Mutations in RNA Polymerase Bridge Helix and Switch Regions Affect Active-Site Networks and Transcript-Assisted Hydrolysis

In bacterial RNA polymerase (RNAP), the bridge helix and switch regions form an intricate network with the catalytic active centre and the main channel. These interactions are important for catalysis, hydrolysis and clamp domain movement. By targeting conserved residues in Escherichia coli RNAP, we are able to show that functions of these regions are differentially required during σ⁷⁰-dependent and the contrasting σ⁵⁴-dependent transcription activations and thus potentially underlie the key mechanistic differences between the two transcription paradigms. We further demonstrate that the transcription factor DksA directly regulates σ⁵⁴-dependent activation both positively and negatively. This finding is consistent with the observed impacts of DksA on σ⁷⁰-dependent promoters. DksA does not seem to significantly affect RNAP binding to a pre-melted promoter DNA but affects extensively activity at the stage of initial RNA synthesis on σ⁵⁴-regulated promoters. Strikingly, removal of the σ⁵⁴ Region I is sufficient to invert the action of DksA (from stimulation to inhibition or vice versa) at two test promoters. The RNAP mutants we generated also show a strong propensity to backtrack. These mutants increase the rate of transcript-hydrolysis cleavage to a level comparable to that seen in the Thermus aquaticus RNAP even in the absence of a non-complementary nucleotide. These novel phenotypes imply an important function of the bridge helix and switch regions as an anti-backtracking ratchet and an RNA hydrolysis regulator.