STRUCTURAL DISSECTION OF THE DNA-BINDING DOMAIN OF THE YEAST TRANSCRIPTIONAL ACTIVATOR GAL4 REVEALS AN ALPHA-HELICAL REGION RESPONSIBLE FORDIMERIZATION

Limited proteolysis of the DNA-binding domain (residues 1-147) of the yeast transcriptional activator GAL4 has been used to define more prec isely the subdomain structure required for DNA binding and dimerizatio n. Two regions of the protein were found to be resistant to proteolysi s: the cysteine-rich, zinc-binding region (residues 6-43) and a hydrop hobic sequence between residues 52 and 97. Carboxy-terminal deletion f ragments of the DNA-binding domain were generated and assayed by DNase 1 footprinting. This showed that the affinity of DNA binding depends on the sequence between residues 65 and 94. Structural comparisons by UV circular dichroism (CD) were made and the difference CD spectra ind icate that strong ct-helical content is found specifically in the regi on between residues 65 and 94, which previous studies have shown to en able dimerization and in this study the formation of a stable protein- DNA complex.