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

Abstract

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.