The carboxy-terminal portion of the aflatoxin pathway regulatory protein AFLR of Aspergillus parasiticus activates GAL1 :: lacZ gene expression in Saccharomyces cerevisiae

AFLR, a DNA-binding protein of 444 amino acids, transactivates the. express ion of aflatoxin biosynthesis genes in Aspergillus parasiticus and Aspergil lus flavus, as well as the sterigmatocystin synthesis genes in Aspergillus nidulans. We show here by fusion of various aflR coding regions to the GAL4 DNA-binding coding region that the AFLR carboxyl terminus contained a regi on that activated GAL1::lacZ gene expression in Saccharomyces cerevisiae an d that the AFLR internal region was required for the activation activity. C ompared to the AFLR carboxy-terminal fusion protein (AFLRC), a mutant AFLRC retained approximately 75% of the activation activity after deletion of th ree acidic amino acids, Asp365, Glu366, and Glu367, in a previously identif ied acidic: stretch. Removal of the carboxy-terminal amino acid, Glu444, di d not affect the activation activity. Substitutions of acidic. Glu423, Asp4 39, or Asp436/Asp439 with basic, amino acids, Lys and His, resulted in 10- to 15-fold-lower activation activities. Strikingly, the Asp436His mutation abolished the activation activity. Substitutions of basic His428 and His442 with acidic Asp resulted in 20 and. 40% decreases in the activation activi ties, respectively. Simultaneous substitutions of Arg427, Arg429, and Arg43 1 with Leu also significantly decreased the activation activity; the decrea se was approximately 50-fold. Results suggest that the AFLR carboxy-termina l region is involved in transcription activation and that total acidity in this region is not a major determinant of;AFLR's activation ability in S. c erevisiae.