Influence of a mutation in the putative nucleotide binding site of the nitrogen regulatory protein NTRC on its positive control function

Abstract

A mutation, serine 170 to alanine, in the proposed ATP binding site of the activator protein NTRC prevents transcriptional activation at σ54-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 wildtype NTRC. The phosphorylated mutant protein shows only a slight decrease in affinity (around 2-fold) for tandem NTRC binding sites In the Klebslella pneumonlae 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 nlfL promoters. In vitro footprintlng experiments reveal that the mutant protein is unable to catalyse isomerisatlon of closed promoter complexes between σ54-RNA polymerase and the nlfL promoter to open promoter complexes. However, the mutant protein retains the ability to increase the occupancy of the -24, -12 region by σ54-RNA polymerase, forming closed complexes at the nlfL 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 nlfL promoter In addition to its role in catalysing open complex formation. © 1991 Oxford University Press.