Mechanism of Transcript Elongation in Chromatin by RNA Polymerase II

描述（由申请人提供）：RNA聚合酶II （pol II）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核小体结构。最近的研究表明，染色质结构对体外转录pol II具有很强的障碍，并且在中等活性基因的转录延伸过程中，dna与组蛋白的相互作用仅部分和短暂地中断。此外，延长pol II复合体是基因调控的主要靶点之一。核小体屏障和与组蛋白和pol II相互作用的蛋白因子参与了这一调控。这些研究提出了以下问题：(1)真核生物聚合酶如何克服核小体屏障？(2)在pol II通过染色质进展过程中，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子如何改变pol II通过染色质的进展速度？本课题的长期研究目标是了解染色质中pol II对转录本伸长的调控机制。具体问题将在体外高度纯化的转录系统中解决。我们将使用生物化学，荧光，分子遗传学和单分子技术的组合来分析均匀和明确的单核体和多核体染色质模板的转录。我们的实验将集中在真核生物pol II的分析上。我们的初步研究表明，通过染色质的转录伴随着组蛋白的短暂DNA解卷和组蛋白八聚体的展开。这些中间体的结构和它们之间相互转换的速率在很大程度上决定了通过染色质转录过程的结果。因此，具体目标是：1。为pol II通过染色质转录过程中dna -组蛋白相互作用的变化以及中间产物之间的相互转换速率提供分子描述。2. 鉴定组蛋白八聚体转录依赖性展开过程中蛋白-蛋白相互作用的干扰，以及参与这一过程的因素。小灵通398/2590 （Rev. 06/09）页延续格式页

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 PUBLIC HEALTH RELEVANCE: Analysis of transcript elongation is important for human health because: (a) expression of at least some human proto-oncogenes (e.g. c-myc and c-fos) is regulated at the level of transcript elongation, (b) elongation factors play important roles in numerous human diseases, including HIV and leukemia, (c) histone modifications associated with transcribed chromatin are changed in human cancers. (d) Chromatin recovery during transcript elongation is essential for normal aging.