RAV1, a novel DNA-binding protein, binds to bipartite recognition sequencethrough two distinct DNA-binding domains uniquely found in higher plants

We have cloned and characterized two novel DNA binding proteins designated RAV1 and RAV2 from Arabidopsis thaliana, RAV1 and RAV2 contain two distinct amino acid sequence domains found only in higher plant species. The N-term inal regions of RAV1 and RAV2 are homologous to the AP2 DNA-binding domain present in a family of transcription factors represented by the Arabidopsis APETALA2 and tobacco EREBP proteins, while the C-terminal region exhibits homology to the highly conserved C-terminal domain, designated B3, of VP1/A B13 transcription factors. Binding site selection assays using a recombinan t glutathione S-transferase fusion protein have revealed that RAV1 binds sp ecifically to bipartite recognition sequences composed of two unrelated mot ifs, 5'-CAACA-3' and 5'-CACCTG-3', separated by various spacings in two dif ferent relative orientations, Analyses using various deletion derivatives o f the RAV1 fusion protein show that the AP2 and B3-like domains of RAV1 bin d autonomously to the CAACA and CACCTG motifs, respectively, and together a chieve a high affinity and specificity of binding, From these results, we s uggest that the AP2 and BS-like domains of RAV1 are connected by a highly f lexible structure enabling the two domains to bind to the CAACA and CACCTG motifs in various spacings and orientations.