Molecular analysis of eukaryotic transcription

总结 转录调控是许多信号传导和发育途径的主要靶点，并且转录调控是细胞周期的调控。 转录是控制细胞生长、分化和发育的关键步骤之一。发表的研究 表明至少两类核心启动子，它们在是否含有共有TATA元件方面不同 以及它们对共活化剂复合物TFIID和佐贺的需求。现有证据表明， 这些启动子类型在与基础因子的相互作用机制、染色质的调节 及其修饰，以及它们对转录调节因子的反应。由于迄今为止的大多数机械研究 虽然已经对含TATA的启动子进行了研究，但对启动机制了解甚少 大约80%缺乏TATA的基因受到调控，在理解大多数RNA是如何被调控方面留下了很大的空白。 聚合酶（Pol）II转录的基因受到调节。该项目的长期目标是确定 RNA Pol II转录起始的机制以及这些机制如何被用作基因转录的靶点。 调控这项工作的基本原理是，确定启动及其调节中使用的机制 将形成理解导致许多类型的转录障碍的缺陷的分子基础。 人类疾病本申请的目的是确定用于引发的机制， TATA-少启动子的调控，共激活因子TFIID和佐贺的特异性，功能和重叠 以及DNA开放、转录起始位点扫描和转录起始位点识别的机制。 转录起始的动态过程。我们的研究将利用生物化学，分子遗传学， 基因组学和生物物理方法来研究S.酿酒因为 该系统在分子遗传学和生物化学方面的优势允许快速和精确的机制 调查事务所TFIID和佐贺是酵母系统的基本组成部分，它们都是保守的 与人类基因调控机制直接相关。最近的研究结果和新方法呼吁 质疑启动子的初始TFIID和SAGA调控特征，并提出了一个潜在的 由这些因素调节的基因组之间存在大量重叠。我们将采用一种新的方法， 开发了这两种共激活因子的全基因组定位，特异性和功能重要性， 全基因组表达。我们将利用一个新的体外系统，转录TATA少启动子， 研究这些辅助因子的生化机制以及它们是如何被调节的。我们还将使用 创新的单分子方法来研究Pol中转录起始的动力学机制 II系统在含TATA的启动子。我们提出的研究是重要的，因为它将导致一个垂直的 在理解两个保守的共激活因子的作用方面取得了进展，这两个因子共同调节几乎所有的Pol II 转录，阐明在Pol II启动子的主要类别中使用的机制，并揭示不稳定的 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.