Elongation and termination of transcripts by RNA pol II

描述(由申请人提供)：通过RNA聚合酶I(PolII)调节转录来控制基因表达是细胞存活的基础，在疾病中经常被破坏。转录循环包括起始、延伸和终止三个阶段。这一建议侧重于后两个步骤，因为管理它们的机制不如启动机制得到很好的阐明。我们的实验室使用的策略是将遗传学与使用萌芽酵母和哺乳动物细胞进行体内伸长和终止的生化分析相结合，在PolII和延伸因子中进行突变。RNA聚合酶的循环和防止相邻基因之间的干扰需要终止，但触发基因末端极其稳定的转录延伸复合体分解的机制尚不完全清楚。我们将通过询问PolII磷酸化和转录延伸率如何影响它来研究这一机制。我们还将通过两种方法寻找新的终止参与者：1)基于我们鉴定的与终止因子Xrn2相互作用的蛋白质因子的候选方法，以及2)抑制终止的shRNA的无偏基因筛选。我们还将测试我们最近提出的通过提前终止来控制伸长的模型，我们称之为“解封和鱼雷”模型。这一模型表明，PolII从大多数人类基因开始的停顿位置进入基因体的流量受到转录物的去掉和随后Xrn2及其相关因素提前终止转录的限制。我们将测试这一模型的细节，并询问解帽和终止因子是否在转录周期的延伸阶段起调节作用。我们将集中于解帽和终止因子在通过激活HIV Tat、c-myc、热休克因子和低氧诱导因子调控转录中的潜在作用。

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.