A novel basic region/helix-loop-helix protein binds to a G-box motif CACGTG of the bean seed storage protein β- phaseolin gene

Abstract

Expression of the bean seed storage protein β-phaseolin is under strict developmental control primarily at the level of transcription. A G-box motif CACGTG has been shown to be a major positive cis-acting element of the β- phaseolin gene and to be recognized by a DNA binding protein from bean seed nuclei. To understand the molecular nature of the G-box-binding protein, we screened a cDNA expression library from immature bean seed and identified a positive clone based on binding activity of the proteins expressed in Escherichia coli to an oligodeoxyribonucleotide probe. DNA sequence analysis showed that the cDNA for the phaseolin G-box-binding protein (PG1) encodes a basic region/helix-loop-helix (bHLH) domain near the carboxyl terminus. Sequence comparison with other known plant bHLH proteins such as maize R B and Antirrhinum DEL suggests that PG1 represents a new member of bHLH protein family in plants. Electrophoresis mobility-shift and DNase I footprinting assays demonstrated that PG1 protein binds preferentially to the G-box (CA CG TG) and not to other two phaseolin E-box motifs (CA CC TG) and CA TA TG). Expression of PG1 RNA was detectable in all stages of seed development as well as inflower, root, and leaf. We discuss the implications of the bHLH protein for transcriptional regulation of the β- phaseolin gene.