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Ethylene-responsive element-binding proteins (EREBPs)have novel DNA-binding domains (ERF domains), which are widely conserved in plants, and interact specifically with sequences containing AGCCGCC motifs (GCC box). Deletion experiments show that some flanking region at the N terminus of the conserved 59-amino acid ERF domain is required for stable binding to the GCC box. Three ERF domain-containing fragments of EREBP2, EREBP4, and AtERF1 from tobacco and Arabidopsis, bind to the sequence containing the GCC box with a high binding affinity in the pM range. The high affinity binding is conferred by a monomeric ERF domain fragment, 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 6-base pair GCCGCC region of the GCC box, and the first G, the fourth G, and the sixth C exhibit highest binding specificity common in all three ERF domain-containing fragments studied. Other bases within the GCC box exhibit modulated binding specificity varying from protein to protein, implying that these positions are important for differential binding by different EREBPs. The conserved N-terminal half is likely responsible for formation of a stable complex with the GCC box and the divergent C-terminal half for modulating the specificity.

Little is known about the mechanisms that target transcripts for rapid degradation in plants. In mammalian cells, sequences with a high AU content and multiple AUUUA motifs have been shown to cause mRNA instability when present in the 3' untranslated regions of several transcripts. This precedent, coupled with the poor accumulation of AU-rich foreign transcripts in plants (e.g., BT-toxin mRNAs), prompted us to test whether AU sequences could destabilize transcripts in tobacco. To address this question, we made a set of constructs containing sequences with high AU content inserted into the 3' untranslated regions of reporter genes. The stability of the corresponding transcripts was then assayed in stably transformed cell lines of tobacco. These experiments showed that a 60-base sequence containing 11 copies of the AUUUA motif (AUUUA repeat) markedly destabilized a beta-glucuronidase reporter transcript compared to a no-insert control or a 60-base spacer sequence (GC control). Another sequence with an identical A+U content had little effect. The same results were obtained when each sequence was assayed within the 3' untranslated region of a beta-globin reporter transcript. In regenerated transgenic plants, the AUUUA repeat decreased the accumulation of the beta-globin transcript by approximately 14-fold, compared to the GC control. Taken together, our results indicate that the AUUUA repeat is recognized as an instability determinant in plant cells and that the effect is due to the sequence of the element, not simply to the high AU content.