Molecular Analysis of a Yeast Transcriptional Regulator

酵母转录调节因子的分子分析

• 批准号: 8515445
• 负责人: David T. Auble
• 金额: $ 37.48万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-05-01 至 2016-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8515445
• 关键词: ATP Hydrolysis; ATP phosphohydrolase; ATPase Domain; Address; Behavior; Binding; Binding Proteins; Biochemical; C-terminal; Cells; Chromatin; Chromatin Structure; Cockayne Syndrome; Complex; Coupled; Couples; Cues; DNA; DNA-Protein Interaction; Data; Defect; Development; Dissociation; Ensure; Environment; Enzymes; Event; Family; Gene Expression; Genetic; Genetic Transcription; Genomics; Goals; Growth and Development function; Half-Life; Human; In Vitro; Life; Link; Malignant Neoplasms; Measurement; Mediating; Modeling; Molecular Analysis; Movement; N-terminal; Noise; Normal Cell; Nucleosomes; Play; Population; Population Dynamics; Property; Protein Binding; RNA Polymerase II; Reaction; Regulation; Role; Saccharomycetales; Stress; Structure; System; TATA-Box Binding Protein; Testing; Time; Transcriptional Regulation; Variant; Work; X-Linked Mental Retardation; Yeasts; abstracting; alpha-Thalassemia; cell growth; human disease; in vivo; insight; member; novel; promoter; protein complex; residence; response; scaffold; tool

DESCRIPTION (provided by applicant): Project Summary/Abstract The broad, long-term objectives of the proposed project are to elucidate mechanisms responsible for assembly and activity of RNA polymerase II (Pol II) preinitiation complexes (PICs) at promoters. The TATA- binding protein (TBP), a central component of the PIC, can bind to DNA in vitro with exceptional stability. However, essentially all of the TBP in yeast cells is highly dynamic, with residence times on chromatin of just a few seconds. Prevailing models derived from in vitro observations posit that a stable promoter-bound complex facilitates transcription reinitiation and thereby perpetuates the activated state. In contrast, we find that dynamic turnover is critical for global transcriptional control in vivo. TBP's dynamic behavior is a consequence of the activity of Mot1, an essential, conserved, Snf2/Swi2-related ATPase that uses the energy of ATP hydrolysis to dissociate TBP from DNA. The goals of this proposal are to use Mot1 as a model to better understand how members of this critical class of enzymes use ATP hydrolysis to disrupt protein-DNA interactions, and to understand why Mot1-mediated dynamics are essential for transcription in vivo. In the first Aim, the Mot1 biochemical system and recent structural data will be exploited to test a specific model for the Mot1 catalytic mechanism. Emerging evidence indicates that cell-to-cell gene expression variation ("noise") impacts a wide range of cellular responses to stress and developmental cues, but mechanisms regulating noise are not well understood. Preliminary results show that Mot1 has a previously unappreciated role in suppressing transcriptional noise across a cell population. In Aim 2, we will define the scope and function of Mot1 and other related global regulators in establishing noise levels, and correlate this variation with PIC dynamics. In Aim 3, we will define a newly discovered functional relationship between Mot1 and FACT, an essential regulator of chromatin structure and dynamics. In addition to determining how Mot1 and FACT work together in vivo, new functions of Mot1 in chromatin structure regulation will be addressed, including the unexpected ability of Mot1 to bind to nucleosomes and to regulate the interaction of FACT with nucleosomes. Precise transcriptional control is essential for normal cell growth and development, and numerous transcriptional defects have been linked to human diseases including cancer. Defects in human Snf2/Swi2- related protein complexes are known contributors to certain cancers, Cockayne's Syndrome, alpha- thalassemia, and the most common form of X-linked mental retardation.

描述(由申请人提供)： 项目概述/摘要拟议项目的广泛、长期目标是阐明负责在启动子处组装和活性RNA聚合酶II(POL II)预起始复合体(PIC)的机制。TATA结合蛋白(TBP)是PIC的中心成分，在体外可以与DNA结合，具有极高的稳定性。然而，酵母细胞中的TBP基本上都是高度动态的，在染色质上的停留时间只有几秒钟。从体外观察得出的流行模型假设，稳定的启动子结合的复合体促进转录重新启动，从而使激活状态永久化。相反，我们发现动态周转对活体内的全局转录控制至关重要。TBP的动态行为是Mot1活性的结果，Mot1是一种基本的、保守的、与Snf2/Swi2相关的ATPase，它利用ATP水解的能量将TBP从DNA中解离出来。这项建议的目标是使用Mot1作为模型，以更好地了解这一关键类别的酶如何利用ATP水解来破坏蛋白质-DNA相互作用，并了解为什么Mot1介导的动力学对体内转录是必不可少的。在第一个目标中，将利用Mot1生化系统和最新的结构数据来测试Mot1催化机理的特定模型。越来越多的证据表明，细胞间的基因表达差异(“噪音”)会影响细胞对压力和发育线索的广泛反应，但对噪音的调控机制还不是很清楚。初步结果表明，Mot1在抑制细胞群体中的转录噪音方面具有以前未被认识到的作用。在目标2中，我们将定义Mot1和其他相关全球监管机构在确定噪声水平方面的范围和功能，并将这种变化与PIC动态联系起来。在目标3中，我们将定义新发现的Mot1和FACT之间的功能关系，FACT是染色质结构和动力学的重要调节因子。除了确定Mot1和FACT在体内如何协同工作外，还将讨论Mot1在染色质结构调节中的新功能，包括Mot1意外地与核小体结合以及调节FACT与核小体的相互作用。精确的转录调控对细胞的正常生长和发育至关重要，许多转录缺陷与包括癌症在内的人类疾病有关。人类Snf2/Swi2相关蛋白复合体的缺陷是已知的某些癌症、Cockayne综合征、阿尔法地中海贫血以及最常见的X连锁智力低下的诱因。