BIOCHEMISTRY OF EUKARYOTIC MESSENGER RNA SYNTHESIS

真核信使 RNA 合成的生物化学

• 批准号: 2392068
• 负责人: Ronald C. Conaway
• 金额: $ 31.6万
• 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/2392068
• 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， 启动真核生物蛋白质编码基因的转录， 监管.本提案中所述研究的目标是 确定哺乳动物细胞催化转录的酶机制 RNA聚合酶II和一组一般的转录因子， 合成的转录物的准确起始和有效延伸 大量的推广者。我们以大鼠肝脏为模型， 用于研究RNA聚合酶II的转录酶学 由于一般因子可以从大鼠肝脏中纯化至均一， 其数量足以对其进行严格的生化分析， 行动的结构和机制。 指导本建议中所述研究的总体目标仍然是 基本上与原来的建议相同：（i） 用纯化的酶在体外重建，忠实地转录 真核生物蛋白质编码基因和（ii）使用这种重构的酶 系统来阐明控制转录的生化机制 通过哺乳动物RNA聚合酶II的起始和延伸。研究 将沿着以下路线组织：（1）详细分析 一种新RNA聚合酶II延伸因子的结构和功能 命名为延伸蛋白（SIll）及其与血管生成因子的产物的相互作用 Hippel-Lindau（VHL）肿瘤抑制基因。(2)纯化和分析 一种新发现的延伸因子命名为5-IV (3)详细 分析TFIIF在RNA聚合酶II选择性结合至 的推动者。(4)研究TFIIH在ATP依赖性 转录起始的激活。 这些研究为今后的调查研究奠定了坚实的基础 真核生物信使RNA合成的机制， 也应该产生重要的见解的分子基础冯 Hippel-Lindau病及其相关癌症