A REB1P-BINDING SITE IS REQUIRED FOR EFFICIENT ACTIVATION OF THE YEAST RAP1 GENE, BUT MULTIPLE BINDING-SITES FOR RAP1P ARE NOT ESSENTIAL

The Saccharomyces cerevisiae RAP1 protein (Rap1p) is a key multifunctional transcription factor. Using gel retardation analysis, four binding sites for Rap1p have been identified within the promoter of the RAP1 gene. These sites are located downstream of a binding site for the transcription factor Reb1p. The Reb1p site and an associated AT-rich region are important for transcriptional activation, but deletion of three of the Rap1p-binding sites had little effect on promoter activity. The activity of the RAP1 promoter has been analysed in a yeast strain (YDS410) that contains a temperature-sensitive mutation in the RAP1 gene. This mutation renders the DNA-binding activity of Rap1p temperature dependent. When YDS410 was grown at a semi-permissive temperature (30 degrees C), the activity of the RAP1 promoter increased by approximately 170%, compared with the same strain grown at the permissive temperature (25 degrees C). A RAP1 promoter in which three of the four Rap1p-binding sites had been deleted, showed only a small increase in activity in the same experiment. These data confirm that Rap1p is not required for activation of the RAP1 gene, and suggest a role for Rap1p in negative autoregulation.