Structure/Function Analysis of E. coli NRII

描述：（改编自研究者摘要）：NRI/NRII 大肠杆菌的双组分调控系统调节 参与氮同化反应的基因， 氮素状况这些信号调节PH和GInK信号的活性 转导蛋白，这是反过来调节激酶和磷酸酶 NIRII的活动。NRII的激酶和磷酸酶活性控制着细胞的生长， NRI的磷酸化状态，其仅能够激活转录， 磷酸化我们建议研究该激酶的机制和调控 和NRH的磷酸酶活性。这些研究将推动我们的 了解E.信号转导机制 通过双组分调节系统的转导。 提出了四种主要方法。第一，NRJI和域的结构 将沿着P11和NRII的复合物一起研究从NRII衍生的P11 以及PH与NRII的分离激酶结构域的复合物。二是 负责改变激酶的突变效应的机制， 将研究NIR 11的磷酸酶活性。这些研究将 涉及使用NRH的分离的中心结构域的生化方法， NRII的全长突变体版本，以及 磷酸酶活性的调节。第三，对完整细胞的研究将 用于表征PIT和G1 nK与 NRII。第四，影响激酶的突变和 将研究NRII的磷酸酶活性，并研究NRII的磷酸酶活性的能力。 影响磷酸酶活性的突变将相互补充， 研究了总之，这些方法应该导致一种机械的 了解NRII激酶和磷酸酶活性及其 通过PH和G1 nK调节。

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.