BIOCHEMISTRY OF EUKARYOTIC MESSENGER RNA SYNTHESIS

我们的长期目标是在分子水平上了解 真核蛋白质编码基因的转录被启动并 受监管的。这项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和一组通用的转录因子 合成转录本的准确起始性和高效延伸性 来自一大批发起人。我们将重点放在大鼠肝脏作为模型 为我们研究RNA聚合酶II转录的酶 因为可以从大鼠肝脏中提纯到均一的一般因子 其数量足以对其进行认真的生化分析 结构和作用机制。 指导本提案中描述的研究的总体目标仍然是 基本与原建议相同：(I) 体外重组，用纯化的酶，忠实地转录 真核蛋白质编码基因和(Ii)使用该重组酶 用于阐明转录的生化机制的系统 哺乳动物RNA聚合酶的启动和延伸II.研究 将按照以下思路组织：(1)详细分析 一种新的RNA聚合酶II延伸因子的结构和功能 指定的Elongin(SILL)及其与冯氏产物的相互作用 Hippel-Lindau(VHL)肿瘤抑制基因。(2)提纯和分析 一种新发现的延伸率为5-IV。(3)详细说明 TFIIF在RNA聚合酶II选择性结合中的作用分析 它的推动者。(4)探讨TFIIH在ATP依赖中的作用 转录启动的激活。 这些研究应为今后的调查提供坚实的基础。 控制真核信使RNA合成的机制，以及它们 也应该对von的分子基础有重要的见解 希佩尔-林道病及其相关癌症。

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.