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.

这个项目的目标是更好地理解真核rna对转录启动的激活作用。 聚合酶II(RNAPII)，这一过程在癌细胞中通常是异常的。拟议中的实验将 联合共定位单分子光谱(COSMOS)，一种TIRF显微技术 用酿酒酵母核抽提物同时分析数百个单分子事件 支持强大的转录激活。提取物将从表达两到三个基因的菌株中制备 每个转录因子都用不同的颜色进行荧光标记。这些提取物将与一种 用另一种颜色标记转录激活剂(Gal4-vp16或Gcn4)，并将DNA模板固定在 显微镜载玻片。COSMOS可以精确测量启动子DNA、 激活子、共激活子和RNAPII转录机制。具体目标1将衡量时间 激活剂、RNAPII和共激活剂介体之间的关系。这些结果将表明， Mediator和RNAPII作为一个复合体到达和离开启动者，或者Mediator是否可以保持绑定到 支持多种RNAPII绑定事件。同样，它们将揭示激活者招聘调解人 涉及协同热力学相互作用，或者相反，如果激活剂以动力学方式加速形成 调解人-不再需要绑定激活剂的PIC复合体。《特定目标2》类似地分析了 激活剂影响共激活剂Swi/Snf、SAGA和NuA4的启动子结合。这三个因素作用于 因此，对比实验将在裸模板和显色模板上进行。 标记不同的辅助激活剂组合的实验将揭示它们的结合是否独立， 连续的、同时的或相互排斥的最后，《特定目标3》将比较单身的效果。 而不是结合在启动子上的多个激活剂。一组实验将监测匹配的启动子 具有单个而不是多个Gal4结合位点。Gal4-vp16也将与Gal4-vp64进行比较， 更强的激活剂，携带四个串联的vp16激活域与Gal4 DNA结合域融合。这些 实验将揭示转录协同是否反映结合频率、持续时间、 和/或共用共激活器和调解器/RNAPII。加在一起，这些单分子实验将 揭示有关转录激活的基本信息，这些信息是无法从系综中收集到的 生化或基因组技术。酵母系统被公认为是所有人的优秀模型系统。 真核生物，这里的发现将提供对哺乳动物同源物的更深层次的理解，这些同源物非常 在癌症中经常发生突变。