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

J. Christopher Corton, Evelyn Moreno, Stephen Albert Johnston
1998 Journal of Biological Chemistry  
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 Zn 2 Cys 6 domain). In an effort to characterize the interactions between the Zn 2 Cys 6 class transcriptional activator proteins and their DNAbinding sites, we have replaced in the full-length GAL4 protein small regions of the Zn 2 Cys 6 domain with the analogous regions of another Zn 2 Cys 6 protein called
more » ... R1 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 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) Nature 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. UAS G , UAS of GAL; CAT, chloramphenicol acetyltransferase; DBD, DNA-binding domain; UAS u , UAS of URA.
doi:10.1074/jbc.273.22.13776 pmid:9593720 fatcat:o3reatzrwngkdhmyuc4qqt6mmu