Structure/function analysis of the E. coli NIR/NRII signal transduction system

描述（由申请人提供）：我们建议继续研究NRI-NRII双组分信号转导系统的遗传学，生理学和生物化学，该系统在控制大肠杆菌的氮同化中起主要作用。该系统为通过碳氮状态信号调控中枢代谢提供了一个范例。我们在NRI/NRII系统上的工作也为理解双组分信号转导系统提供了一个范例。双组分信号转导系统是细菌中最常见的信号转导系统类型，也存在于低等真核生物和植物中。在细菌中，这些系统控制着细胞周期、发育、毒力、趋化性、对环境压力的众多反应以及新陈代谢的各个方面。我们的工作还将为PII蛋白的信号转导提供一个范例，PII蛋白是自然界中分布最广泛的信号转导蛋白。我们的研究探讨了PII蛋白如何整合不同的信号并控制作为信号转导酶的受体。因此，本研究将直接探讨自然界中广泛存在并对人类健康和福祉有重大影响的信号转导机制。拟开展大肠杆菌NRI蛋白和NRII蛋白的结构/功能研究。这些方法使用多种遗传、生化和生物物理方法，旨在提供有关调节机制的详细信息。具体目的包括：(1)确定Pll调控NRII激酶和磷酸酶活性的机制；(2)确定NRI“自磷酸酶”活性的机制，以及NRII和Pll复合物对其的控制。在这两个特定目标中，生化方法被用来识别信号蛋白的相互作用表面，活性的化学反应，以及导致催化活性调节的蛋白质结构域的相互作用。遗传方法用于定义蛋白质的功能，并将特定功能映射到蛋白质的区域。生物物理学的方法被用来确定蛋白质的结构。鉴于它们在自然界中广泛存在，并与许多公共卫生和农业问题直接相关，了解双组分系统和Pll信号蛋白使用的信号转导机制是一个重要的目标。我们对模型系统的研究应该会有快速的进展。

DESCRIPTION (provided by applicant): We propose to continue our study of the genetics, physiology, and biochemistry of the NRI-NRII two-component signal transduction system that plays a major role in controlling nitrogen assimilation in E. coli. This system provides a paradigm for the regulation of central metabolism by signals of carbon and nitrogen status. Our work on the NRI/NRII system also provides a paradigm for understanding two-component signal transduction systems. Two component signal transduction systems are the most common type of signal transduction system in bacteria and are also present in lower eukaryotes and plants. In bacteria, these systems control the cell cycle, development, virulence, chemotaxis, numerous responses to environmental stress, and various aspects of metabolism. Our work will also provide a paradigm for signal transduction by PII proteins, which are the most widely distributed signal transduction proteins in nature, Our studies investigate how PII proteins integrate distinct signals and control receptors that are signal-transduction enzymes. Thus, the proposed research will directly investigate signal transduction mechanisms that are widely-occuring in nature and have significant impact on human health and well-being. The proposed work will focus on structure/function studies of the NRI and NRII proteins of E. coli. The approaches use a variety of genetic, biochemical, and biophysical methods and are designed to provide detailed information on the mechanisms of regulation. The Specific Aims include: (1) Determining the mechanism of regulation of NRII kinase and phosphatase activities by Pll, and (2) Determining the mechanism of the NRI "autophosphatase" activity, and its control by the complex of NRII and Pll. In both Specific Aims, biochemical approaches are used to identify interacting surfaces of the signalling proteins, the chemistry of the activities, and the interactions of protein domains that result in regulation of the catalytic activities. Genetic approaches are used to define the functions of the proteins, and map specific functions to regions of proteins. Biophysical approaches are used to determine the structures of proteins. Given their widespread occurance in nature and direct link to numerous public health and agricultural issues, understanding the mechanisms of signal transduction used by two-component systems and Pll signalling proteins is an important objective. Our studies with a model system should allow rapid progress.