A positive regulatory gene is required for accumulation of the functional messenger RNA for the glucose-repressible alcohol dehydrogenase from Saccharomyces cerevisiae

Abstract

The amount of glucose-repressible alcohol dehydrogenase is regulated by the amount of its functional messenger RNA. ADHII † † Abbreviations used: ADHII, alcohol dehydrogenase, isoenzyme II: ADHI, alcohol dehydrogenase, isoerizyme 1: SDS, sodium dodecyl sulfate. protein was detected by a radioimmune assay and differentiated from ADHI, the classical ADH isozyme, by limited proteolysis with Staphylococcus aureus protease. When yeast containing the wild-type alleles for ADR2 (the ADH II structural locus) and for ADR1 (its positive regulatory gene) were pulse-labeled with [ 35S]methionine during derepression, radioactive label accumulated in the antibody-precipitated ADHII coterminously with the appearance of ADHII activity. The kinetics of functional ADHII mRNA appearance during derepression in this strain were shown to be the same as those for ADHII protein synthesis in vivo when RNA, extracted from derepressed cells, was translated in a wheat germ cell-free translation system. The role of the positive regulatory gene, ADR1, in ADHII expression was analyzed using two strains mutated at that locus. Yeast containing the adr1-1 allele are incapable of derepressing ADHII activity. When this strain was pulselabeled with [ 35S]methionine during derepression, approximately one-tenth to one-twentieth the level of ADHII protein synthesis was detected as in the wild-type strain. When RNA was extracted during derepression from cells containing the udr1-1 allele and translated in a wheat germ cell-free system, little functional ADHII mRNA was found to be present. The role of the ADR1 gene was further analyzed using a strain containing the ADR1-5 c allele, which allows constitutive synthesis of ADHII activity. In this strain during glucose repression. ADHII protein synthesis and amount of functional mRNA were at levels comparable to those found for the wild-type strain after complete derepression. Similar kinetics of ADHII protein synthesis and of mRNA accumulation during derepression were observed in the strain carrying the ADR1-5 c allele when compared to that carrying the ADR1 allele, but the absolute amounts were greater by three- to fourfold in cells containing the ADR1-5 c allele. These results indicate that the ADR1 gene acts to increase the level of functional ADHII mRNA during derepression.