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

项目摘要 适当的基因表达控制对于正确的细胞功能是必不可少的，但这一点被打乱了 在许多人类疾病中。RNA聚合酶II(Pol II)是基因表达的引擎， 其职能的几乎每一个方面的监管目标。在细胞的核中，PolII是 被招募到启动子，定位转录起始点，并进入转录 染色质上下文。这发生在侧翼的核小体中，核小体是 染色质。很明显，控制转录的最早阶段是基因的一个主要方面 真核生物的调控，从简单的酵母到人类。基因组研究表明， POL II与紧邻启动子下游的第一个核小体相遇 +1核小体)在这一调控过程中很重要，但这些研究的结果是 相关性，基于分布总体的平均值。这些因素的意义 相关性很少通过直接的生化分析来检验。这项提案将提供这些测试 通过采用LUSE实验室开发的生化方法直接解决功能 启动子近端核小体对Pol II转录组装的重要性 复杂和最初的过渡到文字记录的延伸。我们还将确定 转录水平提高对+1核小体的影响 机械设备。我们的体外系统具有独特的能力来揭示POL的基本性质 II/核小体相互作用，额外的因子可以调节或调节。我们将同时聘用这两名员工 人类和酵母转录系统。这些是最被理解的真核生物 生物化学水平的转录系统，它们为我们的 实验。我们将整合真核启动子的全部复杂性(两者都包含 以及缺少塔塔主题)，这使我们能够解决真正的可能性，即 POL II对+1核小体的反应取决于启动子的结构。我们将在 两位研究人员进行了广泛的早期研究，以开始测试拟议的RNA模型 聚合酶II-核小体与导致机械性结论的方法的相互作用。

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.