BACTERIAL REGULATORY SWITCHES--STRUCTURE AND MECHANISM

细菌对环境胁迫的反应是通过激活 基因来改变生物体以适应新的环境。它最近 变得明显，普遍的机械反应占主导地位， 外部信号通过所谓的 双组分调节开关。 这些组件系统由一个 含组氨酸的蛋白激酶，当被ATP激活时， 外部刺激，会磷酸化一个特定的区域，通常是" 调节蛋白的氨基末端结构域，其反过来影响 基因的转录 这种系统存在于芽孢杆菌属中。 枯草芽孢杆菌和控制从休眠到孢子形成形式的过渡。 这项建议旨在确定 激酶激活B中的应答调节因子（RR）。枯草杆菌。 的 已经鉴定了一种激酶（KinA）和两种RR蛋白（SpoOF和SpoOA 并在大肠杆菌中以毫克量表达， 磷酸化系统已经开发。 使用合成肽 类似物，RR蛋白的突变形式，各种化学分析 技术，水解裂解和X射线晶体学分析，它是 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， 鼠伤寒沙门氏菌磷酸化的SpooF具有长的半衰期（> 12 h）。 因此，将鉴定磷酸化位点， 衍生物也将结晶，以允许构象比较 和游离蛋白质。 结构研究将扩展到 更复杂的SpoOA 基因工程截短的最小组氨酸 含有KinA的区域将被产生和结晶， 分子图形学研究将用于确定相互作用 SpoOF/SpoOA-KinA区域之间。 了！磷酸化 反应将进行动力学分析，核苷酸结合位点，动力学 确定金属离子的常数和参与度。 此外该 将检查反向磷酸化反应的发生， 通过荧光、UV/维斯和CD等方法研究了分子的构象变化 光谱测量。 从这些观察中， 将在以下背景下开发和放置激酶-RR相互作用的研究： 整体B。枯草杆菌调节 结果将用于 巩固了更一般的理论，两个组成部分的开关，形成 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.