Rice Promoter Binding Microarray for cis-Acting Elements from Rice Transcription Factors

Protein binding microarray (PBM) was introduced to conveniently determine protein-DNA interactions in vitro. Previously we designed Q9-PBM, in such a way that target probes are quadruples of all possible 9-mer combinations. The Q9-PBM can provide highly consistent and concrete results for consensus binding motifs. To confirm the binding activity in the natural promoter region we designed organism specific PBM, rice protein binding microarray (RPBM). Probes of RPBM were designed from 19,480 rice promoter sequences containing 40 bp long probe with 20 bp overlap (covering 1kb from 5’ upstream). RPBM technology was tested with RISBZ1 transcription factor, an endosperm specific basic leucine zipper transcription factor. The binding motif of RISBZ1 already was identified as GCN4 motif (TGAGTCA). RPBM shows RISBZ1 could bind to ACGT motif (CCACGTCA) as well as GCN4 motif known binding site. In RPBM binding affinity to ACGT motif was highly significant, compared to GCN4 motif. We also confirmed the binding affinity with EMSA experiments. RPBM is expected to open a new era to identify cognate binding site of transcription factors along with Q9-PBM.