Nucleotide-dependent triggering of RNA polymerase-DNA interactions by an AAA regulator of transcription.
Enhancer-dependent activator proteins, which act upon the bacterial RNA polymerase containing the sigma54 promoter specificity factor, belong to the AAA superfamily of ATPases. Activator-sigma54 contact is required for the sigma54-RNAP to isomerize and engage the DNA template for transcription. How ATP hydrolysis is used to trigger changes in sigma54-RNA polymerase and promoter DNA that lead to DNA opening is poorly understood. Here, band shift and footprinting assays were used to investigate the DNA binding activities of sigma54 and sigma54-RNA polymerase in the presence of the activator protein PspF bound to poorly hydrolysable analogues of ATP and the ATP hydrolysis transition-state analogue ADP.AlFx. Results show that different nucleotide-bound forms of PspF can change the interactions between sigma54, sigma54-RNA polymerase, and a DNA fork junction structure present within closed promoter complexes. This provides evidence that in the activation transduction pathway, several functional states of the activator, prior to ATP hydrolysis, can serve to alter the fork junction binding activity of sigma54 and sigma54-RNA polymerase that precede full DNA opening. A sequential set of nucleotide-dependent transitions in sigma54-RNA polymerase promoter complexes needed for productive open complex formation may therefore depend upon different nucleotide-bound forms of the activator.
Cannon
Wendy
W
Department of Biological Sciences, Sir Alexander Fleming Building, Imperial College London, South Kensington Campus, United Kingdom.
Bordes
Patricia
P
Wigneshweraraj
Siva R
SR
Buck
Martin
M
D000227:Adenine Nucleotides / Q000502:physiology*; D000255:Adenosine Triphosphate / Q000378:metabolism; D001483:Base Sequence; D004247:DNA / Q000378:metabolism*; D018983:DNA Footprinting; D015342:DNA Probes; D004268:DNA-Binding Proteins; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D011485:Protein Binding; D051877:RNA Polymerase Sigma 54; D012808:Sigma Factor / Q000378:metabolism*; D014158:Transcription, Genetic
D000227:Adenine Nucleotides; D015342:DNA Probes; D004268:DNA-Binding Proteins; D012808:Sigma Factor; D000255:Adenosine Triphosphate; D004247:DNA; D012321:DNA-Directed RNA Polymerases; D051877:RNA Polymerase Sigma 54