INTER AND INTRAMOLECULAR COMMUNICATIONS IN TRANSCRIPTION

描述：(改编自申请者的摘要)笔录是第一 基因表达的步骤和大多数基因表达的步骤 监管就会发生。申请者研究的长期目标是 在分子水平上理解导致转录启动的步骤。 这样的认识是非常重要的，不仅对于推进我们的基本 了解这一基本的生物过程，也是为了实用 应用；例如，针对转录的药物的合理设计 引发复合体或设计更好的表达系统。这项建议 旨在填补导致以下步骤的知识中的一些重要空白 转录启动，并集中在大肠杆菌RNA的sigma70亚基上 聚合酶。Sigma70亚单位对于初始启动子识别是必不可少的， 也参与了启动子DNA的融化，很可能是一个重要的 生产性全长转录合成的决定因素。眼前的目标 这项建议的目的是(1)确定初始承认的机制 用RNA聚合酶全酶检测启动子DNA，(2)确定一种 启动子-聚合酶复合体的限速构象变化，以及(3) 确定Sigma70亚单位在转录形成中的作用 伸长模式中的复杂性。 申请者将使用分子生物学、蛋白质化学 和荧光光谱学方法来设计组件 转录起始复合体，以允许选择性地观察和研究 转录启动的各个步骤。预计这些数据将提供 第一个三维描述与时间相关的结构变化 转录起始复合体，因为它从初始启动子进化而来 识别复合体到能够产生全长的伸长复合体 文字记录合成。申请者希望确定关键的决定因素 启动子的初始识别和产生性延伸，形成复合体。

DESCRIPTION: (adapted from applicant's abstract) Transcription is the first step of gene expression and the step at which the majority of gene expression regulation occurs. The long-term goal of the applicant's research is to understand the steps leading to transcription initiation at a molecular level. Such an understanding is of great importance, not only for advancing our basic knowledge of this fundamental biological process, but also for practical applications; e.g., the rational design of drugs targeted to transcription initiation complexes or the design of better expression systems. This proposal is aimed at filling some important voids in knowledge of steps leading to transcription initiation and is focused on the sigma70 subunit of E. coli RNA polymerase. The sigma70 subunit is essential for initial promoter recognition, is also involved in melting of promoter DNA, and is likely to be an important determinant of productive full-length transcript synthesis. The immediate goals of this proposal are to (1) determine the mechanism of initial recognition of promoter DNA by RNA polymerase holoenzyme, (2) determine the nature of a rate-limiting conformational change in promoter-polymerase complex, and (3) determine the role of sigma70 subunit in the formation of a transcribing complex in the elongation mode. The applicant will use a combination of molecular biology, protein chemistry and fluorescence spectroscopy methods to engineer the components of transcription initiation complexes, to allow selective observation and study of individual steps of transcription initiation. The data are expected to provide the first three-dimensional description of time-dependent structural changes in transcription initiation complex, as it evolves from the initial promoter recognition complex to an elongation complex capable of productive full-length transcript synthesis. The applicant hopes to identify critical determinants of initial promoter recognition and productive elongation, complex formation.