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GENETIC AND BIOCHEMICAL STUDIES OF PHOB ACTIVATION

PHOB 激活的遗传和生物化学研究

基本信息

批准号：
6667944
负责人：
WILLIAM R MCCLEARY
金额：
$ 15万
依托单位：
BRIGHAM YOUNG UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2003
资助国家：
美国
起止时间：
2003-08-01 至 2007-07-31
项目状态：
已结题

项目摘要

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.

描述（由申请人提供）：拟议研究的目的是了解信号如何通过大肠杆菌中的PhoB-PhoR双组分信号转导途径传递。双组分信号转导系统控制微生物生理学的许多方面。磷酸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模型。磷酸盐信号传导途径由磷酸盐转运蛋白PstSCAB、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和作为转录激活因子的反应调节因子PhoB组成。PhoB是一种多结构域蛋白，其N-末端结构域在天冬氨酸残基上磷酸化，其C-末端结构域结合DNA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物化学技术，以确定氨基酸残基，参与传播的乙酰基磷酸化为基础的信号从接收域的输出域。此外，该研究还将通过进行定点突变实验和分析突变体的表型来研究连接两个结构域的6-7个氨基酸的接头片段。将尝试理解接头区中每个残基的作用。关于PhoU蛋白质的研究知之甚少。其结构域结构将通过使用部分蛋白水解实验和通过遗传缺失分析进行研究。PhoU的细胞定位将使用GFP-PhoU融合蛋白和/或通过免疫电子显微镜来确定。此外，将通过定量蛋白质印迹分析测定不同生长条件下PhoU、PhoR、PhoB和PstSCAB转运蛋白的量。PhoU表达不足和过度的影响也将在phoU减去遗传背景中确定。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。这一特征，加上它们在高等真核生物中的缺失，使它们成为开发新抗生素的目标。对这些信号转导蛋白的进一步了解将有助于合理设计药物来对抗病原体。