ANALYSIS OF THE CHICKEN GPAT AIRC BIDIRECTIONAL PROMOTER FOR DE-NOVO PURINE NUCLEOTIDE SYNTHESIS

GPAT and AIRC encode two enzymes that catalyze steps 1 and 6 plus 7, r espectively, of the de novo purine biosynthetic pathway. The chicken g enes are closely linked and divergently transcribed from an similar to 230-base pair intergenic region, The promoter was scanned by deletion mutagenesis in a bireporter vector that allowed assay of transcriptio nal activity in both directions in transfected HepG2 and chicken LMH c ells, Three classes of deletions were obtained: those affecting bidire ctional transcription, those predominantly affecting GPAT transcriptio n, and those predominantly affecting AIRC transcription, Defects in bi directional transcription resulted from removal of an initiator-like e lement overlapping the AIRC transcription start site, as well as delet ions removing a series of GC and CCAAT boxes from the AIRC proximal ha lf of the promoter and a CCAAT-containing segment from the GPAT side. Several regions in the GPAT proximal half of the promoter, including a n octamer-like motif downstream from the transcription start site, wer e required predominantly for GPAT expression. Evidence for interaction of HeLa nuclear proteins with some of these sites was obtained by gel retardation, DNase I, and methylation interference assays. Overall, t he results showed that the intergenic region is an integrated bidirect ional promoter and that a novel initiator-like element plays a central role in coordinating expression of the divergently transcribed AIRC a nd GPAT genes.