IDENTIFICATION OF SEQUENCES RESPONSIBLE FOR TRANSCRIPTIONAL REGULATION OF THE STRONGLY EXPRESSED METHANOL OXIDASE-ENCODING GENE IN HANSENULA-POLYMORPHA

The methylotrophic yeasts have been the subject of intensive studies, because of their highly regulated methanol metabolism and the biogenes is of peroxisomes. We investigated the 5' regulatory region of the MOX gene from the yeast, Hansemula polymorpha, encoding the peroxisomal m ethanol oxidase, the key enzyme of methanol metabolism. This tightly r egulated yeast promoter of approximately 1.5 kb is unusually large, an d also of remarkable strength under inducing conditions, belonging to the strongest yeast promoters yet described. Deletion analyses reveale d a complex promoter structure composed of several sequence elements w ith positive and negative regulatory effects on reporter gene expressi on and a pronounced cooperation between the elements. Specific binding of several factors was detected in vitro by gel retardation and DNase I footprinting experiments. On the basis of deletion data, two bindin g sites could be identified as upstream activation sequences (UAS1 and UAS2) and one binding site as an upstream repressing sequence (URS1).