Replacement of invariant bZip residues within the basic region of the yeast transcriptional activator GCN4 can change its DNA binding specificity.

Two residues are invariant in all bZip basic regions: asparagine -18 and arginine -10 (we define the first leucine of the leucine zipper of GCN4 as +1). X-ray structures of two specific GCN4-DNA complexes (Ellenberger et al., Cell, 71, 1223-1237, 1992; König & Richmond, J. Mol. Biol., 233, 139-154, 1993) demonstrate the involvement of both residues in specific base pair recognition. We replaced either asparagine -18 or arginine -10 with all other amino acids and tested the DNA binding properties of the resulting mutant peptides by gel mobility shift assays. Peptides with histidine -18 or tyrosine -10 bind with changed specificities to variants of the ATF/CREB site 5'A4T3G2A1C0*G0'T1'C2'A3'T4'3' with symmetric exchanges in positions 2/2' or 0/0', respectively. The double mutant with histidine -18 and tyrosine -10 combines the features of the parental single mutants and binds specifically to the respective double exchange target. Furthermore, the tyrosine -10 mutant clearly prefers the palindrome 5'ATGATATCAT3' over the corresponding pseudo-palindrome 5'ATGATTCA-T3', whereas the lysine -10 mutant binds better to the pseudo-palindromic AP1 site 5'ATGACTCAT3' than to the palindromic ATF/CREB site. Thus, although invariant within natural bZip proteins, asparagine -18 or arginine -10 can be functionally replaced by other amino acids, and their replacement can lead to new DNA binding specificities.