2 UPSTREAM ACTIVATION SEQUENCES CONTROL THE EXPRESSION OF THE XPR2 GENE IN THE YEAST YARROWIA-LIPOLYTICA

We have initiated a study of the promoter region of the alkaline extracellular protease gene (XPR2) from Yarrowia lipolytica to identify upstream sequences possibly involved in carbon, nitrogen, and peptone control of XPR2 expression. Deletion analysis showed that the TATA box and two major upstream activation sequences (UASs) were essential for promoter activity under conditions of repression or full induction. Within the distal UAS (UAS1), in vivo footprinting studies with dimethyl sulfate (DMS) identified two sequences similar to Saccharomyces cerevisiae GCN4 (-800 to -792)- and TUF/RAP1 (-790 to -778)-binding sites and two sequences which partially overlap a repeated sequence (-778 to -771 and -720 to -713) similar to the CAR1 upstream repression sequence of S. cerevisiae. Oligonucleotides carrying the TUF/RAP1-like-binding site and adjacent downstream nucleotides restored full transcriptional activity of a UAS1-deleted promoter. Within the proximal UAS (UAS2), a directly repeated decameric sequence (-146 to -137 and -136 to -127) was protected against DMS in vivo. Sequences identical to the ABF1 -binding site of S. cerevisiae (-121 to -109) or similar to the GCN4-binding site (-113 to -105) were not clearly protected from DMS in vivo. An oligomer (-150 to -106) carrying these three sequences, inserted into a UAS2-deleted promoter, increased the transcriptional activity. The results from footprints under different physiological conditions suggested that protein binding to both UASs was constitutive. Deletion of both UASs greatly reduced XPR2 expression without abolishing its regulation. Our results strongly suggest that these UASs are targets for transcriptional factors required for assisting specific regulatory proteins.