Transcription initiation at a consensus bacterial promoter proceeds via a ‘bindunwindload-and-lock’ mechanism

Abhishek Mazumder; Richard H. Ebright; Achillefs N. Kapanidis

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

Transcription initiation at a consensus bacterial promoter proceeds via a ‘bindunwindload-and-lock’ mechanism

Abhishek Mazumder, Richard H. Ebright, Achillefs N. Kapanidis

Waksman Institute of Microbiology

Rutgers, The State University

Research output: Contribution to journal › Article › peer-review

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 analyse 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 activecentre 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.

Original language	English (US)
Article number	e70090
Journal	eLife
Volume	10
DOIs	https://doi.org/10.7554/eLife.70090
State	Published - Oct 2021

ASJC Scopus subject areas

Neuroscience(all)
Biochemistry, Genetics and Molecular Biology(all)
Immunology and Microbiology(all)

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

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@article{5425513bd87c4eb8b97cbc3e72d272ae,

title = "Transcription initiation at a consensus bacterial promoter proceeds via a {\textquoteleft}bindunwindload-and-lock{\textquoteright} mechanism",

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 analyse 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 activecentre cleft. Our work defines a new, {\textquoteleft}bind-unwind-load-and-lock{\textquoteright}, 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.",

author = "Abhishek Mazumder and Ebright, {Richard H.} and Kapanidis, {Achillefs N.}",

note = "Funding Information: We thank Dr Anssi Malinen for discussions and early work on the development of real-time smFRET assays, and Dr Horst Steuer for the development of custom software. This work was supported by the Wellcome Trust [110164/Z/15/Z to ANK] and NIH [GM041376 to RHE]. Publisher Copyright: ‍‍Copyright Mazumder et al. This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.",

year = "2021",

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doi = "https://doi.org/10.7554/eLife.70090",

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N1 - Funding Information: We thank Dr Anssi Malinen for discussions and early work on the development of real-time smFRET assays, and Dr Horst Steuer for the development of custom software. This work was supported by the Wellcome Trust [110164/Z/15/Z to ANK] and NIH [GM041376 to RHE]. Publisher Copyright: ‍‍Copyright Mazumder et al. This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

PY - 2021/10

Y1 - 2021/10

N2 - 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 analyse 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 activecentre 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.

AB - 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 analyse 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 activecentre 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.

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Transcription initiation at a consensus bacterial promoter proceeds via a ‘bindunwindload-and-lock’ mechanism

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