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 sigma(54) promoter specificity factor, belong to the AAA superfamily of ATPases. Activator-sigma(54) contact is required for the sigma(54)-RNAP to isomerize and engage the DNA template for transcription. How ATP hydrolysis is used to trigger changes in sigma(54)-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 sigma(54) and sigma(54)-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 sigma(54), sigma(54)-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 sigma(54) and sigma(54)-RNA polymerase that precede full DNA opening. A sequential set of nucleotide-dependent transitions in sigma(54)-RNA polymerase promoter complexes needed for productive open complex formation may therefore depend upon different nucleotide-bound forms of the activator.