Activation of transcription at sigma 54-dependent bacterial promoters proceeds via a mechanism that is independent of recruitment of RNA polymerase to the promoter, but is instead totally dependent on activator-driven conformational changes in the promoter-bound RNA polymerase. Understanding of the activation mechanism first requires a detailed description of the interactions taking place in the polymerase holoenzyme and closed complex. The interactions of sigma 54 with core RNA polymerase and promoter DNA were investigated using enzymatic and chemical (hydroxyl radical) protease footprinting of sigma. Regions of sigma were identified that are in direct contact with ligands, or whose conformation changes following ligand binding. A comparison of wild-type sigma and a mutant bearing a deletion of conserved Region I, which is required for response to activator proteins and regulated initiation, revealed differences in the protease sensitivity of free sigma indicating that Region I affects sigma conformation. Comparison of the holoenzyme and closed complex hydroxyl radical footprints revealed that residues of wild-type sigma protected by promoter DNA overlap, to a large extent, the residues of Region I-deleted sigma protected by core polymerase. Region I could thus modify DNA-binding by changing conformation of the DNA-binding domain of sigma 54 in a core polymerase-dependent manner. These differences can account for the modified promoter binding of the Region I-deleted sigma holoenzyme observed by DNA footprinting, and are likely of significance to the Region I-dependent activation of transcription.
Copyright 1998 Academic Press.