DNA distortion and nucleation of local DNA unwinding within sigma-54 (sigma N) holoenzyme closed promoter complexes.
The sigma N (sigma 54) RNA polymerase holoenzyme has the distinctive property of binding to promoters to form a closed promoter complex, which only isomerizes to the open complex when acted upon by an enhancer binding activator protein. We probed promoter complexes that form between sigma N and its holoenzyme with the conformationally sensitive footprinting reagents ortho-copper phenanthroline, potassium permanganate, and diethylpyrocarbonate. Results from these experiments indicate that the contacts sigma N makes at the -12 promoter element are necessary to promote a local DNA distortion immediately adjacent to this promoter element when the holoenzyme but not sigma N alone binds promoter DNA. Complexes in which this local distortion is not detected are not activatable, and the altered DNA conformation is diminished in the activated complex. We propose that a barrier to open complex formation in the sigma N holoenzyme closed complex is at some step or steps after the initial nucleation of DNA strand separation, which is detected as an altered DNA conformation stably maintained within the closed complex. Thus the activator protein may promote a conformational change in the sigma N holoenzyme to allow propagation of the altered DNA conformation, probably local unwinding, which we propose is necessary for formation of the melted DNA state, characteristic of the open promoter complex.
Morris
L
L
Agricultural and Food Research Council, University of Sussex, Brighton, United Kingdom.
Cannon
W
W
Claverie-Martin
F
F
Austin
S
S
Buck
M
M
D001426:Bacterial Proteins / Q000378:metabolism*; D017104:Bacteriophage M13 / Q000378:metabolism; D001483:Base Sequence; D004269:DNA, Bacterial / Q000737:chemistry / Q000378:metabolism*; D004279:DNA, Viral / Q000737:chemistry / Q000378:metabolism*; D004268:DNA-Binding Proteins; D012321:DNA-Directed RNA Polymerases; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins; D005798:Genes, Bacterial; D007202:Indicators and Reagents; D007711:Klebsiella pneumoniae / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D051877:RNA Polymerase Sigma 54; D012808:Sigma Factor / Q000378:metabolism*
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004279:DNA, Viral; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D007202:Indicators and Reagents; D012808:Sigma Factor; C497751:rpoN protein, E coli; D012321:DNA-Directed RNA Polymerases; D051877:RNA Polymerase Sigma 54