799 @default.
• W2549271860 cites W2139694420 @default.
• W2549271860 cites W2142672816 @default.
• W2549271860 cites W2145583354 @default.
• W2549271860 cites W2146801942 @default.
• W2549271860 cites W2149360962 @default.
• W2549271860 cites W2150790075 @default.
• W2549271860 cites W2151619201 @default.
• W2549271860 cites W2152562710 @default.
• W2549271860 cites W2154714625 @default.
• W2549271860 cites W2156118208 @default.
• W2549271860 cites W2156571424 @default.
• W2549271860 cites W2157881623 @default.
• W2549271860 cites W2159211495 @default.
• W2549271860 cites W2159637373 @default.
• W2549271860 cites W2165048399 @default.
• W2549271860 cites W2180229411 @default.
• W2549271860 cites W2196232857 @default.
• W2549271860 cites W2269683575 @default.
• W2549271860 cites W2950967305 @default.
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