SEQUENCE-SPECIFIC BINDING BY ASPERGILLUS-NIDULANS AFLR, A C-6 ZINC CLUSTER PROTEIN REGULATING MYCOTOXIN BIOSYNTHESIS

The Aspergillus nidulans aflR gene is found within a 60 kb gene cluste r that includes approximate to 24 other genes that putatively function in the production of the aflatoxin-related mycotoxin sterigmatocystin . Previous work showed that AflR is a C-6 zinc binuclear cluster prote in that is conserved across Aspergillus spp. and functions as a pathwa y-specific transcription factor in activating expression of other clus ter genes. In this report, we demonstrate that A. nidulans AflR (AnAfl R) is a 45 kDa protein that binds to the palindromic sequence 5'-TCG(N -5)CGA-3' found in the promoter regions of several aflatoxin and steri gmatocystin cluster genes (ste genes). The in vivo relevance of this A nAflR binding site was assessed by examining the contribution of the t hree TCG(N-5)CGA elements in the 1.1 kb promoter region of stcU using gene fusions with the bacterial uidA gene encoding beta-glucuronidase (GUS). By mutating one, two or all three of the AnAflR-binding element s and examining GUS activity in wild-type aflR or Delta aflR A. nidula ns strains, we found that ste gene activation required both AnAflR and at least one TCG(NF5)CGA AflR binding site.