Mechanism, Activation, and Control of rRNA Transcription

描述（由申请人提供）：负责制造蛋白质（例如核糖体RNA，核糖体蛋白，翻译因子和trna）的机器是所有生物体生长和发育的核心，其合成控制已成为分子微生物学50多年来的核心问题。最近，对大肠杆菌中负责rRNA和tRNA转录的机制的理解也变得清晰起来，可以为一般的转录机制提供基本的见解。提案中涉及的问题分为五个具体目标。在第一个目标中，我们将继续研究RNA聚合酶α亚基的c端结构域在细菌启动子功能中的作用，特别是它如何在转录起始过程中刺激异构化步骤，以及它在转录因子Fis激活中的作用。在第二个目标中，我们建议确定所谓的鉴别区（-10元件下游启动子的一部分）如何与RNAP相互作用以调节转录并确定转录起始位点的身份。在第三个目标中，我们建议确定RNAP与ppGpp和DksA这两个对细菌转录起始调控至关重要的分子的相互作用位点，以深入了解小分子转录调控的机制。在第四个目标中，我们建议确定ppGpp， DksA和转录本中初始NTP浓度调节的其他启动子，从而揭示翻译机制的合成与其他细胞过程机制的合成之间以前未被怀疑的联系。在最终目标中，我们建议确定Crl的结构和机制，这是我们最近提出的可能作为sigma亚基伴侣的小蛋白质。由于这些因子中的许多也是毒力因子，调节在细菌发病机制中起重要作用的基因的表达，近年来越来越清楚，这些研究不仅对理解转录起始机制（所有生物体基因表达的基本步骤）很重要，而且对理解传染病及其预防也很重要。

DESCRIPTION (provided by applicant): The machinery responsible for making proteins (e.g. ribosomal RNA, ribosomal proteins, translation factors, and tRNAs) is central to growth and development of all organisms, and the control of its synthesis has been a central issue in molecular microbioogy for over 50 years. More recently, it has also become clear that an understanding of the mechanisms responsible for rRNA and tRNA transcription in Escherichia coli can provide fundamental insights into the mechanism of transcription in general. The questions addressed in the proposal are divided into five specific aims. In the first aim, we will continue to study the role of the C-terminal domain of the alpha subunit of RNA polymerase in bacterial promoter function, specifically how it stimulates isomerization steps in the process of transcription initiation, as well as its role in activation by the transcription factor Fis. In the second aim, we propose to determine how the so-called Discriminator Region (the part of the promoter just downstream of the -10 element) interacts with RNAP to regulate transcription and to determine the identity of the transcription start site. In the third aim, we propose to determine the sites of interaction of RNAP with ppGpp and DksA, two molecules crucial for regulation of bacterial transcription initiation, in order to gain insights into mechanisms of transcription regulation by small molecules. In the fourth aim, we propose to determine other promoters regulated by ppGpp, DksA, and the concentration of the initial NTP in the transcript and thus to uncover previously-unsuspected connections between the synthesis of the translation machinery and the synthesis of the machineries for other cellular processes. In the final aim, we propose to determine the structure and mechanism of Crl, a small protein that we have recently proposed might function as a sigma subunit chaperone. Since many of these factors are also virulence factors, regulating expression of genes that play important roles in bacterial pathogenesis, it has become clear in recent years that these studies are important not only for understanding the mechanism of transcription initiation in general, a fundamental step in gene expression in all organisms, but also for an understanding of infectious disease and its prevention.