MOLECULAR CHARACTERIZATION OF PORIN GENE REGULATION

孔蛋白基因调控的分子表征

• 批准号: 6519947
• 负责人: Linda J. Kenney
• 金额: $ 22.65万
• 依托单位: OREGON HEALTH & SCIENCE UNIVERSITY
• 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 2003-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6519947
• 关键词: DNA binding protein; DNA directed RNA polymerase; DNA footprinting; Escherichia coli; bacterial genetics; bacterial proteins; binding sites; biological signal transduction; environmental adaptation; genetic regulation; membrane proteins; osmotic pressure; phosphorylation

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 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 is repressed and ompC is activated. A model arising from genetic studies predicts that phospho- OmpR (OmpR-P) binds with high affinity to activate ompF and with low affinity to repress ompF and activate ompC. Recent work by the PI indicates that this currently accepted hypothesis is not sufficient to account for porin gene regulation. This application contains three aims. The first is to use in vitro footprinting to examine the pattern of binding as a function of OmpR and OmpR-P concentration. If the binding is sufficiently different, it would lead to the rejection of the affinity hypothesis and alternatives would need to be considered. The aim further examines binding at low and high osmolarity both in vitro and in vivo, to correlate occupancy with osmoregulated expression of ompF and ompC. The second aim is to determine whether the linker of OmpR is required for communicating between the amino-terminal phosphorylation domain and the carboxyl-terminal DNA-binding domain and whether it plays an active or passive role in this process. If the linker is required, studies to determine the length and amino acid requirements (if any) will be conducted. The use of site-specific spectroscopic probes will explore the conformational changes that occur during signaling. The final aim is to determine the contact sites and to understand the interactions between OmpR and the alpha subunit of RNA polymerase (RpoA) that are important for transcriptional activation. This application begins with DNA-binding of OmpR and OmpR-P to the regulatory regions of ompF and ompC. It then focuses on the response regulator OmpR and examines conformational changes that are important for signaling. It lastly considers the role of OmpR interactions with RNA polymerase in stimulating transcription. Information gained in studying the porin regulon is relevant to systems in animal cells in which transmembrane signaling-dependent changes in kinase activity result in a phosphorylation cascade and ultimately to changes in gene expression. It is also relevant to pathogenic systems that use two-component systems to modulate their virulence properties.

双元监管体系已成为适应性应对的典范。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介质渗透压的变化做出反应。EnvZ，渗透传感器被细胞内的三磷酸腺苷磷酸化，然后磷酸化OmpR。在低渗透压下，外膜上的主要孔蛋白是OmpF，在高渗透压下，OmpF被抑制，OMPC被激活。一个来自遗传学研究的模型预测，磷酸化OmpR(OmpR-P)以高亲和力结合以激活OmpF，而以低亲和力结合以抑制OmpF和激活OMPC。PI最近的工作表明，这一目前被接受的假说不足以解释孔蛋白基因的调控。这个应用程序包含三个目标。第一种是使用体外足迹来研究结合模式作为OmpR和OmpR-P浓度的函数。如果结合足够不同，将导致对亲和力假设的拒绝，需要考虑替代方案。其目的是进一步检查体外和体内低渗透压和高渗透压下的结合，以将占有率与OmpF和OMPC的渗透调节表达相关联。第二个目的是确定OmpR的连接子是否需要在氨基末端的磷酸化结构域和羧基末端的DNA结合结构域之间进行通讯，以及它在这一过程中是主动的还是被动的。如果需要接头，将进行研究以确定长度和氨基酸需要量(如果有)。使用特定部位的光谱探针将探索在信号传递过程中发生的构象变化。最终目标是确定接触部位，并了解OmpR和RNA聚合酶阿尔法亚单位(RpoA)之间的相互作用，这对转录激活是重要的。这一应用始于OmpR和OmpR-P与OmpF和OMPC的调节区的DNA结合。然后，它将重点放在反应调节因子OmpR上，并研究对信号传递重要的构象变化。它最后考虑了OmpR与RNA聚合酶相互作用在刺激转录中的作用。在研究孔蛋白调节子中获得的信息与动物细胞中的系统有关，在该系统中，依赖于跨膜信号的激酶活性的变化导致磷酸化级联反应，并最终导致基因表达的变化。它也与使用双组分系统来调节其毒力特性的致病系统有关。