Sequence-specific interactions of a nuclear protein factor with the promoter region of a rice gene for alpha-amylase, RAmy3D.

The expression of a rice gene for alpha-amylase, RAmy3D, in suspension-cultured cells is induced at the transcriptional level by the deprivation of sugars. Binding of a nuclear protein from suspension-cultured rice cells to the promoter region of the RAmy3D gene was studied by gel-retardation and DNase I footprinting assays. Gel-retardation assays indicated that a 358-bp fragment of the promoter region interacted specifically with a protein factor from suspension-cultured cells. DNase I footprinting analysis allowed us to define three protein-binding regions. Each of these protein-binding sequences contained the GCCG G/C CG motif, which is specifically present in the promoter region of the sugar-regulated gene, RAmy3D, for rice alpha-amylase and not in that of the gibberellin-regulated RAmy1A gene. Subsequent cross-competition experiments using gel-retardation assay and synthetic oligonucleotides showed that the GCCG G/C CG motifs directly mediated the binding of a nuclear protein. These observations are discussed in relation to expression of the gene for alpha-amylase in suspension-cultured cells.