Transcription initiation at a consensus bacterial promoter proceeds via a 'bind-unwind-load-and-lock' mechanism

  1. Abhishek Mazumder  Is a corresponding author
  2. Richard H Ebright
  3. Achillefs Kapanidis  Is a corresponding author
  1. University of Oxford, United Kingdom
  2. Rutgers University, United States

Abstract

Transcription initiation starts with unwinding of promoter DNA by RNA polymerase (RNAP) to form a catalytically competent RNAP-promoter complex (RPO). Despite extensive study, the mechanism of promoter unwinding has remained unclear, in part due to the transient nature of intermediates on path to RPo. Here, using single-molecule unwinding-induced fluorescence enhancement to monitor promoter unwinding, and single-molecule fluorescence resonance energy transfer to monitor RNAP clamp conformation, we analyze RPo formation at a consensus bacterial core promoter. We find that the RNAP clamp is closed during promoter binding, remains closed during promoter unwinding, and then closes further, locking the unwound DNA in the RNAP active-centre cleft. Our work defines a new, 'bind-unwind-load-and-lock' model for the series of conformational changes occurring during promoter unwinding at a consensus bacterial promoter and provides the tools needed to examine the process in other organisms and at other promoters.

Data availability

All information for replication is included in the submission and data corresponding to each figure are provided as source data files. MATLAB software packages TwoTone and ebFRET are available on Github (https://github.com/annawang692/TwoTone2018 and http://ebfret.github.io/).

Article and author information

Author details

  1. Abhishek Mazumder

    Department of Physics, University of Oxford, Oxford, United Kingdom
    For correspondence
    abhishek.mazumder@physics.ox.ac.uk
    Competing interests
    The authors declare that no competing interests exist.
  2. Richard H Ebright

    Department of Chemistry, Rutgers University, Piscataway, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-8915-7140
  3. Achillefs Kapanidis

    Department of Physics, University of Oxford, Oxford, United Kingdom
    For correspondence
    kapanidis@physics.ox.ac.uk
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-6699-136X

Funding

Wellcome Trust (110164/Z/15/Z)

  • Achillefs Kapanidis

NIH Office of the Director (GM041376)

  • Richard H Ebright

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Copyright

© 2021, Mazumder et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

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  1. Abhishek Mazumder
  2. Richard H Ebright
  3. Achillefs Kapanidis
(2021)
Transcription initiation at a consensus bacterial promoter proceeds via a 'bind-unwind-load-and-lock' mechanism
eLife 10:e70090.
https://doi.org/10.7554/eLife.70090

Share this article

https://doi.org/10.7554/eLife.70090

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