title: Nucleotide-dependent interactions between a fork junction-RNA polymerase complex and an AAA+ transcriptional activator protein.
abstract: Enhancer-dependent transcriptional activators that act upon the sigma54 bacterial RNA polymerase holoenzyme belong to the extensive AAA+ superfamily of mechanochemical ATPases. Formation and collapse of the transition state for ATP hydrolysis engenders direct interactions between AAA+ activators and the sigma54 factor, required for RNA polymerase isomerization. A DNA fork junction structure present within closed complexes serves as a nucleation point for the DNA melting seen in open promoter complexes and restricts spontaneous activator-independent RNA polymerase isomerization. We now provide physical evidence showing that the ADP.AlF(x) bound form of the AAA+ domain of the transcriptional activator protein PspF changes interactions between sigma54-RNA polymerase and a DNA fork junction structure present in the closed promoter complex. The results suggest that one functional state of the nucleotide-bound activator serves to alter DNA binding by sigma54 and sigma54-RNA polymerase and appears to drive events that precede DNA opening. Clear evidence for a DNA-interacting activity in the AAA+ domain of PspF was obtained, suggesting that PspF may make a direct contact to the DNA component of a basal promoter complex to promote changes in sigma54-RNA polymerase-DNA interactions that favour open complex formation. We also provide evidence for two distinct closed promoter complexes with differing stabilities.
authors.affiliation: Department of Biological Sciences, Sir Alexander Fleming Building, Imperial College London, South Kensington Campus, London SW7 2AZ, UK.
authors.forename: W V
authors.initials: WV
authors.lastname: Cannon
authors.forename: J
authors.initials: J
authors.lastname: Schumacher
authors.forename: M
authors.initials: M
authors.lastname: Buck
mesh_terms: D000244:Adenosine Diphosphate / Q000378:metabolism*; D000251:Adenosine Triphosphatases / Q000378:metabolism; D017607:Aluminum Compounds / Q000378:metabolism; D001665:Binding Sites; D018983:DNA Footprinting; D004269:DNA, Bacterial / Q000737:chemistry / Q000378:metabolism*; D004268:DNA-Binding Proteins; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism*; D005459:Fluorides / Q000378:metabolism; D046911:Macromolecular Substances; D011401:Promoter Regions, Genetic; D017434:Protein Structure, Tertiary; D051877:RNA Polymerase Sigma 54; D012808:Sigma Factor / Q000378:metabolism*; D015534:Trans-Activators / Q000737:chemistry / Q000378:metabolism*
chemical_list: D017607:Aluminum Compounds; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D046911:Macromolecular Substances; C098954:PspF protein, E coli; D012808:Sigma Factor; D015534:Trans-Activators; C497751:rpoN protein, E coli; D000244:Adenosine Diphosphate; D012321:DNA-Directed RNA Polymerases; D051877:RNA Polymerase Sigma 54; D000251:Adenosine Triphosphatases; D005459:Fluorides; C032311:aluminum fluoride