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

Richard H Ebright,Abhishek Mazumder,Achillefs N Kapanidis

doi:10.7554/elife.70090

Richard H Ebright, Abhishek Mazumder + Show 1 more

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

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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 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.

Highlights

Transcription initiation is the first and most highly regulated step in gene expression [1,2]
These changes in fluorescence emission intensity provide a powerful approach to monitor promoter unwinding and rewinding in solution during transcription initiation [15,28,29]. We have adapted this approach to enable detection of promoter unwinding in solution at the single-molecule level in real-time, and we designate our adaptation of the approach "single-molecule unwinding-induced fluorescence enhancement", to underscore the similarity to the established method of single-molecule protein-induced fluorescence enhancement
Containing promoter DNA fragment to E. coli RNA polymerase (RNAP)- 70 holoenzyme immobilised on a coverslip mounted in a total-internal-reflection fluorescence (TIRF) microscope, we detected the appearance of fluorescence signal from single fluorescent species, indicating binding of single molecules of Cy3

Summary

Introduction

Transcription initiation is the first and most highly regulated step in gene expression [1,2]. RNA polymerase (RNAP), together with the transcription initiation factor , unwinds ~13 bp of promoter DNA to form a "transcription bubble," and places the template-strand ssDNA of the unwound transcription bubble in contact with the RNAP active center, yielding a catalytically competent RNAP-promoter transcription-initiation complex (RPo; 1). High-resolution structures of RPO define the contacts that RNAP and make with promoter DNA, as well as the conformation and interactions of unwound template-strand ssDNA engaging the RNAP active center [3,4,5]. In crystal structures of RNAP- holoenzyme, the RNAP active-center cleft is too narrow to accommodate double-stranded DNA (

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