PROMOTER ANALYSIS OF THE PDA1 GENE ENCODING THE E1-ALPHA SUBUNIT OF THE PYRUVATE-DEHYDROGENASE COMPLEX FROM SACCHAROMYCES-CEREVISIAE

The location and sequence of the PDA1 gene, encoding the E1 alpha subu nit of the pyruvate dehydrogenase (PDH) complex from Saccharomyces cer evisiae, were determined. The PDA1 gene was located on a 6.2 kb fragme nt of chromosome V, approximately 18 kb centromere distal to RAD3. Con sistent with this, the PDA1 gene was genetically mapped at 4 cM from R AD3. A part of the 6.2 kb fragment of chromosome V was sequenced. The nucleotide sequence contained the PDA1 open reading frame and the enti re putative promoter. Computer analysis revealed a putative GCN4 bindi ng motif in the PDA1 promoter. The presence of transcriptional element s was experimentally determined by deletion analysis. To this end, Exo III deletions were constructed in the 5' to 3' direction of the PDA1 p romoter and effects on transcription were determined by Northern analy sis. Transcription was unaffected upon deletion to position -190 relat ive to the ATG start codon. Deletions from position -148 and beyond, h owever, reduced promoter activity at least 40-fold. Apparently the 42 bp between nucleotides -190 and -148 contain an element essential for transcription. Inactivation of the PDA1 promoter could not be attribut ed to deletions of a recognizable TATA element or any known yeast regu latory motifs. The possible role of the CCCTT sequence present in the 42 bp region and also in the promoters of the other genes encoding sub units of the PDH complex is discussed.