Abstract
Gal4-VP16 is an artificial transcription activator with a Gal4 DNA binding domain and aVP16 activation domain, helpful in studying eukaryotic transcription mechanism due to its high
transcriptional potency. While numerous papers have shown that Gal4-VP16 has a synergistic effect on transcriptional response when multiple binding sites are present on target DNA, its mechanism of synergy has not been well established. I investigated whether Gal4-VP16 binds cooperatively in vitro, since cooperative binding mechanism may contribute to transcriptional synergy. I expressed and fluorescently labeled a tagged Gal4-VP16 construct designed to have minimal effect on protein function. I then conducted single-molecule experiments with fluorescently labeled Gal4-VP16 to observe activators binding to target DNA molecules with five binding sites. Experimental traces of activator behavior qualitatively agreed with numerical simulation results that modeled independent binding. This suggests that synergy does not arise from cooperative Gal4-VP16 binding, but instead occurs later in the transcription process.