NOVEL CONTROL MECHANISMS IN ENDOSPORE FORMATION

描述（由申请人提供）：本研究将通过称为衰减或抗终止的一类重要机制研究细菌基因的调节。这些机制通过调节转录起始（mRNA合成）和被调控基因编码区之间的转录终止信号来控制基因表达。它们在细菌中非常普遍。瑞士实验室已经发现了两个这样的系统；这些将被详细的生物化学表征。在第一个系统中，衰减调节蛋白PyrR通过结合pyr mRNA上的特定位点来调节嘧啶生物合成（pyr）基因。对PyrR-RNA相互作用的详细研究将通过使用表面等离子体共振的结合研究和PyrR-RNA复合物的高分辨率x射线晶体学进行。转录暂停在PyrR作用中的作用将通过遗传学方法进行研究。PyrR对枯草芽孢杆菌pyr操纵子的三个终止位点的综合调控将通过体内pyr RNA种类的定量测量来表征。在第二个系统中，已经证明CTP直接调节枯草芽孢杆菌pyg RNA（编码CTP合成酶）的5'引线终止，而不涉及调节蛋白。这一调控机制将通过pyrG体外转录的生化分析来研究。比较基因组学表明，在许多不同的细菌属中都发现了pyr依赖过程和类似枯草芽孢杆菌的pyr抗终止对pyr基因的调控，包括许多抗生素耐药性日益成为临床问题的致病细菌和其他在发酵和生物技术中重要的细菌。本研究的最终目的是表征分枝杆菌中pyr基因的调控，其中PyrR似乎作为蛋白质合成的抑制剂。计划在这些调查中使用非致病性物种，但分枝杆菌是结核病和麻风病的病原体。

DESCRIPTION (provided by applicant): This research will study the regulation of bacterial genes by an important class of mechanisms called attenuation or antitermination. Such mechanisms control gene expression by regulating transcription termination signals that lie between the start of transcription (mRNA synthesis) and the coding regions of the genes being regulated. They are very widespread in bacteria. Two such systems have been discovered in the Switzer laboratory; these will be biochemically characterized in detail. In the first system the attenuation regulatory protein PyrR regulates pyrimidine biosynthetic (pyr) genes by binding to specific sites on pyr mRNA. A detailed study of PyrR-RNA interaction will be undertaken by binding studies using surface plasmon resonance and by high-resolution x-ray crystallography of PyrR-RNA complexes. The role of transcriptional pausing in PyrR action will be studied by genetic methods. The integrated regulation of the B. subtilis pyr operon by PyrR at three termination sites in the operon will be characterized by quantitative measurements of pyr RNA species in vivo. In the second system direct regulation of termination in the 5' leader of B. subtilis pyrG RNA (encoding CTP synthetase) by CTP without involvement of a regulatory protein has been demonstrated. The mechanism of this regulation will be studied by biochemical analysis of pyrG transcription in vitro. Comparative genomics demonstrates that the regulation of pyr genes by PyrR-dependent processes and pyrG antitermination similar to B. subtilis are found in many diverse bacterial genera, including many disease-causing bacteria in which antibiotic resistance is a growing clinical problem and others that are important in fermentation and biotechnology. A final objective of this research is to characterize the regulation of pyr genes in Mycobacteria, in which PyrR appears to act as an inhibitor of protein synthesis. It is planned to use nonpathogenic species for these investigations, but Mycobacteria are the agents of tuberculosis and leprosy.