Alterations in the GAL4 DNA-binding domain can affect transcriptional activation independent of DNA binding

The GAL4 protein belongs to a large class of fungal transcriptional activator proteins encoding within their DNA-binding domains (DBD) six cysteines that coordinate two atoms of zinc (the Zn₂Cys₆ domain). In an effort to characterize the interactions between the Zn₂Cys₆ class transcriptional activator proteins and their DNA- binding sites, we have replaced in the full-length GAL4 protein small regions of the ZneCys₆ domain with the analogous regions of another Zn₂Cys₆ protein called PPR1 an activator of pyrimidine biosynthetic genes. Alterations between the first and third cysteines abolished binding to GAL4 (upstream activation sequence of GAL (UAS(G))) or PPR1 (upstream acitvation sequence of UAS) DNA-binding sites and severely reduced transcriptional activation in yeast. In contrast, alterations be- tween the third and fourth cysteines had only minor effects on binding to UAS(G) but led to substantial decreases in activation in both yeast and a mammalian cell line. In the crystal structure of the GAL4 DBD- UAS(G) complex (Marmorstein, R., Carey, M., Ptashne, M., and Harrison, S.C. (1992) Nature 356, 408-414), this region is facing away from the DNA, making it likely that there exists within the GAIA DBD an accessible domain important in activation.