B SUBTILIS PHO SIGNAL TRANSDUCTION NETWORK

描述：双分量信号是信号的主要形式 在真核生物中有独特的例子。 对枯草芽孢杆菌的相当大的兴趣集中在鉴定 启动这一过程的环境和生理信号 孢子形成 营养和细胞周期相关的信号产生期间 晚期生长是通过相互关联的调节途径进行的， 哪些基因被表达，通常在启动时达到高潮， 孢子形成 PI对B的表征。枯草硷性 磷酸酶（APase）多基因家族的研究导致了这样的假设， 不同的环境信号由特定的双分量接收， 整合输入信息以确定响应的系统 B。枯草芽孢杆菌对磷酸盐利用率下降的影响。 本提案旨在了解三个独立的 双组分系统（PhoP-PhoR、ResD-ResE和SpoOA），每个接收 他们的具体环境信号，沟通，以实现最终的Pho 反应 PI处于幸运的位置， 信号传导电路的结构。 这提供了 测试和完善她的假设的框架。 平行路径 通过PhoR-PhoR正向调节Pho反应。 一种途径包括 ResD-ResE系统，而另一个涉及过渡状态调节器， AbrB. SpoO系统通过负调节磷酸化酶的活性来抑制Pho反应。 两条路。 将继续进行遗传学研究，以进一步确定 ResD到PhoP-PhoR活化途径中的中间体和生物化学 计划进行研究以探讨它们之间的相互作用。 阻遏物 SpoOA对AbrB活化途径的作用已被充分理解。 的PI 将使用遗传和生物化学技术来确定 SpoOA抑制resA启动子，该启动子负责 resDE基因的转录。 她将使用生物化学方法， 探索PhoR中的两个结构域以寻找可能的信号机制。 启动子 将分析已知需要PhoP激活的直接相互作用 与PhoP和可能的功能保守序列在这些 发起人。 这项建议分若干阶段。 第一是利用基因 选择或筛选以鉴定该复合物的新组分， 相互作用调节子 phoPR操纵子中有四个RNA起始位点 通过引物延伸确定的启动子区域，以及详细的 在各种突变背景下对这些进行定量分析将确定 哪些位点受哪条途径的调控。 删除分析也是 计划好了 为了表征PhoP和PhoR本身，凝胶阻滞和DNA 足迹实验都计划评估直接监管， 各种Pho调控的启动子。 PhoR包含两个区域， 与外界的相互作用（两个跨膜区域之间的区域） 和内部（第二跨膜区域和第二跨膜区域之间的区域）。 组氨酸激酶结构域）信号。 它们会发生变异， 被其他传感器激酶的相应结构域取代， 评估诱导对磷酸盐饥饿的影响。 相互作用 将使用交联来寻找蛋白质。

DESCRIPTION: Two-Component signaling is the principle form of signal transduction in procaryotes with distinctive examples in eucaryotes. Considerable interest in Bacillus subtilis has been focused on identifying the environmental and physiological signals that initiate the process of sporulation. Nutritional and cell cycle related signals produced during late growth are processed by interconnected regulatory pathways that control which genes are expressed, often culminating in the initiation of sporulation. The PI's characterization of the B. subtilis alkaline phosphatase (APase) multigene family has led to the hypothesis that different environmental signals are received by specific two-component systems that integrate the incoming information to determine the response of B. subtilis to declining phosphate availability. The present proposal is designed to understand how three separate two-component systems (PhoP-PhoR, ResD-ResE, and SpoOA), each receiving their specific environmental signals, communicate to achieve the final Pho response. The PI is in the fortunate position of having the basic architecture of the signal transduction circuitry in place. This provides a framework for testing and refining her hypothesis. Parallel pathways positively regulate the Pho response via PhoR-PhoR. One pathway includes the ResD-ResE system, while the other involves a transition state regulator, AbrB. The SpoO system represses the Pho response by negatively regulating both pathways. Continuing genetic studies will be used to further identify intermediates in the ResD to PhoP-PhoR activation pathway and biochemical studies are planned to probe the interactions between them. The repressor function of SpoOA on the AbrB activation pathway is well udnerstood. The PI will use genetic and biochemical techniques to determine the mechanism of SpoOA repression of the resA promoter which is responsible for the transcription of the resDE genes. She will use biochemical approaches to explore two domains in the PhoR for pssible signaling mechanisms. Promoters known to require PhoP for activation will be analyzed for direct interaction with PhoP and for possible function of conserved sequences within these promoters. This proposal has a number of phases. The first is to employ genetic selections or screenings to identify new components of this complex, interacting regulon. There are four RNA start sites in the phoPR operon promoter region as determined by primer extension, and a detailed quantitative analysis of these in various mutant backgrounds will determine which sites are regulated by which pathway. A deletion analysis is also planned. To characterize PhoP and PhoR themselves, gel retardation and DNA footprinting experiments are all planned to assess direct regulation on various Pho regulated promoters. PhoR contains two regions where interaction with external (the region between two membrane-spanning regions) and internal (the region between the second membrane-spanning region and the histidine kinase domain) signals could occur. These will be mutated or substituted with corresponding domains of other sensor kinases and the effect of the induction upon phosphate starvation assessed. The interacting proteins will be sought using cross-linking.