Single-molecule analysis of eukaryotic transcription activation

真核转录激活的单分子分析

• 批准号: 10544151
• 负责人: Stephen Buratowski
• 金额: $ 40.37万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-02-07 至 2025-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10544151
• 关键词: Acceleration; Acetyl Coenzyme A; Affect; Behavior; Binding; Binding Proteins; Binding Sites; Biochemical; Biological Models; Cells; Chromatin; Color; Complement; Complex; DNA; DNA Binding Domain; Disease; Dissociation; Enhancers; Eukaryota; Event; Fluorescence; Frequencies; Gal-VP16; Gene Expression; Gene Expression Regulation; Genetic Transcription; Genomics; Glean; Goals; Histone Acetylation; Homologous Gene; Immobilization; Kinetics; Label; Malignant Neoplasms; Measurement; Measures; Mediator; Methods; Microscope; Microscopy; Modeling; Molecular; Monitor; Mutate; Normal Cell; Nuclear Extract; Nucleosomes; Oncogenes; Pathway interactions; Pharmaceutical Preparations; Process; Property; Proteins; Proto-Oncogenes; RNA Polymerase II; RNA Splicing; Reaction; SAGA; Saccharomyces cerevisiae; Slide; Spectrum Analysis; System; TP53 gene; Techniques; Technology; Testing; Thermodynamics; Total Internal Reflection Fluorescent; Transcription Coactivator; Transcription Initiation; Transcriptional Activation; Tumor Suppressor Proteins; Yeasts; cancer cell; comparative; experience; experimental study; histone acetyltransferase; light microscopy; mRNA Precursor; promoter; recruit; residence; single molecule; synergism; therapy design; transcription factor

The goal of this project is to better understand activation of transcription initiation by eukaryotic RNA polymerase II (RNApII), a process that is often abnormal in cancer cells. The experiments proposed will combine Colocalization Single-Molecule Spectroscopy (CoSMoS, a TIRF microscopy technique for simultaneously analyzing hundreds of single-molecule events) with Saccharomyces cerevisiae nuclear extracts that support robust transcription activation. Extracts will be prepared from strains expressing two or three transcription factors each fluorescently labeled with a different color. These extracts will be combined with a transcription activator (Gal4-vp16 or Gcn4) labeled with yet another color, and DNA templates immobilized on the microscope slide. CoSMoS allows precise measurements of interaction dynamics between promoter DNA, activators, co-activators, and the RNApII transcription machinery. Specific Aim 1 will measure temporal relationships between activator, RNApII, and the co-activator Mediator. These results will show whether Mediator and RNApII arrive at and leave promoters as a complex, or whether Mediator can stay bound to support multiple RNApII binding events. Similarly, they will reveal whether activator recruitment of Mediator involves cooperative thermodynamic interactions, or instead if activator kinetically accelerates formation of a Mediator-PIC complex that no longer requires bound activator. Specific Aim 2 is a similar analysis of how activator affects promoter binding of the coactivators Swi/Snf, SAGA, and NuA4. These three factors act upon nucleosomes, so comparative experiments will be carried out on naked versus chromatinized templates. Experiments labeling different combinations of coactivators will reveal if their binding is independent, sequential, simultaneous, or mutually exclusive. Finally, Specific Aim 3 will compare effects of having single versus multiple activators bound at the promoter. One set of experiments will monitor matched promoters having single versus multiple Gal4 binding sites. Gal4-vp16 will also be compared to Gal4-vp64, an even stronger activator that carries four tandem vp16 activation domains fused to Gal4 DNA binding domain. These experiments will reveal whether transcriptional synergy reflects increased binding frequencies, durations, and/or co-occupancy of coactivators and Mediator/RNApII. Together, these single molecule experiments will reveal fundamental information about transcription activation that has been impossible to glean from ensemble biochemical or genomic techniques. The yeast system is well established as an excellent model system for all eukaryotes, and findings here will provide deeper understanding of the mammalian homologs that are very frequently mutated in cancer.

该项目的目标是更好地了解真核 RNA 转录起始的激活 聚合酶 II (RNApII)，癌细胞中经常出现异常的过程。所提出的实验将 结合共定位单分子光谱（CoSMoS，一种 TIRF 显微镜技术） 使用酿酒酵母核提取物同时分析数百个单分子事件） 支持强大的转录激活。提取物将从表达两种或三种的菌株中制备 每个转录因子都用不同的颜色进行荧光标记。这些提取物将与 转录激活剂（Gal4-vp16 或 Gcn4）用另一种颜色标记，DNA 模板固定在 显微镜载玻片。 CoSMoS 可以精确测量启动子 DNA 之间的相互作用动态， 激活子、共激活子和 RNApII 转录机制。具体目标 1 将测量时间 激活子、RNApII 和共激活子介体之间的关系。这些结果将表明是否 Mediator 和 RNApII 作为复合物到达并离开启动子，或者 Mediator 是否可以与启动子结合 支持多个 RNApII 结合事件。同样，他们将揭示是否招募 Mediator 的激活者 涉及协同热力学相互作用，或者如果活化剂在动力学上加速形成 不再需要结合激活剂的介体-PIC 复合物。具体目标 2 是一个类似的分析 激活剂影响共激活剂 Swi/Snf、SAGA 和 NuA4 的启动子结合。这三个因素作用 核小体，因此将在裸模板和染色模板上进行比较实验。 标记不同的共激活剂组合的实验将揭示它们的结合是否独立， 顺序的、同时的或互斥的。最后，具体目标 3 将比较单一目标的效果 与结合在启动子上的多个激活剂相比。一组实验将监测匹配的启动子 具有单个或多个 Gal4 结合位点。 Gal4-vp16 还将与 Gal4-vp64 进行比较，Gal4-vp64 甚至是 更强的激活剂，携带四个与 Gal4 DNA 结合域融合的串联 vp16 激活域。这些 实验将揭示转录协同作用是否反映了结合频率、持续时间的增加， 和/或共激活子和介体/RNApII 的共同占用。总之，这些单分子实验将 揭示有关转录激活的基本信息，而这些信息是不可能从整体中收集到的 生物化学或基因组技术。酵母系统已成为所有领域的优秀模型系统 真核生物，这里的发现将提供对哺乳动物同源物的更深入的了解，这些同源物是非常重要的 在癌症中经常发生突变。