UNIQUE MODE OF GCC BOX RECOGNITION BY THE DNA-BINDING DOMAIN OF ETHYLENE-RESPONSIVE ELEMENT-BINDING FACTOR (ERF DOMAIN) IN PLANT

Ethylene-responsive element-binding proteins (EREBPs)have novel DNA-bi nding domains (ERF domains), which are widely conserved in plants, and interact specifically with sequences containing AGCCGCC motifs (GCC b ox). Deletion experiments show that some flanking region at the N term inus of the conserved 59-amino acid ERF domain is required for stable binding to the GCC box. Three ERF domain-containing fragments of EREBP 2, EREBP4, and AtERF1 from tobacco and Arabidopsis, bind to the sequen ce containing the GCC box with a high binding affinity in the pM range . The high affinity binding is conferred by a monomeric ERF domain fra gment, and DNA truncation experiments show that only 11-base pair DNA containing the GCC box is sufficient for stable ERF domain interaction . Systematic DNA mutation analyses demonstrate that the specific amino acid contacts are confined within the B-base pair GCCGCC region of th e GCC box, and the first G, the fourth G, and the sixth C exhibit high est binding specificity common in all three ERF domain-containing frag ments studied. Other bases within the GCC box exhibit modulated bindin g specificity varying from protein to protein, implying that these pos itions are important for differential binding by different EREBPs, The conserved N-terminal half is likely responsible for formation of a st able complex with the GCC box and the divergent C-terminal half for mo dulating the specificity.