Elongation and termination of transcripts by RNA pol II

描述（由申请人提供）：通过RNA聚合酶I（pol II）调节转录来控制基因表达是细胞活力的基础，并且通常在疾病中被破坏。转录周期包括起始、延伸和终止阶段。这一建议侧重于后两个步骤，因为支配它们的机制不如启动机制那么清楚。我们的实验室使用联合收割机的策略，使突变的pol II和延伸因子的延长和终止在体内使用芽殖酵母和哺乳动物细胞的生化测定。终止是回收RNA聚合酶和防止相邻基因之间的干扰所必需的，但是触发在基因末端的极其稳定的转录延伸复合物的分解的机制还不完全清楚。我们将通过询问它如何受到pol II磷酸化和转录延伸率的影响来研究这种机制。我们还将通过两种方法寻找终止中的新参与者：1）基于我们对与终止因子Xrn 2（一种RNA核酸外切酶）相互作用的蛋白质因子的鉴定的候选方法; 2）对抑制终止的shRNA进行无偏倚的遗传筛选。 我们还将测试我们最近提出的一个模型，我们称之为“开盖和鱼雷”模型，用于通过过早终止来控制伸长。该模型表明，从大多数人类基因开始进入基因体的暂停位点的pol II的通量受到转录物脱帽和随后由Xrn 2和相关因子过早终止转录的限制。我们将测试这个模型的细节，并询问去帽和终止因子是否在转录周期的延伸阶段的调节中起作用。我们将集中在去帽和终止因子在控制转录的激活剂HIV达特，c-myc，热休克因子和缺氧诱导因子的潜在作用。

DESCRIPTION (provided by applicant): The control of gene expression through regulated transcription by RNA polymerase I (pol II) is fundamental to the cell viability and is often corrupted in disease. The transcription cycle comprises initiation, elongation and termination phases. This proposal focuses on the latter two steps because the mechanisms that govern them are less well elucidated than initiation. Our lab uses strategies that combine genetics to make mutations in pol II and elongation factors with biochemical assays of elongation and termination in vivo using budding yeast and mammalian cells. Termination is required to recycle the RNA polymerase and to prevent interference between adjacent genes but the mechanism that triggers disassembly of the extremely stable transcription elongation complex at the end of genes is quite incompletely understood. We will investigate this mechanism by asking how it is affected by pol II phosphorylation and transcription elongation rate. We will also look for new players in termination by using two approaches: 1) a candidate approach based on our identification of protein factors that interact with the termination factor Xrn2, an RNA exonuclease and 2) an unbiased genetic screen for shRNA that inhibit termination. We will also test a model we recently proposed for control of elongation by premature termination that we call the "decap and torpedo" model. This model suggests that the flux of pol II from the pause site at the beginning of most human genes into the gene body is limited by decapping of the transcript and subsequent premature termination of transcription by Xrn2 and associated factors. We will test the details of this model and ask whether decapping and termination factors function in regulation of the elongation phase of the transcription cycle. We will focus on the potential role of decapping and termination factors in control of transcription by the activators HIV Tat, c-myc, heat shock factor and hypoxia inducible factors.