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

eLife

Achillefs N Kapanidis¹  Abhishek Mazumder¹  Richard H Ebright² 

[1] Biological Physics Research Group, Clarendon Laboratory, Department of Physics, University of Oxford, Oxford, United Kingdom;Waksman Institute and Department of Chemistry, Rutgers University, Piscataway, United States;

关键词: RNA polymerase;    smFRET;    transcription initiation;    E. coli;   

DOI  :  10.7554/eLife.70090

来源: eLife Sciences Publications, Ltd

【 摘 要 】

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.

【 授权许可】

CC BY