MOLECULAR CHARACTERIZATION OF THE DNA-BINDING AND DIMERIZATION DOMAINS OF THE BZIP TRANSCRIPTION FACTOR, EMBP-1

The wheat basic-leucine zipper (bZIP) DNA-binding protein EmBP-1 has b een implicated in the mechanisms of abscisic acid (ABA) mediated gene regulation. Sequence and structural homology to the yeast bZIP protein GCN4 has been used to predict the location of the functional domains of EmBP-1. In order to test these predictions, the presumptive DNA-bin ding and dimerization domains oi EmBP-1 were mapped by producing a ser ies of truncated protein fragments and functionally testing them in vi tro. Deletion of 5 amino acids of the predicted basic domain resulted in a loss of all DNA-binding activity. A fragment containing all six l eucine repeat elements showed strong DNA-binding activity. Sequential deletion of the leucine repeat elements resulted in first an increase in DNA-binding activity (-L6 and -L5) followed by a reduction in bindi ng activity (-L4) and eventually complete elimination of all detectabl e DNA-binding activity (-L3 and -L2). This demonstrates the importance of an intact leucine zipper domain of at least 4 repeat elements for efficient DNA-binding. The smallest polypeptide that retained DNA-bind ing activity is a fragment spanning amino acid residues 248-308 (ca. 8 .4 kDa) consisting of minimal basic and leucine zipper domains. Dimeri zation of EmBP-1 was demonstrated by co-translation of fragments of di ffering molecular weights and identification of a DNA-protein complex with intermediate mobility to that produced by each fragment alone. A unique leucine-proline repeat element found N-terminal to the DNA-bind ing domain of EmBP-1 does not appear to play a role in DNA-binding or dimerization. These results confirm the locations of the functional do mains of EmBP-1 predicted by similarity to GCN4. The high degree of fu nctional conservation of the bZIP proteins spanning organisms from pla nts to fungi highlights the ancient origin of this class of transcript ion factors and of the mechanisms of gene regulation in which they par ticipate.