Structural Basis for Bacterial Ribosome-Associated Quality Control by RqcH and RqcP

•Molecular architecture of RqcH on a peptidyl-tRNA 50S complex revealed•The NFACT-N domain distorts and monitors the anticodon tRNAAla•Discovery involvement RqcP/YabO in ribosome-associated quality control (RQC)•Model for how mediate tRNA movement during polyalanine tailing In all branches life, stalled translation intermediates are recognized processed by (RQC) pathways. RQC begins with splitting ribosomes, leaving an unfinished polypeptide still attached to large subunit. Ancient conserved NEMF family proteins target these incomplete degradation addition C-terminal “tails.” How such can occur without regular suite translational components is, however, unclear. Using single-particle cryo-electron microscopy (EM) native complexes, we show that Bacillus subtilis is mediated protein concert RqcP, Hsp15 protein. Our structures reveal factors across ribosomal subunit synthesize polypeptides absence mRNA or small cells, stalling truncated damaged mRNAs harmful because it sequesters ribosomes from active production result synthesis cytotoxic proteins. Therefore, pathways have evolved domains life disassemble complexes (Inada, 2020Inada T. Quality controls induced aberrant translation.Nucleic Acids Res. 2020; 48: 1084-1096Crossref PubMed Scopus (6) Google Scholar; Joazeiro, 2019Joazeiro C.A.P. Mechanisms functions control.Nat. Rev. Mol. Cell Biol. 2019; 20: 368-383Crossref (107) Scholar). eukaryotes, 80S split into 40S 60S subunits Pelota/Dom34 ABCE1/Rli1 (Franckenberg et al., 2012Franckenberg S. Becker Beckmann R. Structural view recycling archaeal eukaryotic after canonical termination ribosome rescue.Curr. Opin. Struct. 2012; 22: 786-796Crossref (30) The resulting 60S-peptidyl-tRNA then pathway, where NEMF-family proteins—Rqc2p yeast humans—facilitate alanine threonine (CAT) tails nascent chains (Brandman 2012Brandman O. Stewart-Ornstein J. Wong D. Larson A. Williams C.C. Li G.W. Zhou King Shen P.S. Weibezahn al.A ribosome-bound triggers peptides signals stress.Cell. 151: 1042-1054Abstract Full Text PDF (346) Defenouillère Fromont-Racine, 2017Defenouillère Q. Fromont-Racine M. complex: peptide clearance proteostasis maintenance.Curr. Genet. 2017; 63: 997-1005Crossref (19) Inada, Kostova 2017Kostova K.K. Hickey K.L. Osuna B.A. Hussmann J.A. Frost Weinberg D.E. Weissman J.S. CAT-tailing as fail-safe mechanism efficient polypeptides.Science. 357: 414-417Crossref (65) 2015Shen Park Qin Y. X. Parsawar K. M.H. Cox Cheng Lambowitz A.M. al.Protein synthesis. Rqc2p mRNA-independent elongation chains.Science. 2015; 347: 75-78Crossref (160) Sitron Brandman, 2019Sitron C.S. Brandman CAT drive off ribosome.Nat. 26: 450-459Crossref (36) Yan Zaher, 2019Yan L.L. Zaher H.S. do cells cope RNA damage its consequences?.J. Chem. 294: 15158-15171Abstract (42) ubiquitinated Listerin/Ltn1 (Bengtson 2010Bengtson Joazeiro C.A. Role E3 ubiquitin ligase control.Nature. 2010; 467: 470-473Crossref (292) Lyumkis 2014Lyumkis Oliveira dos Passos Tahara E.B. Webb Bennett E.J. Vinterbo Potter Carragher B. basis surveillance subunit-associated complex.Proc. Natl. Acad. Sci. USA. 2014; 111: 15981-15986Crossref (77) Scholar) extracted p97/Cdc48 prior proteasomal (Defenouillère 2013Defenouillère Yao Mouaikel Namane Galopier Decourty L. Doyen Malabat C. Saveanu Jacquier Cdc48-associated bound particles required products.Proc. 2013; 110: 5046-5051Crossref (156) Verma 2013Verma Oania R.S. Kolawa N.J. Deshaies R.J. Cdc48/p97 promotes ribosome.eLife. 2: e00308Crossref (153) Bacterial homologs members FbpA (fibronectin binding A) virulence factors. number Gram-positive bacterial species—including Enterococcus faecalis (Singh 2015Singh K.V. La Rosa S.L. Somarajan S.R. Roh J.H. Murray B.E. fibronectin-binding EfbA contributes pathogenesis protects against infective endocarditis caused faecalis.Infect. Immun. 83: 4487-4494Crossref (28) Scholar), Listeria monocytogenes (Osanai 2013Osanai S.J. Asano Sashinami H. Hu D.L. Nakane Fibronectin-binding protein, FbpA, adhesin responsible infection.Microbiol. Immunol. 57: 253-262Crossref Streptococcus pneumoniae (Pracht 2005Pracht Elm Gerber Bergmann Rohde Seiler Kim K.S. Jenkinson H.F. Nau Hammerschmidt PavA modulates adherence, invasion, meningeal inflammation.Infect. 2005; 73: 2680-2689Crossref (140) (Rodriguez Ayala 2017Rodriguez F. Bauman Bartolini Saball E. Salvarrey Leñini Cogliati Strauch Grau Transcriptional regulation adhesive properties extracellular matrix through YloA.Mol. Microbiol. 104: 804-821Crossref (9) Scholar)—were proposed directly adhesion matrix, although direct experimental demonstration this function has been lacking. A recent study demonstrated homolog, (“bacterial Rqc2 homolog”), bona fide factor (Lytvynenko 2019Lytvynenko I. Paternoga Thrun Balke Muller T.A. Chiang C.H. Nagler Tsaprailis G. Anders Bischofs al.Alanine Tails Signal Proteolysis Ribosome-Associated Control.Cell. 178: 76-90.e22Abstract (27) recruited 50S-peptidyl-tRNA promote polypeptides, targeting ClpXP machinery Although rqcH absent lineages, discovery, along broad distribution archaea, implies were present last universal common ancestor (LUCA) therefore, integral three (Burroughs Aravind, 2014Burroughs Aravind highly related DNA-glycosylase fold helps predict multiple novel modifications.RNA 11: 360-372Crossref (17) Lytvynenko widely distributed kingdoms typically contain, N terminus C terminus, (1) followed (2) two helix-hairpin-helix (HhH) motifs, which homology DNA glycosylases but no known enzymatic activity; (3) coiled-coil motif, consisting long ? helices separated “middle” domain, termed CC-M; (4) NFACT-R predicted bind RNA; (5) NFACT-C unknown Shao 2015Shao Brown Santhanam Hegde Structure assembly pathway complex.Mol. Cell. 433-444Abstract (112) An early structural around P site, likely recognizing (Lyumkis Subsequently, more reported: Rqc2p-60S tRNAs aminoacyl site (A site) peptidyl (P (Shen vitro reconstituted mammalian 60S-Listerin-NEMF P-site (Shao both structures, HhH close tRNA, coiled coil spanned contact stalk base middle CC-M domain. revealed global mode associated due low resolution proteins, at most only partial molecular model could be built detailed understanding interact mechanistic insight catalyze tailing, identity other involved so far remained elusive. Here, obtained affinity purification discover additional factor, RqcP (formerly YabO), was previously not co-distributed many lineages. Surprisingly, our series mimic distinct pre- post-translocational states observed elongation. This provides cooperate movement, thereby processive cycle independent mRNA, subunit, translocase EF-G. To investigate mediates subtilis, determined cryo-EM RqcH-50S isolated purification. purified chromatography expressing C-terminally tagged FLAG3 epitope (Figure S1A). Single-particle analysis extensive silico sorting, yielded four functional states: A–D S1B). State contained A/P-like configuration 1A), whereas state B RqcH, classical P-site-like conformation, well identified YabO (Figures 1B S1B)—a homolog coli Hsp15. similar A, presence exit (E 1C). D implying had dissociated sample preparation States B, C, refined average resolutions 3.5 Å, 2.9 3.1 2.6 respectively S2A–S2D); while resolved, density ligands varied S2E–S2H). exhibited high flexibility A/P better ordered conformation 1A–1C S2E–S2G). We further improved map using multibody refinement sufficient unambiguous fitting individual based X-ray bacteria (Manne 2019Manne Narayana S.V.L. Chattopadhyay Crystal structure N-terminal pneumoniae.Acta Crystallogr. F Commun. 75: 657-662Crossref Musyoki 2016Musyoki Shi Z. Xuan Lu Qi Gao Zheng Zhang Haywood al.Structural anchorless FBPS bacterium suis.Proc. 2016; 113: 13869-13874Crossref (20) 1D; Table 1; Video S1). relatively resolved S2I–S2K), consistent bulky aromatic side chains, exception minimal M resembled hairpin S3L–S3O). appeared flexible less S3I S3J). Nonetheless, short loops, able residues 2 565 use fit refine (Table 1).Table 1Cryo-EM Data Collection, Model Refinement, Validation StatisticsCollection DetailsRqcH ARqcH BRqcH multibodyNumber micrograph movies3,032/4,1453,032/4,1453,032/4,145Electron fluence (e?/Å2)28.3/29.728.3/29.728.3/29.7Defocus range (?m)?0.7 ?1.9?0.7 ?1.9Accession numbersEMDB: 11890,PDB: 7AS9EMDB: 11889,PDB: 7AS8EMDB: 11891,PDB: 7ASAAverage (Å)3.52.94.0Number particles10,70374,21074,210Number non-hydrogen atoms93,14293,8058,110Number residues3,7203,810750Number bases2,9992,997116RefinementMap CC atoms0.840.900.75Map whole volume0.820.900.74Map sharpening (Å2)?66.07?71.99?98.32Root-Mean-Square DeviationBond length (Å)0.0070.0110.013Bond angle (°)1.0501.0102.026ValidationMolProbity score2.081.700.98Clash score8.304.050.66% Poor rotamers0.000.030.18Ramachandran plot% Favored86.7891.2796.19% Allowed13.008.653.54% Disallowed0.220.080.27 Open table new tab https://www.cell.com/cms/asset/1f6b3de2-37e9-4ec8-a65b-5bfff010c299/mmc5.mp4Loading ... Download .mp4 (25.21 MB) Help files S1. Overview RqcH-YabO-P-tRNA-50S Density Model, Related Figures 1 3 best state, located near central protuberance (CP) between E sites, coils span interface uL11 back A-site finger (ASF; H38), positioned 1E; overall homologs, human S3A–S3H); higher reveals details before. predominantly anticodon-stem loop (ASL) 1E S2O) appear establish any rationalizes reported D97A/R98A (DR) E121A/I122A/M123A (EIM) mutations specifically abrogate since motifs within loops approach 2A–2C). good agreement previous report introduction DR EIM did destabilize interaction 2E). Indeed, one volume sorting (termed hereinafter B?) little domains, indicating There contacts namely, ASF 1E) distal portion uL11/H44 1F). Both interactions necessary DWH motif proximity ASF, leads loss assessed sucrose gradient centrifugation cellular lysates 2D 2E); treatment thiostrepton, antibiotic overlapping 2F–2I), abrogated association 2J). (Osuna 2017Osuna Howard C.J. Kc activities insights tailing.eLife. 6: e27949Crossref antibiotics peptidyl-transferase center (lincomycin), (viomycin), GTPase EF-Tu (kirromycin) EF-G (fusidic acid) perturb Puromycin, releases chain if accessible, mild effect, density—positioned 23S rRNA 68 69—that correspond component 3A 3B ). assigned mass spectrometry S1) excellent features fitted crystal template (Staker 2000Staker B.L. Korber P. Bardwell J.C. Saper M.A. RNA-binding motif.EMBO 2000; 19: 749-757Crossref (44) 3C). binds (Korber 2000Korber Stahl J.M. Nierhaus K.H. Hsp15: heat shock protein.EMBO 741-748Crossref (63) translocate (Jiang 2009Jiang Schaffitzel Bingel-Erlenmeyer Ban N. Koning R.I. de Geus D.C. Plaisier J.R. Abrahams J.P. Recycling aborted subunit-nascent chain-tRNA Hsp15.J. 2009; 386: 1357-1367Crossref While CP instead adjacent H69 S3I–S3K). Gammaproteobacteria contain suggesting may differently than YabO. Additionally, extension (CTE) 3D S3L). YabO/Hsp15 diverse clades divide either those having CTE, Hsp15, not, 3D); strikingly, division strongly RqcH. Presence CTE entirely mutually exclusive and, few exceptions, lacking 3D; S2). Together Proteobacteria YabO/Hsp15, suggests functionally equivalent orthologs rather divergent paralogs. supported observation that, unlike expression (Nicolas 2012Nicolas Mäder U. Dervyn Rochat Leduc Pigeonneau Bidnenko Marchadier Hoebeke Aymerich al.Condition-dependent transcriptome high-level regulatory subtilis.Science. 335: 1103-1106Crossref (544) Collectively, findings suggest containing they raise question role used genetic assess RQC. As shown earlier, does strong phenotype grown laboratory conditions rich medium However, when mutation combined tmRNA, resultant ?ssrA ?rqcH strain particularly sensitive inhibitors tetracycline displays growth defect elevated temperatures 3E). Consistent RQC, ?yabO defect, simultaneous tmRNA phenocopies double deletion. Importantly, heterologous Hsp15—either full-length 3F) version alone S3M)—does counter sensitivity strain. being specialized dissect affinity-purified tag, yielding YabO-50S S4A). co-purified confirmed S4B), also pull-outs E, P- E-site tRNAs, S4B). D, pull-out dataset 3.2 S5A–S5C). states, S5D–S5F) established defined ASL 4A–4D). essential recruitment 50S, migrated vice versa 4E). By contrast, critical function, Arg16 Ala (R16A) completely abolished 3D, 4E, S4A; result, substitution yabO R16A background deletion 3E), thus reinforcing crucial residue functionality. Comparison (RqcH A/P-site YabO; Figure 4F) tRNA; 4G) shows translocation-like 4H) 4I), proposals riboso