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.

该项目的目的是更好地了解真核RNA转录启动的激活 聚合酶II（RNAPII），这一过程在癌细胞中通常异常。提出的实验将 结合共定位单分子光谱（宇宙，一种TIRF显微镜技术，用于 同时用酿酒酵母核提取物分析数百个单分子事件） 这支持强大的转录激活。提取物将通过表达两三个的菌株制备 转录因子每个荧光用不同的颜色标记。这些提取物将与 带有另一种颜色的标记的转录激活剂（GAL4-VP16或GCN4），DNA模板固定在 显微镜载玻片。宇宙允许精确测量启动子DNA之间的相互作用动力学 激活剂，共激活器和RNAPII转录机械。特定目标1将测量时间 激活剂，RNAPII和共激活器介体之间的关系。这些结果将显示是否 调解员和RNAPII到达并将发起人作为复合物离开，或者调解员是否可以与 支持多个RNAPII结合事件。同样，他们将揭示激活者是否招募中介者 涉及合作热力学相互作用，或者如果激活因激活剂会加速A的形成 不再需要绑定激活剂的介体-PIC复合物。特定目标2是对如何的类似分析 激活因子会影响共激活器SWI/SNF，SAGA和NUA4的启动子结合。这三个因素对 核小体，因此将在裸模板上与染色体化模板上进行比较实验。 标记共激活因子不同组合的实验将揭示其结合是否独立， 顺序，同时或相互排斥。最后，特定的目标3将比较单身的效果 与启动子上绑定的多个激活剂相对于多个激活剂。一组实验将监视匹配的启动子 具有单个GAL4结合位点。 GAL4-VP16也将与GAL4-VP64进行比较， 更强的激活因子携带四个串联VP16激活结构域融合到GAL4 DNA结合域。这些 实验将揭示转录协同作用是否反映了增加的结合频率，持续时间， 和/或共同激活因子和中介/rnapii的共占者。这些单分子实验将在一起 揭示有关转录激活的基本信息，这些信息是不可能从合奏中收集的 生化或基因组技术。酵母系统已成为所有人的出色模型系统 真核生物和这里的发现将为哺乳动物的同源物提供更深入的了解 经常在癌症中突变。