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An ensemble of interconverting conformations of the elemental paused transcription complex creates regulatory options

Transcriptional pausing underpins the regulation of cellular RNA synthesis, but its mechanism remains incompletely understood. Sequence-specific interactions of DNA and RNA with the dynamic, multidomain RNA polymerase (RNAP) trigger reversible conformational changes at pause sites that temporarily i...

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Autores principales: Kang, Jin Young, Mishanina, Tatiana V., Bao, Yu, Chen, James, Llewellyn, Eliza, Liu, James, Darst, Seth A., Landick, Robert
Formato: Online Artículo Texto
Lenguaje:English
Publicado: National Academy of Sciences 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9974457/
https://www.ncbi.nlm.nih.gov/pubmed/36795753
http://dx.doi.org/10.1073/pnas.2215945120
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author Kang, Jin Young
Mishanina, Tatiana V.
Bao, Yu
Chen, James
Llewellyn, Eliza
Liu, James
Darst, Seth A.
Landick, Robert
author_facet Kang, Jin Young
Mishanina, Tatiana V.
Bao, Yu
Chen, James
Llewellyn, Eliza
Liu, James
Darst, Seth A.
Landick, Robert
author_sort Kang, Jin Young
collection PubMed
description Transcriptional pausing underpins the regulation of cellular RNA synthesis, but its mechanism remains incompletely understood. Sequence-specific interactions of DNA and RNA with the dynamic, multidomain RNA polymerase (RNAP) trigger reversible conformational changes at pause sites that temporarily interrupt the nucleotide addition cycle. These interactions initially rearrange the elongation complex (EC) into an elemental paused EC (ePEC). ePECs can form longer-lived PECs by further rearrangements or interactions of diffusible regulators. For both bacterial and mammalian RNAPs, a half-translocated state in which the next DNA template base fails to load into the active site appears central to the ePEC. Some RNAPs also swivel interconnected modules that may stabilize the ePEC. However, it is unclear whether swiveling and half-translocation are requisite features of a single ePEC state or if multiple ePEC states exist. Here, we use cryo-electron microscopy (cryo-EM) analysis of ePECs with different RNA–DNA sequences combined with biochemical probes of ePEC structure to define an interconverting ensemble of ePEC states. ePECs occupy either pre- or half-translocated states but do not always swivel, indicating that difficulty in forming the posttranslocated state at certain RNA–DNA sequences may be the essence of the ePEC. The existence of multiple ePEC conformations has broad implications for transcriptional regulation.
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spelling pubmed-99744572023-08-16 An ensemble of interconverting conformations of the elemental paused transcription complex creates regulatory options Kang, Jin Young Mishanina, Tatiana V. Bao, Yu Chen, James Llewellyn, Eliza Liu, James Darst, Seth A. Landick, Robert Proc Natl Acad Sci U S A Biological Sciences Transcriptional pausing underpins the regulation of cellular RNA synthesis, but its mechanism remains incompletely understood. Sequence-specific interactions of DNA and RNA with the dynamic, multidomain RNA polymerase (RNAP) trigger reversible conformational changes at pause sites that temporarily interrupt the nucleotide addition cycle. These interactions initially rearrange the elongation complex (EC) into an elemental paused EC (ePEC). ePECs can form longer-lived PECs by further rearrangements or interactions of diffusible regulators. For both bacterial and mammalian RNAPs, a half-translocated state in which the next DNA template base fails to load into the active site appears central to the ePEC. Some RNAPs also swivel interconnected modules that may stabilize the ePEC. However, it is unclear whether swiveling and half-translocation are requisite features of a single ePEC state or if multiple ePEC states exist. Here, we use cryo-electron microscopy (cryo-EM) analysis of ePECs with different RNA–DNA sequences combined with biochemical probes of ePEC structure to define an interconverting ensemble of ePEC states. ePECs occupy either pre- or half-translocated states but do not always swivel, indicating that difficulty in forming the posttranslocated state at certain RNA–DNA sequences may be the essence of the ePEC. The existence of multiple ePEC conformations has broad implications for transcriptional regulation. National Academy of Sciences 2023-02-16 2023-02-21 /pmc/articles/PMC9974457/ /pubmed/36795753 http://dx.doi.org/10.1073/pnas.2215945120 Text en Copyright © 2023 the Author(s). Published by PNAS. https://creativecommons.org/licenses/by-nc-nd/4.0/This article is distributed under Creative Commons Attribution-NonCommercial-NoDerivatives License 4.0 (CC BY-NC-ND) (https://creativecommons.org/licenses/by-nc-nd/4.0/) .
spellingShingle Biological Sciences
Kang, Jin Young
Mishanina, Tatiana V.
Bao, Yu
Chen, James
Llewellyn, Eliza
Liu, James
Darst, Seth A.
Landick, Robert
An ensemble of interconverting conformations of the elemental paused transcription complex creates regulatory options
title An ensemble of interconverting conformations of the elemental paused transcription complex creates regulatory options
title_full An ensemble of interconverting conformations of the elemental paused transcription complex creates regulatory options
title_fullStr An ensemble of interconverting conformations of the elemental paused transcription complex creates regulatory options
title_full_unstemmed An ensemble of interconverting conformations of the elemental paused transcription complex creates regulatory options
title_short An ensemble of interconverting conformations of the elemental paused transcription complex creates regulatory options
title_sort ensemble of interconverting conformations of the elemental paused transcription complex creates regulatory options
topic Biological Sciences
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9974457/
https://www.ncbi.nlm.nih.gov/pubmed/36795753
http://dx.doi.org/10.1073/pnas.2215945120
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