Molecular Analysis of a Yeast Transcriptional Regulator

项目摘要/摘要 基因的表达在转录启动水平上受到广泛的控制，这些过程 在启动子处协调多亚单位RNA聚合酶II(POL II)预起始复合体(PIC)的组装。 从体外观察得出的主流模型假定了PIC和PIC的组装的明确途径 形成稳定的启动子结合的复合体，促进转录的重新启动并使 激活状态。然而，PIC在体内组装的途径还不是很清楚，正在出现的证据 提示PIC组件和启动子之间的动态相互作用比观察到的要多得多 在试管中。拟议项目的广泛、长期目标是阐明途径和机制。 负责PICS在体内的组装和活动。为了实现这一目标，在本提案中，我们将应用 我们前期建立的测定芽殖酵母染色质结合动力学的方法 细胞。我们的多学科方法提供了转录因子(TF)结合的定量估计 单拷贝基因座的动力学，包括开启和关闭速率以及Tf对DNA位点的占有率 在细胞群体中。在目标1中，我们将测量关键转录的染色质结合动力学 模型激活基因的成分和RNA合成动力学，以及突变分析将用于 确定它们之间的关系。在目标2中，我们将测量Key的染色质结合动力学 基因组范围内的PIC的组成部分，并确定动力学行为和基因之间的关系 调节特性、RNA合成速率、染色质环境等特性。这一景观 当PIC组装过程在细胞中展开时，动力学特性将揭示它们在体内的范围和规模。 我们之前的工作表明，称为TATA结合蛋白的PIC组件的动态性质 (TBP)由一种名为Mot1的基本ATPase控制。在目标3中，我们将使用组合方法来测试 Mot1的S在基因激活中发挥作用的特定模型，通过这样做揭示了如何调节 染色质结合动力学可以在全球范围内影响基因表达。 在Tf结合部位存在广泛的等位基因变异，这种变异以及Tf的改变 表达水平，可以导致染色质占有率的差异，从而导致大量和普遍的 人类疾病，包括肥胖、心血管疾病、精神疾病和癌症。TFS本身 在很大程度上难以进行药物干预；相反，我们提出了一种定量的 了解体内TF结合和PIC组装动力学将识别动力学瓶颈， 为最终开发治疗人类转录缺陷的全新方法奠定了基础 发展和疾病。

Project Summary/Abstract Gene expression is extensively controlled at the level of transcription initiation by processes that orchestrate assembly of the multi-subunit RNA polymerase II (Pol II) preinitiation complex (PIC) at promoters. Prevailing models derived from in vitro observations posit a defined pathway for assembly of the PIC and formation of stable promoter-bound complexes that facilitate transcription reinitiation and perpetuate the activated state. However, pathways for PIC assembly in vivo are not well understood, and emerging evidence suggests much more dynamic interactions between PIC components and promoters than have been observed in vitro. The broad, long-term objectives of the proposed project are to elucidate pathways and mechanisms responsible for assembly and activity of PICs in vivo. To address this goal, in this proposal we will apply novel methods that we developed in the prior period for measuring chromatin-binding dynamics in budding yeast cells. Our multi-disciplinary approaches provide quantitative estimates of transcription factor (TF) binding kinetics to single-copy loci including on- and off-rates as well as fractional occupancies of a DNA site by a TF in a cell population. In Aim 1, we will measure both chromatin binding dynamics of critical transcriptional components and RNA synthesis dynamics at a model activated gene, and mutational analyses will be used to determine the relationships between them. In Aim 2, we will measure chromatin-binding dynamics of key components of the PIC, genome-wide, and determine the relationships between kinetic behavior and gene regulatory properties, RNA synthesis rates, chromatin environment, and other properties. This landscape of kinetic properties will reveal the in vivo scope and scale of PIC assembly processes as they unfold in cells. Our prior work indicates that the dynamic properties of a PIC component called the TATA-binding protein (TBP) are controlled by an essential ATPase called Mot1. In Aim 3, we will use combined approaches to test specific models for Mot1's function in gene activation, and in so doing shed light on how regulation of chromatin binding dynamics can impact gene expression on a global scale. There is widespread allelic variation in TF binding sites, and such variation, as well as alterations in TF expression levels, can lead to differences in chromatin occupancy that contribute to numerous and prevalent human diseases, including obesity, cardiovascular disease, mental illnesses, and cancer. TFs themselves have been largely refractory to pharmacologic intervention; instead, we propose that a quantitative understanding of TF binding and PIC assembly dynamics in vivo will identify kinetic bottlenecks that will provide a foundation for ultimately developing entirely new approaches to treat transcriptional defects in human development and disease.