IN-VITRO RECOGNITION OF SPECIFIC DNA TARGETS BY ALCR, A ZINC BINUCLEAR CLUSTER ACTIVATOR DIFFERENT FROM THE OTHER PROTEINS OF THIS CLASS

AlcR is the transactivator mediating transcriptional induction of the ale gene cluster in Aspergillus nidulans. The AlcR DNA-binding domain consists of a zinc binuclear cluster different from the other members of the Zn(2)Cys(6) family by several features, In particular, it is ab le to bind to symmetric and asymmetric sites with the same affinity, w ith both sites being functional in A, nidulans. Here, we show that unl ike the other proteins of the Zn(2)Cys(6) binuclear cluster family, Al cR, binds most probably as a monomer to its cognate targets, Two molec ules of the AlcR protein can simultaneously bind in a noncooperative m anner to inverted repeats, The consensus core has been determined prec isely (5'-CCGCN-3'), and the AlcR-binding site in the aldA promoter ha s been localized, The sequence downstream of the zinc cluster is neces sary for high affinity binding, Furthermore, our data show that the us e of the carrier protein glutathione S-transferase in AlcR binding exp eriments introduces an important bias in the recognition of DMA sites due to its tertiary dimeric structure.