Effects of insertions and deletions in glnG (ntrC) of Escherichia coli on nitrogen regulator I-dependent DNA binding and transcriptional activation.

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RESUMO

Phosphorylated nitrogen regulator I (NRI, also called NTRC), encoded by glnG (ntrC), stimulates transcription in Escherichia coli and other enteric bacteria from sites analogous to eukaryotic enhancers. We isolated 30 mutants, obtained without phenotypic selection, that have either a small insertion or deletion within glnG. Mutants were classified by the ability of NRI to repress the glnAp1 and glnL promoters and to activate two versions of the nitrogen-regulated glnAp2 promoter; each activity was measured in cells grown with three concentrations of NRI. The results were interpreted within the framework of the three-domain hypothesis of NRI structure. NRI is thought to contain a phosphorylated regulatory domain that controls binding of ATP, a central domain that hydrolyzes ATP and interacts with RNA polymerase, and a DNA-binding region of unknown extent. Our results suggest that the 70 amino acids from residue 400 to the carboxyl terminus constitute a DNA-binding domain that includes residues for specific contacts and dimerization. Our results also suggest that (i) transcription from glnAp2 without specific NRI-binding sites requires binding to sites with some similarity to the specific sites, and (ii) if an NRI variant can stimulate transcription, then increasing the concentration of NRI diminishes glnA expression for all mutants but one.

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