Cloning, nucleotide sequence, and regulation of MET14, the gene encoding the APS kinase of Saccharomyces cerevisiae

Abstract

The MET14 gene of Saccharomyces cerevisiae, encoding APS kinase (ATP:adenylylsulfate-3'-phosphotransferase, EC 2.7.1.25), has been cloned. The nucleotide sequence predicts a protein of 202 amino acids with a molecular mass of 23,060 dalton. Translational fusions of MET14 with the beta-galactosidase gene (lacZ) of Escherichia coli confirmed the results of primer extension and Northern blot analyses indicating that the ca. 0.7 kb mRNA is transcriptionally repressed by the presence of methionine in the growth medium. By primer extension the MET14 transcripts were found to start between positions -25 and -45 upstream of the initiator codon. Located upstream of the MET14 gene is a perfect match (positions -222 to -229) with the previously proposed methionine-specific upstream activating sequence (UASMet). This is the same as the consensus sequence of the Centromere DNA Element I (CDEI) that binds the Centromere Promoter Factor I (CPFI) and of two regulatory elements of the PHO5 gene to which the yeast protein PHO4 binds. The human oncogenic protein c-Myc also has the same recognition sequence. Furthermore, in the 270 bp upstream of the MET14 coding region there are several matches with a methionine-specific upstream negative (URSMet) control element. The significance of these sequences was investigated using different upstream deletion mutations of the MET14 gene which were fused to the lacZ gene of E. coli and chromosomally integrated. We find that the methionine-specific UASMet and one of the URSMet lie in regions necessary for strong activation and weak repression of MET14 transcription, respectively. We propose that both types of control are exerted on MET14.