A PHYSICOCHEMICAL INVESTIGATION OF THE SELF-ASSOCIATION OF THE DNA-BINDING DOMAIN OF THE YEAST TRANSCRIPTIONAL ACTIVATOR GAL-4

It has previously been suggested that the DNA binding domain (residues 1 to 147) of the yeast transcriptional activator GAL 4 exists in solu tion in dimeric form, with the region responsible for dimerisation som ewhere between residues 74 and 147. In this study limited proteolysis and carboxy-terminal deletions of the DNA binding domain (residues 1 t o 147) of the yeast transcriptional activator GAL 4 followed by subseq uent characterization by equilibrium sedimentation in the analytical u ltracentrifuge have been used to define more precisely the regions req uired for DNA binding and protein self-association. Sedimentation equi librium analyses confirmed that the 'hydrophobic region' of the protei n (residues 54-97, which contains a larger proportion of alpha-helix), is essential for dimerisation, with an apparent dissociation constant K-D,K-app, of approximate to 50 mu M for the 1-94 residue peptide and approximate to 20 mu M for the 1-147 residue peptide. Our studies do not rule out the possible formation of small amounts of additional hig her order complexes.