THE ROLE OF GCR1P IN THE TRANSCRIPTIONAL ACTIVATION OF GLYCOLYTIC GENES IN YEAST SACCHAROMYCES-CEREVISIAE

To study the interdependence of Gcr1p and Rap1p, we prepared a series of synthetic regulatory sequences that contained various numbers and c ombinations of CT-boxes (Gcr1p-binding sites) and RPG-boxes (Rap1p-bin ding sites). The ability of the synthetic oligonucleotides to function as regulatory sequences was tested using an ENO1-lacZ reporter gene. As observed previously synthetic oligonucleotides tides containing bot h CT-and RPG-boxes conferred strong UAS activity. Likewise, a lone CT- box did not show any UAS activity. By contrast, oligonucleotides conta ining tandem CT-boxes but no RPG-box conferred strong promoter activit y. This UAS activity was not dependent on position or orientation of t he oligonucleotides in the 5' noncoding region. However, it was depend ent on both GCR1 and GCR2. These results suggest that the ability of G cr1p to bind Gcr1p-binding sites in vivo is not absolutely dependent o n Rap1p. Eleven independent mutants of GCR1 were isolated that conferr ed weak UAS activity to a single CT-box. Five mutants had single mutat ions in Gcr1p's DNA-binding domain and displayed slightly higher affin ity for the CT-box. These results support the hypothesis that Gcr1p an d Gcr2p play the central role in glycolytic gene expression and that t he function of Rap1p is to facilitate the binding of Gcr1p to its targ et.