ACTIVATION MECHANISM OF THE MULTIFUNCTIONAL TRANSCRIPTION FACTOR REPRESSOR-ACTIVATOR PROTEIN-1 (RAP1P)

Transcriptional activation in eukaryotic organisms normally requires c ombinatorial interactions of multiple transcription factors. In most c ases, the precise role played by each transcription factor is not know n. The upstream activating sequence (UAS) elements of glycolytic enzym e genes in Saccharomyces cerevisiae are excellent model systems for th e study of combinatorial interactions. The yeast protein known as Rap1 p acts as both a transcriptional repressor and an activator, depending on sequence context. Rap1p-binding sites are found adjacent to Gcr1p- binding sites in the UAS elements of glycolytic enzyme genes. These UA S elements constitute some of the strongest activating sequences known in S. cerevisiae. In this study, we have investigated the relationshi p between Rap1p- and Gcr1p-binding sites and the proteins that bind th em. In vivo DNA-binding studies with rap1(ts) mutant strains demonstra ted that the inability of Rap1p to bind at its site resulted in the in ability of Gcr1p to bind at adjacent binding sites. Synthetic oligonuc leotides, modeled on the UAS element of PYK1, in which the relative po sitions of the Rap1p- and Gcr1p-binding sites were varied were prepare d and tested for their ability to function as UAS elements. The abilit y of the oligonucleotides to function as UAS elements was dependent no t only on the presence of both binding sites but also on the relative distance between the binding sites. In vivo DNA-binding studies showed that the ability of Rap1p to bind its site was independent of Gcr1p b ut that the ability of Gcr1p to bind its site was dependent on the pre sence of an appropriately spaced and bound Rap1p-binding site. In vitr o binding studies showed Rap1p-enhanced binding of Gcr1p on oligonucle otides modeled after the native PYK1 UAS element but not when the Rap1 p- and Gcr1p-binding sites,vere displaced by 5 nucleotides. This work demonstrates that the role of Rap1p in the activation of glycolytic en zyme genes is to bind in their UAS elements and to facilitate the bind ing of Gcr1p at adjacent binding sites.