Abstract

ATP-driven remodelting 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 (sigma(54)-RNAP, E sigma(54)) and a slowly hydrolysed ATP analogue (ATP gamma S), we provide evidence for a nucleotide-dependent temporal pathway leading to DNA melting involving a small set of sigma(54)-DNA conformational states. We demonstrate that the ATP hydrolysis-dependent remodelling of E sigma(54) occurs in at least two distinct temporal steps. The first detected remodelling phase results in changes in the interactions between the promoter specificity sigma(54) factor and the promoter DNA. The second detected remodelling phase causes changes in the relationship between the promoter DNA and the core RNAP catalytic beta/beta' subunits, correlating with the loading of template DNA into the catalytic cleft of RNAP. It would appear that, for E sigma(54) promoters, loading of template DNA within the catalytic cleft of RNAP is dependent on fast ATP hydrolysis steps that trigger changes in the beta' jaw domain, thereby allowing acquisition of the open complex status. (C) 2009 Elsevier Ltd. All rights reserved.