PMID:2041769

Citation	Austin, S, Kundrot, C and Dixon, R (1991) Influence of a mutation in the putative nucleotide binding site of the nitrogen regulatory protein NTRC on its positive control function. Nucleic Acids Res. 19:2281-7
Abstract	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.
Links	PubMed PMC329431
Keywords	Adenosine Triphosphate/metabolism; Amino Acid Sequence; Bacterial Proteins; Binding Sites; DNA, Bacterial/metabolism; DNA-Binding Proteins/genetics; DNA-Binding Proteins/metabolism; Gene Expression Regulation; Genes, Bacterial; Klebsiella pneumoniae/genetics; Models, Molecular; Molecular Sequence Data; Mutation; Nitrogen Fixation/genetics; PII Nitrogen Regulatory Proteins; Phenotype; Phosphorylation; Promoter Regions, Genetic; Sigma Factor/metabolism; Trans-Activators; Transcription Factors/genetics; Transcription Factors/metabolism
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Annotations

Gene product	Qualifier	GO Term	Evidence Code	with/from	Aspect	Extension	Notes	Status
KLEPN:NTRC	involved_in	GO:0006355: regulation of transcription, DNA-templated	ECO:0000315: mutant phenotype evidence used in manual assertion		P		Seeded From UniProt	complete
KLEPN:NTRC		GO:0006355: regulation of transcription, DNA-dependent	ECO:0000315:		P		Figure 4: NTRC often prohibits transcriptional activation at promoters that need o54. Both the wild-type NTRC-phosphate and the mutant phenotype yielded the protection of -25, -24, and -13 guanine residues; these resides may be necessary for Eo54 recognition. However, -8 and -9 may be responsible for open promoter complexes, and the mutant phenotype is unable to express high levels of -8 and -9, causing the promoter to remain in closed form.	complete CACAO 2719

References

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