SEPARATION OF TRANSCRIPTIONAL ACTIVATION AND SILENCING FUNCTIONS OF THE RAP1-ENCODED REPRESSOR ACTIVATOR PROTEIN-1 - ISOLATION OF VIABLE MUTANTS AFFECTING BOTH SILENCING AND TELOMERE LENGTH

The repressor/activator protein 1 (RAP1) binds to the upstream activating sites of many genes, the silencer elements flanking the unexpressed mating-type loci HMR and HML, and the poly(C1-3A) sequences at telomeres, suggesting that RAP1 might have three distinct regulatory functions. To determine the in vivo role of RAP1 in repression of the HMR silent locus, we developed a screen to isolate rap1 mutants specifically defective in silencing. Fifteen independent mutants defining four different rap1 alleles were isolated. These alleles are defective to different extents in repression of an HMR locus containing a mutated, but fully functional, silencer. All four alleles are missense mutations in only three codons within a small C-terminal region of the gene. These silencing-defective mutants have no apparent growth defects, indicating that expression of the large number of essential genes that have promoters containing RAP1-binding sites is normal. A transcriptional silencing function of RAP1 can therefore be genetically separated from its presumably essential activation functions. Surprisingly, three of the silencing-defective rap1 alleles have significantly longer telomeres, suggesting that the function of RAP1 in both transcriptional silencing and telomere-length regulation may be related. In addition, we have demonstrated that increased gene dosage of either SIR1 or SIR4, two other factors required for silencing, suppresses the silencing defect of the rap1 mutants. The properties of SIR4 dosage suppression suggest that SIR4 protein may interact directly with RAP1 at silencers.