DISSECTION OF A CARBOXY-TERMINAL REGION OF THE YEAST REGULATORY PROTEIN RAP1 WITH EFFECTS ON BOTH TRANSCRIPTIONAL ACTIVATION AND SILENCING

RAP1 is an essential sequence-specific DNA-binding protein in Saccharomyces cerevisiae whose binding sites are found in a large number of promoters, where they function as upstream activation sites, and at the silencer elements of the HMR and HML mating-type loci, where they are important for repression. We have examined the involvement of specific regions of the RAP1 protein in both repression and activation of transcription by studying the properties of a series of hybrid proteins containing RAP1 sequences fused to the DNA-binding domain of the yeast protein GAL4 (amino acids 1 to 147). GAL4 DNA-binding domain/RAP1 hybrids containing only the carboxy-terminal third of the RAP1 protein (which lacks the RAP1 DNA-binding domain) function as transcriptional activators of a reporter gene containing upstream GAL4 binding sites. Expression of some hybrids from the strong ADHI promoter on multicopy plasmids has a dominant negative effect on silencers, leading to either partial or complete derepression of normally silenced genes. The GAL4/RAP1 hybrids have different effects on wild-type and several mutated but functional silencers. Silencers lacking either an autonomously replicating sequence consensus element of the RAP1 binding site are strongly derepressed, whereas the wild-type silencer containing a deletion of the binding site for another silencer-binding protein, ABF1, are only weakly affected by hybrid expression. By examining a series of GAL4 DNA-binding domain/RAP1 hybrids, we have mapped the transcriptional activation and derepression functions to specific parts of the RAP1 carboxy terminus. Activation requires only a small region from amino acids 630 to 695, whereas derepression is observed only when the carboxy-terminal 150 amino acids (678 to 827) are present in the hybrids. Several GAL4 DNA-binding domain/RAP1 hybrids function only as activators or as derepressors, supporting the idea that at least some activation and silencing functions of RAP1 residue in separate parts of the protein.