BIOCHEMISTRY OF EUKARYOTIC MESSENGER RNA SYNTHESIS

真核信使 RNA 合成的生物化学

• 批准号: 2180969
• 负责人: Ronald C. Conaway
• 金额: $ 26.66万
• 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/2180969
• 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理解 真核蛋白编码基因的转录是如何启动的 并受到监管。这项提案中描述的研究目标是 确定催化转录起始的酶机制 由RNA聚合酶II和一组五种基本的启动因子 都是从肝脏中提纯的。这些因子指导转录 从许多启动子的核心区启动，包括那些 病毒、淋巴因子和肝脏基因。以大鼠肝脏为模型 因为启动因子可以被提纯到 从大鼠肝脏提取的同质性足以满足严重的 对它们的结构和作用机制进行生化分析。 这项提案的总体目标基本上与 在前一项提议中描述的：在体外重建，与 纯化的酶，真核生物转录的准确启动 蛋白编码基因，并利用这个重组系统阐明 RNA聚合酶引发的生化机制II. 研究将按照以下思路组织：(1)详细 精华的结构和活性的表征 各种因素。(2)制定提纯高铁的程序 分子量、内源性TATA因子。(3)调查 ATP激活启动的机制。(4)详细分析 RNA聚合酶II及其启动因子与核糖核酸的相互作用 在预引发复合体的组装过程中，相互作用于DNA。 这些研究应为今后对 真核蛋白编码基因转录的机制 受监管的。它们还应提供有关 一大类具有生物医学重要性的基因。