Gal80-Gal80 interaction on adjacent Gal4p binding sites is required for complete GAL gene repression

Regulation of the GAL genes of Saccharomyces cerevisiae is determined by th e interplay of the transcriptional activator Gal4p and the repressor Gal80p , which binds and masks the activation domain of Gal4p under non-inducing c onditions. Here we demonstrate that Gal80p dimerizes with high affinity and that this dimerization appears to stabilize the Gal4p-Gal80p interaction a nd also, indirectly, the Gal4p-DNA interaction in a (Gal4p)(2)(Gal80p)(2)DN A complex. In addition, Gal80 dimers transiently interact with each other t o form higher order multimers, We provide evidence that adjacent Gal4p bind ing sites, when correctly spaced, greatly stabilize Gal80p dimer-dimer inte ractions and that this stabilization results in the complete repression of GAL genes with multiple Gal4p binding sites. In contrast, GAL genes under t he control of a single Gal4p binding site do not stabilize Gal80p multimers , resulting in significant and biologically important transcriptional leaka ge. Cooperative binding experiments indicate that Gal80p dimer-dimer intera ction probably does not lead to a stronger Gal4p-Gal80p interaction, but mo st likely to a more complete shielding of the Gal4p activation domain.