Regulation of the Metabolic Processes of the single cell at the Molecular level

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The object of the proposed research is to elucidate the mechanism responsible for the activation of transcription at the nitrogen-regulated promoter glnAp2 of the structural gene for glutamine synthetase in Escherichia coli. We have shown that core RNA polymerase with sigma54, the specific o factor for nitrogen regulated promoters, combines with glnAp2 in a closed complex. The activator NRI, after phosphorylation by the modulator NRII stimulates the isomerization to the open complex. We plan to separate the open complexes from other proteins and small molecules and to probe the interaction of NRI- phosphate with the DNA of the promoter and with sigma54. We shall also attempt to discover the mechanism responsible for the ability of NRI to activate in intact cells the transcription of glnAp2 in the absence of NRII by the isolation and study of mutants altered in this ability. We shall attempt to identify the DNA sites in the vicinity of glnAp2 that are responsible for the dampening of glnAp2 transcription by an increased intracellular concentration of NRI.