Nucleotide-dependent interactions between a fork junction-RNA polymerase complex and an AAA plus transcriptional activator protein

Enhancer-dependent transcriptional activators that act upon the sigma(54) 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 sigma(54) 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.AlFx bound form of the AAA+ domain of the transcriptional activator protein PspF changes interactions between sigma(54)-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 sigma(54) and sigma(54)-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 sigma(54)-RNA polymerase-DNA interactions that favour open complex formation. We also provide evidence for two distinct closed promoter complexes with differing stabilities.