INTER AND INTRAMOLECULAR COMMUNICATIONS IN TRANSCRIPTION

描述：（改编自申请人的摘要）转录是第一个 基因表达的步骤和大多数基因表达的步骤 调节发生。申请人研究的长期目标是 了解导致分子水平转录起始的步骤。 这种理解非常重要，不仅对于推进我们的基础 了解这一基本的生物过程，也可用于实践 应用程序；例如，针对转录的药物的合理设计 起始复合物或更好的表达系统的设计。这个提议 旨在填补导致以下步骤的知识中的一些重要空白 转录起始并集中于大肠杆菌 RNA 的 sigma70 亚基 聚合酶。 sigma70 亚基对于初始启动子识别至关重要， 也参与启动子 DNA 的解链，并且可能是一个重要的 高效全长转录本合成的决定因素。近期目标 该提案的主要内容是（1）确定初始认可机制 启动子DNA通过RNA聚合酶全酶，（2）确定a的性质 启动子-聚合酶复合物中的限速构象变化，以及 (3) 确定 sigma70 亚基在转录形成中的作用 复杂的伸长模式。 申请人将结合分子生物学、蛋白质化学 和荧光光谱方法来设计组件 转录起始复合物，以允许选择性观察和研究 转录起始的各个步骤。数据预计将提供 第一个对随时间变化的结构变化的三维描述 转录起始复合物，从初始启动子进化而来 识别复合体到能够生产全长的延伸复合体 转录合成。申请人希望确定关键的决定因素 初始启动子识别和生产性延伸，复合物形成。

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.