INTER & INTRAMOLECULAR COMMUNICATIONS IN TRANSCRIPTION

描述（由申请人提供）：转录起始是细胞过程调节的一个主要点。本应用程序的长期目标是详细了解转录起始的所有步骤，在这个过程中，DNA依赖酶RNA聚合酶首先定位启动子，然后在随后的异构化步骤中熔化转录起点附近的DNA双链片段，以暴露DNA模板链。在分子水平上理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，由于细菌RNA聚合酶具有显著的结构和功能特性，细菌转录机制是药物发现的一个有吸引力的靶点。因此，了解细菌转录将有助于发现新的抗生素，由于耐药微生物的问题日益严重，这是一个重要的健康相关问题。该提案的重点是大肠杆菌RNA聚合酶一个亚基，在最近的研究中显示在转录起始的所有步骤中起着密切的作用。三个目标将解决有关该亚基在RNA聚合酶发现和熔化启动子DNA中的作用的基本问题：目标#1。确定转录泡形成起始的分子机制。目标# 2。确定西格玛区4 - β亚基“皮瓣”结构域接触的功能作用。目标# 3。确定RNA聚合酶搜索启动子时sigma - DNA接触的相对重要性。

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.