Characterization of OmpR Gene Regulation

OmpR 基因调控的表征

• 批准号: 7282373
• 负责人: Linda J. Kenney
• 金额: $ 28.29万
• 依托单位: UNIVERSITY OF ILLINOIS AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-04-01 至 2010-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7282373
• 关键词: ATP phosphohydrolase; Address; Adopted; Affinity; Binding; Biochemical Genetics; Biological Assay; C-terminal; Cells; Chimera organism; Complex; Culture Media; DNA; DNA Binding; Escherichia coli; Eukaryota; Eukaryotic Cell; Event; Fluorescence Resonance Energy Transfer; Funding; Gene Expression Regulation; Genes; Genetic Transcription; Head; Helix (Snails); In Vitro; Length; Measurement; Measures; Membrane; Methods; Modeling; Molecular; N-terminal; Osmolar Concentration; Pathogenesis; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphotransferases; Play; Prokaryotic Cells; Protein Dephosphorylation; Regulation; Research; Role; Salmonella; Salmonella enterica; Shigella; Signal Pathway; Signal Transduction; Spheroplasts; Structure; System; Systemic infection; Tail; Testing; Transcriptional Activation; Uncertainty; Ursidae Family; Virulence Factors; Work; Yang; Yersinia; base; crosslink; ear helix; genetic regulatory protein; in vivo; pathogen; porin; promoter; reconstitution; research study; response; sensor; transcription factor

DESCRIPTION (provided by applicant): Two component regulatory systems have emerged as a paradigm for adaptive responses. The simplest systems consist of a sensor and a response regulator. The two-component system in E. coli that regulates the porin genes responds to changes in osmolarity of the growth medium. EnvZ, the presumed osmosensor is phosphorylated by intracellular ATP and then phosphorylates OmpR. At low osmolarity, the major porin in the outer membrane is OmpF and at higher osmolarity, ompF transcription is repressed and ompC is activated. Two-component systems are intimately involved in the coordinate expression of virulence factors in many different pathogens and OmpR is an important global regulatory protein. In Salmonella, OmpR lies at the first step of a regulatory cascade that turns on a downstream two-component regulatory system and activates expression of a type three secretion system required for systemic infection. In the present application, the hypothesis to be tested is that EnvZ controls the concentration of OmpR approximately P by adjusting its phosphatase activity in response to the osmotic signal. Prior to the previous funding period, we discovered that DNA binding stimulates OmpR phosphorylation. This observation may have important mechanistic implications for signaling and it suggests that response regulators that function as transcription factors may be phosphorylated while bound to the DNA. In the first aim, we will attempt to isolate and characterize such an EnvZ/OmpR/DNA complex. An EnvZ-GFP fusion will be employed to examine signaling and transcription in intact cells and in spheroplasts to determine the role (if any) of the outer membrane. An OmpR approximately P dephosphorylation assay has been developed and will be used to test the role of EnvZ in OmpR-P turnover. In the previous funding period, we discovered that OmpR is capable of binding to DNA in a head-to-head orientation rather than the previously proposed head-to-tail mode. Our new model predicts that the recognition helix is actually helix 2 rather than helix 3. We will determine the role of helices 2 and 3 in DNA recognition and test the hypothesis that OmpR can bind to DNA in more than one orientation. In aim three, we propose to solve the full-length structure of OmpR, the structures of the isolated N- and C-terminal domains, the phosphorylated N-terminal domain and the C-terminal domain bound to DNA by NMR.

描述（由申请人提供）：两个组成部分的调节系统已经作为适应性响应的范例出现。最简单的系统由传感器和响应调节器组成。大肠杆菌中调节孔蛋白基因的双组分系统对生长培养基渗透压的变化做出反应。 EnvZ（假定的渗透传感器）被细胞内 ATP 磷酸化，然后磷酸化 OmpR。在低渗透压下，外膜中的主要孔蛋白是 OmpF，在较高渗透压下，ompF 转录受到抑制，ompC 被激活。双组分系统与许多不同病原体中毒力因子的协调表达密切相关，OmpR 是一种重要的全局调节蛋白。在沙门氏菌中，OmpR 位于调节级联的第一步，该级联打开下游两部分调节系统并激活全身感染所需的三型分泌系统的表达。在本申请中，要测试的假设是EnvZ通过响应渗透信号调节其磷酸酶活性来控制OmpR大约P的浓度。在上一个资助期之前，我们发现 DNA 结合会刺激 OmpR 磷酸化。这一观察结果可能对信号传导具有重要的机制意义，并且表明充当转录因子的反应调节因子在与 DNA 结合时可能会被磷酸化。第一个目标是，我们将尝试分离和表征这样的 EnvZ/OmpR/DNA 复合物。 EnvZ-GFP 融合体将用于检查完整细胞和原生质球中的信号传导和转录，以确定外膜的作用（如果有）。一种 OmpR 大约 P 去磷酸化测定已经开发出来，并将用于测试 EnvZ 在 OmpR-P 周转中的作用。在之前的资助期间，我们发现OmpR能够以头对头的方式与DNA结合，而不是之前提出的头对尾的模式。我们的新模型预测识别螺旋实际上是螺旋 2，而不是螺旋 3。我们将确定螺旋 2 和 3 在 DNA 识别中的作用，并测试 OmpR 可以以多个方向结合 DNA 的假设。在目标三中，我们建议通过NMR解析OmpR的全长结构、分离的N端和C端结构域、磷酸化的N端结构域和与DNA结合的C端结构域的结构。