Integrating Cell Envelope and Starvation Stress: Regulation of Sigma(E) by ppGpp

描述（由申请人提供）：E细胞包膜感应通路是细菌维持包膜完整性的关键调控系统。？E对几种重要的细菌病原体的生存能力和其他病原体的毒力都是必需的。？E的活性受两个主要信号系统的调控，这两个信号系统整合了不同的信号，即细胞包膜应激和营养限制。作为对包膜应力的响应，？E通过其专用的抗sigma因子的调节蛋白水解而激活。在营养限制期间？在没有明显包膜胁迫的情况下，E被警报器ppGpp激活，提前为包膜生存做好准备。每个系统的激活程度决定了系统的总体活动。细胞中的E，使t与细胞的需要相匹配。除了激活？E， ppGpp激活其他替代sigma因子和应激途径，同时抑制快速生长所需的基因。实际上，ppGpp介导了从快速生长的转录程序到优化应激生存的转录程序的转换。ppGpp调控的机制是什么？E的活性尚不清楚。这些知识对于我们理解这种重要且高度保守的替代sigma因子如何响应不断变化的环境以维持细胞包膜完整性至关重要。本建议的目的是确定通过何种机制？E的活性受全局调控因子ppGpp调控，从而协调包膜和细胞质对饥饿的反应。提出的研究的中心假设是ppGpp与共同调节因子DksA和潜在的其他因素一起控制？通过直接激活？E全酶(E？E)间接地通过增加E？E通过影响核心RNA聚合酶（RNAP）的sigma因子之间的竞争。这一假设将通过三个具体目标来解决。(1)明确E?E起始转录的生化特性。？E是一个高度保守的，广泛分布的可选sigma因子的成员，缺乏几个已知对其他sigma因子转录起始的特定步骤重要的保守区域和氨基酸。这些差异如何改变转录起始还没有被研究。E？在评估pGp的影响之前，必须先确定E。(2)阐明ppGpp和DksA调控E？E活动。因为E？70和ppGpp/DksA的替代sigma因子尚不清楚，所提出的工作结果将对这些全球调控因子转录激活的生化基础的整体理解产生强烈的积极影响。(3)确定ppGpp如何调控？E活性独立于DksA。初步研究表明，一种新的调控？ppGpp的E存在，目前的模型无法解释，这需要DksA与ppGpp一起作用。我们将探讨这种新的监管策略。因为两者？E和ppGpp系统对重要细菌病原体的毒力至关重要，更好地了解这些反应的交集可能会为药物发现和疫苗设计带来新的途径。

DESCRIPTION (provided by applicant): The ?E cell envelope-sensing pathway is a key regulatory system used by bacteria to maintain cell envelope integrity. ?E is essential for viabiliy of several important bacterial pathogens and required for virulence of others. ?E activity is regulated by two major signaling systems that integrate distinct signals, cell envelope stress and nutrient limitation. In response to envelope stress, ?E is activated by regulated proteolysis of it dedicated antisigma factor. During nutrient limitation ?E is activated by the alarmone ppGpp, in the absence of apparent envelope stress, preparing the envelope for survival in advance. The extent of activation by each system serves to set the overall activity of ?E in the cell, matching t to cellular needs. In addition to activating ?E, ppGpp activates other alternative sigma factors and stress pathways, while repressing genes required for rapid growth. In effect, ppGpp mediates a switch from a transcriptional program for rapid growth to one optimized for stress survival. The mechanism by which ppGpp regulates ?E activity is not wel understood. Such knowledge is critical for our understanding of how this important and highly conserved alternative sigma factor responds to changing environments to maintain cell envelope integrity. The objective of this proposal is to determine the mechanism(s) by which ?E activity is regulated by the global regulator, ppGpp, thereby coordinating envelope and cytoplasmic responses to starvation. The central hypothesis of the proposed research is that ppGpp, in conjunction with the coregulator DksA and potentialy other factors, controls ?E activity by directly activating ?E holoenzyme (E?E) and indirectly by increasing the amount of E?E through its influence on the competition among sigma factors for core RNA polymerase (RNAP). This hypothesis will be addressed through three specific aims. (1) Define the biochemical properties of transcription initiation by E?E. ?E is a member of a highly conserved, widely distributed class of alternative sigma factors that lack several conserved regions and amino acids known to be important for specific steps in transcription initiation by other sigma factors. How these differences alter transcription initiation has not been investigated. The fundamental parameters of transcription initiation by E?E must be established before the influence of pGp can be assessed. (2) Elucidate the mechanism by which ppGpp and DksA regulate E?E activity. Since activation of E?70 and alternative sigma factors by ppGpp/DksA is not well understood, results from the proposed work will have a strong positive impact on the overall understanding of the biochemical basis of transcriptional activation by these global regulators. (3) Determine how ppGpp regulates ?E activity independently of DksA. Preliminary work suggests that a novel mechanism of regulation of ?E by ppGpp exists that cannot be explained by current models, which require DksA to act with ppGpp. This novel regulatory strategy will be explored. Because both the ?E and ppGpp systems are critical for virulence in important bacterial pathogens, a better understanding of the intersection of these responses may lead to new avenues for drug discovery and vaccine design.