Structural Studies of RNA Polymerase II transcription initiation and elongation

描述（由申请人提供）：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，该信使RNA将在核糖体中翻译以产生所有细胞蛋白质。转录的起始阶段需要Pol I与一般转录因子或GTF（包括TFIIB、TBP、TFIIF、TFIIH和TFIIE）之间的及时相互作用。起始过程是高度动态的;生化实验暗示可能的离散阶段，其中GTF识别、熔化和装载核酸支架。初始启动子解链由TFIIH解旋酶触发，其产生7-9个碱基的转录泡（TB）;该泡进一步解绕至约18-25个碱基，并下降至Pol II的活性位点，其中短DNA-RNA杂合物（转录本）被合成; 10个或更多个核苷酸的转录物导致成熟TB的启动子逃逸和稳定化，最终导致GTF的移位，准备进入生产性伸长的Pol II。这一过程是普遍的，对于所有真核生物物种，是基因调控的核心;了解其分子细节将 提供了可能导致基因表达的药理学操纵的基本线索。该提案的目的是使用生物化学和X射线晶体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负载和稳定中发挥的作用。

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.