Molecular analysis of eukaryotic transcription

摘要 转录调控是许多信号和发育途径的主要靶点， 转录是控制细胞生长、分化和发育的关键步骤之一。已发表的研究 建议至少两类核心启动子，它们是否包含共识TATA元素 以及他们对共激活剂络合物TFIID和SAGA的要求。现有证据表明 这些启动子类型与基本因子相互作用的机制不同，也不同于染色质的调节 及其修饰，以及它们对转录调节因子的反应。因为到目前为止大多数机械论研究 已经在含有TATA的启动子上进行了研究，对其引发机制知之甚少 以及对~80%缺乏TATA的基因的调控，在理解大多数RNA是如何 聚合酶(POL)II转录基因受调控。该项目的长期目标是确定 RNA POL II转录启动机制及其作为基因靶标的研究进展 监管。这项工作的基本原理是确定在启动过程中使用的机制及其调节 将形成分子基础，以了解导致多种类型的转录紊乱的缺陷 人类疾病。本申请的目标是确定用于启动和 TATA非启动子的调控，共激活因子TFIID和SAGA的特异性、功能和重叠 DNA开放、转录起始点(TSS)扫描和TSS识别的机制 转录启动的动态过程。我们提议的研究将利用生化、分子遗传学、 基因组学和生物物理方法来研究酿酒酵母的转录调控，因为 该系统在分子遗传学和生物化学方面的优势使快速和精确的机械 调查。基础机械、TFIID和SAGA都是保守的，因此来自酵母系统的结果 与人类基因调控机制直接相关。最近的发现和新的方法呼吁 质疑TFIID和SAGA对启动子的初始调控特征，并提出潜在的 受这些因素调控的一组基因之间有很大的重叠。我们将使用一种新的方法，我们 开发用于绘制这两个共激活因子在全基因组中的位置、特异性和功能重要性的图 全基因组表达。我们将利用一种新的体外系统将不含TATA的启动子转录成 研究这些辅活化子的生化机制以及它们是如何被调控的。我们还将使用 创新的单分子方法研究极体转录启动的动力学机制 系统中含有塔塔的发起人。我们提出的研究具有重要意义，因为它将导致 共同调控几乎所有POL II的两个保守的共激活因子的作用研究进展 转录，阐明了Pol II启动子的主要类别的机制，并揭示了不稳定 转录起始途径中的中间体和限速步骤。

Summary Transcriptional regulation is a primary target of many signaling and developmental pathways and regulation of transcription is one of the key steps in control of cell growth, differentiation and development. Published studies suggest at least two classes of core promoters that differ in whether they contain a consensus TATA element and in their requirements for the coactivator complexes TFIID and SAGA. Available evidence suggests that these promoter types differ in the mechanism of interaction with the basal factors, in regulation by chromatin and its modifications, and in their response to transcription regulators. Since most mechanistic studies to date have been conducted on TATA-containing promoters, little is understood about the mechanisms of initiation and regulation at the ~80% of genes lacking TATAs, leaving a large gap in understanding how most RNA polymerase (Pol) II transcribed genes are regulated. The long term goals of this project are to determine the mechanisms of RNA Pol II transcription initiation and how these mechanisms are utilized as targets for gene regulation. The rationale for this work is that determining the mechanisms used in initiation and its regulation will form the molecular basis for understanding defects in transcription disorders leading to many types of human disease. The objectives of this application are to determine the mechanisms utilized for initiation and regulation at TATA-less promoters, the specificity, function, and overlap of the coactivators TFIID and SAGA and the mechanism of DNA opening, transcription start site (TSS) scanning and TSS recognition during the dynamic process of transcription initiation. Our proposed research will utilize biochemical, molecular genetic, genomics, and biophysical approaches to examine transcriptional regulation in S. cerevisiae because the advantages in molecular genetics and biochemistry in this system allow rapid and precise mechanistic investigations. The basal machinery, TFIID and SAGA are all conserved so that results from the yeast system are directly relevant to human gene regulatory mechanisms. Recent findings and new approaches have called into question the initial TFIID and SAGA-regulated characterization of promoters and suggest a potentially large overlap between the sets of genes modulated by these factors. We will use a new approach we developed to map the genome-wide location, specificity and functional importance of these two coactivators on genome-wide expression. We will utilize a new in vitro system that transcribes TATA-less promoters to investigate biochemical mechanisms of these coactivators and how they are regulated. We will also use an innovative single molecule approach to investigate the dynamic mechanism of transcription initiation in the Pol II system at TATA-containing promoters. Our proposed research is significant because it will lead to a vertical advance in understanding the role of two conserved coactivators that together regulate nearly all Pol II transcription, illuminate mechanisms used at the major class of Pol II promoters, and reveal unstable intermediates and rate-limiting steps in the transcription initiation pathway.