MECHANISMS OF EUKARYOTIC TRANSCRIPTION INITIATION

真核转录起始机制

• 批准号: 9335931
• 负责人: Eric A Galburt
• 金额: $ 30.12万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-09-01 至 2020-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9335931
• 关键词: Active Sites; Address; Biochemical; Biological; Biological Assay; Biophysics; Cells; Chromatin Loop; Collaborations; Complex; DNA; DNA-Directed RNA Polymerase; Data; Defect; Dependence; ERCC3 gene; Enzymes; Eukaryota; Exhibits; Foundations; Gene Expression; Gene Expression Regulation; Gene Targeting; Genetic Transcription; Goals; Homo sapiens; Human; Hydrolysis; Image; In Vitro; Knowledge; Lead; Location; Magnetism; Malignant Neoplasms; Measures; Mission; Modeling; Molecular; Molecular Conformation; Monitor; Motor; Mutation; National Institute of General Medical Sciences; Organism; Outcome; Pathway interactions; Phenotype; Play; Polymerase; Process; Property; RNA Polymerase II; Recruitment Activity; Regulation; Regulator Genes; Research; Resolution; Role; Saccharomyces cerevisiae; Scanning; Site; Stream; Structural Models; Structure; Superhelical DNA; System; TATA-Box Binding Protein; Techniques; Testing; Time; Transcription Coactivator; Transcription Elongation; Transcription Factor TFIIB; Transcription Initiation; Transcription Initiation Site; Transcription Process; Work; Yeasts; base; biophysical techniques; cell growth regulation; design; ds-DNA; experimental study; human disease; innovation; insight; laser tweezer; melting; novel; promoter; single molecule; transcription factor; transcription factor TFIIH; translocase

Project Summary/Abstract Transcription initiation plays a pivotal role in the establishment and regulation of cellular phenotypes in all organisms, yet the molecular mechanism of initiation in eukaryotes remains unclear. The process involves the recruitment of RNA polymerase (RNAP), DNA melting, transcription start-site scanning, the initiation of NTP hydrolysis by RNAP, and the transition into processive transcription elongation. In Eukaryotes, transcription is initiated by the pre-initiation complex (PIC) which minimally consists of of TATA-box binding protein (TBP), TFIIB, IIE, IIF, IIH, and RNAP II. Due to the indisputable biological significance of this complex, a plethora of biochemical and structural data have been collected on PICs with the goal of understanding mechanism. These studies have identified and described PIC components and have provided illuminating images of the complex that are exceptionally useful for developing mechanistic hypotheses. However, as structures are snapshots, they represent static images and lack information regarding the dynamics of the PIC factors and the DNA template during initiation. Ultimately, knowledge of how the complex works requires understanding how the PIC transitions between defined structural states along the pathway to initiation. We have begun to successfully observe the dynamic transitions between intermediate states using single-molecule assays well- suited to following transcription initiation. Our long-term goal is to determine the mechanisms behind transcrip- tion initiation and its regulation by promoter sequence, transcription factors, and transcriptional activators. This proposal aims to determine the structural transitions of the DNA template induced by PIC activity by applying single-molecule biophysical assays to purified transcription factors from both Saccharomyces cer- evisiae and Homo sapiens. The combination of our magnetic tweezers and optical tweezers studies will allow us to make distinct insights into a complex and unsynchronized process. The use of both yeast and human systems will uniquely allow us to compare initiation mechanisms between these highly homologous complexes that exhibit distinct activities in relation to their propensity to scan for start-sites and their ability to be activated by superhelical DNA. We will test competing models for the mechanism of start-site scanning, directly measure the rate and processivity of the recently demonstrated dsDNA translocase activity of Ssl2 in the context of TFIIH, and will test the hypothesis that the 5 bp open complex we have observed in preliminary work on yeast PICs is a conserved feature of eukaryotic initiation by monitoring DNA opening by human PICs. The proposed research is innovative because it uses high-resolution single-molecule techniques to di- rectly measure PIC-dependent conformational changes of promoter DNA and the dsDNA translocation activity of TFIIH in real-time. The results from the project will have a large impact as they will answer fundamental mechanistic questions regarding the dynamic processes that lead to transcription initiation in Eukaryotes.

项目摘要/摘要 转录倡议在细胞表型的建立和调节中起关键作用 所有生物，但是真核生物中启动的分子机制尚不清楚。该过程涉及 募集RNA聚合酶（RNAP），DNA熔化，转录起始位点扫描，NTP的主动性 RNAP的水解，并过渡到过程转录伸长。在真核生物中，转录为 由原始络合物（PIC）发起 Tfiib，IIE，IIF，IIH和RNAP II。由于这种复合物的无可争议的生物学意义 已经收集了对图片的生化和结构数据，以理解机制。 这些研究已经确定并描述了PIC组件，并提供了照明图像 复合物对于开发机械假设非常有用。但是，由于结构是 快照，它们代表静态图像，并且缺乏有关图片因素和动态的信息 启动过程中的DNA模板。最终，了解建筑群的工作需要理解 PIC如何沿着启动途径定义的结构状态之间的过渡。我们已经开始 成功观察中间状态之间的动态转变，使用单分子测定法 适合以下转录计划。我们的长期目标是确定转录本背后的机制 启动子序列，转录因子和转录激活剂的调节及其调节。 该建议旨在确定PIC活性引起的DNA模板的结构过渡 通过将单分子生物物理测定应用于两种糖含量的纯粹转录因子 Evisiae和Homo Sapiens。我们的磁性镊子和光学镊子研究的结合将允许 我们对复杂且不同步的过程有不同的见解。酵母和人的使用 系统将允许我们比较这些高度同源复合物之间的主动机制 与他们承诺扫描起始站点及其被激活的能力有关的揭示了不同的活动 由超螺旋DNA。我们将测试竞争模型的起点扫描机制，直接测量 在 TFIIH，并将检验以下假设：我们在酵母初期观察到的5 bp开放式复合物 图片是通过监测人类图片开放DNA的真核计划的保守特征。 拟议的研究具有创新性，因为它使用高分辨率的单分子技术来进行。 直接测量启动子DNA和DSDNA易位活性的PIC依赖性咨询变化 实时的TFIIH。该项目的结果将产生很大的影响，因为他们将回答基本 有关导致真核生物转录启动的动态过程的机械问题。