Structural Studies of RNA Polymerase II transcription initiation and elongation

RNA 聚合酶 II 转录起始和延伸的结构研究

基本信息

批准号：
8798014
负责人：
Guillermo Alberto Calero
金额：
$ 29.65万
依托单位：
UNIVERSITY OF PITTSBURGH AT PITTSBURGH
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-01-15 至 2019-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8798014
关键词：
Active Sites Architecture Area Award Binding Biochemical Cells Communities Complex Crystallization Crystallography DNA DNA Polymerase I DNA-Directed RNA Polymerase Data Face Gene Expression Gene Expression Regulation General Transcription Factors Genes Genetic Transcription Hybrids Intention Knowledge Laboratories Lead Left Maps Membrane Messenger RNA Methodology Molecular Nucleic Acids Nucleotides Play Polymerase Positioning Attribute Process Proteins RNA RNA Polymerase II Research Resolution Resources Ribosomes Roentgen Rays Role Staging Structural Biologist Structure Techniques Time Transcript Transcription Elongation Transcription Factor TFIIB Transcription Initiation Translating Transmission Electron Microscopy Work X-Ray Crystallography base cell dimension crosslink density electron density free-electron laser helicase human GTF2B protein human disease improved melting nano nanocrystal nanoscale novel novel strategies prevent promoter protein complex public health relevance receptor reconstitution research study scaffold stoichiometry transcription factor TFIIE transcription factor TFIIF transcription factor TFIIH

项目摘要

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.

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，以产生将在核糖体中翻译以产生所有细胞蛋白的信使RNA。转录的起始阶段需要Pol I与一般转录因子或GTF之间的及时相互作用，包括TFIIB，TBP，TFIIF，TFIIH，TFIIH和TFIIE。启动过程是高度动态的。生化实验暗示了GTF识别，融化和加载核酸支架的可能离散阶段。初始启动子熔化是由产生7-9个基碱基转录气泡（TB）的TFIIH解旋酶触发的。气泡进一步解开了大约18-25个底座，并降落到Pol II的活性位点，在该位点合成了短的DNA-RNA杂交（转录本）。 10个或多个核苷酸的转录物导致启动子逃脱和成熟的结核病的稳定，最终导致GTF驱逐出去，使固定的Pol II进入生产性延伸。对于所有真核物种，这个过程都是普遍的，并且是基因调节的核心。了解其分子细节将提供可能导致基因表达的药理学操纵的基本线索。该建议的目的是使用生化和X射线晶体学技术来了解启动子与Pol II的分子细节以及TFIIB和TFIIF在TB负载和稳定中的作用。