Abstract
It is long established that the presence of multiple binding sites for transcription activator proteins can synergistically stimulate RNA polymerase II (RNApII) transcription at a promoter. Multiple mechanisms for activator synergy that act at different stages in the transcription pathway have been proposed, but none are fully established. In this work, I investigated synergy at stages of activated RNApII transcription leading up to preinitiation complex (PIC) formation by using multi-wavelength colocalization single-molecule spectroscopy (“CoSMoS”). Experiments were performed in an in vitro budding yeast nuclear extract system and driven by the recombinant activator Gal4-VP16. In experiments that used combinations of labeled Gal4-VP16, Mediator (labeled on the core Med7 subunit), and RNApII (labeled on the Rpb1 subunit), we observed the binding and unbinding of the labeled proteins with individual surface-tethered template molecules in real-time. In Chapter 2, I show that association of the proteins with the template was hierarchical: essentially all Mediator binding required the presence of Gal4-VP16 on the DNA and similarly RNApII binding required the presence of Mediator. Mediator initial recruitment to DNA occurs via interaction with the UAS rather than the core promoter, consistent with the idea that forming an UAS-bound activator-tethered “pre-PIC” containing Mediator, RNApII, and one or more general transcription factors is a precursor to core promoter-bound PIC formation, at least at some RNApII promoters. We developed a new kinetic scheme to model association events; the derivation is expanded in Chapter 3.I also analyzed the fluorescence spot intensities in the CoSMoS images using the image physics-based Bayesian machine learning program Tapqir. This allowed us to quantitate the distributions of the numbers of activator and Mediator molecules per DNA. The number of activator dimers on a DNA was on average higher on DNAs where Mediator was detected then at equivalent timepoints on DNAs where Mediator was absent, suggesting that the presence of multiple activator proteins help the recruitment and/or stabilization of Mediator. This model is further supported by additional measurements showing that Mediator occupancy on DNA had a highly cooperative dependence on activator concentration, with a Hill coefficient = 4.39 [95% CI: 4.35, 4.43]. Simultaneous involvement of multiple activator molecules linking Mediator to DNA at the UAS is consistent with our quantitative data and could contribute to activator synergy. Taken together, our results offer insight into the real-time actions by which activator and Mediator regulate RNApII recruitment. We propose that multiple UAS-bound activators cooperatively recruit Mediator and RNApII sequentially to UAS, after which they are transferred to the core promoter for PIC assembly and initiation.