Cryo- EM structure of the mycobacterial 70S ribosome in complex with ribosome hibernation promotion factor RafH, reveals the unique mode of mycobacterial ribosome hibernation

Cryo- EM structure of the mycobacterial 70S ribosome in complex with ribosome hibernation promotion factor RafH, reveals the unique mode of mycobacterial ribosome hibernation | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Cryo- EM structure of the mycobacterial 70S ribosome in complex with ribosome hibernation promotion factor RafH, reveals the unique mode of mycobacterial ribosome hibernation Prem Kaushal, Niraj Kumar, Shivani Sharma This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2837054/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 20 Jan, 2024 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Abstract Ribosome hibernation is a key survival strategy bacteria adopt under environmental stress, where a protein, hibernation promotion factor (HPF), transitorily inactivates the ribosome and slows down its overall protein synthesis. The mechanism is well studied in enteric bacteria, which mainly hibernate its ribosome in 100S disome form through a dual domain, long HPF (HPF long ) or a single domain, short HPF (HPF short ) in concert with another ribosome modulation factor. Mycobacterium tuberculosis encounters hypoxia (low oxygen) as a major stress in the host macrophages, and it overexpresses RafH protein, which is critical for its survival. The RafH, a dual domain HPF, an orthologue of bacterial HPF long , hibernates ribosome in 70S monosome form only. Here we report the cryo- EM structure of Mycobacterium smegmatis , a close homologue of M. tuberculosis , 70S ribosome in complex with the RafH factor at an overall 2.8 Å resolution. The RafH N- terminus domain (NTD) is conserved and binds to the decoding center of the ribosomal small subunit, a similar binding for HPF long NTD, but additionally it also interacts with the inter subunit bridge, B2a. Contrary to the HPF long C- terminus domain (CTD), the RafH CTD, which is larger, binds to a unique site at the platform binding center of the ribosomal small subunit and sandwiches between bS1 and uS11 ribosomal proteins. The two domain connecting linker regions, which remain mostly disordered in earlier reported HPF long structures, interacts mainly with the anti-Shine Dalgarno sequence of the 16S rRNA. The helix H54a of 23S rRNA, unique to the mycobacterial ribosome, adopts a different conformation and come close to RafH CTD, suggesting its role in ribosome hibernation. RafH inhibits in-vitro protein synthesis in a concentration dependent manner. Further, the modeling studies provided the structural basis for the incompatibility of mycobacterial ribosomes to form 100S like disome architecture. Biological sciences/Structural biology/Electron microscopy/Cryoelectron microscopy Biological sciences/Molecular biology/Translation/Ribosome Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Full Text Additional Declarations There is NO Competing Interest. Supplementary Files SupplementryMovie1.mp4 Supplementary Movie 1 SupplementryMovie2.mp4 Supplementary Movie 2 SupplementryMovie3.mp4 Supplementary Movie 3 SupplementryMovie4.mp4 Supplementary Movie 4 SupplementryMovie5.mp4 Supplementary Movie 5 SupplementryMovie6.mp4 Supplementary Movie 6 Table1cryoemmodeldataFINAL.docx Table2RafHintreactionsfinal.pdf SupplementaryFiguresapril823.docx Supplementary Figures Cite Share Download PDF Status: Published Journal Publication published 20 Jan, 2024 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-2837054","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":205985160,"identity":"d01294b3-219f-46fa-8825-50ad8d2504b2","order_by":0,"name":"Prem Kaushal","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA8ElEQVRIie2QsQrCMBCGTwqZgl0L4jsUhFYH+ywNhboUERwdFFzFWXDwFeruEDnUpQ+QwUEQOgcEcXAwVRBBG3RzyAe54Scf93MABsPfwtVzoMIlQPhI3O8UWM9+VpA+FQ1+DTcpZAFbzMcHbK+CLtQQJfT2pUprGscCRMTS/dbFJI/6UI1jB9y8VHEz6gmQFkudUCmcsxGlnspRo9hnpQzZYtaR2Lwr9knqFUpUMWQjkahvjy3qFBqlNSGeCLNdIxVJbz3hUZ9Q4hUlSxWfWrmQ20FdFVseLjzo2tQ6SnnVFCtG+JqQt+STYjAYDAYtN+NeXBOA6vlyAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0001-6433-1368","institution":"UNESCO-DBT Regional Centre for Biotechnology","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Prem","middleName":"","lastName":"Kaushal","suffix":""},{"id":205985161,"identity":"7895340c-2630-4ffd-a2bc-e90ea229cf3d","order_by":1,"name":"Niraj Kumar","email":"","orcid":"","institution":"UNESCO-DBT Regional Centre for Biotechnology","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Niraj","middleName":"","lastName":"Kumar","suffix":""},{"id":205985162,"identity":"0b973802-a3a2-4590-ae4d-0d06b58c0309","order_by":2,"name":"Shivani Sharma","email":"","orcid":"","institution":"UNESCO-DBT Regional Centre for Biotechnology","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Shivani","middleName":"","lastName":"Sharma","suffix":""}],"badges":[],"createdAt":"2023-04-19 13:53:04","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2837054/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2837054/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41467-024-44879-y","type":"published","date":"2024-01-20T05:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":38183837,"identity":"7e5f5eb3-6184-4970-b985-d5b381e49e71","added_by":"auto","created_at":"2023-06-07 18:33:51","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":736231,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e70S ribosome RafH complex. \u003c/strong\u003e(a-c) The 10-40% sucrose density gradient fractionation profile and corresponding peaks analysis on agarose gel are shown for initial ribosome purification (a), after dissociation (b), and after re-association (c). (d) the 70 ribosomes RafH complex formation and sucrose density pelleting analyzed on 12% SDSPAGE, lane 1 - marker, lane 2 - pure RafH protein, lane 3, 4 - input, lane 5 to 8 - supernatant and pellet fraction after pelleting on a sucrose cushion. (e) In-vitro protein synthesis assay by titrating ribosome and RafH at different stoichiometric ratios. (f) The 2D cryo- EM micrograph collected during the initial grid screening stage in JEOL 2200 FS microscope with a Gatan K2 Summit camera.\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-2837054/v1/2a15555d7acaf06670aa1283.png"},{"id":38183299,"identity":"3acdcac6-6cb2-49d9-8cbb-dd2f24771bef","added_by":"auto","created_at":"2023-06-07 18:25:51","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2295702,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCryo- EM structure of Mycobacterium smegmatis 70S ribosome RafH complex.\u003c/strong\u003e (a, b) the overall architecture of the 70S RafH complex is shown in the mRNA entry site (a) and mRNA exit site (b) by a rotation through a diagonal axis. The SSU 16S rRNA (khaki), SSU r-proteins (dark golden), RafH (maroon), tRNA (pink), the LSU 23S rRNA and 5S rRNA (cornflower blue), LSU r- proteins (royal blue), bS1 (dark salmon) and uS2 (orange) are labeled. The single particle reconstruction data processing summary is shown in Supplementary Fig. S1, gold standard FSC and local resolution of final maps is shown in Supplementary Fig. S2, The cryo- EM maps for individual r-proteins, bS1 and uS2 and E-site tRNA and their model is shown in Supplementary Fig. S3, a full RafH model is shown in Supplementary Fig. S4. (c) the RafH NTD cryo- EM density (left panel) and model in ribbon (right panel), the top panel is rotated by 180° along X-axis, and shown in the bottom panel, the secondary structures are labeled. (d) the cryo- EM density in mesh and model in stick style corresponds to RafH linker region residues, 111-124 (maroon) and a-SD region of 16S rRNA nucleotides, 1518-1522 (khaki), are shown. For more clarity, an animation is provided Supplementary Movie 1.\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-2837054/v1/ef78804c6e394c67107c70db.png"},{"id":38183301,"identity":"4951e5f5-8b50-4e21-98b2-663183923145","added_by":"auto","created_at":"2023-06-07 18:25:51","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":3085498,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRibosome and RafH NTD interaction. \u003c/strong\u003eThe cryo- EM density in surface view for the small subunit with RafH at the centre, and its magnified regions where the cryo- EM density in mesh and model in stick and ribbon are shown. For clarity, the ribosomal large subunit is not shown. The RafH 16S rRNA interaction in counterclockwise from the bottom left for α1 R75 with Bridge B2a, α1 with h44, α3 W96 with h23 G673, α2 with C1382- C1383, residues from β2, β3, and β4 with h31 U947, G948 are shown. For more detail see Supplementary Movie 2-6, Supplementary Fig. S5, Supplementary Table S2.\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-2837054/v1/bedd2128d4d7d8ffc9a627d6.png"},{"id":38183836,"identity":"c9455f6b-e75b-49b6-8e3f-645ea38420f8","added_by":"auto","created_at":"2023-06-07 18:33:51","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2469123,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRafH CTD structure and its binding site on the ribosome.\u003c/strong\u003e (a) The RafH CTD binding site present in cryo- EM map in surface style for 70S RafH complex is shown, and the same color scheme of Fig. 2a, b is used. A thumbnail for the 70S is shown on the left. (b) cryo- EM density corresponding to RafH CTD in mesh, model in ribbon, and stick is shown. The thumbnail is shown on the left. (c) the structure of HPFlong CTD dimer (PDB ID; 6T7O) with its first monomer (A) (gray) and second monomer (B) (black) are shown. (d) the pretranslation initiation structure SSU (PDB ID; 5LMT) docked into the ribosome RafH complex SSU structure. For clarity, only the RafH in ribbon and 95% transparent surface, initiation complex factors: mRNA (blue), a-SD (green), IF1 (cornflower blue), IF3 (cyan), and P- tRNA (dark olive green) are shown.\u003c/p\u003e","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-2837054/v1/59f13b41583f144b9fce68f3.png"},{"id":38183303,"identity":"6e9e2e96-9297-4c64-8c6f-756c68d19963","added_by":"auto","created_at":"2023-06-07 18:25:51","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1435281,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eComparison of RafH binding in 70S ribosome with HPFlong binding in 100S ribosome. \u003c/strong\u003e(a) RafH, bS1, uS2, and h40 of 16S rRNA and H54a of 23S rRNA are shown with LSU and SSU in the 95% transparent background. (b) the corresponding position of HPFlong, uS2, and h40 of 16S rRNA in one of the ribosomes of the Staphylococcus aureus 100S structure (PDB ID; 5NGM) is shown with LSU and SSU in the 95% transparent background. (c) two 70S ribosome RafH complex structures docked into the corresponding positions in Staphylococcus aureus 100S dimer structure (PDB ID; 6FXC) and RafH CTD interacting components are shown in 80% transparent background on the left side and magnified view with a white background are shown in the box on the right side. One 70S ribosome is labeled as A, and the other 70S ribosome is labeled as B. (d) the HPF\u003csup\u003elong\u003c/sup\u003e interacting components uS2 and h40 of 16S rRNA in Staphylococcus aureus 100S ribosome dimer interface (PDB ID; 6FXC) are shown on the right side with 30S and 50S in 80% transparent background, and a magnified view with white background is shown in the box on the left side. Similar to (c), one 70S ribosome is labeled as A, and the other 70S ribosome is labeled as B.\u003c/p\u003e","description":"","filename":"Fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-2837054/v1/3f3202ae1fb74b5c105a1878.png"},{"id":38183839,"identity":"b020d401-c2c2-4fe5-accb-4b8036508696","added_by":"auto","created_at":"2023-06-07 18:33:51","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":549837,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDifferent modes of ribosome hibernation. \u003c/strong\u003eA schematic presentation for the different modes of ribosome hibernation. Top, RafH mediated hibernation in 70S form (from this study). Second from top, HPF\u003csup\u003elong \u003c/sup\u003einduces ribosome dimerization and formation of 100S disome\u003csup\u003e20,21,22,23,24\u003c/sup\u003e. Third, from the top, HPF\u003csup\u003eshort\u003c/sup\u003e and RMF\u003csup\u003e18,19\u003c/sup\u003e induce ribosome dimerization and 100S ribosome formation. Bottom, YfiA hibernates ribosome in the 70S form\u003csup\u003e26,27\u003c/sup\u003e.\u003c/p\u003e","description":"","filename":"Fig6.png","url":"https://assets-eu.researchsquare.com/files/rs-2837054/v1/f31a3e95debe87365fb5aed5.png"},{"id":49925156,"identity":"27531aa4-4324-40d1-8f00-4af61ac331f2","added_by":"auto","created_at":"2024-01-21 08:13:47","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1058199,"visible":true,"origin":"","legend":"","description":"","filename":"ribosomeRafHmanuscriptapril19FINAL.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2837054/v1_covered_f7636ec2-b17a-4541-866a-64375f72c3b3.pdf"},{"id":38183314,"identity":"2d1c10a1-47ab-48d2-bab5-60d55ff6c608","added_by":"auto","created_at":"2023-06-07 18:25:52","extension":"mp4","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":48126952,"visible":true,"origin":"","legend":"Supplementary Movie 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hibernation","fulltext":[],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":true,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":true,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-2837054/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2837054/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Ribosome hibernation is a key survival strategy bacteria adopt under environmental stress, where a protein, hibernation promotion factor (HPF), transitorily inactivates the ribosome and slows down its overall protein synthesis. The mechanism is well studied in enteric bacteria, which mainly hibernate its ribosome in 100S disome form through a dual domain, long HPF (HPF\u003csup\u003elong\u003c/sup\u003e) or a single domain, short HPF (HPF\u003csup\u003eshort\u003c/sup\u003e) in concert with another ribosome modulation factor. \u003ci\u003eMycobacterium tuberculosis\u003c/i\u003e encounters hypoxia (low oxygen) as a major stress in the host macrophages, and it overexpresses RafH protein, which is critical for its survival. The RafH, a dual domain HPF, an orthologue of bacterial HPF\u003csup\u003elong\u003c/sup\u003e, hibernates ribosome in 70S monosome form only. Here we report the cryo- EM structure of \u003ci\u003eMycobacterium smegmatis\u003c/i\u003e, a close homologue of \u003ci\u003eM. tuberculosis\u003c/i\u003e, 70S ribosome in complex with the RafH factor at an overall 2.8 Å resolution. The RafH N- terminus domain (NTD) is conserved and binds to the decoding center of the ribosomal small subunit, a similar binding for HPF\u003csup\u003elong\u003c/sup\u003e NTD, but additionally it also interacts with the inter subunit bridge, B2a. Contrary to the HPF\u003csup\u003elong\u003c/sup\u003e C- terminus domain (CTD), the RafH CTD, which is larger, binds to a unique site at the platform binding center of the ribosomal small subunit and sandwiches between bS1 and uS11 ribosomal proteins. The two domain connecting linker regions, which remain mostly disordered in earlier reported HPF\u003csup\u003elong\u003c/sup\u003e structures, interacts mainly with the anti-Shine Dalgarno sequence of the 16S rRNA. The helix H54a of 23S rRNA, unique to the mycobacterial ribosome, adopts a different conformation and come close to RafH CTD, suggesting its role in ribosome hibernation. RafH inhibits \u003ci\u003ein-vitro\u003c/i\u003e protein synthesis in a concentration dependent manner. Further, the modeling studies provided the structural basis for the incompatibility of mycobacterial ribosomes to form 100S like disome architecture.","manuscriptTitle":"Cryo- EM structure of the mycobacterial 70S ribosome in complex with ribosome hibernation promotion factor RafH, reveals the unique mode of mycobacterial ribosome hibernation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-06-07 18:25:46","doi":"10.21203/rs.3.rs-2837054/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"f5cf64f5-cc94-4954-b995-0aaa91131cef","owner":[],"postedDate":"June 7th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":22024628,"name":"Biological sciences/Structural biology/Electron microscopy/Cryoelectron microscopy"},{"id":22024629,"name":"Biological sciences/Molecular biology/Translation/Ribosome"}],"tags":[],"updatedAt":"2024-01-21T08:13:33+00:00","versionOfRecord":{"articleIdentity":"rs-2837054","link":"https://doi.org/10.1038/s41467-024-44879-y","journal":{"identity":"nature-communications","isVorOnly":false,"title":"Nature Communications"},"publishedOn":"2024-01-20 05:00:00","publishedOnDateReadable":"January 20th, 2024"},"versionCreatedAt":"2023-06-07 18:25:46","video":"","vorDoi":"10.1038/s41467-024-44879-y","vorDoiUrl":"https://doi.org/10.1038/s41467-024-44879-y","workflowStages":[]},"version":"v1","identity":"rs-2837054","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2837054","identity":"rs-2837054","version":["v1"]},"buildId":"FbvkV6FR0MCFSLy54lSbu","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}