BACTERIAL REGULATORY SWITCHES--STRUCTURE AND MECHANISM

细菌通过激活转录因子来响应环境压力 使生物体适应新环境的基因。最近是这样的 很明显，普遍的机械性反应占上风，其中 外部信号通过所谓的 双部件调节开关。这些组件系统由一个 含有蛋白激酶的组氨酸，当被ATP激活时，在 外部刺激，将使一个特定的区域磷酸化，通常是 调节蛋白的氨基末端结构域，进而影响 基因的转录。这种系统存在于芽孢杆菌中。 枯草杆菌，并控制从休眠到产孢型的转变。 这项提议旨在确定 激酶激活枯草杆菌中的反应调节因子(RR)。这个 已鉴定了两种RR蛋白(Spoof和SpoOA) 并在大肠杆菌和体外培养条件下以毫克量表达 已开发了磷酸化体系。使用合成肽 RR蛋白的类似物、突变形式、各种化学分析 技术、水解解理和X射线结晶学分析 旨在明确描述Spoof/SpoOA的结构及其 磷酸化的对应物。与磷酸化的Chey相比， 鼠伤寒沙门氏菌的磷酸化恶搞有很长的半衰期(&gt； 12h)。因此，将确定磷酸化的位置，并且这 衍生物也将被结晶，以进行构象比较 用免费的蛋白质。然后，结构研究将扩展到 更复杂的SpoOA。基因工程截短最小组氨酸 将产生并结晶包含KENA的区域，并 分子图形学研究将被用于确定相互作用 在保守的Spoof/SpoOA-kinase区之间。这个!磷酸化 将对反应进行动力学分析，核苷酸结合部位，动力学 测定了金属离子的常数和参与程度。此外， 将检查反向磷酸化反应的发生情况并 构象变化将通过荧光、UV/Vis和CD来揭示 光谱测量。从这些观察中可以看到一幅详细的图景 一系列的激酶-RR相互作用将被发展并置于 总体而言，枯草杆菌的调控。结果将被用来，用来 巩固形成两个组件开关的更一般的理论 细菌对环境变化的调节反应的基础。

Bacteria respond to environmental stress by activating transcription of genes to modify the organism for the new environment. It has recently become apparent that general mechanistic response prevails in which an external signal induces the transcriptional change through a so-called two component regulatory switch. These component systems consist of a histidine containing protein kinase that when activated by ATP, after external stimulus, will phosphorylate a particular region, usually the' amino-terminal domain of a regulatory protein which in turn influences the transcription of genes. Such a system is present in Bacillus subtilis and controls the transition from dormant to sporulating forms. This proposal is designed to determine the precise mechanism by which the kinase activates the response regulators (RRs) in B. subtilis. The kinase (KinA) and two RR proteins (SpoOF and SpoOA) have been identified and expressed in milligram quantities in Escherichia coli and an in vitro phosphorylation system has been developed. Using synthetic peptide analogs, mutant forms of the RR proteins, various chemical analytical techniques, hydrolytic cleavage and X-ray crystallographic analysis it is intended to definitively describe the structure of SpoOF/SpoOA and their phosphorylated counterparts. In contrast to the phosphorylated CheY from Salmonella typhimurium the phosphorylated SpoOF has a long half life (> 12 h). Thus the site of phosphorylation will be identified and this derivative will also be crystallized to allow conformational comparisons with the free protein. Structural studies will then be extended to the more complex SpoOA. Genetically engineered truncated minimal histidine containing regions of KinA will be generated and crystallized and molecular graphics studies will be employed to, determine interactions between conserved SpoOF/SpoOA-KinA regions. The! phosphorylation reactions will be analyzed kinetically, nucleotide binding sites, kinetic constants and participation of metal ions determined. In addition, the occurrence of reverse phosphorylation reactions will be examined and conformational changes will be revealed by fluorescence, UV/Vis and CD spectrometric measurements. From these observations a detailed picture of kinase-RR interactions will be developed and placed in the context of overall B. subtilis regulation. The results will then be, used to consolidate the more general theory of two component switches forming the basis of bacterial regulatory responses to environmental changes.