Monitoring RNA dynamics in native transcriptional complexes
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Cotranscriptional RNA folding is crucial for the timely control of biological processes, but because of its transient nature, its study has remained challenging. While single-molecule Förster resonance energy transfer (smFRET) is unique to investigate transient RNA structures, its application to cotranscriptional studies has been limited to nonnative systems lacking RNA polymerase (RNAP)–dependent features, which are crucial for gene regulation. Here, we present an approach that enables site-specific labeling and smFRET studies of kilobase-length transcripts within native bacterial complexes. By monitoring Escherichia coli nascent riboswitches, we reveal an inverse relationship between elongation speed and metabolite-sensing efficiency and show that pause sites upstream of the translation start codon delimit a sequence hotspot for metabolite sensing during transcription. Furthermore, we demonstrate a crucial role of the bacterial RNAP actively delaying the formation, within the hotspot sequence, of competing structures precluding metabolite binding. Our approach allows the investigation of cotranscriptional regulatory mechanisms in bacterial and eukaryotic elongation complexes.
Chauvier , A , St-Pierre , P , Nadon , J-F , Hien , E , Perez Gonzalez , D C , Eschbach , S H , Lamontagne , A-M , Penedo , C & Lafontaine , D A 2021 , ' Monitoring RNA dynamics in native transcriptional complexes ' , Proceedings of the National Academy of Sciences of the United States of America , vol. 118 , no. 45 , e2116155118 . https://doi.org/10.1073/pnas.2106564118
Proceedings of the National Academy of Sciences of the United States of America
Copyright © 2021 the Author(s). Published by PNAS. This open access article is distributed under Creative Commons Attribution License 4.0 (CC BY).
DescriptionThis work was supported by grants from the Canadian Institutes of Health Research, the Natural Sciences and Engineering Research Council of Canada. JCP wishes to thank the Scottish Universities Physics Alliance (SUPA) and the Engineering and Physical Sciences Research Council for support. C. P. G. thanks EPSRC and the University of St Andrews for a PhD scholarship.
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