{"paper_id":"2e0dc4e1-5119-4d46-aace-35197f642e4e","body_text":"Codon decoding by split-tRNA: revisiting the tRNA selection mechanism | 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 Codon decoding by split-tRNA: revisiting the tRNA selection mechanism Sergey Mureev, Kirill Alexandrov, Yue Wu, Zhen-ling Cui This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6894815/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 The translation machinery is required to process all codon triplets without exception while maintaining high speed and accuracy, despite orders-of-magnitude differences in cognate pairing stability. For stability-based selection to be efficient, the range of pairing stabilities must be narrowed by raising the lower bound and lowering the upper bound. The constrained structure and intramolecular cooperativity of tRNA complicate understanding of how it modulates codon–anticodon stability and whether it affects selection kinetics beyond codon recognition. To address these questions, we engineered functional split-tRNAs bearing a dangling anticodon in place of the anticodon loop. Our results demonstrate that split-tRNA supports in vitro translation nearly as efficiently as intact synthetic tRNA, challenging the notion that tRNA strain is essential for triggering GTP hydrolysis in response to codon recognition. Using split-tRNA architecture, we found that codon–anticodon stability is likely modulated by the dipole moments of adjacent nucleobases. Our kinetic modeling aligns with a conformational selection mechanism, where the decoding site fluctuates between open and closed states, and the correct codon–anticodon minihelix acts as an allosteric effector that permits its spontaneous closure and stabilizes the closed state. Overall, our data challenge the notion that tRNA is an active player in the selection process. Biological sciences/Biochemistry/RNA Biological sciences/Molecular biology/Translation Full Text Additional Declarations There is NO Competing Interest. Supplementary Files Supplementaryncbv8.pdf Supplementary notes, figures and tables 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. 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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-6894815\",\"acceptedTermsAndConditions\":true,\"allowDirectSubmit\":false,\"archivedVersions\":[],\"articleType\":\"Article\",\"associatedPublications\":[],\"authors\":[{\"id\":479410174,\"identity\":\"b1c18699-d7a4-4f41-a212-2ba44e49c273\",\"order_by\":0,\"name\":\"Sergey 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