MOLECULAR CHARACTERIZATION OF PORIN GENE REGULATION

孔蛋白基因调控的分子表征

• 批准号: 6127990
• 负责人: Linda J. Kenney
• 金额: $ 20.68万
• 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 2005-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6127990
• 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.

双组分调节系统已成为适应性反应的范例。最简单的系统由一个传感器和一个响应调节器组成。E.调节孔蛋白基因的大肠杆菌对生长培养基的渗透压的变化作出反应。EnvZ是OmpR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被抑制，ompC被激活。由遗传研究产生的模型预测磷酸化OmpR（OmpR-P）以高亲和力结合以激活ompF，并且以低亲和力结合以抑制ompF并激活ompC。PI最近的工作表明，目前接受的假设是不足以解释孔蛋白基因调控。这项申请包含三个目标。第一种是使用体外足迹法来检查作为OmpR和OmpR-P浓度的函数的结合模式。如果结合是充分不同的，这将导致拒绝的亲和力假设和替代品将需要考虑。目的是进一步检查在体外和体内低和高渗透压下的结合，以将占据与ompF和ompC的E2调节表达相关联。第二个目的是确定OmpR的接头是否需要氨基端磷酸化结构域和羧基端DNA结合结构域之间的通信，以及它是否在这个过程中起着主动或被动的作用。如果需要接头，将进行研究以确定长度和氨基酸要求（如有）。位点特异性光谱探针的使用将探索信号传导过程中发生的构象变化。最终目的是确定接触位点，并了解OmpR和RNA聚合酶α亚基（RpoA）之间的相互作用，这对转录激活很重要。本申请开始于OmpR和OmpR-P与ompF和ompC的调节区的DNA结合。然后，它集中在响应调节OmpR和检查构象变化是重要的信号。最后考虑OmpR与RNA聚合酶的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，在该系统中激酶活性的跨膜信号依赖性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。