Characterization of OmpR Gene Regulation

描述(申请人提供)：作为适应性反应的范例，出现了两个组成部分的监管系统。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，推测的渗透传感器被细胞内的ATP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转录被抑制，OMPC被激活。两组分系统密切参与多种不同病原菌毒力因子的协同表达，OmpR是一种重要的全球调控蛋白。在沙门氏菌中，OmpR处于调节级联反应的第一步，该调节级联反应启动下游的两组分调节系统，并激活全身感染所需的3型分泌系统的表达。在目前的应用中，需要检验的假设是EnvZ通过调节其磷酸酶活性来响应渗透压信号来控制OmpR的浓度约为P。在前一个资金阶段之前，我们发现DNA结合刺激OmpR磷酸化。这一观察结果可能对信号传递具有重要的机制意义，并表明作为转录因子的反应调节因子可能在与DNA结合时被磷酸化。在第一个目标中，我们将尝试分离和鉴定这样一个EnvZ/OmpR/DNA复合体。EnvZ-GFP融合将被用于检测完整细胞和球体中的信号和转录，以确定外膜的作用(如果有的话)。已经建立了一种OmpR约P去磷酸化实验，并将用于测试EnvZ在OmpR-P周转中的作用。在之前的资助期，我们发现OmpR能够以头对头的方式与DNA结合，而不是以前提出的头到尾的方式。我们的新模型预测识别螺旋实际上是螺旋2而不是螺旋3。我们将确定螺旋2和3在DNA识别中的作用，并检验OmpR可以以多个方向与DNA结合的假设。在第三个目标中，我们提出了用核磁共振技术解决OmpR的全长结构，分离的N-末端和C-末端结构域，磷酸化N-末端结构域和与DNA结合的C-末端结构域的结构。

DESCRIPTION (provided by applicant): 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 presumed 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 transcription is repressed and ompC is activated. Two-component systems are intimately involved in the coordinate expression of virulence factors in many different pathogens and OmpR is an important global regulatory protein. In Salmonella, OmpR lies at the first step of a regulatory cascade that turns on a downstream two-component regulatory system and activates expression of a type three secretion system required for systemic infection. In the present application, the hypothesis to be tested is that EnvZ controls the concentration of OmpR approximately P by adjusting its phosphatase activity in response to the osmotic signal. Prior to the previous funding period, we discovered that DNA binding stimulates OmpR phosphorylation. This observation may have important mechanistic implications for signaling and it suggests that response regulators that function as transcription factors may be phosphorylated while bound to the DNA. In the first aim, we will attempt to isolate and characterize such an EnvZ/OmpR/DNA complex. An EnvZ-GFP fusion will be employed to examine signaling and transcription in intact cells and in spheroplasts to determine the role (if any) of the outer membrane. An OmpR approximately P dephosphorylation assay has been developed and will be used to test the role of EnvZ in OmpR-P turnover. In the previous funding period, we discovered that OmpR is capable of binding to DNA in a head-to-head orientation rather than the previously proposed head-to-tail mode. Our new model predicts that the recognition helix is actually helix 2 rather than helix 3. We will determine the role of helices 2 and 3 in DNA recognition and test the hypothesis that OmpR can bind to DNA in more than one orientation. In aim three, we propose to solve the full-length structure of OmpR, the structures of the isolated N- and C-terminal domains, the phosphorylated N-terminal domain and the C-terminal domain bound to DNA by NMR.