Influence of a mutation in the putative nucleotide binding site of the nitrogen regulatory protein NTRC on its positive control function.
A mutation, serine 170 to alanine, in the proposed ATP binding site of the activator protein NTRC prevents transcriptional activation at sigma 54-dependent promoters both in vivo and in vitro. The rate of phosphorylation of the mutant protein by NTRB and the stability of mutant NTRC-phosphate were similar to those of wild-type NTRC. The phosphorylated mutant protein shows only a slight decrease in affinity (around 2-fold) for tandem NTRC binding sites in the Klebsiella pneumoniae nifL promoter suggesting that the mutation primarily influences the positive control function of NTRC. Moreover the mutant protein is trans dominant to the wild-type protein with respect to transcriptional activation at both the glnAp2 and nifL promoters. In vitro footprinting experiments reveal that the mutant protein is unable to catalyse isomerisation of closed promoter complexes between sigma 54-RNA polymerase and the nifL promoter to open promoter complexes. However, the mutant protein retains the ability to increase the occupancy of the -24, -12 region by sigma 54-RNA polymerase, forming closed complexes at the nifL promoter, which are not detectable in the absence of NTRC. These data support a model in which the activator influences the formation of closed complexes at the nifL promoter in addition to its role in catalysing open complex formation.
Austin
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S
AFRC IPSR Nitrogen Fixation Laboratory, University of Sussex, Brighton, UK.
Kundrot
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C
Dixon
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R
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D001665:Binding Sites; D004269:DNA, Bacterial / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D005786:Gene Expression Regulation; D005798:Genes, Bacterial; D007711:Klebsiella pneumoniae / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D009586:Nitrogen Fixation / Q000235:genetics; D051922:PII Nitrogen Regulatory Proteins; D010641:Phenotype; D010766:Phosphorylation; D011401:Promoter Regions, Genetic; D012808:Sigma Factor / Q000378:metabolism; D015534:Trans-Activators; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D051922:PII Nitrogen Regulatory Proteins; D012808:Sigma Factor; D015534:Trans-Activators; D014157:Transcription Factors; D000255:Adenosine Triphosphate