RNA POLYMERASE II ELONGATION COMPLEX--STRUCTURE/FUNCTION

RNA 聚合酶 II 延伸复合物——结构/功能

• 批准号: 6519456
• 负责人: Daniel Reines
• 金额: $ 26.6万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-07-01 至 2004-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6519456
• 关键词: DNA directed RNA polymerase; RNA biosynthesis; enzyme mechanism; enzyme structure; enzyme substrate complex; gene expression; genetic promoter element; genetic regulatory element; genetic transcription; laboratory mouse; nucleic acid sequence; nucleic acid structure; protein structure function; transcription factor

DESCRIPTION (adapted in part from applicant's description): This project examines the mechanism of action of the TFIIS (SII) transcript cleavage factor and its role in transcriptional regulation. SII stimulates transcription by binding to paused or arrested RNA polymerase II and activating a latent nuclease in the enzyme that cleaves the nascent transcript near its 3' end to enable readthrough of various blocks to chain elongation. This project will continue to explore SII's mechanism with a focus on its interaction with nascent RNA. The contact site of RNA on SII will be precisely mapped. Mutant SII proteins eliminating RNA contact will be generated and tested for in vitro activity and an ability to complement the phenotypes of an SII gene deletion in yeast. SII contact sites on the polymerase will be mapped by protease footprinting. Direct evidence for arrested RNA polymerase II on chromatin in vivo will be sought using nuclear run-on and chromatin immunoprecipitation assays with anti-polymerase antibodies. Genes with biased base content will be surveyed to see if NTP pools regulate elongation efficiency in a gene-specific manner in vivo. Another aim is to determine the mechanism used to regulate PUR5 expression in response to NTP pool reduction in yeast. This will include measurement of NTP pools under various growth conditions and in response to 6-azauracil, an inhibitor of UTP and GTP synthesis. Finally, mice disrupted for two SII family genes (TceaI and Tcea3) will be generated and analyzed for developmental phenotypes.

描述（部分改编自申请人的描述）：该项目 检查TFIIS（SII）转录物切割因子的作用机制 及其在转录调控中的作用。SII刺激转录， 与暂停或停滞的RNA聚合酶II结合并激活潜伏的 酶中的核酸酶，其在新生转录物的3'末端附近切割新生转录物， 使各种块的通读能够链延伸。该项目将 继续探索SII的机制，重点是其与 新生RNA RNA在SII上的接触位点将被精确定位。突变体 将生成消除RNA接触的SII蛋白，并进行体外检测 活性和补充SII基因缺失的表型的能力， 酵母聚合酶上的SII接触位点将通过蛋白酶作图 脚印RNA聚合酶II在细胞核染色质上被抑制的直接证据 体内将寻求使用核运行和染色质免疫沉淀 用抗聚合酶抗体进行测定。具有偏向性碱基含量的基因将被 调查，看看NTP池是否调节基因特异性 体内的方式。另一个目的是确定用于调节PUR 5的机制 在酵母中响应NTP池减少的表达。这将包括 在各种生长条件下和响应于 6-azauracil，UTP和GTP合成的抑制剂。最后，小鼠被打乱， 将产生两个SII家族基因（Tcea 1和Tcea 3），并分析其 发育表型