INTER AND INTRAMOLECULAR COMMUNICATIONS IN TRANSCRIPTION

转录中的分子间和分子内通讯

• 批准号: 2903188
• 负责人: TOMASZ HEYDUK
• 金额: $ 26.47万
• 依托单位: SAINT LOUIS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-08-01 至 2003-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2903188
• 关键词: DNA; DNA directed RNA polymerase; biophysics; chemical binding; chemical kinetics; conformation; enzyme mechanism; enzyme structure; enzyme substrate complex; fluorescence resonance energy transfer; fluorescence spectrometry; genetic promoter element; genetic transcription; nucleic acid structure; stop flow technique; thermodynamics

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.

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 规则发生。申请人研究的长期目标是 理解导致分子水平转录起始的步骤。 这样的理解是非常重要的，不仅是为了推进我们的基本 这一基本生物过程的知识，但也为实际 应用;例如，转录靶向药物的合理设计 起始复合物或设计更好的表达系统。这项建议 旨在填补知识中的一些重要空白， 转录起始，并集中在E.杆菌RNA 聚合酶。σ 70亚基对于初始启动子识别是必需的， 也参与启动子DNA的解链，并且可能是一个重要的 生产性全长转录本合成的决定因素。近期目标 （1）确定初始承认机制， 启动子DNA通过RNA聚合酶全酶，（2）确定一个 启动子-聚合酶复合物中的限速构象变化，和（3） 确定sigma 70亚基在转录形成中的作用， 复合体的伸长模式。 申请人将使用分子生物学、蛋白质化学 和荧光光谱学方法来设计 转录起始复合物，允许选择性观察和研究 转录起始的各个步骤。预计这些数据将提供 第一个三维描述的时间相关的结构变化， 转录起始复合物，因为它从最初的启动子 识别复合物转化为能够生产全长的延伸复合物 转录物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。