Coupling σ Factor Conformation to RNA Polymerase Reorganisation for DNA Melting

ATP-driven remodelling of initial RNA polymerase (RNAP) promoter complexes occurs as a major post recruitment strategy used to control gene expression. Using a model-enhancer-dependent bacterial system (σ54–RNAP, Eσ54) and a slowly hydrolysed ATP analogue (ATPγS), we provide evidence for a nucleotide-dependent temporal pathway leading to DNA melting involving a small set of σ54–DNA conformational states. We demonstrate that the ATP hydrolysis-dependent remodelling of Eσ54 occurs in at least two distinct temporal steps. The first detected remodelling phase results in changes in the interactions between the promoter specificity σ54 factor and the promoter DNA. The second detected remodelling phase causes changes in the relationship between the promoter DNA and the core RNAP catalytic β/β′ subunits, correlating with the loading of template DNA into the catalytic cleft of RNAP. It would appear that, for Eσ54 promoters, loading of template DNA within the catalytic cleft of RNAP is dependent on fast ATP hydrolysis steps that trigger changes in the β′ jaw domain, thereby allowing acquisition of the open complex status.