FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2766089724> ?p ?o ?g. }

• W2766089724 endingPage "527.e6" @default.
• W2766089724 startingPage "515" @default.
• W2766089724 abstract "•Polyproline-containing peptides stall translation by destabilizing the P-site tRNA•Elongation factor EF-P recognizes the P-site tRNA and E-site mRNA codon•The lysine modification of EF-P stabilizes the CCA end of the P-site tRNA•EF-P promotes a favorable geometry of the P-site for peptide bond formation Ribosomes synthesizing proteins containing consecutive proline residues become stalled and require rescue via the action of uniquely modified translation elongation factors, EF-P in bacteria, or archaeal/eukaryotic a/eIF5A. To date, no structures exist of EF-P or eIF5A in complex with translating ribosomes stalled at polyproline stretches, and thus structural insight into how EF-P/eIF5A rescue these arrested ribosomes has been lacking. Here we present cryo-EM structures of ribosomes stalled on proline stretches, without and with modified EF-P. The structures suggest that the favored conformation of the polyproline-containing nascent chain is incompatible with the peptide exit tunnel of the ribosome and leads to destabilization of the peptidyl-tRNA. Binding of EF-P stabilizes the P-site tRNA, particularly via interactions between its modification and the CCA end, thereby enforcing an alternative conformation of the polyproline-containing nascent chain, which allows a favorable substrate geometry for peptide bond formation. Ribosomes synthesizing proteins containing consecutive proline residues become stalled and require rescue via the action of uniquely modified translation elongation factors, EF-P in bacteria, or archaeal/eukaryotic a/eIF5A. To date, no structures exist of EF-P or eIF5A in complex with translating ribosomes stalled at polyproline stretches, and thus structural insight into how EF-P/eIF5A rescue these arrested ribosomes has been lacking. Here we present cryo-EM structures of ribosomes stalled on proline stretches, without and with modified EF-P. The structures suggest that the favored conformation of the polyproline-containing nascent chain is incompatible with the peptide exit tunnel of the ribosome and leads to destabilization of the peptidyl-tRNA. Binding of EF-P stabilizes the P-site tRNA, particularly via interactions between its modification and the CCA end, thereby enforcing an alternative conformation of the polyproline-containing nascent chain, which allows a favorable substrate geometry for peptide bond formation. Ribosomes catalyze the synthesis of proteins in cells by providing a platform for the binding of tRNAs. There are three tRNA binding sites on the ribosome, the A, P, and E sites. During translation elongation, aminoacyl-tRNAs (aa-tRNAs) binding at the A site undergo peptide bond formation with the peptidyl-tRNA located at the P site. The rate of peptide bond formation is influenced by the chemical nature of the amino acid substrates in both the A and P sites. Among other amino acids, proline is a particularly poor substrate both as donor and acceptor during peptide bond formation (Pavlov et al., 2009Pavlov M.Y. Watts R.E. Tan Z. Cornish V.W. Ehrenberg M. Forster A.C. Slow peptide bond formation by proline and other N-alkylamino acids in translation.Proc. Natl. Acad. Sci. USA. 2009; 106: 50-54Crossref PubMed Scopus (221) Google Scholar, Johansson et al., 2011Johansson M. Ieong K.W. Trobro S. Strazewski P. Åqvist J. Pavlov M.Y. Ehrenberg M. pH-sensitivity of the ribosomal peptidyl transfer reaction dependent on the identity of the A-site aminoacyl-tRNA.Proc. Natl. Acad. Sci. USA. 2011; 108: 79-84Crossref PubMed Scopus (103) Google Scholar, Muto and Ito, 2008Muto H. Ito K. Peptidyl-prolyl-tRNA at the ribosomal P-site reacts poorly with puromycin.Biochem. Biophys. Res. Commun. 2008; 366: 1043-1047Crossref PubMed Scopus (49) Google Scholar, Wohlgemuth et al., 2008Wohlgemuth I. Brenner S. Beringer M. Rodnina M.V. Modulation of the rate of peptidyl transfer on the ribosome by the nature of substrates.J. Biol. Chem. 2008; 283: 32229-32235Crossref PubMed Scopus (109) Google Scholar, Doerfel et al., 2013Doerfel L.K. Wohlgemuth I. Kothe C. Peske F. Urlaub H. Rodnina M.V. EF-P is essential for rapid synthesis of proteins containing consecutive proline residues.Science. 2013; 339: 85-88Crossref PubMed Scopus (313) Google Scholar, Doerfel et al., 2015Doerfel L.K. Wohlgemuth I. Kubyshkin V. Starosta A.L. Wilson D.N. Budisa N. Rodnina M.V. Entropic contribution of elongation factor P to proline positioning at the catalytic center of the ribosome.J. Am. Chem. Soc. 2015; 137: 12997-13006Crossref PubMed Scopus (66) Google Scholar). In fact, ribosomes become stalled when synthesizing proteins containing consecutive proline residues (Doerfel et al., 2013Doerfel L.K. Wohlgemuth I. Kothe C. Peske F. Urlaub H. Rodnina M.V. EF-P is essential for rapid synthesis of proteins containing consecutive proline residues.Science. 2013; 339: 85-88Crossref PubMed Scopus (313) Google Scholar, Ude et al., 2013Ude S. Lassak J. Starosta A.L. Kraxenberger T. Wilson D.N. Jung K. Translation elongation factor EF-P alleviates ribosome stalling at polyproline stretches.Science. 2013; 339: 82-85Crossref PubMed Scopus (294) Google Scholar, Woolstenhulme et al., 2013Woolstenhulme C.J. Parajuli S. Healey D.W. Valverde D.P. Petersen E.N. Starosta A.L. Guydosh N.R. Johnson W.E. Wilson D.N. Buskirk A.R. Nascent peptides that block protein synthesis in bacteria.Proc. Natl. Acad. Sci. USA. 2013; 110: E878-E887Crossref PubMed Scopus (108) Google Scholar). To alleviate the ribosome stalling and allow translation to continue, a specialized translation factor is required, elongation factor P (EF-P) in bacteria or initiation factor 5A (IF5A) in archaea and eukaryotes (Doerfel et al., 2013Doerfel L.K. Wohlgemuth I. Kothe C. Peske F. Urlaub H. Rodnina M.V. EF-P is essential for rapid synthesis of proteins containing consecutive proline residues.Science. 2013; 339: 85-88Crossref PubMed Scopus (313) Google Scholar, Ude et al., 2013Ude S. Lassak J. Starosta A.L. Kraxenberger T. Wilson D.N. Jung K. Translation elongation factor EF-P alleviates ribosome stalling at polyproline stretches.Science. 2013; 339: 82-85Crossref PubMed Scopus (294) Google Scholar, Gutierrez et al., 2013Gutierrez E. Shin B.S. Woolstenhulme C.J. Kim J.R. Saini P. Buskirk A.R. Dever T.E. eIF5A promotes translation of polyproline motifs.Mol. Cell. 2013; 51: 35-45Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar). IF5A has been shown to be essential in eukaryotes (Dever et al., 2014Dever T.E. Gutierrez E. Shin B.S. The hypusine-containing translation factor eIF5A.Crit. Rev. Biochem. Mol. Biol. 2014; 49: 413-425Crossref PubMed Scopus (105) Google Scholar), and deletion of efp in some bacteria leads to growth defects and avirulence (Lassak et al., 2016Lassak J. Wilson D.N. Jung K. Stall no more at polyproline stretches with the translation elongation factors EF-P and IF-5A.Mol. Microbiol. 2016; 99: 219-235Crossref PubMed Scopus (54) Google Scholar). Both EF-P and IF5A bear post-translational modifications that are essential for their rescue activity (Doerfel et al., 2013Doerfel L.K. Wohlgemuth I. Kothe C. Peske F. Urlaub H. Rodnina M.V. EF-P is essential for rapid synthesis of proteins containing consecutive proline residues.Science. 2013; 339: 85-88Crossref PubMed Scopus (313) Google Scholar, Ude et al., 2013Ude S. Lassak J. Starosta A.L. Kraxenberger T. Wilson D.N. Jung K. Translation elongation factor EF-P alleviates ribosome stalling at polyproline stretches.Science. 2013; 339: 82-85Crossref PubMed Scopus (294) Google Scholar, Gutierrez et al., 2013Gutierrez E. Shin B.S. Woolstenhulme C.J. Kim J.R. Saini P. Buskirk A.R. Dever T.E. eIF5A promotes translation of polyproline motifs.Mol. Cell. 2013; 51: 35-45Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar, Peil et al., 2013Peil L. Starosta A.L. Lassak J. Atkinson G.C. Virumäe K. Spitzer M. Tenson T. Jung K. Remme J. Wilson D.N. Distinct XPPX sequence motifs induce ribosome stalling, which is rescued by the translation elongation factor EF-P.Proc. Natl. Acad. Sci. USA. 2013; 110: 15265-15270Crossref PubMed Scopus (119) Google Scholar). In Escherichia coli, lysine 34 (K34) of EF-P is post-translationally modified by the combined action of EpmA (YjeA), EpmB (YjeK), and EpmC (YfcM). EpmB converts (S)-α-lysine to (R)-β-lysine (Behshad et al., 2006Behshad E. Ruzicka F.J. Mansoorabadi S.O. Chen D. Reed G.H. Frey P.A. Enantiomeric free radicals and enzymatic control of stereochemistry in a radical mechanism: the case of lysine 2,3-aminomutases.Biochemistry. 2006; 45: 12639-12646Crossref PubMed Scopus (34) Google Scholar), and EpmA ligates the (R)-β-lysine to the ε-amino group of K34 (Yanagisawa et al., 2010Yanagisawa T. Sumida T. Ishii R. Takemoto C. Yokoyama S. A paralog of lysyl-tRNA synthetase aminoacylates a conserved lysine residue in translation elongation factor P.Nat. Struct. Mol. Biol. 2010; 17: 1136-1143Crossref PubMed Scopus (116) Google Scholar, Navarre et al., 2010Navarre W.W. Zou S.B. Roy H. Xie J.L. Savchenko A. Singer A. Edvokimova E. Prost L.R. Kumar R. Ibba M. Fang F.C. PoxA, yjeK, and elongation factor P coordinately modulate virulence and drug resistance in Salmonella enterica.Mol. Cell. 2010; 39: 209-221Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). EpmC recognizes the modified form of EF-P and hydroxylates the C5(δ) of K34 (Peil et al., 2012Peil L. Starosta A.L. Virumäe K. Atkinson G.C. Tenson T. Remme J. Wilson D.N. Lys34 of translation elongation factor EF-P is hydroxylated by YfcM.Nat. Chem. Biol. 2012; 8: 695-697Crossref PubMed Scopus (69) Google Scholar); however, the hydroxylation is not required for the rescue activity of EF-P (Doerfel et al., 2013Doerfel L.K. Wohlgemuth I. Kothe C. Peske F. Urlaub H. Rodnina M.V. EF-P is essential for rapid synthesis of proteins containing consecutive proline residues.Science. 2013; 339: 85-88Crossref PubMed Scopus (313) Google Scholar, Ude et al., 2013Ude S. Lassak J. Starosta A.L. Kraxenberger T. Wilson D.N. Jung K. Translation elongation factor EF-P alleviates ribosome stalling at polyproline stretches.Science. 2013; 339: 82-85Crossref PubMed Scopus (294) Google Scholar). Surprisingly, the resulting ε(R)-β-lysylhydroxylysine modification of E. coli EF-P and the enzymes associated with this modification are not conserved across all bacteria (Bailly and de Crécy-Lagard, 2010Bailly M. de Crécy-Lagard V. Predicting the pathway involved in post-translational modification of elongation factor P in a subset of bacterial species.Biol. Direct. 2010; 5: 3Crossref PubMed Scopus (55) Google Scholar, Lassak et al., 2015Lassak J. Keilhauer E.C. Fürst M. Wuichet K. Gödeke J. Starosta A.L. Chen J.M. Søgaard-Andersen L. Rohr J. Wilson D.N. et al.Arginine-rhamnosylation as new strategy to activate translation elongation factor P.Nat. Chem. Biol. 2015; 11: 266-270Crossref PubMed Scopus (89) Google Scholar). Instead, unrelated enzymes and/or modifications have been identified in other bacteria. In Pseudomonas aeruginosa and Shewanella oneidensis, EarP catalyzes the addition of rhamnose to arginine 32 (R32) of EF-P (Lassak et al., 2015Lassak J. Keilhauer E.C. Fürst M. Wuichet K. Gödeke J. Starosta A.L. Chen J.M. Søgaard-Andersen L. Rohr J. Wilson D.N. et al.Arginine-rhamnosylation as new strategy to activate translation elongation factor P.Nat. Chem. Biol. 2015; 11: 266-270Crossref PubMed Scopus (89) Google Scholar, Rajkovic et al., 2015Rajkovic A. Erickson S. Witzky A. Branson O.E. Seo J. Gafken P.R. Frietas M.A. Whitelegge J.P. Faull K.F. Navarre W. et al.Cyclic rhamnosylated elongation factor P establishes antibiotic resistance in Pseudomonas aeruginosa.MBio. 2015; 6: e00823Crossref PubMed Scopus (47) Google Scholar), whereas Bacillus subtilis is reported to bear a 5-aminopentanol moiety attached to K32 (Rajkovic et al., 2016Rajkovic A. Hummels K.R. Witzky A. Erickson S. Gafken P.R. Whitelegge J.P. Faull K.F. Kearns D.B. Ibba M. Translation control of swarming proficiency in Bacillus subtilis by 5-amino-pentanolylated elongation factor P.J. Biol. Chem. 2016; 291: 10976-10985Crossref PubMed Scopus (34) Google Scholar). In eukaryotes, a conserved lysine residue is post-translationally modified to hypusine by the action of deoxyhypusine synthase (DHS) and deoxyhypusine hydroxylase (DOHH) (Dever et al., 2014Dever T.E. Gutierrez E. Shin B.S. The hypusine-containing translation factor eIF5A.Crit. Rev. Biochem. Mol. Biol. 2014; 49: 413-425Crossref PubMed Scopus (105) Google Scholar, Lassak et al., 2016Lassak J. Wilson D.N. Jung K. Stall no more at polyproline stretches with the translation elongation factors EF-P and IF-5A.Mol. Microbiol. 2016; 99: 219-235Crossref PubMed Scopus (54) Google Scholar). The structure of bacterial EF-P revealed a three-domain architecture, with the modified residue located at the tip of domain 1 (Hanawa-Suetsugu et al., 2004Hanawa-Suetsugu K. Sekine S. Sakai H. Hori-Takemoto C. Terada T. Unzai S. Tame J.R. Kuramitsu S. Shirouzu M. Yokoyama S. Crystal structure of elongation factor P from Thermus thermophilus HB8.Proc. Natl. Acad. Sci. USA. 2004; 101: 9595-9600Crossref PubMed Scopus (86) Google Scholar). aIF5A and eIF5A are homologous to bacterial EF-P domains 1 and 2 but lack the bacterial-specific domain 3 (Dever et al., 2014Dever T.E. Gutierrez E. Shin B.S. The hypusine-containing translation factor eIF5A.Crit. Rev. Biochem. Mol. Biol. 2014; 49: 413-425Crossref PubMed Scopus (105) Google Scholar, Lassak et al., 2016Lassak J. Wilson D.N. Jung K. Stall no more at polyproline stretches with the translation elongation factors EF-P and IF-5A.Mol. Microbiol. 2016; 99: 219-235Crossref PubMed Scopus (54) Google Scholar). The X-ray structure of unmodified Thermus thermophilus EF-P in complex with T. thermophilus 70S ribosome bearing a deacylated tRNAfMet at the P site revealed that EF-P binds within the E site of the ribosome with the unmodified arginine 32 (R32) of EF-P interacting with the CCA end of the P-site tRNA (Blaha et al., 2009Blaha G. Stanley R.E. Steitz T.A. Formation of the first peptide bond: the structure of EF-P bound to the 70S ribosome.Science. 2009; 325: 966-970Crossref PubMed Scopus (167) Google Scholar). Similarly, structures of modified eIF5A on the yeast ribosome also visualized the hypusine modification extending into the peptidyltransferase center (PTC) of the ribosome (Melnikov et al., 2016bMelnikov S. Mailliot J. Shin B.S. Rigger L. Yusupova G. Micura R. Dever T.E. Yusupov M. Crystal structure of hypusine-containing translation factor eIF5A bound to a rotated eukaryotic ribosome.J. Mol. Biol. 2016; 428: 3570-3576Crossref PubMed Scopus (37) Google Scholar, Schmidt et al., 2016Schmidt C. Becker T. Heuer A. Braunger K. Shanmuganathan V. Pech M. Berninghausen O. Wilson D.N. Beckmann R. Structure of the hypusinylated eukaryotic translation factor eIF-5A bound to the ribosome.Nucleic Acids Res. 2016; 44: 1944-1951Crossref PubMed Scopus (72) Google Scholar), where it interacts with the CCA end of the P-site tRNA (Schmidt et al., 2016Schmidt C. Becker T. Heuer A. Braunger K. Shanmuganathan V. Pech M. Berninghausen O. Wilson D.N. Beckmann R. Structure of the hypusinylated eukaryotic translation factor eIF-5A bound to the ribosome.Nucleic Acids Res. 2016; 44: 1944-1951Crossref PubMed Scopus (72) Google Scholar). However, to date, no structures exist of EF-P or eIF5A in complex with polyproline-stalled ribosomes; therefore, it remains unclear how the proline residues stall translation and how EF-P/IF5A alleviates these stalled ribosomes. To investigate how polyproline stretches cause translational arrest, we employed a previously used reporter mRNA coding for NlpD-PPP protein bearing three consecutive proline (71PPP73) residues (Starosta et al., 2014Starosta A.L. Lassak J. Peil L. Atkinson G.C. Virumäe K. Tenson T. Remme J. Jung K. Wilson D.N. Translational stalling at polyproline stretches is modulated by the sequence context upstream of the stall site.Nucleic Acids Res. 2014; 42: 10711-10719Crossref PubMed Scopus (63) Google Scholar) (Figure 1A), which was translated in an E. coli lysate-based translation system derived from an E. coli efp deletion strain (see STAR Methods). As expected (Starosta et al., 2014Starosta A.L. Lassak J. Peil L. Atkinson G.C. Virumäe K. Tenson T. Remme J. Jung K. Wilson D.N. Translational stalling at polyproline stretches is modulated by the sequence context upstream of the stall site.Nucleic Acids Res. 2014; 42: 10711-10719Crossref PubMed Scopus (63) Google Scholar), ribosomes with peptidyl-tRNA stalled at the PPP stretch could be alleviated by the exogenous addition of purified modified EF-P protein (Figure 1A). Previous biochemical studies (Doerfel et al., 2013Doerfel L.K. Wohlgemuth I. Kothe C. Peske F. Urlaub H. Rodnina M.V. EF-P is essential for rapid synthesis of proteins containing consecutive proline residues.Science. 2013; 339: 85-88Crossref PubMed Scopus (313) Google Scholar, Ude et al., 2013Ude S. Lassak J. Starosta A.L. Kraxenberger T. Wilson D.N. Jung K. Translation elongation factor EF-P alleviates ribosome stalling at polyproline stretches.Science. 2013; 339: 82-85Crossref PubMed Scopus (294) Google Scholar, Woolstenhulme et al., 2013Woolstenhulme C.J. Parajuli S. Healey D.W. Valverde D.P. Petersen E.N. Starosta A.L. Guydosh N.R. Johnson W.E. Wilson D.N. Buskirk A.R. Nascent peptides that block protein synthesis in bacteria.Proc. Natl. Acad. Sci. USA. 2013; 110: E878-E887Crossref PubMed Scopus (108) Google Scholar), as well as toeprinting assays using the same NlpD-PPP template (Starosta et al., 2014Starosta A.L. Lassak J. Peil L. Atkinson G.C. Virumäe K. Tenson T. Remme J. Jung K. Wilson D.N. Translational stalling at polyproline stretches is modulated by the sequence context upstream of the stall site.Nucleic Acids Res. 2014; 42: 10711-10719Crossref PubMed Scopus (63) Google Scholar), indicate that ribosomes stall in the absence of EF-P because of slow peptide bond formation between the peptidyl-Pro-Pro-tRNA in the P site and the incoming Pro-tRNA in the A site (Figure 1B). These PPP-stalled ribosomes were purified using the 6x-Histidine tag located at the N terminus of the nascent peptide (Figure 1B) and subjected to cryo-electron microscopy (cryo-EM) analysis (see STAR Methods). In silico sorting of the cryo-EM images yielded two subpopulations of non-rotated ribosomes bearing a P-site tRNA but differing by the absence or presence of A-site tRNA (44% and 17%, respectively; Figure S1A). The cryo-EM structures were refined to yield average resolutions of 3.6 Å and 3.9 Å, respectively (Figures 1C and 1D; Figures S1B–S1E; Table 1). In addition, a large population (30%) of vacant ribosomes was observed, as well as a small population (9%) of 70S ribosomes in a rotated state lacking EF-P but containing hybrid A/P-site and P/E-site tRNAs (Figure S1A), the latter presumably representing a post-peptide bond formation state.Table 1Cryo-EM Data Collection, Refinement, and Validation Statistics#1 P-site tRNA only (EMDB: 3898, PDB: 6ENF)#2 A- and P-site tRNA + EF-P (EMDB: 3899, PDB: 6ENJ)#3 P-site tRNA + EF-P (EMDB: 3903, PDB: 6ENU)Data CollectionMicroscopeFEI Titan KriosFEI Titan KriosFEI Titan KriosCameraFalcon IIFalcon IIFalcon IIMagnification129,151129,151129,151Voltage (kV)300300300Electron dose (e–/Ǻ2)282828Defocus range (μm)−0.8 to −2.5−0.8 to −2.5−0.8 to −2.5Pixel size (Ǻ)1.0841.0841.084Initial particles (no.)229,613229,613229,455Final particles (no.)75,08921,65569,761Model CompositionProtein residues5,5315,9515,944RNA bases4,5474,6934,613RefinementResolution range (Å)3.33.93.2Map CC (around atoms)0.780.720.80Map CC (whole unit cell)0.760.750.75FSCaverage0.850.850.85Map sharpening B factor (Ǻ2)−62,88−66,61−60,10RMS Deviations Bond lengths (Å)0.0110.0030.007 Bond angles (°)0.7290.5940.932ValidationMolProbity score1.771.641.77Clashscore4.293.444.11Poor rotamers (%)00.040.41Ramachandran PlotFavored (%)92.0691.3388.83Allowed (%)7.768.3710.74Disallowed (%)0.180.310.43 Open table in a new tab The density quality and resolution for the A-site and P-site tRNAs were generally poorer and less uniform than observed in previous ribosomal complexes (Arenz et al., 2014aArenz S. Meydan S. Starosta A.L. Berninghausen O. Beckmann R. Vázquez-Laslop N. Wilson D.N. Drug sensing by the ribosome induces translational arrest via active site perturbation.Mol. Cell. 2014; 56: 446-452Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, Arenz et al., 2014bArenz S. Ramu H. Gupta P. Berninghausen O. Beckmann R. Vázquez-Laslop N. Mankin A.S. Wilson D.N. Molecular basis for erythromycin-dependent ribosome stalling during translation of the ErmBL leader peptide.Nat. Commun. 2014; 5: 3501Crossref PubMed Scopus (89) Google Scholar, Arenz et al., 2016aArenz S. Bock L.V. Graf M. Innis C.A. Beckmann R. Grubmüller H. Vaiana A.C. Wilson D.N. A combined cryo-EM and molecular dynamics approach reveals the mechanism of ErmBL-mediated translation arrest.Nat. Commun. 2016; 7: 12026Crossref PubMed Scopus (73) Google Scholar). In particular, the density was well resolved for the anticodon stem loop (ASL) of the tRNA on the 30S subunit and progressively deteriorated toward the elbow and acceptor arm of the tRNAs on the 50S subunit (Figures 1E and 1F; Figures S2A–S2G). In fact, density for the CCA end of the P- and A-site tRNAs at the PTC was only present at low thresholds (Figures 1G and 1H). Local resolution calculations also confirmed the flexible nature of the CCA end, particularly with respect to the terminal A76 nucleotide (Figures S2H–S2J). In the structure containing only P-site tRNA, no significant density was observed for the nascent polypeptide chain (Figure 1G), whereas in the structure with both A- and P-site tRNAs, the density attributable to the nascent chain was fragmented and disconnected from the tRNAs (Figure 1H). The density for the CCA end of the A-site tRNA was worse than the one of the P-site tRNA (Figure 1D; Figures S2D–S2G), suggesting that the Pro-tRNA had severe problems to accommodate at the A site of the PTC. Consistent with this notion, the N terminus of ribosomal protein L27, which becomes stabilized upon A-site tRNA accommodation (Polikanov et al., 2014Polikanov Y.S. Steitz T.A. Innis C.A. A proton wire to couple aminoacyl-tRNA accommodation and peptide-bond formation on the ribosome.Nat. Struct. Mol. Biol. 2014; 21: 787-793Crossref PubMed Scopus (131) Google Scholar, Voorhees et al., 2009Voorhees R.M. Weixlbaumer A. Loakes D. Kelley A.C. Ramakrishnan V. Insights into substrate stabilization from snapshots of the peptidyl transferase center of the intact 70S ribosome.Nat. Struct. Mol. Biol. 2009; 16: 528-533Crossref PubMed Scopus (285) Google Scholar), remained disordered (Figure S2K). Collectively, our findings suggest that the presence of the polyproline stretch within the nascent polypeptide chain leads to destabilization of the peptidyl-tRNA and prevents accommodation of the aa-tRNA at the A site, thereby causing translational stalling. To investigate structurally how EF-P relieves the translation arrest caused by polyproline stretches, we incubated PPP-stalled ribosomes with fully modified E. coli EF-P (Figure 2A) and analyzed the resulting complexes by cryo-EM. In silico sorting of the cryo-EM data yielded two major subpopulations of ribosomes bearing P-site tRNA, distinguished by the presence (30%) or absence (33%) of EF-P (Figure S1F). The EF-P-containing subpopulation was extremely heterogeneous, and only a stable subpopulation containing A- and P-site tRNAs with EF-P bound in the E site (Figure 2B) could be refined further, yielding an average resolution of 3.7 Å (Figures S1G and S1H; Table 1). Despite multiple attempts, we were unable to obtain a clean subpopulation containing P-site tRNA and EF-P but lacking A-site tRNA. For completeness, we also refined the major P-site tRNA subpopulation lacking EF-P (Figure 2C) to an average resolution of 3.2 Å (Figures S1I and S1J; Table 1). As before (Figure 1G), little density was observed for the nascent polypeptide chain attached to the P-site tRNA in the EF-P-lacking structure (Figure 2D) despite the improved quality of the density for the CCA end of the P-site tRNA. By contrast, additional nascent chain density was observed when EF-P was present (Figure 2E); however, this density fused directly to the A-site tRNA rather than the P-site tRNA (Figure 2F). Therefore, we concluded that the EF-P-containing subpopulation represents a post-peptide bond formation state with deacylated tRNA in the P site and peptidyl-tRNA in the A site. We also observe that the N terminus of L27 was ordered (Figure 2G), which, as mentioned, is diagnostic for accommodation of the aa-tRNA at the A site (Polikanov et al., 2014Polikanov Y.S. Steitz T.A. Innis C.A. A proton wire to couple aminoacyl-tRNA accommodation and peptide-bond formation on the ribosome.Nat. Struct. Mol. Biol. 2014; 21: 787-793Crossref PubMed Scopus (131) Google Scholar, Voorhees et al., 2009Voorhees R.M. Weixlbaumer A. Loakes D. Kelley A.C. Ramakrishnan V. Insights into substrate stabilization from snapshots of the peptidyl transferase center of the intact 70S ribosome.Nat. Struct. Mol. Biol. 2009; 16: 528-533Crossref PubMed Scopus (285) Google Scholar). In order to capture EF-P bound to polyproline-stalled ribosomes in a pre-peptide bond formation state, we employed a modified version of the NlpD-PPP mRNA that was truncated directly after the codon for the second proline of the PPP motif (Figure 3A). Ribosomes translating the truncated NlpD-PP mRNA become stalled after the PP motif because the absence of an A-site codon precludes binding of the next aa-tRNA; thus, the ribosomes cannot catalyze peptide bond formation even when EF-P is present (Figure 3A). The purified truncated NlpD-PP-stalled ribosomes were then incubated with active modified E. coli EF-P (Figure 3A), and the resulting complexes were analyzed by cryo-EM. In silico sorting of the cryo-EM data yielded two major subpopulations of ribosomes bearing either P- and E-site tRNAs (22%) or P-site tRNA with EF-P bound in the E site (74%) (Figure S1K). The EF-P-containing subpopulation could be further segregated into ribosome populations that differed with respect to the L1 stalk adopting an “in” (30%) or “out” (44%) conformation. The “in” position of the L1 stalk significantly improved the quality of the EF-P density, and therefore this population was further refined, yielding a final cryo-EM structure (Figure 3B) with an average resolution of 3.1 Å (Figures S1L and S1M; Table 1). Similarly, we could also refine the major P- and E-site tRNA-containing ribosome subpopulation that lacked EF-P (Figure 3C) to a final average resolution of 3.2 Å (Figures S1N and S1O). Local resolution calculations indicate less flexibility of the P-site tRNA in the presence of EF-P (Figure 3D) when compared to ribosomes bound with E-site tRNA (Figure 3E) or having a vacant E site (Figure 3F), thus supporting the hypothesis that EF-P stabilizes the P-site peptidyl-tRNA on the ribosome. The well-resolved density for E. coli EF-P bound to the ribosome population with the L1 “in” conformation enabled a complete molecular model to be generated (Figure 4A; Figure S3A). The overall conformation of E. coli EF-P on a polyproline-stalled ribosome is very similar to that observed by X-ray crystallography for T. thermophilus EF-P bound to a T. thermophilus 70S ribosome with a deacylated-tRNAfMet in the P site (Blaha et al., 2009Blaha G. Stanley R.E. Steitz T.A. Formation of the first peptide bond: the structure of EF-P bound to the 70S ribosome.Science. 2009; 325: 966-970Crossref PubMed Scopus (167) Google Scholar), whereas it deviates more significantly from the binding position observed for the yeast homolog eIF5A bound to the 80S ribosome (Schmidt et al., 2016Schmidt C. Becker T. Heuer A. Braunger K. Shanmuganathan V. Pech M. Berninghausen O. Wilson D.N. Beckmann R. Structure of the hypusinylated eukaryotic translation factor eIF-5A bound to the ribosome.Nucleic Acids Res. 2016; 44: 1944-1951Crossref PubMed Scopus (72) Google Scholar, Melnikov et al., 2016bMelnikov S. Mailliot J. Shin B.S. Rigger L. Yusupova G. Micura R. Dever T.E. Yusupov M. Crystal structure of hypusine-containing translation factor eIF5A bound to a rotated eukaryotic ribosome.J. Mol. Biol. 2016; 428: 3570-3576Crossref PubMed Scopus (37) Google Scholar) (Figures S3B and S3C). We observe that the backbone of Asp69 of E. coli EF-P is within hydrogen bonding distance of U17a within the D-loop of the peptidyl-tRNAPro in the P site (Figure S3D). This interaction is also observed in the T. thermophilus EF-P-ribosome structure (Blaha et al., 2009Blaha G. Stanley R.E. Steitz T.A. Formation of the first peptide bond: the structure of EF-P bound to the 70S ribosome.Science. 2009; 325: 966-970Crossref PubMed Scopus (167) Google Scholar) (Figure S3E) but is not possible for tRNAs containing shorter D-loops (Figure S3F), thus providing a specificity determinant for EF-P to recognize tRNAfMet and tRNAPro (Katoh et al., 2016Katoh T. Wohlgemuth I. Nagano M. Rodnina M.V. Suga H. Essential structural elements in tRNA(Pro) for EF-P-mediated alleviation of translation stalling.Nat. Commun. 2016; 7: 11657Crossref PubMed Scopus (50) Google Scholar) (Figures S3D and S3E). By contrast, such a specific interaction between yeast eIF5A and the P-site tRNA was not observed (Schmidt et al., 2016Schmidt C. Becker T. Heuer A. Braunger K. Shanmuganathan V. Pech M. Berninghausen O. Wilson D.N. Beckmann R. Structure of the hypusinylated eukaryotic translation factor eIF-5A bound to the ribosome.Nucleic Acids Res. 2016; 44: 1944-1951Crossref PubMed Scopus (72) Google Scholar, Melnikov et al., 2016bMelnikov S. Mailliot J. Shin B.S. Rigger L. Yusupova G. Micura R. Dever T.E. Yusupov M. Crystal structure of hypusine-containing trans" @default.
• W2766089724 created "2017-11-10" @default.
• W2766089724 creator A5000054179 @default.
• W2766089724 creator A5001331353 @default.
• W2766089724 creator A5003327550 @default.
• W2766089724 creator A5003691907 @default.
• W2766089724 creator A5007441071 @default.
• W2766089724 creator A5010441616 @default.
• W2766089724 creator A5012407865 @default.
• W2766089724 creator A5031556547 @default.
• W2766089724 creator A5040732268 @default.
• W2766089724 creator A5045212144 @default.
• W2766089724 creator A5058863190 @default.
• W2766089724 creator A5067284224 @default.
• W2766089724 creator A5081814635 @default.
• W2766089724 creator A5084266902 @default.
• W2766089724 creator A5084511288 @default.
• W2766089724 creator A5084871178 @default.
• W2766089724 creator A5090860538 @default.
• W2766089724 creator A5091287470 @default.
• W2766089724 date "2017-11-01" @default.
• W2766089724 modified "2023-10-06" @default.
• W2766089724 title "Structural Basis for Polyproline-Mediated Ribosome Stalling and Rescue by the Translation Elongation Factor EF-P" @default.
• W2766089724 cites W1894942982 @default.
• W2766089724 cites W1899616654 @default.
• W2766089724 cites W1966866477 @default.
• W2766089724 cites W1968202347 @default.
• W2766089724 cites W1968759973 @default.
• W2766089724 cites W1970474745 @default.
• W2766089724 cites W1976954198 @default.
• W2766089724 cites W1981225934 @default.
• W2766089724 cites W1996868085 @default.
• W2766089724 cites W1998185437 @default.
• W2766089724 cites W2010693453 @default.
• W2766089724 cites W2012499642 @default.
• W2766089724 cites W2015688756 @default.
• W2766089724 cites W2022955211 @default.
• W2766089724 cites W2025445920 @default.
• W2766089724 cites W2028293439 @default.
• W2766089724 cites W2031880123 @default.
• W2766089724 cites W2031899577 @default.
• W2766089724 cites W2032648965 @default.
• W2766089724 cites W2033306225 @default.
• W2766089724 cites W2035266068 @default.
• W2766089724 cites W2035687084 @default.
• W2766089724 cites W2049475242 @default.
• W2766089724 cites W2050610109 @default.
• W2766089724 cites W2057477511 @default.
• W2766089724 cites W2058464545 @default.
• W2766089724 cites W2059013803 @default.
• W2766089724 cites W2063742123 @default.
• W2766089724 cites W2069524886 @default.
• W2766089724 cites W2070171578 @default.
• W2766089724 cites W2073895233 @default.
• W2766089724 cites W2081693079 @default.
• W2766089724 cites W2086344973 @default.
• W2766089724 cites W2097604487 @default.
• W2766089724 cites W2104234755 @default.
• W2766089724 cites W2105751124 @default.
• W2766089724 cites W2114536619 @default.
• W2766089724 cites W2114833790 @default.
• W2766089724 cites W2127386189 @default.
• W2766089724 cites W2128824543 @default.
• W2766089724 cites W2131994638 @default.
• W2766089724 cites W2132589721 @default.
• W2766089724 cites W2132629607 @default.
• W2766089724 cites W2132717858 @default.
• W2766089724 cites W2136441728 @default.
• W2766089724 cites W2144081223 @default.
• W2766089724 cites W2144773160 @default.
• W2766089724 cites W2152425069 @default.
• W2766089724 cites W2152745366 @default.
• W2766089724 cites W2154714625 @default.
• W2766089724 cites W2158527336 @default.
• W2766089724 cites W2160544821 @default.
• W2766089724 cites W2161605421 @default.
• W2766089724 cites W2163959161 @default.
• W2766089724 cites W2180229411 @default.
• W2766089724 cites W2220104844 @default.
• W2766089724 cites W2223861440 @default.
• W2766089724 cites W2235780920 @default.
• W2766089724 cites W2269683575 @default.
• W2766089724 cites W2305154643 @default.
• W2766089724 cites W2395051652 @default.
• W2766089724 cites W2395410212 @default.
• W2766089724 cites W2464297396 @default.
• W2766089724 cites W2474908907 @default.
• W2766089724 cites W2555829444 @default.
• W2766089724 cites W2593511418 @default.
• W2766089724 cites W2604467178 @default.
• W2766089724 cites W2617303727 @default.
• W2766089724 cites W2618385378 @default.
• W2766089724 cites W2625010928 @default.
• W2766089724 cites W2953320394 @default.
• W2766089724 doi "https://doi.org/10.1016/j.molcel.2017.10.014" @default.
• W2766089724 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/29100052" @default.
• W2766089724 hasPublicationYear "2017" @default.
• W2766089724 type Work @default.

• next