NOVEL CONTROL MECHANISMS IN ENDOSPORE FORMATION

描述(申请人提供)：这项研究将研究一类重要的机制，称为衰减或抗终止对细菌基因的调节。这种机制通过调节转录终止信号来控制基因的表达，转录终止信号位于转录开始(mRNA合成)和被调控基因的编码区之间。它们在细菌中非常普遍。斯威策实验室已经发现了两个这样的系统；这两个系统将进行详细的生化表征。在第一个系统中，衰减调节蛋白PYRR通过与PYR基因上的特定位点结合来调节PYR基因。将通过使用表面等离子激元共振的结合研究和通过高分辨率的PyrR-RNA络合物的X射线结晶学来详细研究PyrR-RNA相互作用。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在操纵子的三个末端对B.subtilis PYR操纵子的整合调控将通过体内PYR RNA物种的定量测量来表征。在第二个系统中，已经证明了CTP不参与调控蛋白的参与，而直接调节枯草杆菌pyrG RNA(编码CTP合成酶)5‘前导的终止。这种调控的机制将通过体外转录的生物化学分析来研究。比较基因组学表明，在许多不同的细菌属中都发现了通过依赖于PyrR的过程来调节pyr基因的过程和类似于枯草杆菌的pyrG抗终止作用，包括许多致病细菌，其中抗生素耐药性是一个日益严重的临床问题，以及其他在发酵和生物技术中很重要的细菌。这项研究的最后一个目标是表征分枝杆菌中PYR基因的调节，其中PYR似乎是蛋白质合成的抑制因子。计划使用非致病物种进行这些研究，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.