GENETIC STUDIES OF GLUTAMINE SYNTHETASE IN BACTERIA

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The products of genes ntrA, ntrB, and ntrC are required for nitrogen-regulated transcription in enteric bacteria. The product of gene ntrA (called NTRA) is an alternate sigma subunit for RNA polymerase: NTRA plus core RNA polymerase (referred to as NTRA holoenzyme) can bind to a "strong" nitrogen-regulated promoter in the absence of other factors. The ntrC product (called NTRC), which is a DNA binding protein, is required for efficient initiation of transcription by NTRA holoenzyme. Binding sites for NTRC in two nitrogen-regulated promoter regions lie 100-150bp upstream of the promoter (76,101) and appear to resemble transcriptional enhancers in eukaryotes (76). To activate transcription NTRC must be phosphorylated. It is phosphorylated--and dephosphorylated--by the product of the ntrB gene (NTRB), which is a bifunctional protein kinase- phosphoprotein phosphatase (69,50). The degee of phosphorylation of NTRC is metabolically regulated. Metabolic regulation controls the activities of the bifunctional enzyme NTRB and involves at least two additional proteins, one of which appears to be a primary sensor of the status of cellular nitrogen nutrition. We will undertake biochemical studies and a structure-function analysis of NTRC in order to determine the precise mechanism by which this enhancer activating protein stimulates initiation of transcription. We will also perform a structure-function analysis of the sigma factor NTRA to determine the nature of its interactions with NTRC, core polymerase and promoter DNA. These studies will be facilitated by the fact that NTRA is not essential for cell viability. Finally, we will study in detail the nature of metabolic controls of NTRB activity by other proteins and by small molecules. Our eventual goal in this regard is to determine whether pool sizes of the metabolites glutamine and 2- oxoglutarate correlate directly with rates of transcription by NTC and NTRA holoenzyme and, if so, the extent to which these pool sizes must vary to give the full physiological range of transcription rates.