Studies of Elongation Arrest and Transcript Hydrolysis by RNA Polymerase II

9317613霍利这个项目将对蛋白质和脱氧核糖核酸序列进行生化分析，这些蛋白质和脱氧核糖核酸序列决定了人类核糖核酸聚合酶II的体外延伸行为。这些实验的主要目标是回答三个一般领域的问题。首先，PolII为什么以及如何在转录上被阻止在停滞位置，PolII在这些位置的行为揭示了关于延长复合体的功能和结构？针对这些问题的初步研究主要集中在腺病毒主要晚期停滞部位的体外转录停滞机制。正在定义和描述导致该地点被捕的DNA序列要素。这些研究将扩展到其他几个观察到逮捕和/或终止的地点，特别强调了解该地点的哪些特征规定了文字记录的发布。第二组问题涉及两个延伸率因素SII和TFIIF的作用。SII已被证明促进阻止部位的通读并刺激PolII的3‘-5’外核溶解活性，而TFIIF已被证明提高了总的延长率。这些活动的生化特征将继续，重点放在几个具体问题上，这些问题涉及核酸外切酶活性可能的生理作用，以及TFIIF活性对于调节该活性以促进有效延伸的可能性。最后，PolII伸长复合体在模板上向后移位的能力将被用来提出和回答有关伸长复合体结构的问题，特别是关注RNA二级结构通过抑制DNA：RNA杂交体的改造来抑制备份反应的可能性，以及PolII可能对备份时遇到的终止位点做出反应。%生物学中最重要和最基本的问题之一是，在遗传上，细胞如何不同地利用遗传信息，成为有机体中不同功能和结构角色的特化细胞。细胞的具体形态和功能由该细胞正在产生的蛋白质决定；指定该细胞所能产生的所有可能蛋白质的信息编码在基因中。因此，在每种细胞类型中，一些基因必须保持关闭(不表达)，而另一些基因则处于打开(表达)状态。基因表达的第一步是合成基因的副本。这种复制过程被称为转录，产生一种RNA分子，然后细胞的机器使用它来指导由该基因编码的蛋白质的生产。转录是由一种名为RNA聚合酶的酶执行的，它不仅必须选择要在特定细胞中表达的基因，而且必须复制该基因的完整副本。转录的启动和完成都受到调控，这种调控是不同细胞产生不同蛋白质组机制的重要组成部分。这项研究将集中于了解转录是如何在复制过程的继续和完成的水平上被调节的。具体地说，在一些基因中存在导致转录停止的信号，并且已经确定了一些蛋白质，它们改变了RNA聚合酶对这些信号做出反应或忽略这些信号的能力。这个项目是对RNA聚合酶与这些信号和蛋白质相互作用的生化分析，旨在了解这些相互作用的分子细节及其对基因表达调控的重要性。***

9317613 Hawley This project will be a biochemical analysis of the proteins and DNA sequences that determine the in vitro elongation behavior of human RNA polymerase II. The major goals of these experiments are to answer questions in three general areas. First, why and how does pol II become transcriptionally blocked at arrest sites and what does the behavior of pol II at such sites reveal about the function and structure of elongation complexes? Initial studies addressing these questions have focused on the mechanism of transcription arrest in vitro at the adenovirus major late arrest site. The DNA sequence elements contributing to arrest at this site are being defined and characterized. These studies will be extended to several additional sites at which arrest and/or termination is observed, with a particular emphasis on understanding what features of the site specify transcript release. The second set of questions addresses the roles of two elongation factors, SII and TFIIF. SII has been shown to promote readthrough of arrest sites and to stimulate a 3' -- 5' exonucleolytic activity of pol II, whereas TFIIF has been shown to enhance the overall rate of elongation. The biochemical characterization of these activities will continue, focusing on several specific questions that address possible physiological roles for the exonuclease activity and the possibility that TFIIF activity is important for moderating that activity to promote efficient elongation. Finally, the ability of pol II elongation complexes to translocate backwards on the template will be used to pose and answer questions about the structure of the elongation complex, focusing particularly on the possibility that RNA secondary structures can inhibit the backing up reaction by inhibiting reformation of the DNA:RNA hybrid and that pol II may respond to termination sites encountered while backing up. %%% One of the most important and fundamental questions in biology is how genetically i dentical cells differentially use the genetic information to become specialized for different functional and structural roles in the organism. The specific morphology and function of a cell are determined by what proteins that cell is making; the information specifying production of all the possible proteins the cell could make is encoded in the genes. Thus, in each cell type, some genes must remain "off" (not expressed), while others are turned on (expressed). The first step in gene expression is the synthesis of a copy of the gene. This copying process, called transcription, produces an RNA molecule that is then used by the cell's machinery to direct production of the protein encoded by that gene. Transcription is performed by an enzyme called RNA polymerase, which must not only select the genes that are to be expressed in a certain cell but then must make a complete copy of that gene. Both the initiation and the completion of transcription are regulated, and this regulation is an important part of the mechanism by which different cells make different sets of proteins. This research will focus on understanding how transcription is regulated at the level of continuation and completion of the copying process. Specifically, there are signals within some genes that cause transcription to stop, and there are proteins that have been identified that alter the ability of the RNA polymerase to respond to or ignore these signals. This project is a biochemical analysis of the interaction of the RNA polymerase with these signals and proteins and is directed at understanding the molecular details of these interactions and their importance to the regulation of gene expression. ***