The effect of nucleosomes on the earliest stages of RNA polymerase II transcription

核小体对 RNA 聚合酶 II 转录最早阶段的影响

• 批准号: 9766312
• 负责人: Donal Luse
• 金额: $ 33.25万
• 依托单位: CLEVELAND CLINIC LERNER COM-CWRU
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-09-01 至 2022-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9766312
• 关键词: Active Sites; Address; Adopted; Affect; Architecture; Biochemical; Biological Assay; Cell Nucleus; Cell physiology; Chromatin; Chromatin Structure; Complex; Correlation Studies; DNA; DNA Polymerase II; DNA-Directed RNA Polymerase; Drug Targeting; ERCC3 gene; Elements; Eukaryota; Event; Gene Expression; Gene Expression Regulation; Genetic Transcription; Genomics; Health; Histones; Human; In Vitro; Lead; Location; Modeling; Movement; Nature; Nucleosomes; Orthologous Gene; Pattern; Population; Positioning Attribute; Process; Property; Regulation; Research Personnel; Role; Saccharomyces cerevisiae; Scanning; Shapes; Signal Transduction; Site-Directed Mutagenesis; Surveys; System; TAF1 gene; Testing; Transcript; Transcription Initiation; Transcription Initiation Site; Variant; Work; Yeasts; base; experimental study; human disease; in vivo; mutant; promoter; recruit; response; transcription factor; transcription factor TFIIH; translocase

Project Summary Appropriate control of gene expression is essential for correct cellular function and is disrupted in many human diseases. RNA polymerase II (Pol II) is the engine of gene expression and the target of regulation for almost every facet of its function. In the nucleus of the cell, Pol II is recruited to promoters, locates transcription start sites, and enters into transcription in a chromatin context. This occurs amidst flanking nucleosomes, which are the primary unit of chromatin. It is clear that control of the earliest stages of transcription is a major aspect of gene regulation in eukaryotes from simple yeast to humans. Genomic studies have suggested that the encounter of Pol II with the first nucleosome immediately downstream of the promoter (the +1 nucleosome) is important in this regulatory process, but the results of those studies are correlations, based on averages of distributed populations. The significance of those correlations is rarely tested by direct biochemical assays. This proposal will provide those tests by adopting biochemical approaches developed in the Luse lab to directly address the functional importance of the promoter proximal nucleosome for assembly of the Pol II transcription complex and the initial transition into transcript elongation. We will also determine the consequences for the +1 nucleosome resulting from the advance of the transcriptional machinery. Our in vitro systems have the unique ability to reveal the fundamental nature of Pol II/nucleosome interactions that additional factors can modulate or regulate. We will employ both the human and yeast transcription systems. These are the best understood eukaryotic transcription systems at the biochemical level and they provide complementary strengths for our experiments. We will incorporate the full complexity of eukaryotic promoters (both containing and lacking the TATA motif) in our work, which allows us to address the real possibility that the response of Pol II to the +1 nucleosome depends on promoter architecture. We will build on the extensive earlier studies by both investigators to begin testing proposed models of RNA polymerase II-nucleosome interactions with approaches that lead to mechanistic conclusions.

项目摘要 基因表达的适当控制对于正确的细胞功能至关重要， 在许多人类疾病中。RNA聚合酶II（Pol II）是基因表达的引擎， 几乎在其职能的每一个方面都是监管的目标。在细胞核中，Pol II是 募集到启动子上，定位转录起始位点，并在转录起始位点中进入转录。 染色质背景。这发生在侧翼的核小体中，核小体是蛋白质的主要单位。 染色质很明显，控制转录的最早阶段是基因调控的一个主要方面。 从简单的酵母到人类的真核生物中的调节。基因组研究表明， Pol II与紧邻启动子下游的第一个核小体的相遇（ +1核小体）在这一调控过程中很重要，但这些研究的结果是 基于分布人口的平均值的相关性。这些的意义 相关性很少通过直接的生物化学测定来测试。本提案将提供这些测试 通过采用Luse实验室开发的生物化学方法， 启动子近端核小体对Pol II转录组装的重要性 复合物和转录延长的初始过渡。我们还将确定 结果为+1核小体所造成的进展的转录 机械.我们的体外系统具有独特的能力，可以揭示Pol的基本性质。 II/核小体相互作用，额外的因素可以调节或调节。我们将同时雇用两者 人类和酵母的转录系统这些是最好理解的真核生物 转录系统在生化水平，他们提供了互补的优势，我们的 实验我们将整合真核启动子的全部复杂性（两者都含有 缺乏TATA主题），这使我们能够解决真实的可能性， Pol II对+1核小体的反应取决于启动子结构。我们将继续努力， 两名研究人员开始对提出的RNA模型进行了广泛的早期研究， 聚合酶II-核小体相互作用的方法，导致机械的结论。