Binding of the C6-zinc cluster protein, AFLR, to the promoters of aflatoxin pathway biosynthesis genes in Aspergillus parasiticus

AFLR is a Zn₂Cys₆-type sequence-specific DNA-binding protein that is thought to be necessary for expression of most of the genes in the aflatoxin pathway gene cluster in Aspergillus parasiticus and A. flavus, and the sterigmatocystin gene cluster in A. nidulans. However, it was not known whether AFLR bound to the promoter regions of each of the genes in the cluster. Recently, A. nidulans AFLR was shown to bind to the motif 5′-TCGN₅CGA-3′. In the present study, we examined the binding of AFLR to promoter regions of 11 genes in the A. parasiticus cluster. Based on electrophoretic mobility shift assays, the genes nor1, pksA, adhA, norA, ver1, omtA, ordA, and, vbs, had at least one 5′-TCGN₅CGA-3′ binding site within 200 bp of the translation start site, and pksA and ver1 had an additional binding site further upstream. Although the promoter region of avnA lacked this motif, AFLR bound weakly to the sequence 5′-TCGCAGCCCGG-3′ at −110 bp. One region in the promoter of the divergently transcribed genes aflR/aflJ bound weakly to AFLR even though it contained a site with at most only 7 bp of the 5′-TCGN₅CGA-3′ motif. This partial site may be recognized by a monomeric form of AFLR. Based on a comparison of 16 possible sites, the preferred binding sequence was 5′-TCGSWNNSCGR-3′.     

Alteration of Different Domains in AFLR Affects Aflatoxin Pathway Metabolism inAspergillus parasiticusTransformants

AFLR, a zinc binuclear cluster DNA-binding protein, is required for activation of genes comprising the aflatoxin biosynthetic pathway inAspergillusspp. Transformation ofAspergillus parasiticuswith plasmids containing the intactaflRgene gave clones that produced fivefold more aflatoxin pathway metabolites than did the untransformed strain. When a 13-bp region in theaflRpromoter (positions −102 to −115 with respect to the ATG) was deleted, including a portion of a palindromic site previously shown to bind recombinant AFLR, metabolite production was 40% that of transformants with intactaflR.This result provides further evidence that this site may be involved in the autoregulation ofaflR.Overexpression of pathway genes could also result from increased quantities of AFLR titrating out a putative repressor protein. In AFLR, a 20-amino-acid acidic region near its carboxy-terminus resembles the region in yeast GAL4 required for GAL80 repressor binding. When 3 of the acidic amino acids in this region were deleted, levels of metabolites were even higher than those produced by transformants with intactaflR,as would be expected if repressor binding was suppressed in transformants containing this altered protein. Transformation with plasmids mutated at the AFLR zinc cluster (Cys to Trp at amino acid position 49) or at a putative nuclear localization signal region (RRARK deleted) gave clones with one-fifth the metabolite production of the untransformed fungus in spite of the transformants making the same or moreaflRmRNA. Since these transformants retained a copy of intactaflR,the latter results can be explained best by assuming that AFLR activates genes involved in aflatoxin production as a dimeric protein and that heterodimers containing both mutant and intact AFLR strands are inactive.