Single-molecule analysis of eukaryotic transcription activation

本项目的目的是更好地了解真核RNA对转录起始的激活作用 RNA聚合酶II（RNApII），这是一个在癌细胞中经常异常的过程。提出的实验将 联合收割机共定位单分子光谱学（CoSMoS，一种TIRF显微镜技术，用于 同时分析数百个单分子事件）与酿酒酵母核提取物 支持强大的转录激活。将从表达两种或三种 每个转录因子用不同的颜色荧光标记。这些提取物将与 用另一种颜色标记的转录激活因子（Gal 4-vp 16或Gcn 4）和固定在其上的DNA模板， 显微镜载玻片。CoSMoS允许精确测量启动子DNA之间的相互作用动态， 激活子、辅激活子和RNApII转录机制。具体目标1将测量时间 激活剂、RNApII和共激活剂介体之间的关系。这些结果将显示， Mediator和RNApII到达启动子并使其作为复合物离开，或者Mediator是否可以保持与启动子的结合。 支持多个RNApII结合事件。同样，他们将揭示是否激活剂招募调解员 涉及协同的热力学相互作用，或者相反，如果活化剂动力学地加速 不再需要结合激活剂的介体-PIC复合物。具体目标2是一个类似的分析如何 激活子影响辅激活子Swi/Snf、佐贺和NuA 4的启动子结合。这三个因素作用于 因此，将在裸模板与染色质化模板上进行比较实验。 标记辅激活因子的不同组合的实验将揭示它们的结合是否是独立的， 顺序的、同时的或互斥的。最后，具体目标3将比较具有单一 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Gal 4结合位点。Gal 4-vp 16也将与Gal 4-vp 64进行比较， 更强的激活剂，其携带融合至Gal 4 DNA结合结构域的四个串联vp 16激活结构域。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/或共激活子和介体/RNApII的共占据。这些单分子实验将 揭示了转录激活的基本信息，这是不可能从集合中收集到的 生物化学或基因组技术。酵母系统是一个很好的模式系统， 真核生物，这里的发现将提供更深入的了解哺乳动物同源物， 在癌症中经常发生突变

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.