MOLECULAR CHARACTERIZATION OF PORIN GENE REGULATION

双组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响应生长介质渗透压的变化。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，外膜的主要孔蛋白是OmpF，在高渗透压下，OmpF被抑制，而ompC被激活。一个由遗传学研究产生的模型预测，phospho- 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，这种目前被接受的假设不足以解释孔蛋白基因的调控。此应用程序包含三个目标。第一个是使用体外足迹来检查结合模式作为OmpR和OmpR- p浓度的函数。如果结合有足够的不同，就会导致拒绝亲和假设，需要考虑替代方案。目的是进一步检查体外和体内低和高渗透压下的结合，以确定占用与渗透调节的ompF和ompC表达之间的关系。第二个目的是确定OmpR的连接子是否需要在氨基端磷酸化结构域和羧基端dna结合结构域之间进行通信，以及它在这一过程中是主动还是被动的作用。如果需要连接体，将进行研究以确定长度和氨基酸需求（如果有的话）。使用位点特异性光谱探针将探索信号传递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与RNA聚合酶(RpoA) α亚基之间的相互作用，这对转录激活很重要。这一应用始于将OmpR和OmpR- p的dna结合到ompF和ompC的调控区域。然后将重点放在反应调节因子OmpR上，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。在研究孔蛋白调控中获得的信息与动物细胞中激酶活性的跨膜信号依赖性变化导致磷酸化级联并最终导致基因表达变化的系统有关。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。

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.