Multiple roles of the RNA polymerase β subunit flap domain in σ54-dependent transcription

Recent determinations of the structures of the bacterial RNA polymerase (RNAP) and promoter complex thereof establish that RNAP functions as a complex molecular machine that contains distinct structural modules that undergo major conformational changes during transcription. However, the contribution of the RNAP structural modules to transcription remains poorly understood. The bacterial core RNAP (alpha(2)betabeta' omegaa, E) associates with a sigma (sigma) subunit to form the holoenzyme (Esigma). A mutation removing the beta subunit flap domain renders the Escherichia coli or sigma(70) RNAP holoenzyme unable to recognize promoters. sigma(54) is the major variant sigma subunit that utilizes enhancer-dependent promoters. Here, we determined the effects of beta flap removal on sigma(54)-dependent transcription. Our analysis shows that the role of the beta flap in sigma(54)-dependent and sigma(54)-dependent transcription is different. Removal of the beta flap does not prevent the recognition of sigma(54)-dependent promoters, but causes multiple defects in sigma(54) -dependent transcription. Most importantly, the beta flap appears to orchestrate the proper formation of the Esigma(54) regulatory center at the start site proximal promoter element where activator binds and DNA melting originates.