Mechanism of Virulence Regulation by Membrane Activators

描述（由申请人提供）：霍乱弧菌的毒力基因调控需要四种不寻常的转录调节蛋白的作用，即成对的膜定位转录激活因子/效应分子（称为ToxR/ToxS和TcpP/TcpH）。这些蛋白质的作用机制的工作模型认为，ToxR和TcpP合作，以激活蛋白质ToxT的表达，激活编码霍乱毒素和毒素共调节菌毛的毒力因子的基因。相比之下，ToxR单独-独立于TcpP -可以调节两种外膜蛋白OmpU和OmpT的表达。预计激活剂和效应物之间的相互作用发生在周质空间，虽然在一般情况下，效应物的作用是不太好的特点。TcpH用于阻断靶向TcpP周质结构域的蛋白水解机制。本研究将评估TcpH是否在TcpP功能中发挥任何其他作用。FoxS可能有助于赋予ToxR对其功能至关重要的高级结构。将测试从这些膜定位激活剂功能的机制模型产生的特定假设。我们对这些蛋白质的了解大部分来自遗传和生物化学研究，这些研究将在本文提出的工作中继续进行。随着研究原核细胞的进展和成像和细胞学资源，还提出了旨在开发用于研究转录复合物膜定位的试剂和实验方法的研究。 具体目标 I. 确定ToxR激活ompU和toxT的机制。 二. 通过TcpP确定DNA结合和RNA聚合酶相互作用对toxT激活的作用。 伊利诺伊州 定义激活物/效应物周质相互作用的机制和结果。 四.发展细胞学方法分析膜定位激活因子功能。

DESCRIPTION (provided by applicant): Virulence gene regulation in Vibrio cholerae requires the action of four unusual transcription regulatory proteins, pairs of membrane localized transcription activator/effector molecules called ToxR/ToxS and TcpP/TcpH. Working models for the mechanisms of action of these proteins hold that ToxR and TcpP collaborate to activate expression of a protein ToxT, which activates genes encoding virulence factors cholera toxin and toxin co-regulated pilus. By contrast, ToxR alone - independently of TcpP - can regulate expression of OmpU and OmpT, two outer membrane proteins. Interactions between the activators and effectors are predicted to take place in the periplasmic space, although in general the roles of the effectors are less well characterized. TcpH acts to block a proteolytic mechanism that targets periplasmic domain of the TcpP. Whether TcpH plays any other role in the function of TcpP will be assessed in this study. FoxS may serve to confer higher order structure on ToxR essential for its function. Specific hypotheses generated from these mechanistic models of membrane-localized activator function will be tested. Much of what we understand about these proteins has come from genetic and biochemical studies, and these will continue in the work proposed herein. With advances and imaging and cytological resources available for studying the prokaryotic cell, studies aimed at developing reagents and experimental approaches for studying membrane localization of transcription complexes are also proposed. Specific Aims I. Determine the mechanism of ompU and toxT activation by ToxR. II. Determine the role of DNA binding and RNA polymerase interaction by TcpP for toxT activation. Ill. Define the mechanisms and consequences of activator/effector periplasmic interactions. IV. Develop cytological methods for analyzing membrane localized activator function.