MOLECULAR GENETICS OF BACILLUS SUBTILIS RNA POLYMERASE

应激反应是致病的重要组成部分 革兰氏阴性菌的作用机制。关于这一点我们知之甚少 革兰氏阳性细菌的应激反应，一组包含 许多重要的人类病原体。这样做的长期目标是 建议建立具有关键特征的控制机制 模式革兰氏杆菌枯草芽孢杆菌的应激反应转录 积极的制度。枯草杆菌的一般应激反应是 受另一种转录因子西格玛B的控制，它 与RNA聚合酶的催化核心结合以指导 40多个应激基因的转录。西格玛B的活性是 由信号转导网络进行翻译后控制 包括至少七个编码在sig B操纵子中的RSB蛋白。 RSB网络对两种不同类别的压力做出响应：(I) 能量压力的信号，如进入静止相，以及 (Il)环境压力信号，如盐、热或 酒精休克。这个网络是基于新认识的 “合伙人转换”机制，有别于以下两种机制： 通常用于细菌信号转导的组件系统。 然而，在这两种机制中，关键的蛋白质接触都是由 可逆的磷酸化。RSB网络由两个链接的 合作伙伴交换模块，每个模块包含一个磷酸酶、一个 拮抗剂蛋白和开关蛋白-激酶。这些模块传递 并将两种不同类别的应力信号整合到 SigmaB回应道。需要上行模块进行传输 环境压力信号传给下游模块。这个 然后，下行模块将这些上行信号与 能量应力信号，并通过RsbW将它们传输到Sigma B 下游的开关蛋白激酶--抗Sigma因子 模块。具体目标涉及以下重要内容 问题：(L)合作伙伴的关键方面是什么 开关机制？(2)环境压力信号是如何 进入RSB网络？以及(3)西格玛B如何与 核糖核酸聚合酶核心酶？我们将确定活动和 纯化的野生型和突变型RSB蛋白在体内的相互作用 在体外，并将建立这些活动的体内相关性 以及通过遗传分析的相互作用。最近的数据表明， 存在额外的、未发现的RSB监管机构，我们将 通过基因筛查来确定身份。我们还将使用组合的 用生物化学和遗传学方法鉴定RNA中的残基 聚合酶核心亚单位对a-核心区很重要 协会。我们的结果应该与压力有直接的关系 对其他革兰氏阳性菌的反应，其中Sigma B 最近发现了同源基因和RSB调节器。

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.