MECHANISM OF 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。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。这就提出了一些问题：1.聚合酶如何通过染色质屏障转录？2.该屏障能否改变转录本的伸长率？这些问题将在体外高纯度的转录系统中得到解决。我们将利用生物化学和分子遗传学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，重点分析真核RNA聚合酶II。其具体目的是：1.确定RNA聚合酶II通过染色质转录的机制：组蛋白八聚体的命运和核小体转录障碍的性质。将在这里获得的信息将为通过染色质及其限速步骤理解转录提供一个结构框架(S)。2.确定伸长因子、染色质结构的改变和RNA聚合酶II的修饰在促进染色质转录中的作用。这些实验将有助于在转录延伸水平上更好地理解转录调控。一些延伸因子在肿瘤发生中发挥重要作用的发现，突显了分析转录延伸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. 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 chromatin barrier? 2. Can the barrier 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 and molecular genetic techniques, focusing on analysis of eukaryotic RNA polymerase II. The specific aims are: 1. To ascertain the mechanism of transcription through chromatin by RNA polymerase II: the fate of the histone octamer and the nature of nucleosomal barrier to transcription. Information to be obtained here will provide a structural framework for understanding of transcription through chromatin and its rate-limiting step(s). 2. To ascertain the rote of elongation factors, modifications of chromatin structure, and modifications of RNA polymerase II in facilitating transcription through chromatin. These experiments will lead to better understanding of regulation of transcription at the level of transcript elongation. The discovery that some elongation factors play important roles in oncogenesis underscores the potential clinical significance of analysis of the mechanism of transcript elongation.