Directional co-transcriptional folding and pausing create kinetic checkpoints for riboswitch-controlled gene expression

Directional co-transcriptional folding and pausing create kinetic checkpoints for riboswitch-controlled gene expression | 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 Directional co-transcriptional folding and pausing create kinetic checkpoints for riboswitch-controlled gene expression Nils Walter, Adrien Chauvier, Javier Cabello-Villegas, Edward Nikonowicz This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8735691/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 Transcriptional riboswitches typically regulate gene expression by sensing small metabolites or ions that cannot be fluorophore-labeled without perturbing recognition, limiting direct observation of ligand binding during transcription. The GlyQS T-box riboswitch instead senses the uncharged 3′ end of a macromolecular ligand, tRNAGly, enabling visualization of co-transcriptional ligand binding. Using single-molecule fluorescence microscopy, we show that tRNAGly dynamically samples discrete structural domains of the nascent riboswitch as transcription proceeds. Productive recognition depends on a hierarchical 5′-to-3′ folding pathway shaped by transcription rate and pausing. Pausing enhances tRNA anchoring, stabilizes the riboswitch–tRNA complex, and selectively promotes RNA polymerase readthrough when an uncharged 3′ end is presented. These results support a kinetic checkpoint model in which early recruitment increases local ligand concentration, while pause-mediated stabilization converts transient encounters into committed regulatory decisions. More broadly, our findings identify directional co-transcriptional folding and transcriptional pausing as principles synergistically enabling selective and robust riboswitch function. Biological sciences/Biophysics/Single-molecule biophysics Biological sciences/Biochemistry/RNA Co-transcriptional RNA folding ligand-sensing riboswitch RNA polymerase single molecule fluorescence microscopy transcription Full Text Additional Declarations There is NO Competing Interest. Supplementary Files ChauvieretalDirectionalTBoxRiboswitchFoldingXPausingDriveRegulationSIFinal.docx Chauvier_et_al_DirectionalT-BoxRiboswitchFolding&PausingDriveRegulation_SI_Final 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-8735691","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":605174048,"identity":"b9c76a24-c75a-4988-a90c-f87c5435b33d","order_by":0,"name":"Nils 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