ISOLATION OF 2 SOYBEAN G-BOX BINDING-FACTORS WHICH INTERACT WITH A G-BOX SEQUENCE OF AN AUXIN-RESPONSIVE GENE

G-box binding factors (GBFs) constitute a family of plant DNA-binding proteins that bind to the G-box motif, a regulatory cis element presen t in many plant genes with a palindromic DNA motif of CACGTG. Previous ly TCCACGTGTC, a G-box motif, from an auxin responsive gene GmAux28 ha s been identified as a sequence-specific protein-binding site. Here th e isolation of two soybean cDNA clones, referred to as SGBF-1 and SGBF -2, encoding proteins which bind to the G-box motif is reported. The p rimary structure of SGBF-1 and SGBF-2 predicts that these proteins con tain a basic leucine zipper (bZIP) DNA-binding domain and an N-termina l proline-rich domain. A dramatic difference in the pattern of protein -DNA complex formation was observed when recombinant SGBF-1 and SGBF-2 proteins were analyzed by electrophoretic mobility shift assays (EMSA s). The SGBF-1 binding pattern obtained with the G-box probe resulted in three major retarded bands while the SGBF-2 formed a single complex . This shows that the characteristically diffuse banding pattern of pl ant nuclear proteins interacting with the G-box is also observed in a binding assay using only one recombinant GBF. EMSAs were performed wit h a few selected binding sequences to study the effect of flanking nuc leotides to the hexanucleotide G-box core motif. The binding specifici ty of the SGBF proteins resembles that described for type A cauliflowe r nuclear G-box binding proteins which bind class I G-box elements [(( G)/(T))((C)/(A))CACGTG((G)/(T)) ((A)/(C))]. Phylogenetic analysis of 1 3 GBF-like proteins from various plant species reveals that the SGBF-1 and SGBF-2 proteins belong to different lineages, suggesting that the y may have distinct functions in activating transcription.