Alterations in the GAL4 DNA-binding Domain Can Affect Transcriptional Activation Independent of DNA Binding

Abstract

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 Zn₂Cys₆ 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 <i>GAL</i> (UAS_G)) or PPR1 (upstream acitvation sequence of <i>UAS</i>) DNA-binding sites and severely reduced transcriptional activation in yeast. In contrast, alterations between 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) <i>Nature</i>356, 408–414), this region is facing away from the DNA, making it likely that there exists within the GAL4 DBD an accessible domain important in activation.