MOLECULAR CHARACTERIZATION OF PORIN GENE REGULATION

双成分监管系统已成为适应性反应的范例。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，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 被抑制，ompC 被激活。遗传研究产生的模型预测磷酸化 OmpR (OmpR-P) 以高亲和力结合激活 ompF，以低亲和力结合抑制 ompF 并激活 ompC。 PI 最近的工作表明，目前接受的假设不足以解释孔蛋白基因调控。该应用程序包含三个目标。第一种方法是使用体外足迹法来检查作为 OmpR 和 OmpR-P 浓度函数的结合模式。如果结合足够不同，则将导致亲和力假设的拒绝，并且需要考虑替代方案。该目的进一步检查体外和体内低渗透压和高渗透压下的结合，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 的连接子是否是氨基末端磷酸化结构域和羧基末端 DNA 结合结构域之间的通讯所必需的，以及它在此过程中是否发挥主动或被动作用。如果需要接头，将进行研究以确定长度和氨基酸要求（如果有）。使用位点特异性光谱探针将探索信号传导过程中发生的构象变化。最终目标是确定接触位点并了解 OmpR 和 RNA 聚合酶 (RpoA) α 亚基之间的相互作用，这对于转录激活非常重要。该应用始于 OmpR 和 OmpR-P 与 ompF 和 ompC 调控区域的 DNA 结合。然后，它重点关注响应调节剂 OmpR 并检查对信号传导很重要的构象变化。最后考虑了 OmpR 与 RNA 聚合酶相互作用在刺激转录中的作用。研究孔蛋白调节子时获得的信息与动物细胞系统相关，在动物细胞系统中，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。 它还与使用双组分系统调节其毒力特性的致病系统有关。

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.