Cooperative DNA binding of the yeast transcriptional activator GAL4 (upstream activating sequence/GAL/in vivo "footprinting")

The yeast transcriptional regulatory protein GAL4 binds to four sites in the GAL upstream activating sequence and stimulates transcription of the adjacent GALI and GALIO genes. We show here that binding to at least two of these sites is cooperative in vivo. We also measure stimulation of transcription by pairs of GAL4 binding sites and find that the activities of low-affinity binding sites combine synergisti- cally, whereas the activities of high-affinity binding sites combine only additively. We suggest that the synergistic transcriptional stimulatory activity of the naturally occurring GAL4 binding sites is solely a manifestation of cooperative binding of GAL4 protein to DNA and that the activity of a GAL upstream activating sequence is roughly proportional to the number of GAL4 molecules bound.