BIOCHEMISTRY OF EUKARYOTIC MESSENGER RNA SYNTHESIS

真核信使 RNA 合成的生物化学

• 批准号: 2900705
• 负责人: Ronald C. Conaway
• 金额: $ 34.16万
• 依托单位: OKLAHOMA MEDICAL RESEARCH FOUNDATION
• 依托单位国家: 美国
• 财政年份: 1989
• 资助国家: 美国
• 起止时间: 1989-09-01 至 2000-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2900705
• 关键词: DNA binding protein; DNA directed RNA polymerase; DNA footprinting; RNA biosynthesis; SDS polyacrylamide gel electrophoresis; adenosine triphosphate; adenosinetriphosphatase; chemical binding; enzyme mechanism; gel mobility shift assay; genetic promoter element; genetic regulation; genetic transcription; helicase; high performance liquid chromatography; intermolecular interaction; laboratory rabbit; laboratory rat; messenger RNA; protein kinase; protein purification; protein structure function; transcription factor

Our long range goal is to understand, at the molecular level, how transcription Of eukaryotic protein-coding genes is initiated and regulated. The goal of the research described in this proposal is to determine the enzymatic mechanism of transcription catalyzed by mammalian RNA polymerase II and a set of general transcription factors that direct accurate initiation and efficient elongation of transcripts synthesized from a large number of promoters. We have focused on rat liver as a model for our studies of the enzymology of transcription by RNA polymerase II because the general factors can be purified to homogeneity from rat liver in quantities sufficient for serious biochemical analyses of their structures and mechanisms of action. The general aims guiding the research described in this proposal remain essentially the same as those in the original proposal: (i) to reconstitute in vitro, with purified enzymes, faithful transcription of eukaryotic protein-coding genes and (ii) to use this reconstituted enzyme system to elucidate the biochemical mechanisms governing transcription initiation and elongation by mammalian RNA polymerase II. The research will be organized along the following lines: (1) Detailed analysis of the structure and function of a novel RNA polymerase II elongation factor designated Elongin (SIll) and its interactions with the product of the von Hippel-Lindau (VHL) tumor suppressor gene. (2) Purification and analysis of a newly identified elongation factor designated 5-IV. (3) Detailed analysis of the role of TFIIF in selective binding of RNA polymerase II to its promoters. (4) Investigation of the role of TFIIH in ATP-dependent activation of transcription initiation. These studies should provide a solid foundation for future investigations of the mechanisms governing eukaryotic messenger RNA synthesis, and they should also yield significant insights into the molecular basis of von Hippel-Lindau disease and its associated cancers.

我们的长期目标是在分子水平上了解如何 真核蛋白编码基因的转录被启动并且 受监管。本提案中描述的研究目标是 确定哺乳动物催化转录的酶机制 RNA 聚合酶 II 和一组指导的通用转录因子 合成转录本的准确起始和有效延伸 来自大量的发起人。我们重点关注大鼠肝脏作为模型 用于研究 RNA 聚合酶 II 的转录酶学 因为一般因子可以从大鼠肝脏中纯化至同质 其数量足以对其进行严格的生化分析 结构和作用机制。 本提案中描述的指导研究的总体目标仍然是 与原提案中的内容基本相同： (i) 用纯化的酶在体外重构，忠实转录 真核蛋白编码基因和 (ii) 使用这种重组酶 阐明控制转录的生化机制的系统 哺乳动物 RNA 聚合酶 II 的起始和延伸。研究 具体内容如下：（一）具体分析 新型RNA聚合酶II延伸因子的结构和功能 指定为 Elongin (SIll) 及其与 von 产物的相互作用 Hippel-Lindau (VHL) 肿瘤抑制基因。 (2) 纯化与分析 一个新确定的伸长因子，命名为 5-IV。 （三）详细内容 分析 TFIIF 在 RNA 聚合酶 II 选择性结合中的作用 它的发起人。 (4) TFIIH在ATP依赖性中的作用研究 转录起始的激活。 这些研究应该为未来的调查提供坚实的基础 控制真核生物信使RNA合成的机制，以及它们 还应该对 von 的分子基础产生重要的见解 希佩尔-林道病及其相关癌症。