RNA POLYMERASE II ELONGATION COMPLEX STRUCTURE/FUNCTION

RNA 聚合酶 II 延伸复合物结构/功能

• 批准号: 2444797
• 负责人: Daniel Reines
• 金额: $ 20.81万
• 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 2000-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2444797
• 关键词: DNA directed RNA polymerase; RNA biosynthesis; conformation; crosslink; cytogenetics; enzyme mechanism; enzyme structure; enzyme substrate complex; fluorescent in situ hybridization; gene expression; genetic mapping; genetic promoter element; genetic regulatory element; genetic transcription; human genetic material tag; laboratory rat; molecular cloning; mutant; nonhistone nucleoprotein; nucleic acid sequence; nucleic acid structure; polymerase chain reaction; site directed mutagenesis; transcription factor

Gene expression is controlled in part by regulating the ability of RNA polymerase II to transcribe full length primary transcripts. Elongation is not simply the addition of single ribonucleotide units to a growing chain; instead, it is characterized by a striking plasticity in structural and functional alternatives that RNA polymerases can assume. Recent advances have identified new structural and functional intermediates that are the target of regulatory events. The transcription process can be blocked by specific DNA elements called arrest sites within genes and DNA binding ligands that interrupt elongation. Only a few arrest sites are known and the defining DNA sequence has not been resolved. Specific elongation factors improve the efficiency of RNA chain synthesis. Two such factors, TFIIF and SIII (elongin), are implicated in human diseases including cancer, demonstrating that perturbation of transcript polymerization is potentially deleterious. These two factors increase the average chain elongation rate of RNA polymerase II. An additional elongation factor, SII, rescues "arrested" RNA polymerase iI that is unable to elongate RNA chains but remains template engaged. It does so by activating a newly described ribonuclease activity found in elongation complexes ranging from bacteria to humans. TFIIF, SII, and SIII can potentially control the output of many genes, yet virtually nothing is known about the spectrum of genes whose expression is dependent upon these, and other, elongation factors. We propose to define arrest sties by systematic mutagenesis and to establish an assay that measures their function in living cells. Mapping experiments will enable us to define the molecular architecture of the portions of RNA polymerase Ii that ar important for catalyzing chain elongation and harboring the nascent RNA. Experiments are planned to observe the changing relationship between the growing RNA chain and RNA polymerase Ii proposed to take place in complexes as they lose elongation competence and come to rely on elongation factor rescue. We will use an arrest-prone mutant RNA polymerase II identified in yeast, and yeast with other known mutations in the elongation machinery, to define the in vivo requirements for elongation factors and DNA sequences that precipitate a requirement for elongation factor assistance during gene expression. The cytogenetic location of the human SII gene will be identified to determine if it s a candidate gene for any known inheritable diseases. This study will provide valuable insight into the fundamental process of RNA synthesis which will improve our understanding of normal and disease states at the molecular level.

基因表达部分通过调节RNA的能力来控制 聚合酶II转录全长主要成绩单。 伸长是 不仅是将单个核糖核苷酸单元添加到一个生长的链中； 相反，它的特征是结构性的震撼人头 RNA聚合酶可以假设的功能替代方法。 最近的进步 已经确定了新的结构和功能性中间体 监管事件的目标。 转录过程可以阻止 特定的DNA元素在基因和DNA结合中称为逮捕位点 中断伸长的配体。 只有几个逮捕站点， 定义DNA序列尚未解决。 具体的伸长 因素提高了RNA链合成的效率。 两个这样的因素， TFIIF和SIII（弹性）与人类疾病有关 癌症，表明转录本聚合的扰动是 潜在有害。 这两个因素增加了平均链 RNA聚合酶II的伸长率。 另一个伸长因素， SII，营救“被捕”的RNA聚合酶II，无法拉长RNA 链但仍然参与模板。 它通过激活新的 描述的核糖核酸酶活性在伸长率复合物中发现 对人类的细菌。 TFIIF，SII和SIII可以潜在地控制 许多基因的输出，但实际上对频谱一无所知 表达取决于这些和其他伸长的基因 因素。 我们建议通过系统的诱变定义逮捕和 建立一个测量其在活细胞中功能的测定法。 映射 实验将使我们能够定义 RNA聚合酶II的部分对催化链很重要 伸长和携带新生的RNA。 计划进行实验 观察生长的RNA链和RNA之间的关系变化 聚合酶II提议在复合物中失去伸长时发生 能力并依靠伸长因子救援。 我们将使用 在酵母中鉴定出的容易逮捕突变体RNA聚合酶II，酵母与 伸长机械中的其他已知突变，以定义体内 对沉淀A的伸长因子和DNA序列的要求 基因表达期间伸长因子辅助的要求。 这 将确定人类SII基因的细胞遗传学位置以确定 如果它是任何已知遗传疾病的候选基因。 这项研究 将为RNA合成的基本过程提供宝贵的见解 这将提高我们对正常和疾病状态的理解 分子水平。