MOLECULAR GENETICS OF BACILLUS SUBTILIS RNA POLYMERASE

应激反应是发病机制的重要组成部分， 革兰氏阴性菌的机制。关于这一点， 革兰氏阳性菌之间的应激反应，一组包含 许多重要的人类病原体。这一长期目标 建议是建立控制机制的关键特征， 模式革兰氏菌枯草芽孢杆菌胁迫应答转录 正系统B的一般应激反应。枯草 由选择性转录因子σ B控制， 与RNA聚合酶的催化核心结合， 超过40个胁迫基因的转录。sigma B的活性为 由信号转导网络进行事后控制 包含至少七种在sig B操纵子中编码的Rsb蛋白。 Rsb网络对两种不同类型的压力做出响应：（i） 能量应力信号，如进入稳定期，和 (il)环境压力的信号，例如作为盐、热或 酒精休克这个网络是基于新认识到的 “伙伴转换”机制，这是从两个不同的- 通常用于细菌信号转导的组分系统。 然而，在这两种机制中，关键的蛋白质接触是由 可逆磷酸化Rsb网络由两个链接的 伴侣转换模块，每个包含磷酸酶， 拮抗蛋白和开关蛋白激酶。模块传达 并将两种不同类别的压力信号整合到一起， sigmaB响应。上游模块需要传输 环境压力信号发送到下游模块。的 下游模块然后将这些上游信号与 能量应力信号，并通过RsbW将其传输到sigma B 抗σ因子，下游的开关蛋白激酶， module.具体目标涉及以下重要问题： 问题：（l）合作伙伴的关键方面是什么 切换机制？(2)环境压力的信号 进入RSB网络？以及（3）sigma B如何与 RNA聚合酶核心酶？我们将确定活动和 纯化的野生型和突变型Rsb蛋白在 体外，并将建立这些活动的体内相关性 和相互作用。最近的数据显示， 存在额外的，未发现的Rsb监管机构，我们将 通过基因筛选鉴定。我们还将使用组合 生物化学和遗传学方法来鉴定RNA中的残基 聚合酶核心亚基对于α-核心是重要的 协会我们的研究结果应该与压力有直接关系 在其他革兰氏阳性菌中的反应，其中sigma B 最近发现了同系物和Rsb调节剂。

Stress responses are important components of pathogenetic mechanisms in Gram negative bacteria. Less is known regarding stress responses among Gram positive bacteria, a group containing many significant human pathogens. The long term objective of this proposal is to establish key features of the mechanism controlling stress-responsive transcription in Bacillus subtilis, a model Gram positive system. The general stress response of B. subtilis is governed by the alternative transcription factor sigma B, which associates with the catalytic core of RNA polymerase to direct transcription of over 40 stress genes. Activity of sigma B is controlled post-translationally by a signal transduction network comprising at least seven Rsb proteins encoded in the sig B operon. The Rsb network responds to two different classes of stress: (i) signals of energy stress, such as entry into stationary phase, and (il) signals of environmental stress, such as salt, heat, or ethanol shock. This network is based on the newly recognized "partner switching" mechanism which is distinct from the two- component systems commonly used for bacterial signal transduction. However, in both mechanisms, key protein contacts are controlled by reversible phosphorylation. The Rsb network consists of two linked partner switching modules, each containing a phosphatase, an antagonist protein, and a switch protein-kinase. The modules convey and integrate the two different classes of stress signals to which sigmaB responds. The upstream module is required to transmit environmental stress signals to the downstream module. The downstream module then integrates these upstream signals with energy stress signals and transmits them to sigma B via the RsbW anti-sigma factor, the switch protein-kinase of the downstream module. The specific aims address the following significant questions: (l) What are the critical aspects of the partner switching mechanism? (2) How do signals of environmental stress enter the Rsb network? and (3) How does sigma B associate with the RNA polymerase core enzyme? We will determine the activities and interactions of purified wild type and mutant Rsb proteins in vitro, and will establish the in vivo relevance of these activities and interactions by genetic analysis. Recent data suggests the existence of additional, undiscovered Rsb regulators, which we will identify by a genetic screen. We will also use a combined biochemical and genetic approach to identify residues in the RNA polymerase core subunits that are impOrtant for the a-core association. Our results should have direct relevance to stress response in other Gram positive bacteria, in which sigma B homologues and Rsb regulators have recently been discovered.