REGULATION OF THE MAJOR OUTER MEMBRANE PORIN PROTEINS

肠道细菌产生一组蛋白质，称为孔蛋白， 作为被动扩散孔，以允许小的亲水分子 穿过外膜渗透屏障。 大肠杆菌 K12含有两种主要的非特异性孔蛋白OmpF和OmpC。 在 通常，细胞中存在的这两种孔蛋白的总量 是恒定的。 然而，相对水平在响应中波动， 适应各种环境条件。 一个环境因素 似乎特别重要的是介质渗透压。 如果 渗透压高，OmpC占优势。 相反，在稀释 发现OmpF。认为通过感测渗透压， E.大肠杆菌可以区分其共同的栖息地，即，内部或 在宿主动物之外。 我们希望了解分子 这一监管体系的基础。 我们实验室以前的工作已经确定了两个基因，ompR和envZ， 其指定激活孔蛋白基因所需的蛋白质 表情 EnvZ似乎起膜结合信号的作用 传感器监测环境并指导效应器 蛋白，OmpR（DNA结合蛋白），以适当的顺式作用 在ompF和ompC启动子上的位点。 OmpR和EnvZ共享 与其他的传感器和效应蛋白的显著同源性 在多种生物体中发现的双组分调节系统。 本论文将通过对OmpR的结构和功能的研究， 70个ompR的详细遗传和生化特征 错义突变，其赋予多种孔蛋白表型。 获得的结果应该澄清遗传开关的性质 它控制孔蛋白的波动，并为每个孔蛋白分配角色。 ompF和ompC启动子处的多个顺式作用位点。 另外的实验试图验证一种 OmpR和RNA聚合酶α亚基之间的相互作用， 探索其机械含义。 这些研究涉及 问题是积极的激活剂，如OmpR，如何发挥作用， 刺激转录。 最后，详细分析了 envZ计划类似于，并将补充，我们的 ompR的遗传分析。 本分析的目的是获得 深入了解EnvZ的感觉功能及其作用方式 机械地作为信号转换器来启动基因开关。

Enteric bacteria produce a set of proteins, termed porins, that act as passive diffusion pores to allow small hydrophilic molecules to cross the outer membrane permeability barrier. Escherichia coli K12 contains two major, non-specific porins, OmpF and OmpC. In general, the total amount of these two porins present in the cell is constant. However, the relative levels fluctuate in response to a variety of environmental conditions. One environmental factor that appears to be particularly important is media osmolarity. If osmolarity is high, OmpC predominates. Conversely, in dilute media, OmpF is found. It is thought that by sensing osmolarity, E. coli can distinguish its common habitats, i.e., inside or outside of a host animal. We wish to understand the molecular basis for this regulatory system. Previous work from our lab has identified two genes, ompR and envZ, which specify proteins that are required to activate porin gene expression. EnvZ appears to function as a membrane-bound signal transducer to monitor the environment and direct the effector protein, OmpR (a DNA-binding protein) to the appropriate cis-acting sites at the ompF and ompC promoters. OmpR and EnvZ share significant homology with the sensor and effector proteins of other two-component regulatory systems found in a variety of organisms. The structure and function of OmpR will be probed through a detailed genetic and biochemical characterization of 70 ompR missense mutations which confer a variety of porin phenotypes. Results obtained should clarify the nature of the genetic switch that controls porin fluctuation and assign roles to each of the multiple cis-acting sites at the ompF and ompC promoters. Additional experiments seek to verify the existence of an interaction between OmpR and the a subunit of RNA polymerase and to probe its mechanistic implications. These studies address the question of how positive activators, such as OmpR, function to stimulate transcription. Lastly, a detailed genetic analysis of envZ is planned which is similar to, and will complement, our genetic analysis of ompR. The goal of this analysis is to gain insights into the sensory functions of EnvZ and how it acts mechanistically as a signal transducer to throw the genetic switch.