NOVEL CONTROL MECHANISMS IN ENDOSPORE FORMATION

描述（由申请人提供）：本研究将研究一类称为减毒或抗终止的重要机制对细菌基因的调节。这种机制通过调节位于转录起始（mRNA 合成）和被调节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。瑞士实验室发现了两个这样的系统；这些将被详细地进行生化表征。在第一个系统中，衰减调节蛋白 PyrR 通过与 Pyr mRNA 上的特定位点结合来调节嘧啶生物合成 (pyr) 基因。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 射线晶体学来进行 PyrR-RNA 相互作用的详细研究。将通过遗传方法研究转录暂停在 PyrR 作用中的作用。 PyrR 在操纵子的三个终止位点对枯草芽孢杆菌 Pyr 操纵子的整合调节将通过体内 Pyr RNA 种类的定量测量来表征。在第二个系统中，已经证明了 CTP 对枯草芽孢杆菌 PyG RNA（编码 CTP 合成酶）的 5' 前导序列的终止进行直接调节，而无需调节蛋白的参与。这种调节的机制将通过体外pyrG转录的生化分析来研究。比较基因组学表明，与枯草芽孢杆菌类似的 PyrR 依赖性过程和 PyrG 抗终止对 Pyr 基因的调节存在于许多不同的细菌属中，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重要的细菌。这项研究的最终目标是表征分枝杆菌中pyr基因的调节，其中PyrR似乎充当蛋白质合成的抑制剂。计划使用非致病性物种进行这些研究，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.