Project Summary/Abstract. 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. Our system combines Colocalization Single-Molecule Spectroscopy (CoSMoS, a TIRF microscopy technique for simultaneously analyzing hundreds of single-molecule events) with Saccharomyces cerevisiae nuclear extracts from strains with one or more transcription coactivators engineered for fluorescence labeling. Combining these extracts with a transcription activator (that may be labeled with yet another color), we image binding of up to three factors to DNA templates immobilized on the microscope slide. CoSMoS thus allows precise measurements of interaction dynamics between promoter DNA, activators, co-activators, and the RNApII transcription machinery. Specific Aim 1 extends our work on Mediator from the previous period. Having established the temporal relationships between activator, RNApII, and the co-activator Mediator, we will determine how the Tail and Kinase modules affect the process. We will further test our model that Mediator, RNApII, and a subset of basal factors form a “pre-PIC” while bound to activators, poised for transfer of this activation intermediate to the core promoter. Specific Aim 2 analyzes how activators “recruit” the coactivators SAGA, Swi/Snf, and NuA4. These three factors act upon nucleosomes, so our experiments compare naked versus chromatinized templates, immobilized side-by-side on the same microscope slide. Experiments labeling different combinations of coactivators will reveal if their binding is independent, sequential, simultaneous, or mutually exclusive. Finally, Specific Aim 3 looks at how the characteristics of the activators themselves influence coactivator interactions. As a model system, we compare different activation domains (VP16, Gcn4, Rap1, et al.) fused to the same Gal4 DNA binding domain. We are also studying the natural yeast activator Gcn4. Using matched promoters having single versus multiple Gal4 binding sites, we are uncovering direct evidence for transcription synergy and mechanisms of cooperativity. Interestingly, there are measurable differences in dynamics produced by different activation domains and different coactivators. 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.