BIOCHEMISTRY OF EUKARYOTIC MESSENGER RNA SYNTHESIS

真核信使 RNA 合成的生物化学

• 批准号: 3299909
• 负责人: Ronald C. Conaway
• 金额: $ 23.37万
• 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 1996-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3299909
• 关键词: DNA directed RNA polymerase; DNA footprinting; RNA biosynthesis; SDS polyacrylamide gel electrophoresis; adenosine triphosphate; adenosinetriphosphatase; chemical binding; enzyme mechanism; gel filtration chromatography; gel mobility shift assay; genetic promoter element; genetic regulation; genetic transcription; helicase; high performance liquid chromatography; intermolecular interaction; laboratory rabbit; laboratory rat; liver; messenger RNA; nuclear runoff assay; nucleic acid sequence; phosphorylation; protein purification; protein reconstitution; protein structure function; transcription factor; western blottings

The long-range goal of this research 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 initiation catalyzed by RNA polymerase II and a set of five essential initiation factors that have been purified from liver. These factors direct transcription initiation from the core regions of many promoters, including those of viral, lymphokine, and liver genes. Rat liver has been chosen as a model for these studies because the initiation factors can be purified to homogeneity from rat liver in quantities sufficient for serious biochemical analysis of their structures and mechanisms of action. The overall aims of this proposal remain essentially the same as those described in the previous proposal: to reconstitute in vitro, with purified enzymes, accurate initiation of transcription of eukaryotic protein-coding gene and to use this reconstituted system to elucidate the biochemical mechanism of initiation by RNA polymerase II. The research will be organized along the following lines: (1) Detailed characterization of the structure and activities of the essential factors. (2) Development of procedures for purification of a high molecular weight, endogenous TATA-factor. (3) Investigation of the mechanism by which ATP activates initiation. (4) Detailed analysis of the interactions of RNA polymerase II and the initiation factors with each other an with DNA during assembly of the preinitiation complex. These studies should provide a foundation for future analysis of the mechanisms by which transcription of eukaryotic protein-coding genes is regulated. They should also yield information about the expression of a large class of genes that have biomedical importance.

这项研究的长期目标是在分子水平上了解 水平，真核生物蛋白质编码基因的转录是如何启动的 和监管。本提案中所述研究的目标是 确定转录起始催化的酶机制 由RNA聚合酶II和一组五个必需的起始因子， 已经从肝脏中提纯出来了这些因子指导转录 从许多启动子的核心区域启动，包括 病毒、淋巴因子和肝脏基因。以大鼠肝脏为模型， 因为起始因子可以被纯化， 大鼠肝脏中的均匀性，数量足以进行严重 它们的结构和作用机制的生化分析。 本建议的总体目标与那些建议基本相同， 在先前的提案中描述：在体外重组， 纯化的酶，真核生物转录的准确起始 蛋白质编码基因，并使用此重组系统来阐明 RNA聚合酶II启动的生化机制。的 研究将按照以下思路沿着组织：（1）详细 的结构和活动的基本特征 因素(2)高浓度的苯并咪唑的纯化程序的开发 分子量，内源性TATA因子。(3)调查 ATP激活起始的机制。(4)仔细的分析 RNA聚合酶II和起始因子的相互作用 在前起始复合物的组装过程中，它们彼此和DNA结合。 这些研究将为今后分析 真核生物蛋白质编码基因的转录机制是 监管.它们还应该产生关于 具有生物医学重要性的一大类基因。