Mechanism Transcript Elongation in Chromatin

描述(申请人提供)：转录过程对所有活细胞都是必不可少的。转录分四个步骤完成：启动子结合、RNA链起始、RNA转录物延长和RNA转录物终止。转录调控可能发生在这些步骤中的每一步。最近的研究表明，RNA聚合酶II的转录延长是基因调控的主要目标之一。已经证实，转录延伸水平上的基因表达调控涉及到与组蛋白相互作用的因素。这些因素有助于组蛋白在转录延伸过程中瞬间从染色质上移位，并通过染色质改变转录速率。这一建议的长期研究目标是了解真核RNA聚合酶在染色质中对转录本延长的机制和调节。当真核基因被激活进行转录时，它们的染色质结构会发生变化，以适应转录因子，并允许RNA聚合酶进行有效的转录。然而，对于许多基因来说，有证据表明转录区域被核小体覆盖。这就提出了问题：1.聚合酶如何转录通过核小体屏障？2.与组蛋白相互作用的因素如何改变转录延长的速度？这些问题将在体外高纯度的转录系统中得到解决。我们将使用生化、荧光、分子遗传学和单分子技术分析均一和定义明确的单核小体和多核小体染色质模板的转录。我们的实验将集中在真核RNA聚合酶II的分析上。具体目的是：1.测试组蛋白从核小体上的瞬时置换是否是转录延长过程中的限速步骤。2.确定延伸因子(如FACT)是否通过与聚合酶参与的核小体中的组蛋白相互作用而通过染色质促进转录。一些延长因子在肿瘤发生中发挥重要作用的发现，突显了通过染色质分析转录本延长机制的潜在临床意义。

DESCRIPTION (provided by applicant): The process of transcription is essential for all living cells. Transcription is accomplished in four steps: promoter binding, RNA chain initiation, RNA transcript elongation, and RNA transcript termination. Regulation of transcription can occur at each of these steps. Recent studies suggest that transcript elongation by RNA polymerase II is one of the major targets of gene regulation. It has been established that regulation of gene expression at the level of transcript elongation involves factors interacting with histones. These factors facilitate transient displacement of histones from chromatin during transcript elongation and modify the rate of transcription through chromatin. The long-term research goal of this proposal is to understand the mechanism and the regulation of transcript elongation by eukaryotic RNA polymerases in chromatin. When eukaryotic genes are activated for transcription, their chromatin structure changes to accommodate transcription factors and to allow efficient transcription by RNA polymerases. However, for many genes there is evidence that transcribed regions are covered with nucleosomes. This raises the questions: 1. How do polymerases transcribe through the nucleosome barrier? 2. How do the factors interacting with histones change the rate of transcript elongation? These questions will be addressed in a highly purified transcription system in vitro. We will analyze transcription of homogeneous and well-defined mono- and polynucleosomal chromatin templates using biochemical, fluorescent, molecular genetic and single-molecule techniques. Our experiments will be focused on analysis of eukaryotic RNA polymerase II. The specific aims are: 1. To test whether transient displacement of histones from nucleosome is the rate-limiting step during transcript elongation. 2. To determine whether elongation factors such as FACT facilitate transcription through chromatin by interacting with histones within polymerase-engaged nucleosomes. The discovery that some elongation factors play important roles in oncogenesis underscores the potential clinical significance of analysis of the mechanism of transcript elongation through chromatin.