Protein–DNA Interactions That Govern AAA+ Activator-Dependent Bacterial Transcription Initiation

Transcriptional control at the promoter melting step is not yet well understood. In this study, a site-directed photo-cross-linking method was used to systematically analyse component protein–DNA interactions that govern promoter melting by the enhancer-dependent Escherichia coli RNA polymerase (RNAP) containing the σ54 promoter specificity factor (Eσ54) at a single base pair resolution in three functional states. The σ54-factor imposes tight control upon the RNAP by creating a regulatory switch where promoter melting nucleates, ∼ 12 bp upstream of the transcription start site. Promoter melting by Eσ54 is only triggered upon remodelling of this regulatory switch by a specialised activator protein in an ATP-hydrolysing reaction. We demonstrate that prior to DNA melting, only the σ54-factor directly interacts with the promoter in the regulatory switch within the initial closed Eσ54–promoter complex and one intermediate Eσ54–promoter complex. We establish that activator-induced conformational rearrangements in the regulatory switch are a prerequisite to allow the promoter to enter the catalytic cleft of the RNAP and hence establish the transcriptionally competent open complex, where full promoter melting occurs. These results significantly advance our current understanding of the structural transitions occurring at bacterial promoters, where regulation occurs at the DNA melting step.