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

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 RP...

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Autores principales:	Abhishek Mazumder, Richard H Ebright, Achillefs N Kapanidis
Formato:	article
Lenguaje:	EN
Publicado:	eLife Sciences Publications Ltd 2021
Materias:	RNA polymerase smFRET transcription initiation Medicine R Science Q Biology (General) QH301-705.5
Acceso en línea:	https://doaj.org/article/a30f5b4d3ff2433d975fec7b77a92d47
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spelling	oai:doaj.org-article:a30f5b4d3ff2433d975fec7b77a92d472021-11-30T11:42:37ZTranscription initiation at a consensus bacterial promoter proceeds via a ‘bind-unwind-load-and-lock’ mechanism10.7554/eLife.700902050-084Xe70090https://doaj.org/article/a30f5b4d3ff2433d975fec7b77a92d472021-10-01T00:00:00Zhttps://elifesciences.org/articles/70090https://doaj.org/toc/2050-084XTranscription 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 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.Abhishek MazumderRichard H EbrightAchillefs N KapanidiseLife Sciences Publications LtdarticleRNA polymerasesmFRETtranscription initiationMedicineRScienceQBiology (General)QH301-705.5ENeLife, Vol 10 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	RNA polymerase smFRET transcription initiation Medicine R Science Q Biology (General) QH301-705.5
spellingShingle	RNA polymerase smFRET transcription initiation Medicine R Science Q Biology (General) QH301-705.5 Abhishek Mazumder Richard H Ebright Achillefs N Kapanidis Transcription initiation at a consensus bacterial promoter proceeds via a ‘bind-unwind-load-and-lock’ mechanism
description	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 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.
format	article
author	Abhishek Mazumder Richard H Ebright Achillefs N Kapanidis
author_facet	Abhishek Mazumder Richard H Ebright Achillefs N Kapanidis
author_sort	Abhishek Mazumder
title	Transcription initiation at a consensus bacterial promoter proceeds via a ‘bind-unwind-load-and-lock’ mechanism
title_short	Transcription initiation at a consensus bacterial promoter proceeds via a ‘bind-unwind-load-and-lock’ mechanism
title_full	Transcription initiation at a consensus bacterial promoter proceeds via a ‘bind-unwind-load-and-lock’ mechanism
title_fullStr	Transcription initiation at a consensus bacterial promoter proceeds via a ‘bind-unwind-load-and-lock’ mechanism
title_full_unstemmed	Transcription initiation at a consensus bacterial promoter proceeds via a ‘bind-unwind-load-and-lock’ mechanism
title_sort	transcription initiation at a consensus bacterial promoter proceeds via a ‘bind-unwind-load-and-lock’ mechanism
publisher	eLife Sciences Publications Ltd
publishDate	2021
url	https://doaj.org/article/a30f5b4d3ff2433d975fec7b77a92d47
work_keys_str_mv	AT abhishekmazumder transcriptioninitiationataconsensusbacterialpromoterproceedsviaabindunwindloadandlockmechanism AT richardhebright transcriptioninitiationataconsensusbacterialpromoterproceedsviaabindunwindloadandlockmechanism AT achillefsnkapanidis transcriptioninitiationataconsensusbacterialpromoterproceedsviaabindunwindloadandlockmechanism
_version_	1718406641195417600

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

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