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 sigma(54) promoter specificity factor (E sigma(54)) at a single base pair resolution in three functional states. The sigma(54)-factor imposes tight control upon the RNAP by creating a regulatory switch where promoter melting nucleates, approximately 12 bp upstream of the transcription start site. Promoter melting by E sigma(54) 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 sigma(54)-factor directly interacts with the promoter in the regulatory switch within the initial closed E sigma(54)-promoter complex and one intermediate E sigma(54)-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.
Burrows
Patricia C
PC
Division of Biology, Faculty of Natural Sciences, Sir Alexander Fleming Building, Imperial College London, London SW7 2AZ, UK.
Wigneshweraraj
Siva R
SR
Buck
Martin
M
D001426:Bacterial Proteins / Q000378:metabolism*; D001483:Base Sequence; D003432:Cross-Linking Reagents / Q000494:pharmacology; D004269:DNA, Bacterial / Q000378:metabolism*; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology; D029968:Escherichia coli Proteins; D020035:Holoenzymes; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009154:Mutation; D009692:Nucleic Acid Heteroduplexes; D011401:Promoter Regions, Genetic; D011489:Protein Denaturation; D051877:RNA Polymerase Sigma 54 / Q000235:genetics / Q000378:metabolism; D016962:Sinorhizobium meliloti / Q000235:genetics / Q000378:metabolism; D015534:Trans-Activators / Q000378:metabolism*; D014158:Transcription, Genetic; D014466:Ultraviolet Rays
D001426:Bacterial Proteins; D003432:Cross-Linking Reagents; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D020035:Holoenzymes; D009692:Nucleic Acid Heteroduplexes; D015534:Trans-Activators; D012321:DNA-Directed RNA Polymerases; D051877:RNA Polymerase Sigma 54