Transcription termination and its regulation in E. Coli

描述（由申请人提供）：转录终止是一个过程，伸长复合物（EC）解离成RNA转录物，DNA模板和RNA聚合酶（RNAP）响应内在信号或特定因素。Rho终止因子是调节肠杆菌基因表达所必需的，是特异性抗生素的靶点。在过去的三十年里，Rho已经被深入研究，然而，实际的终止过程，即Rho破坏EC的机制，仍然未知。此外，体内大多数Rho终止位点的身份以及Rho在细胞适应环境变化中的作用仍然未知。这项工作的长期目标是提供大肠杆菌中rho依赖性终止的全面生理和机制描述，以及特定宿主和噬菌体蛋白对其调节的机制。具体来说，我们建议：1)确定RNAP在终止过程中的构象变化，以及某些RNAP结构域在Rho终止中的作用。2)确定大肠杆菌S4和噬菌体N蛋白如何修饰EC，使其对Rho终止具有抗性。3)确定我们在前期研究中发现的新型抗rho因子的生理作用和机制。4)在基因组尺度上确立Rho在基因调控中的作用。拟议的研究对人类健康的意义是多方面的。对终止/抗终止过程的完整结构理解将允许设计小分子模拟物和抑制剂，改变细菌基因表达模式或过早中断必要基因的转录，从而作为新型抗菌剂。已经描述了专门针对Rho的抗生素的例子。此外，更好地了解抗终止机制将为构建过量生产必需膳食补充剂和其他生物活性化合物的菌株提供最佳策略。最后，由于真核RNAP与细菌RNAP具有基本的序列和结构同源性，因此EC稳定和不稳定的基本机制必须相似。因此，提出的实验也将提供真核生物转录终止的基本机制的见解。我们将探讨大肠杆菌中Rho终止和抗终止的机制。拟议的研究对人类健康的意义是多方面的。对终止/反终止过程的完整结构理解将允许设计终止过程的小分子模拟物和抑制剂，从而改变细菌基因表达模式或过早中断必要基因的转录，从而作为新型抗菌剂。此外，更好地了解抗终止机制将为构建过量生产必需膳食补充剂（维生素，氨基酸等）和其他重要生物活性化合物的菌株提供最佳策略。最后，提出的实验也将提供真核生物转录终止的基本机制的见解。

DESCRIPTION (provided by applicant): Transcription termination is a process whereby the elongation complex (EC) dissociates into RNA transcript, DNA template, and RNA polymerase (RNAP) in response to intrinsic signals or specific factors. Rho termination factor is essential in regulating gene expression in Enterobacter and is a target for specific antibiotics. Rho has been intensively studied in the last three decades, however, the actual termination process, i.e. the mechanism by which Rho disrupts the EC, remains unknown. Moreover, the identity of most Rho termination sites in vivo and the role of Rho in cell's adaptation to environmental changes remain unknown. The long-term objective of the proposed work is to provide a comprehensive physiological and mechanistic description of Rho-dependent termination in Escherichia coli and the mechanism of its regulation by particular host and phage proteins. Specifically we propose to: 1) Determine conformational changes in RNAP that accompany the termination process, and the role of certain RNAP domains in Rho termination. 2) Determine how E.coli S4 and phage l N proteins modify the EC rendering it resistant to Rho termination. 3) Determine the physiological role and mechanism of novel anti-Rho factors that we have identified in preliminary studies. 4) Establish the role of Rho in gene regulation on a genomic scale. The significance of proposed research for human health is several-fold. Complete structural understanding of termination/antitermination processes would allow for designing small molecule mimics and inhibitors that change the pattern of bacterial gene expression or interrupt transcription of essential genes prematurely, and thus serve as novel antimicrobials. Examples of antibiotics that specifically target Rho have been already described. Furthermore, better understanding the mechanisms of antitermination would suggest the optimal strategies for constructing bacterial strains that overproduce essential dietary supplements and other biologically active compounds. Finally, since eukaryotic RNAPs share basic sequence and structural homologies with bacterial RNAP, the fundamental mechanism of the EC stabilization and destabilization must be similar. Therefore, proposed experiments will also provide insight to the basic mechanisms of eukaryotic transcription termination. We will explore the mechanism of Rho termination and antitermination in E.coli. The significance of proposed research for human health is several-fold. Complete structural understanding of termination/antitermination processes would allow designing small molecule mimics and inhibitors of the termination process that change the pattern of bacterial gene expression or interrupt transcription of essential genes prematurely, and thus serve as novel antimicrobials. Furthermore, better understanding the mechanisms of antitermination would suggest optimal strategies for constructing bacterial strains that overproduce essential dietary supplements (vitamins, amino acids, etc.) and other important biologically active compounds. Finally, the proposed experiments will also provide insight to the basic mechanisms of eukaryotic transcription termination.