Structural Studies of RNA Polymerase II transcription initiation and elongation

描述（由申请人提供）：dna定向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，这些信使RNA将在核糖体中翻译以产生所有细胞蛋白质。转录起始阶段需要Pol I与TFIIB、TBP、TFIIF、TFIIH、TFIIE等通用转录因子或gtf及时相互作用。启动过程是高度动态的；生化实验暗示了gtf识别、融化和装载核酸支架的可能的离散阶段。初始启动子熔化由TFIIH解旋酶触发，产生7-9个碱基转录泡（TB）；气泡进一步展开到大约18-25个碱基，并下降到Pol II的活性位点，在那里合成短DNA-RNA杂交（转录物）；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脱位，留下一个稳定的Pol II进入生产延伸。这个过程是普遍的，对所有真核生物物种来说，是基因调控的核心；了解它的分子细节

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.