ASSEMBLY OF POLYMERASE SPECIFIC TRANSCRIPTION COMPLEXES

聚合酶特异性转录复合物的组装

• 批准号: 2190083
• 负责人: Michael D. Brenowitz
• 金额: $ 14.91万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE
• 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 1998-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2190083
• 关键词: DNA; DNA binding protein; DNA directed RNA polymerase; DNA footprinting; acidity /alkalinity; analytical ultracentrifugation; chemical binding; chemical kinetics; chemical stability; conformation; divalent cations; fluorescence spectrometry; gel mobility shift assay; genetic promoter element; genetic transcription; hydropathy; intermolecular interaction; ionic bond; ionic strengths; mutant; nucleic acid structure; protein structure function; site directed mutagenesis; thermodynamics; transcription factor

Accurate transcription by each of the three eukaryotic nuclear RNA polymerases (pols) requires the assembly and recognition of unique transcription complexes on the promoters of appropriate genes. These complexes have at least one common component, the TATA box binding protein (TBP). In addition, complexes recognized by pols Il and III contain homologous proteins (TFIIB and TFIIIB70) that are known or believed, respectively, to interact directly with TBP. The properties of these molecules raise an important question: What mechanisms exist to ensure that a transcription complex with the correct polymerase-specificity will assemble on a particular gene? The long term goal of this proposal is to describe in quantitative terms, the biochemical basis for the assembly of RNA polymerase-specific transcription complexes. Initially, we propose to carry out quantitative analyses of the thermodynamics and kinetics of TBP-TFIIB and TBP-TFIIIB70 associations with model pol II and pol III promoters. Specially adapted enzymatic and chemical protection methods will be used for these studies. We will also analyze the assembly of TBP-TFIIIB70 and TBP-TFIIB complexes in solution by analytical ultracentrifugation. These experiments will be facilitated by using spectroscopically labeled TBP. Together, these studies will define quantitatively the chemical interactions that characterize the assembly of RNA polymerase-specific complexes. In the second phase of the work, spectroscopically-labeled transcription factors will be used to follow conformational changes of the TBP-TFIIB and TBP-TFIIIB70 complexes in solution and after binding to specific DNA templates. In the latter case, conformation changes to the DNA will also be followed using appropriate chemical and enzymatic probes. We will then correlate the conformational changes in the proteins and the DNA with specific kinetic or binding events resolved in the initial phase of the proposal. These data will provide knowledge of the mechanistic differences underlying the unique specificity of the complexes.

三个真核核RNA的精确转录 聚合酶（POLS）需要组装和认可独特 适当基因的启动子上的转录复合物。这些 复合物至少具有一个常见的成分，即TATA盒结合蛋白 （TBP）。 此外，由POLS IL和III识别的复合物包含 同源蛋白（TFIIB和TFIIIB70），已知或相信 分别与TBP直接互动。这些特性 分子提出了一个重要的问题：存在哪些机制来确保 具有正确聚合酶特异性的转录复合物将 组装特定基因？该提议的长期目标是 用定量术语描述组装的生化基础 RNA聚合酶特异性转录复合物。 最初，我们建议对 TBP-TFIIB和TBP-TFIIIB70关联的热力学和动力学 与Pol II和Pol III启动子一起使用。特别适应酶促和 这些研究将使用化学保护方法。我们也会 分析溶液中TBP-TFIIIB70和TBP-TFIIB复合物的组装 通过分析性超速离心。这些实验将有助于 通过使用光谱标记的TBP。这些研究将在一起 定量定义表征的化学相互作用 RNA聚合酶特异性复合物的组装。 在第二阶段，光谱标记的转录 因素将用于遵循TBP-TFIIB的构象变化和 溶液中的TBP-TFIIIB70复合物和与特定DNA结合后 模板。 在后一种情况下，DNA的构象也会变化 遵循适当的化学和酶探针。然后我们会 将蛋白质和DNA的构象变化与 特定的动力学或结合事件在初始阶段解决 提议。 这些数据将提供有关机械的知识 复合物独特特异性的差异。