Molecular Analysis of Eukaryotic Transcription

描述(申请人提供)：转录机制是许多信号转导和发育途径的最终目标，转录调控是控制细胞生长、分化和发育的关键步骤之一。转录及其调控的缺陷直接导致人类疾病，如癌症、炎症、心脏病、神经疾病和出生缺陷。这一建议的长期目标是确定真核RNA聚合酶II的转录启动机制。了解转录过程的分子机制将为理解基因控制和转录调节因子的作用奠定基础，其中许多转录调节因子在招募、起始和延伸步骤的不同时间点调节转录机制的活动。这项工作的具体目的是利用生化、分子、遗传和结构方法来研究酿酒酵母RNA POL II的转录启动机制。利用我们开发的用于绘制大复合体结构排列的生化方法，结合纯化的转录系统，我们将绘制转录周期中间阶段的转录机制的结构图：预起始复合体、开放复合体、起始点扫描、起始和启动子逃逸。我们将使用酵母分子遗传学来测试在生化分析中观察到的相互作用的重要性。综上所述，我们的结果将导致一个详细的模型来解释POL II的转录启动机制以及一般转录因子在这一过程中的作用。 公共卫生相关性：这项研究的目的是了解转录的机制和调控，即信使核糖核酸的合成过程。转录调控是控制细胞生长、分化和发育的关键步骤之一，而转录缺陷直接导致许多人类疾病。了解转录及其调控的机制将为理解导致多种癌症以及心脏病、神经疾病和出生缺陷的转录紊乱的分子缺陷奠定基础。

DESCRIPTION (provided by applicant): The transcription machinery is the ultimate target of many signal transduction and developmental pathways and regulation of transcription is one of the key steps in control of cell growth, differentiation and development. Defects in transcription and its regulation directly contribute to human illnesses such as cancer, inflammation, heart disease, neurological disorders, and birth defects. The broad long-term objective of this proposal is to determine the mechanism of transcription initiation by eukaryotic RNA polymerase II. Understanding the molecular mechanisms of the transcription process will form the basis for understanding gene control and the action of transcription regulators, many of which modulate the activity of the transcription machinery at various points in the recruitment, initiation, and elongation steps. The specific aims of this work will utilize biochemical, molecular, genetic, and structural methods to examine the mechanism of transcription initiation by S. cerevisiae RNA Pol II. Using biochemical methods we have developed for mapping the structural arrangement of large complexes, in conjunction with a purified transcription system, we will map the structure of the transcription machinery at intermediate stages of the transcription cycle: Preinitiation Complex, Open Complex, initiation site-scanning, initiation, and promoter escape. We will use yeast molecular genetics to test the significance of interactions observed in biochemical assays. Combined, our results will lead to a detailed model for the mechanism of transcription initiation by Pol II and the role of the general transcription factors in this process. PUBLIC HEALTH RELEVANCE: The objective of this research is to understand the mechanism and regulation of transcription, the process of mRNA synthesis. Regulation of transcription is one of the key steps in control of cell growth, differentiation, and development, and defects in transcription directly contribute to many human illnesses. Understanding the mechanism of transcription and its regulation will form the basis for understanding the molecular defects in transcription disorders leading to many types of cancer, as well as heart disease, neurological disorders, and birth defects.