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Alterations of highly conserved residues in the regulatory domain of nitrogen regulator I (NtrC) of Escherichia coli.

Program in Molecular and Cell Biology, University of Texas at Dallas, Richardson 75083-0688.

ABSTRACT

Transcription of many nitrogen-regulated (Ntr) genes requires the phosphorylated form of nitrogen regulator I (NRI, or NtrC), which binds to sites that are analogous to eukaryotic enhancers. A highly conserved regulatory domain contains the site of phosphorylation and controls the function of NRI. We analyzed the effects of substitutions in highly conserved residues that are part of the active site of phosphorylation of NRI in Escherichia coli. Fourteen substitutions of aspartate 54, the site of phosphorylation, impaired the response to nitrogen deprivation. Only one of these variants, NRI D-54-->E (NRI-D54E), could significantly stimulate transcription from glnAp2, the major promoter of the glnALG operon. Cells with this variant grew with arginine as a nitrogen source. Experiments with purified components showed that unphosphorylated NRI-D54E stimulated transcription. In contrast, substitutions at aspartate 11 were not as deleterious as those at aspartate 54. Finally, we showed that NRI-K103R, in which arginine replaces the absolutely conserved lysine, is functionally active and efficiently phosphorylated. This substitution appears to stabilize the phosphoaspartate of NRI. The differences between our results and those from study of homologous proteins suggest that there may be significant differences in the way highly conserved residues participate in the transition to the activated state.

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