Molecular Genetics of Bacillus subtilis RNA polymerase

描述（由申请人提供）：细菌暴露于不同的生长限制性应激诱导合成大量蛋白质，这些蛋白质保护细胞免受未来可能致命的应激。这种普遍的应激反应带来了一种特殊的生理状态，这种状态增强了在自然环境、食物和某些病原体相互作用中的生存。在枯草芽孢杆菌和相关的革兰氏阳性病原体，这种反应是由？B转录因子。失去？B功能导致对多种应激的敏感性增加，包括酸、抗生素、冷、热、渗透和氧化应激。我们的长期目标是使用B来理解这种反应。枯草杆菌作为一个模型，开始与传感器检测不同的压力，通过信号转导网络的延伸传达这一信息？B，并结束与生理作用下的基因？B控制。该建议针对信号网络本身，其通过“伴侣转换”机制发挥作用，其中关键蛋白质相互作用由丝氨酸或苏氨酸磷酸化控制。这种机制似乎是古老的，非常可塑的，在真细菌中广泛存在。在B.因此，枯草芽孢杆菌应该有助于了解管理广泛的信号通路的原则。在这里启动？B响应两类应激：（i）能量应激，包括碳、磷酸盐或氧的饥饿;和（ii）环境应激，包括酸、乙醇、热或盐。这两个班是传达给？B通过单独的信号通路，每个终止于差异调节的PP 2C磷酸酶和每个汇聚的直接调节？B，RsbV抗-抗？和RsbW反？，其形成一个伙伴交换模块。能量分支由RsbP磷酸酶（具有对信号传导重要的PAS结构域）和RsbQ（一种预测的也是必需的水解酶）组成。环境分支有8个调节器，有些是多余的，所有的调节器都通过第二个非典型的伴侣转换模块激活RsbU磷酸酶。这些调节剂中的六种在大（>700 kDa）复合物中从细胞提取物共纯化，并且三种形成适合于体外和体内分析的简化核心。我们的具体目标是使用遗传学，生物化学和结构相结合的方法来解决三个问题：（1）能量信号的机制是什么;（2）环境信号的机制是什么;（3）积极和消极反馈的作用是什么？B网络？

DESCRIPTION (provided by applicant): Exposure of bacteria to diverse growth-limiting stresses induces the synthesis of a large set of proteins that protect the cell against future, potentially lethal stresses. This general stress response brings about a special physiological state which enhances survival in the natural environment, in foods, and in some pathogenic interactions. Among Bacillus subtilis and related Gram positive pathogens, this response is governed by the ?B transcription factor. Loss of ?B function causes increased sensitivity to multiple stresses, including acid, antibiotic, cold, heat, osmotic, and oxidative stress. Our long term objective is to understand this response using B. subtilis as a model, beginning with the sensors which detect the different stresses, extending through the signal transduction network which conveys this information to ?B, and ending with the physiological role of genes under ?B control. This proposal addresses the signaling network itself, which functions by the "partner switching" mechanism in which key protein interactions are controlled by serine or threonine phosphorylation. This mechanism appears to be ancient, very plastic, and widespread among eubacteria. Study of this mechanism in B. subtilis should therefore help understand principles governing a broad array of signaling pathways. Here it activates ?B in response to two classes of stresses: (i) energy stress, including starvation for carbon, phosphate, or oxygen; and (ii) environmental stress, including acid, ethanol, heat, or salt. These two classes are conveyed to ?B by separate signaling pathways, each terminating with a differentially regulated PP2C phosphatase and each converging on the direct regulators of ?B, the RsbV anti-anti-? and the RsbW anti-?, which form one partner switching module. The energy branch consists of the RsbP phosphatase (with a PAS domain important for signaling) and RsbQ, a predicted hydrolase that is also required. The environmental branch has eight regulators, some redundant, and all join to activate the RsbU phosphatase via a second, atypical partner switching module. Six of these regulators co-purify from cell extracts in a large (>700 kDa) complex, and three form a simplified core amenable to analysis in vitro and in vivo. Our specific aims use a combined genetic, biochemical and structural approach to address three questions: (1) What is the mechanism of energy signaling; (2) What is the mechanism of environmental signaling; and (3) What is the role of positive and negative feedback in the ?B network?