REPLACEMENT OF INVARIANT BZIP RESIDUES WITHIN THE BASIC REGION OF THEYEAST 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 o f GCN4 as +1). X-ray structures of two specific GCN4-DNA complexes (El lenberger et al., Cell, 71, 1223-1237, 1992; Konig and Richmond, J. Mo l. Biol., 233, 139-154, 1993) demonstrate the involvement of both resi dues in specific base pair recognition. We replaced either asparagine - 18 or arginine - 10 with all other amino acids and tested the DNA bi nding properties of the resulting mutant peptides by gel mobility shif t assays. Peptides with histidine - 18 or tyrosine - 10 bind with chan ged specificities to variants of the ATF/CREB site 5'A(4)T(3)G(2)A(1) C-0G(0), T-1, C-2, A(3), T-4, 3' with symmetric exchanges in position s 2/2' or 0/0', respectively. The double mutant with histidine - 18 an d tyrosine - 10 combines the features of the parental single mutants a nd binds specifically to the respective double exchange target. Furthe rmore, the tyrosine - 10 mutant dearly prefers the palindrome 5'ATGATA TCAT3' over the corresponding pseudo-palindrome 5'ATGATTCAT3', whereas the lysine - 10 mutant binds better to the pseudo-palindromic AP1 sit e 5'ATGACTCAT3' than to the palindromic ATF/CREB site. Thus, although invariant within natural bZip proteins, asparagine - 18 or arginine -1 0 can be functionally replaced by other amino acids, and their replace ment can lead to new DNA binding specificities.