Central domain of the positive control protein NifA and its role in transcriptional activation.
The positive control protein NifA of Klebsiella pneumoniae activates transcription by RNA polymerase containing sigma 54 by catalysing open promoter complex formation. We show that the integrity of the putative ATP-binding pocket in the central domain of NifA is necessary for the positive control function of NifA, but is not required for DNA-binding or recognition of NifA by NifL. The inactive mutant NifA proteins are trans dominant to wild-type NifA and are unable to catalyse formation of open promoter complexes irrespective of whether a closed promoter complex at the nifH promoter has preformed. Formation of the closed complex results in a DNA structural distortion adjacent to the DNA region melted in the open promoter complex. This distortion lies at the leading edge of the E sigma 54 footprint. Although unable to catalyse open complex formation, some mutant NifAs altered the chemical reactivity of the distorted base-pair indicating that they retain the ability to recognize the closed promoter complex. The activation phenotype of partially active NifA molecules was sensitive to promoter sequences known to influence closed complex formation, indicating differences in (1) the susceptibility of the closed complexes towards activation and (2) their requirements for NifA during activation.
Cannon
W
W
AFRC Institute of Plant Science Research, Nitrogen Fixation Laboratory, University of Sussex, Brighton, U.K.
Buck
M
M
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D004252:DNA Mutational Analysis; D004269:DNA, Bacterial / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D003850:Deoxyribonuclease I / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics*; D007711:Klebsiella pneumoniae / Q000235:genetics; D008969:Molecular Sequence Data; D009591:Nitrogenase / Q000235:genetics* / Q000378:metabolism; D009690:Nucleic Acid Conformation; D010088:Oxidoreductases; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism; D014158:Transcription, Genetic / Q000235:genetics*; D001616:beta-Galactosidase / Q000235:genetics
D001426:Bacterial Proteins; D004269:DNA, Bacterial; C061338:NifA protein, Bacteria; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D010088:Oxidoreductases; D009591:Nitrogenase; C029130:nitrogenase reductase; D012321:DNA-Directed RNA Polymerases; D003850:Deoxyribonuclease I; D001616:beta-Galactosidase