A COMPARISON OF THE DIFFERENT DNA-BINDING SPECIFICITIES OF THE BZIP PROTEINS C EBP AND GCN4/

The bZip proteins GCN4 and C/EBP differ in their DNA binding specifici ties: GCN4 binds well to the pseudo-palindromic AP1 site 5'-A(4)T(3)G( 2)A(1)C(0)T(1')C(2')A(3')T(4')-3' and to the palindromic ATF/CREB sequ ence 4)T(3)G(2)A(1)C(0)()G(0')T(1')C(2')A(3')T(4')-3'; C/EBP preferen tially recognizes the palindromic sequence 4)T(3)T(2)G(1)C(0)()G(0')C (1')A(2')A(3')T(4')-3'. According to the X-ray structures of GCN4-DNA complexes, five residues of the basic region of GCN4 are involved in s pecific base contacts: asparagine -18, alanine -15, alanine -14, serin e -11 and arginine -10 (numbered relative to the start point of the le ucine zipper, which we define as +1). In the basic region of C/EBP pos ition -14 is occupied by valine instead of alanine, the other four res idues being identical. Here we analyse the role of valine -14 in C/EBP -DNA complex formation, Starting from a C/EBP-GCN4 chimeric bZip pepti de which displays C/EBP specificity, we systematically mutated positio n -14 of its basic region and characterized the DNA binding specificit ies of the 20 possible different peptides by gel mobility shift assays with various target sites, We present evidence that valine -14 of C/E BP interacts more strongly with thymine 2 than with cytosine 1' of the C/EBP binding site, unlike the corresponding alanine -14 of GCN4 whic h exclusively contacts thymine 1' of the GCN4 binding sites.