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.
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/).
- Achillefs Kapanidis
- Richard H Ebright
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
- Maria Spies, University of Iowa, United States
- Received: May 6, 2021
- Accepted: October 6, 2021
- Accepted Manuscript published: October 11, 2021 (version 1)
© 2021, Mazumder et al.
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