Identification of close contacts between the sigma N (sigma 54) protein and promoter DNA in closed promoter complexes.
The complexes forming between the alternative sigma factor protein sigma N (sigma 54), its holoenzyme and promoter DNA were analysed using the hydroxyl radical probe and by photochemical footprinting of bromouridine-substituted DNA. Close contacts between the promoter, sigma N and its holoenzyme appear to be restricted predominantly to one face of the DNA helix, extending from -31 to -5. They all appear attributable to sigma N and no extra close contacts from the core RNA polymerase subunits in the holoenzyme-promoter DNA complex were detected. We suggest that the apparent absence of close core RNA polymerase contacts in the region of the promoter DNA to be melted during open complex formation is important for maintaining the closed complex. Results of the hydroxyl radical footprinting imply that sigma N makes multiple DNA backbone contacts across and beyond the -12, -24 consensus promoter elements, and the photochemical footprints indicate that consensus thymidine residues contribute important major groove contacts to sigma N. Formation of the open complex is shown to involve a major structural transition in the DNA contacted by sigma N, establishing a direct role for sigma N in formation of the activated promoter complex.
Cannon
W
W
AFRC-IPSR Nitrogen Fixation Laboratory, University of Sussex, Falmer, Brighton, UK.
Austin
S
S
Moore
M
M
Buck
M
M
D001483:Base Sequence; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D004268:DNA-Binding Proteins; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D017665:Hydroxyl Radical; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009591:Nitrogenase / Q000235:genetics; D009690:Nucleic Acid Conformation; D009691:Nucleic Acid Denaturation; D010088:Oxidoreductases; D011401:Promoter Regions, Genetic / Q000235:genetics; D051877:RNA Polymerase Sigma 54; D012808:Sigma Factor / Q000378:metabolism*; D016962:Sinorhizobium meliloti / Q000235:genetics; D013936:Thymidine / Q000378:metabolism
D004268:DNA-Binding Proteins; D012808:Sigma Factor; D017665:Hydroxyl Radical; D004247:DNA; D010088:Oxidoreductases; D009591:Nitrogenase; C029130:nitrogenase reductase; D012321:DNA-Directed RNA Polymerases; D051877:RNA Polymerase Sigma 54; D013936:Thymidine