A physico-chemical 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 solution in dimeric form, with the region responsible for dimerisation somewhere between residues 74 and 147. In this study limited proteolysis and carboxy-terminal deletions of the DNA binding domain (residues 1 to 147) of the yeast transcriptional activator GAL 4 followed by subsequent characterization by equilibrium sedimentation in the analytical ultracentrifuge have been used to define more precisely the regions required for DNA binding and protein self-association. Sedimentation equilibrium analyses confirmed that the 'hydrophobic region' of the protein (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 higher order complexes.