Structure/Function Analysis of E. coli NRII

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DESCRIPTION: (Adapted from the Investigator's abstract): The NRI/NRII two-component regulatory system of Escherichia coli regulates the expression of genes involved in nitrogen assimilation in response to signals of carbon and nitrogen status. These signals regulate the activity of the PH and GInK signal transduction proteins, which is turn regulate the kinase and phosphatase activites of NIRII. The kinase and phosphatase activies of NRII control the phosphorylation state of NRI, which is only able to activate transcription when phosphorylated. We propose to study the mechanism and regulation of the kinase and phosphatase activities of NRH. These studies should advance our understanding of metabolic regulation in E. coli and the mechanisms of signal transduction by the two-component regulatory systems. Four main approaches are proposed. First, the structure of NRJI and domains derivied from NRII will be investigated, along with the complex of P11 and NRII and the complex of PH with the isolated kinase domain of NRII. Second, the mechanisms responsible for the effect of mutations altering the kinase and phosphatase activities of NIR11 will be investigated. These studies will involve biochemical approaches using the isolated central domain of NRH and full-length mutant versions of NRII, as well as genetic studies of the regulation of the phosphatase activity. Third, studies with intact cells will be used to characterize the binding of PIT and G1nK to the kinase domain of NRII. Fourth, the epistasis relationships of mutations affecting the kinase and phosphatase activities of NRII will be investigated, and the ability of mutations affecting the phosphatase activity to complement one another will be investigated. Together, these approaches should result in a mechanistic understanding of the NRII kinase and phosphatase activities and their regulation by PH and G1nK.