RNA POLYMERASE II ELONGATION COMPLEX STRUCTURE/FUNCTION

RNA 聚合酶 II 延伸复合物结构/功能

基本信息

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
项目类别：
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

项目摘要

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 转录全长初级转录本。伸长率是不仅仅是将单个核糖核苷酸单元添加到不断增长的链中；相反，它的特点是在结构和结构方面具有惊人的可塑性。 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基因和 DNA 结合中称为捕获位点的特定 DNA 元件中断延伸的配体。仅知道几个逮捕地点定义的 DNA 序列尚未解决。比伸长率因素提高了RNA链合成的效率。两个这样的因素， TFIIF 和 SIII（elongin）与人类疾病有关，包括癌症，证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。额外的伸长系数， SII 拯救了无法延长 RNA 的“被捕”RNA 聚合酶 iI 链但保持模板接合。它通过激活新的描述了在延伸复合物中发现的核糖核酸酶活性，范围为细菌对人类。 TFIIF、SII 和 SIII 可以潜在地控制许多基因的输出，但实际上对基因谱一无所知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巢穴并建立一种测量其在活细胞中功能的测定方法。测绘实验将使我们能够定义分子结构 RNA 聚合酶 Ii 中对催化链很重要的部分延伸并容纳新生RNA。计划进行实验观察不断增长的RNA链与RNA之间关系的变化聚合酶 Ii 预计在复合物失去伸长时发生能力并开始依赖伸长因子救援。我们将使用一个在酵母中发现了易于停滞的 RNA 聚合酶 II 突变体，以及具有伸长机制中的其他已知突变，以定义体内对延伸因子和 DNA 序列的要求基因表达过程中延伸因子辅助的需求。这将鉴定人类 SII 基因的细胞遗传学位置以确定如果它是任何已知遗传性疾病的候选基因。这项研究将为 RNA 合成的基本过程提供有价值的见解这将提高我们对正常和疾病状态的理解分子水平。