Context specificity of translation inhibitors revealed by toe-seq

Context specificity of translation inhibitors revealed by toe-seq | 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 Biological Sciences - Article Context specificity of translation inhibitors revealed by toe-seq Petr Sergiev, Marsel Kabilov, Ekaterina Komarova, Evgeny Pichkur, and 12 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3832009/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract Translation is a critical step in gene expression and a target for multiple antibiotics. Inhibition of protein synthesis by many antibiotics depends on mRNA context, making them selective towards particular mRNAs. Toe-printing is a standard low-throughput method to determine ribosome position along mRNA. Hereby we report the development and application of the Toe-seq method, combining toe-printing with next-generation sequencing. Toe-seq proved to be efficient to monitor translation and its inhibition by antibiotics for the library of over 37 000 mRNAs. Context-specificity of antibiotic action could be determined for different antibiotics via Toe-seq. Previously known sequence dependencies of translation inhibition were reproduced by Toe-seq. Data obtained by Toe-seq demonstrated context-specificity of etamycin A (EtaA) action whose binding site is located in the nascent peptide tunnel. Comparison of EtaA specificity with that of erythromycin (Ery) and tetracenomycin X (TcmX) revealed important differences and commonalities. Structural basis for the context-specificity of translation inhibition by EtaA has been deciphered by cryo-electron microscopy at 2.2 Å resolution. Toe-seq method allows one to monitor ribosome progression and stalling in vitro at a codon resolution, making it possible to assess antibiotics context specificity in a multiple parallel assay. Biological sciences/Molecular biology/Translation/Ribosome Biological sciences/Structural biology/Electron microscopy/Cryoelectron microscopy Full Text Additional Declarations There is NO Competing Interest. Supplementary Files Supplementarytable1.xlsx Supplementary table 1 Supplementarytable2.xlsx Supplementary table 2 Supplementarytable3.xlsx Supplementary table 3 Supplementarytable4.xlsx Supplementary table 4 Supplementarytable5.xlsx Supplementary table 5 Supplementarytable6.xlsx Supplementary table 6 Supplementarytable7.xlsx Supplementary table 7 SupplementaryTable8.xlsx Supplementary Table 8 Supplementarytable9.xlsx Supplementary table 9 Cite Share Download PDF Status: Under Review 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. We do this by developing innovative software and high quality services for the global research community. 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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-3832009","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Biological Sciences - Article","associatedPublications":[],"authors":[{"id":268837924,"identity":"29bd3562-d868-4aa9-8d62-2b44f01bc90f","order_by":0,"name":"Petr Sergiev","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA/klEQVRIiWNgGAWjYNCCigMMfBBWAjHKmYH4zAEGNtK0MLaRosWcvf+YxM95d+TY2NsfPq74k5bYIHb8mQTjnsM4tVj2HGaT7N32zJiN50Cy4dm2nMQG6RwzCYZnuLUY3Ehmk+DddjixTSLhmGRjQwVIC5sEwwE8Wu4/ZpP8O+dwfZtEYvvPhj8gLenP8Gu5wcwmzdtwOIFNIpmNsYEN5LAEM7xaLHuSja1ljj0zbOM5xizZ2JZm3CadY2yRcCAdpxZz9oMPb76puSPPDwyxjw1/kmX7pdMf3vhwwBq3wxgYWCSQBRzbQGQCTg1gLcwfkAXs8SgeBaNgFIyCEQoAmpZVxReRu44AAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0001-8866-1863","institution":"Lomonosov Moscow State University","correspondingAuthor":true,"prefix":"","firstName":"Petr","middleName":"","lastName":"Sergiev","suffix":""},{"id":268837925,"identity":"6166c682-60b1-4a64-bba2-80058d278b1f","order_by":1,"name":"Marsel Kabilov","email":"","orcid":"https://orcid.org/0000-0003-2777-0833","institution":"Institute of Chemical Biology and Fundamental Medicine","correspondingAuthor":false,"prefix":"","firstName":"Marsel","middleName":"","lastName":"Kabilov","suffix":""},{"id":268837926,"identity":"5ef6fe64-0aa7-4f02-8e55-db9fafddea63","order_by":2,"name":"Ekaterina Komarova","email":"","orcid":"https://orcid.org/0000-0002-6285-6395","institution":"Lomonosov Moscow State University, Department of Bioengineering and Bioinformatics, Moscow, 119992","correspondingAuthor":false,"prefix":"","firstName":"Ekaterina","middleName":"","lastName":"Komarova","suffix":""},{"id":268837927,"identity":"e2aaa299-3b2c-45c6-b3dd-5a163f09609d","order_by":3,"name":"Evgeny Pichkur","email":"","orcid":"","institution":"NRC “Kurchatov Institute”","correspondingAuthor":false,"prefix":"","firstName":"Evgeny","middleName":"","lastName":"Pichkur","suffix":""},{"id":268837928,"identity":"41ba6445-f6e0-42bb-a0b1-e32ffc76f8a7","order_by":4,"name":"Polina Zotova","email":"","orcid":"","institution":"Lomonosov Moscow State University","correspondingAuthor":false,"prefix":"","firstName":"Polina","middleName":"","lastName":"Zotova","suffix":""},{"id":268837929,"identity":"35621de1-cf24-4da9-8997-d738b204309f","order_by":5,"name":"Pavel Kasatsky","email":"","orcid":"","institution":"Molecular and Radiation Biophysics Division, Petersburg Nuclear Physics Institute named by B.P. 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