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压力下，诱导大量蛋白质的合成，这些蛋白质保护细胞免受未来潜在的致命压力。这种普遍的应激反应带来了一种特殊的生理状态，提高了在自然环境、食物和一些致病性相互作用中的生存能力。在枯草芽孢杆菌和相关革兰氏阳性病原体中，这种反应是由？B转录因子。损失？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。我们的长期目标是用枯草芽孢杆菌作为模型来理解这种反应，从检测不同压力的传感器开始，通过信号转导网络将信息传递给？B、结束与基因的生理作用下？B控制。这一建议解决了信号网络本身，它通过“伙伴切换”机制起作用，其中关键的蛋白质相互作用由丝氨酸或苏氨酸磷酸化控制。这种机制似乎很古老，可塑性很强，在真细菌中很普遍。因此，对枯草芽孢杆菌这一机制的研究应该有助于理解控制广泛信号通路的原理。在这里激活？B是对两类压力的反应：(i)能量压力，包括对碳、磷酸盐或氧气的饥饿；（ii）环境压力，包括酸、乙醇、热或盐。这两个类是传达给？B通过单独的信号通路，每个信号通路都以差异调节的PP2C磷酸酶终止，每个信号通路都收敛于？B、rbv反反？和RsbW反？，组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组成，RsbQ是一种预测的水解酶，也是必需的。环境分支有8个调节器，有些是冗余的，它们都通过第二个非典型伙伴切换模块连接起来激活RsbU磷酸酶。这些调节因子中有6个从细胞提取物中共同纯化，形成一个大的（>700 kDa）复合物，其中3个形成一个简化的核心，可用于体外和体内分析。我们的具体目标是使用遗传、生化和结构相结合的方法来解决三个问题：(1)能量信号传导的机制是什么；(2)环境信号的机制是什么；(3)积极反馈和消极反馈的作用是什么？网络吗?

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?