GENETIC AND BIOCHEMICAL STUDIES OF PHOB ACTIVATION

描述（由申请人提供）：拟议研究的目的是了解大肠杆菌PhoB-PhoR双组分信号转导途径中信号是如何传递的。双组分信号转导系统控制着微生物生理的许多方面。磷酸盐信号系统存在于许多细菌中，并为理解其他基于磷酸化的信号级联提供了一个模型。磷酸信号通路由磷酸转运蛋白PstSCAB组成；生化功能未知的辅助蛋白PhoU；接收和处理环境信号的组氨酸激酶PhoR；以及作为转录激活因子的反应调节因子PhoB。PhoB是一种多结构域蛋白，其n端结构域在天冬氨酸残基上被磷酸化，其c端结构域与DNA结合并与RNA聚合酶相互作用以激活转录。拟议的实验将采用遗传和生化技术来鉴定参与基于天冬氨酸磷酸化的信号从接收域到输出域传播的氨基酸残基。此外，本研究还将通过定点诱变实验和突变体表型分析，对连接这两个结构域的6-7个氨基酸连接段进行研究。将尝试了解每个残基在连接体区域中的作用。我们对PhoU蛋白知之甚少。它的结构域结构将通过部分蛋白水解实验和基因缺失分析来研究。PhoU的细胞定位将通过GFP-PhoU融合蛋白和/或通过免疫电镜来确定。此外，通过定量Western blot分析，测定不同生长条件下PhoU、PhoR、PhoB和PstSCAB转运体的数量。PhoU过低表达和过表达的影响也将在PhoU负遗传背景下确定。这项工作很重要，因为这些信号蛋白是细菌在不断变化的环境中生存所必需的。这一特点，再加上它们在高等真核生物中不存在，使它们成为开发新抗生素的目标。增加对这些信号转导蛋白的了解将有助于合理设计对抗病原体的药物。

DESCRIPTION (provided by applicant): The objective of the proposed research is to understand how signals are transmitted through the PhoB-PhoR two-component signal transduction pathway in Escherichia coli. Two-component signal transduction systems control many aspects of the physiology of microorganisms. The phosphate signaling system is present in many bacteria and provides a model for understanding other phosphorylation-based signaling cascades. The phosphate signaling pathway is comprised of a phosphate transporter, PstSCAB; an auxiliary protein of unknown biochemical function, PhoU; the histidine kinase, PhoR, which receives and processes environmental signals; and the response regulator, PhoB, which functions as a transcriptional activator. PhoB is a multi-domain protein whose N-terminal domain becomes phosphorylated on an aspartate residue and whose C-terminal domain binds DNA and interacts with RNA polymerase to activate transcription. The proposed experiments will employ genetic and biochemical techniques to identify amino acid residues that are involved in the propagation of the aspartyl phosphorylation-based signal from the receiver domain to the output domain. In addition, the research will also investigate the 6-7 amino acid linker segment that connects the two domains by conducting site-directed mutagenesis experiments and analyzing the phenotypes of the mutants. Attempts will be made to understand the role of each residue in the linker region. Very little is known about the PhoU protein. Its domain structure will be investigated by using partial proteolysis experiments and through a genetic deletion analysis. PhoU's cellular localization will be determined using GFP-PhoU fusion proteins and/or through immunoelectron microscopy. In addition, the amounts of PhoU, PhoR, PhoB, and the PstSCAB transporter under different growth conditions will be determined through quantitative Western blot analysis. The effects of under- and over-expression of PhoU will also be determined in a phoU minus genetic background. The proposed work is important because these signaling proteins are essential for bacteria to survive changing environments. This feature, combined with their absence in higher eukaryotes, makes them targets for the development of new antibiotics. An increased understanding of these signal transduction proteins will assist in the rational design of drugs to combat pathogens.