GENETIC AND BIOCHEMICAL STUDIES OF PHOB ACTIVATION

PHOB 激活的遗传和生物化学研究

• 批准号: 2444891
• 负责人: WILLIAM R MCCLEARY
• 金额: $ 12.01万
• 依托单位: BRIGHAM YOUNG UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-07-01 至 2000-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2444891
• 关键词: DNA binding protein; DNA footprinting; Escherichia coli; bacterial genetics; bacterial proteins; chimeric proteins; conformation; fluorescence spectrometry; gel mobility shift assay; intermolecular interaction; phosphorylation; site directed mutagenesis

DESCRIPTION: The long term objective of the proposed research is to understand how phosphorylation activates response regulator proteins. These proteins control a wide range of adaptive responses in both prokaryotes and eukaryotes. This proposal focuses on the activation of PhoB, a response regulator from E. coli that controls gene expression in response to external phosphate levels. PhoB is made up of two domains: an N-terminal regulatory domain that contains the site of phosphorylation, and a C-terminal transcriptional activator domain that binds DNA and interacts with RNA polymerase. PhoB is a molecular switch that is turned on by auto-phosphorylation and off by dephosphorylation. The regulatory domain of PhoB is composed of two functional modules: a switch and a relay. The switch catalyzes phosphorylation and dephosphorylation whereas the relay transmits the phosphorylation status of the switch to the rest of the protein. Control of the phosphate response occurs by regulation of PhoB phosphorylation and dephosphorylation. To understand the activation of PhoB, this research will study PhoB autophosphorylation and dephosphorylation, will characterize the activated state of PhoB and will test competing hypotheses regarding the mechanism by which phosphorylation of the regulatory domain increases the activity of the C-terminal output domain. The enzymatic mechanisms of PhoB will be investigated through the analysis of mutant proteins. Site directed mutations will be created in PhoB to probe the roles of specific residues. In addition, a genetic screen for randomly generated mutations will be used to identify residues that are involved in the activation of PhoB. Fluorescence spectrophotometric and 32P-based assays will be used to follow PhoB phosphorylation and dephosphorylation. The activated state of PhoB will be characterized in the absence of auxiliary proteins by using a low molecular weight phospho-donor, acetyl phosphate. DNA binding, DNA bending, and PhoB:PhoB interactions will be studied using multiple gel retardation and footprinting assays. Experiments to test how phosphorylation of the regulatory domain of PhoB activates the effector domain involve the generation and analysis of several chimeric CheY/PhoB constructs. We will also employ gel-filtration chromatography and fluorescence spectroscopy to follow potential oligomerization and/or conformational changes within PhoB that occur upon phosphorylation. The proposed work is important for understanding the molecular mechanisms of signal transduction. In particular, it will help elucidate general principals regarding the activation of proteins by phosphorylation. Phosphorylation-activated transcription factors, similar to PhoB, are widespread throughout nature and control responses that regulate the cell cycle, differentiation, development, virulence, metabolite utilization, and transport. Knowledge gained about the mechanisms of PhoB activation should be readily applicable to other systems.

描述：拟议研究的长期目标是 了解磷酸化如何激活反应调节蛋白。这些 蛋白质控制着原核生物和 真核生物。这项提议的重点是激活Phob，这是一种回应 来自大肠杆菌的调节因子，控制基因表达以响应外部环境 磷酸盐水平。PHOB由两个结构域组成：N-末端调节域 含有磷酸化部位的结构域和C-末端 与DNA结合并与RNA相互作用的转录激活因子结构域 聚合酶。PHOB是一种分子开关，通过 自动磷酸化和通过去磷酸化关闭。的监管范围 PHOB由两个功能模块组成：开关和继电器。这个 开关催化磷酸化和去磷酸化，而继电器 将开关的磷酸化状态传输到 蛋白。磷酸盐反应的调控是通过调节PHOB来实现的 磷酸化和去磷酸化。 为了了解PHOB的激活，本研究将研究PHOB 自动磷酸化和去磷酸化，将表征被激活的 并将通过以下方式检验关于该机制的相互竞争的假说 调节结构域的哪一种磷酸化增加了 C端输出域。PHOB的酶机制将是 通过突变蛋白的分析进行了研究。站点定向 将在Phob中产生突变，以探测特定残基的作用。 此外，还将使用随机产生的突变的基因筛查 以确定参与PHOB激活的残基。 随后将使用荧光分光光度分析和基于32P的分析 PHOB的磷酸化和去磷酸化。光子b的激活状态 将在没有辅助蛋白质的情况下使用低分子量 分子量磷供体，乙酰磷酸盐。DNA结合，DNA弯曲， 和PHOB：PHOB相互作用将使用多重凝胶延迟来研究 以及足迹分析。测试磷酸化的实验如何 PHOB的调控结构域激活涉及 几种嵌合Chey/Phob构建物的产生和分析。我们会 还使用凝胶过滤层析和荧光光谱来 跟踪PHOB内潜在的寡聚和/或构象变化 发生在磷酸化过程中。 所提出的工作对于理解分子机制是重要的。 信号转导。特别是，它将有助于澄清一般 关于通过磷酸化激活蛋白质的原理。 磷酸化激活的转录因子，类似于Phob，是 广泛存在于自然界和调控细胞的反应中 循环、分化、发育、毒力、代谢物利用和 运输。关于Phob激活机制的知识应该 可方便地应用于其他系统。