MUTATIONAL ANALYSIS OF THE C-TERMINAL REGION OF AREA, THE TRANSCRIPTION FACTOR MEDIATING NITROGEN METABOLITE REPRESSION IN ASPERGILLUS-NIDULANS

In Aspergillus nidulans the positive-acting, wide domain regulatory ge ne areA mediates nitrogen metabolite repression. Previous analysis dem onstrated that the C-terminal 153 residues of the areA product (AREA) are inessential for at least partial expression of most genes subject to regulation by areA. Paradoxically, ar areA(r)2, a -1 frameshift rep lacing the wild-type 122 C-terminal residues with a mutant peptide of 117 amino acids, leads to general loss of function. To determine the b asis for the are A(r)2 mutant phenotype, and as a means of delineating functional domains within the C-terminal region of AREA, we have sele cted and characterised areA(r)2 revertants. Deletion analysis, utilisi ng direct gene replacement, extended this analysis. A mutant areA prod uct truncated immediately after the last residue of the highly conserv ed GATA (DNA-binding) domain retains partial function. The areA(r)2 pr oduct retains some function with respect to the expression of uaZ (enc oding urate oxidase) and the mutant allele is partially dominant with respect to nitrate reductase levels. Consistent with the areA(r)2 prod uct having a debilitating biological activity, we have demonstrated th at a polypeptide containing both the wild-type DNA-binding domain and the mutant C-terminus of AREA2 is able to bind DNA in vitro but no lon ger shows specificity for GATA sequences.