Mechanism of Transcript Elongation in Chromatin by RNA Polymerase II

描述(申请人提供)：RNA聚合酶II(PolII)转录的重要过程发生在真核细胞的染色质环境中；事实上，适度转录的基因保留了核小体结构。最近的研究表明，染色质结构对体外转录PolII具有很强的屏障作用，DNA-组蛋白相互作用只有部分发生，并在中等活性基因的转录延长过程中短暂中断。此外，延长PolII复合体也是基因调控的主要靶点之一。与组蛋白和PolII相互作用的核小体屏障和蛋白质因子参与了这一调节。这些研究提出了以下问题：(1)真核聚合酶如何克服核小体障碍？(2)在PolII通过染色质进行过程中，组蛋白恢复的机制是什么？(3)与染色质和靶酶相互作用的因素如何通过染色质改变PolII的进展速度？这一建议的长期研究目标是了解PolII在染色质中对转录本延长的机制和调节。具体的问题将在体外高纯度的转录系统中解决。我们将结合生物化学、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生物PolII的分析上。我们的初步研究表明，通过染色质转录伴随着组蛋白的瞬时DNA解卷和组蛋白八聚体的解开。这些中间体的结构和它们之间的相互转化速度在很大程度上决定了通过染色质进行转录的结果。因此，本研究的具体目的是：1.提供PolII通过染色质转录过程中DNA-组蛋白相互作用变化的分子描述以及中间产物之间的相互转化速率。2.确定组蛋白八聚体在转录依赖的去折叠过程中受到干扰的蛋白质相互作用，以及参与这一过程的因素。PHS 398/2590(06/09版)页面续格式页面

DESCRIPTION (provided by applicant): The vital process of transcription by RNA polymerase II (pol II) occurs in chromatin environment in eukaryotic cells; in fact, moderately transcribed genes retain nucleosomal structure. Recent studies suggest that chromatin structure presents a strong barrier for transcribing pol II in vitro, and that DNA-histone interactions are only partialy and transiently disrupted during transcript elongation on moderately active genes. Furthermore, elongating pol II complex is one of the major targets during gene regulation. The nucleosomal barrier and protein factors interacting with histones and pol II participate in this regulation. These studies raise the following questions: (1) How do eukaryotic polymerases overcome the nucleosome barrier? (2) What are the mechanisms of histone recovery during progression of pol II through chromatin? (3) How do the factors interacting with chromatin and with the target enzymes change the rate of pol II progression through chromatin? The long-term research goal of this proposal is to understand the mechanism and the regulation of transcript elongation by pol II in chromatin. The specific 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 a combination of biochemical, fluorescent, molecular genetic and single-molecule techniques. Our experiments will be focused on analysis of eukaryotic pol II. Our preliminary studies have shown that transcription through chromatin is accompanied by transient DNA uncoiling from histones and unfolding of histone octamer. The structures of these intermediates and the rates of interconversion between them largely determine the outcome of the process of transcription through chromatin. Accordingly, the specific aims are: 1. To provide molecular description of the changes in DNA-histone interactions during transcription through chromatin by pol II and the rates of interconversion between the intermediates. 2. To identify protein-protein interactions perturbed during transcription-dependent unfolding of the histone octamer, and factors involved in this process. PHS 398/2590 (Rev. 06/09) Page Continuation Format Page