Bipartite determinants of DNA-binding specificity of plant basic leucine zipper proteins

The basic leucine zipper (bZIP) proteins are one of the largest and most conserved groups of eukaryotic transcription factors/repressors. Two major subgroups among the plant bZIP proteins have been identified as G-box (CCACGTGG) or C-box (TGACGTCA) binding proteins based on their DNA binding specificity and the amino acid sequences of their basic regions. We have investigated how plant bZIP proteins determine their DNA binding specificity by mutation of the basic domain of the G-box-binding protein EmBP-1. Four subregions of the EmBP-1 basic domain that differ from the C-box-binding protein TGA1a were substituted singly or in combination with the corresponding regions of TGA1a. DNA binding experiments with the mutant proteins demonstrated that binding specificity of plant bZIP proteins is determined independently by two regions, the core basic region and the hinge region. These two regions have an additive effect on DNA binding specificity. PCR-assisted binding-site selections using key mutants demonstrated that only G-box and C-box binding specificity can be generated by combinations of amino acids in the basic domains of EmBP-1 and TGA1a. These results suggest that factorial contributions of the amino acid residues in the basic domain combine to determine DNA-binding specificity of bZIP proteins.