INTER & INTRAMOLECULAR COMMUNICATIONS IN TRANSCRIPTION

描述(由申请人提供)：转录启动是细胞过程的一个主要调节点。这项应用的长期目标是详细了解转录启动所涉及的所有步骤，在这个过程中，DNA依赖的酶RNA聚合酶首先定位启动子，并在随后的异构化步骤中熔化转录起始点附近的一段DNA双链，以暴露DNA的模板链。在分子水平上了解转录对于推进这一基本细胞过程的基本知识将是重要的。此外，细菌转录机制是一个有吸引力的药物发现的目标，因为细菌RNA聚合酶的结构和功能特性具有显著的保守性。因此，了解细菌的转录将有助于发现新的抗生素，这是一个重要的健康问题，因为耐药微生物的问题越来越多。该提案的重点是大肠杆菌RNA聚合酶，这是一种在最近的研究中被证明在转录启动的所有步骤中发挥密切作用的亚基。关于该亚基在RNA聚合酶发现和融化启动子DNA中的作用，我们将寻求三个基本问题：目的#1.确定转录泡泡形成的分子机制。目的#2.确定Sigma区4-β亚单位“瓣”结构域接触的功能作用。目的#3.确定Sigma-DNA接触对RNA聚合酶搜索启动子的相对重要性。 转录起始的分子事件是复杂的，涉及到大的多组分复合体。因此，将使用结合生物物理学(荧光和质谱学)、生物化学和分子生物学方法的多学科方法。预计将对几种蛋白质-蛋白质和蛋白质-DNA接触在RNA聚合酶功能中的作用有详细的了解。

DESCRIPTION (provided by applicant): Transcription initiation is a major point of regulation of cellular processes. The long-term goal of this application is to obtain a detailed understanding of all steps involved in initiation of transcription, a process in which DNA-dependent enzyme RNA polymerase first locates promoter and in subsequent isomerization step melts a segment of DNA duplex in the vicinity of transcription start point to expose the template strand of DNA. Understanding transcription at a molecular level will be important for advancing the basic knowledge of this fundamental cellular process. Additionally, bacterial transcription machinery is an attractive target for drug discovery due to a remarkable conservation of structural and functional properties among bacterial RNA polymerases. Understanding bacterial transcription will thus aid in discovery of new antibiotics, an important health related issue due to the increasing problems with drug resistant microorganisms. The proposal is focused on E. coli RNA polymerase a subunit that in recent studies was shown to play intimate roles in all steps of transcription initiation. Three aims addressing fundamental issues concerning the role of this subunit in finding and melting of promoter DNA by RNA polymerase will be pursued: Aim #1. To determine molecular mechanism of initiation of transcription bubble formation. Aim #2. To determine functional roles of sigma region 4 - beta subunit "flap" domain contact. Aim #3. To determine the relative importance of sigma - DNA contacts for promoter search by RNA polymerase. Molecular events of transcription initiation are complex and involve large multi component complexes. Thus, a multi disciplinary approach combining biophysical (fluorescence and mass spectroscopy), biochemical, and molecular biology methods will be used. It is expected that a detailed understanding of the role of several protein-protein and protein-DNA contacts in RNA polymerase function will be obtained.