THE TRANSCRIPTIONAL ACTIVATORS BAS1, BAS2, AND ABF1 BIND POSITIVE REGULATORY SITES AS THE CRITICAL ELEMENTS FOR ADENINE REGULATION OF ADE5,7

Adenine repression of the purine nucleotide biosyn thetic genes in Sac charomyces cerevisiae involves downregulation of the activator protein BAS1 or BASS by an unknown mechanism, To determine the minimal cis-ac ting requirements for adenine regulation, hybrid promoter constructs w ere made between ADE5,7 promoter fragments and a CYC1-lacZ reporter, A 139-nucleotide fragment containing two BAS1 binding sites was suffici ent to confer adenine regulation on the CYC1-lacZ reporter, Analysis o f deletion and substitution mutations led to the conclusion that the p roximal BAS1 binding site is both necessary and sufficient for regulat ion, whereas the distal site augments the function of the proximal sit e, By performing saturation mutagenesis, we found two essential region s that flank the proximal site, An ABF1 consensus sequence is within o ne of these regions, and mutations that impaired in vitro ABF1 binding impaired promoter activity in vivo. A second region is AT-rich and ap pears to bind BASS, No substitution mutations led to high level consti tutive promoter activity as would be expected from removal of an upstr eam repression sequence. Our results indicate that ABF1, BAS1, and BAS S are required for ADE5,7 pro meter function and that adenine repressi on most likely involves activator modification or a negative regulator that does not itself bind DNA.