Structural Studies of RNA Polymerase II transcription initiation and elongation

描述(申请人提供)：DNA指导的RNA聚合酶II(POL II)是细胞中最重要的蛋白质之一。POL II负责转录绝大多数基因以产生信使RNA，信使RNA将被翻译到核糖体中产生所有细胞蛋白质。转录起始阶段需要Pol I与TFIIB、TBP、TFIIF、TFIIH和TFIIE等通用转录因子的适时相互作用。启动的过程是高度动态的；生化实验暗示，在GTF识别、熔化和加载核酸支架的可能离散阶段。最初的启动子熔化由TFIIH解旋酶触发，产生7-9个碱基的转录气泡(TB)；气泡进一步解离到大约18-25个碱基，并下降到Pol II的活性部位，在那里合成短的DNA-RNA杂交(转录本)；10个或更多核苷酸的转录本导致启动子逃逸和成熟TB的稳定，最终导致GTF移位，留下稳定的Pol II进入生产性延伸。这一过程对所有真核物种来说都是普遍的，是基因调控的核心；了解其分子细节将 提供可能导致对基因表达进行药理学操作的基本线索。这项建议的目的是利用生化和X射线结晶学技术来了解启动子与POL II结合的分子细节，以及TFIIB和TFIIF在TB负载和稳定中所起的作用。

DESCRIPTION (provided by applicant): DNA-directed RNA Polymerase II (Pol II) is one of the most important proteins in the cell. Pol II is responsible for transcribing the vast majority of genes to generate messenger RNA that will be translated in the ribosomes to produce all cellular proteins. The initiation stage of transcription requires timely interactions between Pol I and the general transcription factors or GTFs including, TFIIB, TBP, TFIIF, TFIIH and TFIIE. The process of initiation is highly dynamic; biochemical experiments hint at possible discrete stages where the GTFs recognize, melt and load a nucleic acid scaffold. Initial promoter melting is triggered by TFIIH helicases that generate a 7-9 bases transcription bubble (TB); the bubble is further unwound to approximately 18-25 bases and descends to Pol II's active site where a short DNA-RNA hybrid (transcript) is synthesized; transcripts of 10 or more nucleotides result in promoter escape and stabilization of a mature TB that ultimately leads to dislodging of the GTFs leaving a poised Pol II for entry into productive elongation. This process is universal, for all eukaryotic species, and is at the core of gene regulation; understanding its molecular details will provide essential clues that could potentially lead to pharmacological manipulation of gene expression. The intention of this proposal is to use biochemical and X-ray crystallography techniques to understand the molecular details of promoter binding to Pol II and the role that TFIIB and TFIIF play in TB loading and stabilization.