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.

基因表达部分是通过调节RNA的能力来控制的。 聚合酶II转录全长初级转录物。 伸长率 而不是简单地将单个核苷酸单元添加到生长链上; 相反，它的特点是在结构和 RNA聚合酶可以承担的功能替代品。 最新进展 已经确定了新的结构和功能中间体， 监管事件的目标。 转录过程可以被阻断， 特定的DNA元件，称为基因内的阻滞位点和DNA结合 中断延伸的配体。 只有少数几个逮捕地点是已知的， 定义DNA序列还没有解决。 延伸率 这些因素提高了RNA链合成的效率。 这两个因素， TFIIF和SIII（延伸蛋白）与人类疾病有关，包括 癌症，表明转录聚合的扰动是 可能有害。 这两个因素增加了平均链 RNA聚合酶II的延伸率。 附加的伸长因子， SII，拯救不能延长RNA的“被捕”RNA聚合酶iI 链，但保持模板接合。 它通过激活一个新的 描述了在延伸复合物中发现的核糖核酸酶活性， 细菌对人类 TFIIF、SII和SIII可以潜在地控制 许多基因的输出，但几乎没有什么是已知的频谱， 其表达依赖于这些和其他延伸的基因 因素 我们建议通过系统诱变来定义逮捕站， 建立一种检测方法来测量它们在活细胞中的功能。 映射 实验将使我们能够定义的分子结构的 RNA聚合酶Ii的部分，其对于催化链 延伸并携带新生RNA。 实验计划， 观察生长的RNA链与RNA之间的变化关系 聚合酶Ii提出发生在复合物中，因为它们失去延伸 能力和来依靠延长因子救援。 我们将使用 在酵母中鉴定的易于抑制的突变型RNA聚合酶II， 延伸机制中的其他已知突变，以定义体内 对延伸因子和DNA序列的要求， 在基因表达期间需要延伸因子辅助。 的 将鉴定人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。 本研究 将提供有价值的见解的基本过程的RNA合成 这将提高我们对正常和疾病状态的理解， 分子水平。