Molecular Analysis of a Yeast Transcriptional Regulator

项目总结/摘要 基因表达在转录起始水平受到广泛控制， 协调多亚基RNA聚合酶II（Pol II）前起始复合物（PIC）在启动子处的组装。 来自体外观察的流行模型为PIC的组装提供了确定的途径， 形成稳定的启动子结合复合物，促进转录重新启动并使转录持续存在。 激活状态。然而，PIC在体内组装的途径还没有得到很好的理解， 表明PIC组分和启动子之间的动态相互作用比已经观察到的要多得多 体外拟议项目的广泛的长期目标是阐明途径和机制 负责PIC在体内的组装和活性。为了实现这一目标，在本提案中，我们将采用新的 方法，我们开发了在前期测量染色质结合动态芽殖酵母 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，包括结合率和解离率以及TF对DNA位点的部分占据率 在一个细胞群体中。在目标1中，我们将测量两个染色质结合动力学的关键转录， 成分和RNA合成动力学在一个模型激活的基因，和突变分析将用于 确定它们之间的关系。在目标2中，我们将测量关键的染色质结合动力学， PIC的组成部分，全基因组，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、染色质环境和其他特性。这片 动力学性质将揭示PIC组装过程在细胞中展开时的体内范围和规模。 我们先前的工作表明，称为TATA结合蛋白的PIC组分的动力学性质 (TBP)是由一种叫做Mot 1的ATP酶控制的。在目标3中，我们将使用组合方法来测试 Mot 1在基因激活中的功能的特定模型，并在这样做时阐明了如何调节 染色质结合动力学可以在全局范围内影响基因表达。 TF结合位点存在广泛的等位基因变异，这种变异以及TF结合位点的改变， 表达水平的差异，可导致染色质占有率的差异，这有助于许多和普遍的 人类疾病，包括肥胖、心血管疾病、精神疾病和癌症。TF本身 已经在很大程度上难治药物干预;相反，我们建议， 了解TF结合和PIC组装动力学在体内将确定动力学瓶颈， 为最终开发治疗人类基因组转录缺陷的全新方法提供了基础。 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.