Extent of in vivo binding by an upstream activation factor and the role of multiple binding sites in synergistic transcriptional activation.

We have analyzed site occupancy in vivo with constructs containing one or more copies of the binding site for the yeast trans-activator, yIBF. The data indicate only a modest difference in site occupancy (at most 2-fold) even though multiple copies activate transcription several hundredfold more than one copy. Using studies in which we have mutated the IES2 sequence and slightly decreased its affinity for yIBF, we have also shown that synergistic activation of transcription is dependent on overall site occupancy. Finally, synergy declines as yIBF-binding sites are separated, and loss of synergy appears to be correlated with loss of a linking surface. These results are consistent with the simultaneous contact model of synergistic activation.