Structure/Function Analysis of E. coli NRII

描述：(改编自调查人员摘要)：NRI/NRII 大肠埃希菌的双组分调控系统调控蛋白的表达 参与氮同化的基因响应碳信号和 氮气状况。这些信号调节PH和Gink信号的活性 转导蛋白，它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 NRI的磷酸化状态，只有在以下情况下才能激活转录 磷酸化。我们建议研究该激酶的机制和调节。 和NRH的磷酸酶活性。这些研究应该会促进我们的 对大肠杆菌代谢调控及其信号机制的认识 双组分调控系统的转导。 提出了四种主要的方法。首先，NRJI和域名的结构 从NRII衍生的将与P11和NRII的复合体一起被研究 以及PH与分离的NRII激活区的复合体。第二， 突变改变激酶和蛋白水解酶的机制 将对NIR11的磷酸酶活性进行研究。这些研究将 涉及使用分离的NRH和NRH中央结构域的生化方法 NRII的全长突变版本，以及对 磷酸酶活性的调节。第三，对完整细胞的研究将 被用来表征PIT和G1nK与肌动蛋白激动域的结合 NRII.第四，影响激酶的突变的上位性关系和 将研究NRII的磷酸酶活性，并研究其能力 影响磷酸酶活性以相互补充的突变将是 调查过了。这些方法加在一起，应该会产生一种机械性的 对NRII激酶和磷酸酶活性及其相互关系的认识 受PH和G1nK的调节。

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.