Studies of Elongation Arrest and Transcript Hydrolysis by RNA Polymerase II

近年来越来越清楚的是，真核生物的转录不仅在转录本合成开始之前受到调节，而且在RNA链的延伸水平上也受到调节。 关于这种调控是如何实现的，以及RNA聚合酶在沿着DNA模板延伸的过程中的结构和功能，人们知之甚少。 本研究是对决定人RNA聚合酶II体外转录延伸行为的蛋白质和核酸序列的生化分析。 将研究两种改变RNA聚合酶II的延伸特性的蛋白质：TFIIF，其增加延伸速率; SII，其使RNA聚合酶II能够通过DNA停滞位点读取，否则将阻止其进展。 被捕和生产性延长复合物的结构和功能将进行调查，目的是获得机械信息逮捕如何发生在逮捕网站（共享一些功能，更好地了解终止位点），以及如何TFIIF和SII与这些复合物相互作用，以防止或缓解延长逮捕。 通过将由启动子和腺病毒主要晚期阻滞位点组成的DNA模板连接到琼脂糖珠上，并在来自人培养细胞的核蛋白提取物中组装起始、延伸或阻滞复合物，将在体外分离和研究转录复合物。 这种复合物以前被用于显示寡核苷酸与转录复合物中的RNA的碱基配对对复合物的行为具有深远的影响，这取决于碱基配对发生的位置。 这项工作导致的假设，逮捕结果在一个区域的新生RNA被拉回到聚合酶，并将进一步检查通过分析寡核苷酸杂交位置和构象的RNA聚合酶在逮捕网站之间的关系。 其他实验将绘制聚合酶在停滞和生产性延伸复合物中的DNA模板上的上游和下游边界，并将通过测量在DNA模板上的限定位置处单核苷酸掺入RNA链的速率来研究周围DNA序列的贡献和TFIIF刺激的机制。 这项研究将提供一个详细的RNA聚合酶II延伸复合物的结构，并提出可能的机制，这些复合物可以调节。 近年来，越来越清楚的是，真核生物的转录不仅在转录本合成开始之前受到调控，而且在RNA链的延伸水平上也受到调控。 关于这种调节是如何实现的，或者关于RNA聚合酶在沿着DNA模板延伸的过程中的结构和功能，我们知之甚少。 本研究是对决定人类RNA聚合酶II转录延伸行为的蛋白质和核酸序列的生化分析。这项研究将提供一个详细的RNA聚合酶II延伸复合物的结构，并提出可能的机制，这些复合物可以调节。

9604016 Hawley In recent years it has become increasingly clear that eukaryotic transcription is regulated not only before synthesis of the transcript is initiated, but also at the level of elongation of the RNA chains. Little is know about how this regulation is implemented or about the structure and function of RNA polymerase when it is in the process of elongating along the DNA template. This research is a biochemical analysis of the proteins and nucleic acid sequences that determine the transcription elongation behavior of human RNA polymerase II in vitro. Two proteins that alter the elongation properties of RNA polymerase II will be studied: TFIIF which increases the rate of elongation and SII which enables RNA polymerase II to read through DNA arrest sites that would otherwise block it progression. The structure and function of arrested and productive elongation complexes will be investigated with the goal of obtaining mechanistic information about how arrest occurs at arrest sites (which share some features with better understood termination sites), and how TFIIF and SII interact with those complexes to prevent or relieve elongation arrest. Transcription complexes will be isolated and studied in vitro by attaching a DNA template consisting of a promoter and the adenovirus major late arrest site to agarose beads and assembling initiation, elongation or arrested complexes in an extract of nuclear proteins from human cultured cells. Such complexes were used previously to show that the base pairing of an oligonucleotide to the RNA in transcription complex has a profound effect on the behavior of the complex depending on where the base-pairing occurs. This work led to the hypothesis that arrest results in a region of the nascent RNA being pulled back into the polymerase, and will be further examined by analyzing the relationship between oligonucleotide hybridization position and the conformation of the RNA polymerase at the arrest site. Other experiments will map the upstream and do wnstream boundaries of the polymerase on the DNA template in arrested and productive elongation complexes, and will investigate the contribution of the surrounding DNA sequence and the mechanism of TFIIF stimulation by measuring the rate of single nucleotide incorporation into the RNA chain at defined positions on the DNA template. This research will provide a detailed view of the structure of RNA polymerase II elongation complexes and will suggest possible mechanisms by which these complexes can be regulated. In recent years it has become increasingly clear that eukaryotic transcription is regulated not only before synthesis of the transcript is initiated, but also at the level of elongation of the RNA chains. Little is known about how this regulation is implemented or about the structure and function of RNA polymerase when it is in the process of elongating along the DNA template. This research is a biochemical analysis of the proteins and nucleic acid sequences that determine the transcription elongation behavior of human RNA polymerase II. This research will provide a detailed view of the structure of RNA polymerase II elongation complexes and will suggest possible mechanisms by which these complexes can be regulated.