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 (RP O ). 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. Significance statement Transcription initiation, the first step and most important step in gene expression for all organisms, involves unwinding of promoter DNA by RNA polymerase (RNAP) to form an open complex (RPo); this step also underpins transcriptional regulation and serves as an antibiotic target. Despite decades of research, the mechanism of promoter DNA unwinding has remained unresolved. Here, we solve this puzzle by using single-molecule fluorescence to directly monitor conformational changes in the promoter DNA and RNAP in real time during RPo formation. We show that RPo forms via a “ bind-unwind-load-and-lock ” mechanism, where the promoter unwinds outside the RNAP cleft, the unwound template DNA loads into the cleft, and RNAP “locks” the template DNA in place by closing the RNAP clamp module.