Studies of Elongation Arrest and Transcript Hydrolysis by RNA Polymerase II

近年来，人们越来越清楚地认识到，真核生物的转录不仅在转录物合成开始前受到调控，而且在RNA链延伸的水平上受到调控。对于这种调控是如何实现的，以及RNA聚合酶在沿着DNA模板延伸过程中的结构和功能，人们知之甚少。本研究是对体外确定人RNA聚合酶II转录延伸行为的蛋白质和核酸序列进行生化分析。将研究两种改变RNA聚合酶II延伸特性的蛋白质：增加延伸率的TFIIF和使RNA聚合酶II能够读取DNA阻滞位点的SII，否则会阻止其进展。我们将研究阻滞和生产延伸复合物的结构和功能，目的是获得有关阻滞如何在阻滞位点发生的机制信息（它们与更好地理解的终止位点有一些共同特征），以及TFIIF和SII如何与这些复合物相互作用以防止或减轻延伸阻滞。转录复合物将在体外分离和研究，方法是将由启动子和腺病毒主要晚期阻滞位点组成的DNA模板附着在琼脂糖珠上，并在人类培养细胞的核蛋白提取物中组装起始、延伸或阻滞复合物。这种复合物先前被用来证明转录复合物中寡核苷酸与RNA的碱基配对对复合物的行为有深远的影响，这取决于碱基配对发生的位置。这项工作导致了一个假设，即捕获导致新生RNA的一个区域被拉回聚合酶中，并将通过分析寡核苷酸杂交位置与RNA聚合酶在捕获位点的构象之间的关系来进一步研究。其他实验将绘制DNA模板上聚合酶的上游和下游边界，并通过测量DNA模板上特定位置的单核苷酸掺入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RNA聚合酶II延伸复合物结构的详细视图，并将提出这些复合物可以调节的可能机制。近年来，人们越来越清楚地认识到，真核生物的转录不仅在转录物合成开始前受到调控，而且在RNA链延伸的水平上受到调控。对于这种调控是如何实现的，以及RNA聚合酶在沿着DNA模板伸长过程中的结构和功能，人们知之甚少。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进行生化分析。这项研究将提供RNA聚合酶II延伸复合物结构的详细视图，并将提出这些复合物可以调节的可能机制。

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.