"Mechanisms of RNA Polymerase II initiation"

“RNA 聚合酶 II 的启动机制”

• 批准号: 9160425
• 负责人: Craig Kaplan
• 金额: $ 41.3万
• 依托单位: TEXAS A&M AGRILIFE RESEARCH
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-09-23 至 2020-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9160425
• 关键词: Active Sites; Affect; Alleles; Architecture; Biochemical; Biological Models; Chromatin; Complex; Coupled; DNA; DNA Polymerase II; DNA Sequence; DNA Sequencing Facility; Data; Defect; Disease; Drosophila genus; Drosophila melanogaster; Eukaryota; Fission Yeast; Gene Expression; Genes; Genetic; Genetic Transcription; Genomics; Growth; Human; In Vitro; Knowledge; Location; Modeling; Molecular; Motor; Nucleosomes; Organism; Output; Pattern; Peptide Initiation Factors; Polymerase; Positioning Attribute; Probability; Process; Property; Proteins; RNA Polymerase II; Reporter; Saccharomyces; Saccharomyces cerevisiae; Saccharomycetales; Scanning; Shapes; System; Testing; Thinking; Time; Transcription Initiation; Transcription Initiation Site; Variant; Work; Yeasts; base; combinatorial; design; expectation; genetic analysis; genome-wide; human disease; in vitro activity; in vivo; lens; molecular phenotype; mutant; novel; phosphodiester; preference; promoter; research study; tool; transcription factor; transcription factor TFIIH; translocase; whole genome

PROJECT SUMMARY Transcription in eukaryotes is initiated following the coordinated action of dozens to hundreds of proteins. These activities result in the recruitment of RNA Polymerase II (Pol II) to core promoters and the subsequent initiation process. During initiation, Pol II must be directed to the transcription start site (TSS) followed by TSS recognition, initial phosphodiester bond formation, and promoter clearance. The mechanism by which Pol II initiation engages the TSS and how this process determines overall promoter output is surprisingly poorly understood. Most promoters in eukaryotes initiate transcription in a broad or dispersed fashion where multiple TSSs are used. With a collaborative team in place, we wish to determine the mechanism by which Pol II engages TSSs in initiation and how initiation is affected by promoter architecture. In budding yeast, Pol II scans for TSSs and this allows us to rationalize how biochemical activities in initiation and promoter architecture may determine initiation levels. We have rationalized initiation by a model we call the Shooting Gallery that relates initiation probability to the cooperation between the Pol II active site and the activity of TFIIH, the putative motor that drives scanning. We have designed genetic, genomic, and biochemical experiments to explore and test the Shooting Gallery model. Our approaches include quantitative modeling of initiation at all yeast promoters to determine how they might be unique or what promoter features control expression levels. We will establish Schizosaccharomyces pombe and Drosophila melanogaster as models for direct tests of the conservation of promoter scanning as a mechanism for Pol II initiation. Our experiments will reveal whether there are universal initiation mechanisms in eukaryotes or not. We expect to greatly increase our power to predict promoter output based on biochemical properties of Pol II and TFIIH, our understanding of promoter architecture, and their interplay with underlying sequence, each of which are fundamental features of gene expression in all eukaryotes.

项目摘要 在数十个蛋白质的协调作用之后，真核生物中的转录开始。 这些活动导致RNA聚合酶II（POL II）募集到核心启动子和随后的 启动过程。在启动过程中，Pol II必须针对转录起始位点（TSS），然后是TSS 识别，初始磷酸二酯键的形成和启动子清除率。 pol II的机制 启动参与TSS，以及该过程如何确定整体启动子的输出，令人惊讶 理解。真核生物中的大多数发起人以多种多样的方式启动转录 使用TSS。有了一个协作团队，我们希望确定Pol II的机制 吸引TSSS参与启动以及如何受到启动器体系结构的影响。在萌芽的酵母中，pol II 扫描TSSS，这使我们能够合理化启动和启动子中的生化活动 体系结构可能决定起始水平。我们通过一个模型合理化了启动，我们称射击 画廊将启动概率与Pol II活动站点之间的合作与活动的活动相关联 TFIIH，推定的电动机，可驱动扫描。我们设计了遗传，基因组和生化 探索和测试拍摄库模型的实验。我们的方法包括定量建模 在所有酵母启动子中的启动，以确定它们可能是独特的或启动子的控制 表达水平。我们将建立schizosacchomyces pombe和果蝇Melanogaster作为模型 直接测试启动子扫描作为Pol II启动的机制。我们的实验会 揭示真核生物中是否存在普遍的起始机制。我们期望大大增加 我们根据Pol II和TFIIH的生化特性预测启动子产量的能力，我们对 启动子架构及其与基本顺序的相互作用，每个序列都是基本特征 所有真核生物中的基因表达。