A NOVEL BASIC REGION HELIX-LOOP-HELIX PROTEIN BINDS TO A G-BOX MOTIF CACGTG OF THE BEAN SEED STORAGE PROTEIN BETA-PHASEOLIN GENE/

Expression of the bean seed storage protein P-phaseolin is under stric t developmental control primarily at the level of transcription. A G-b ox motif CACGTG has been shown to be a major positive cis-acting eleme nt of the beta-phaseolin gene and to be recognized by a DNA binding pr otein from bean seed nuclei. To understand the molecular nature of the G-box-binding protein, we screened a cDNA expression library from imm ature bean seed and identified a positive clone based on binding activ ity of the proteins expressed in Escherichia coli to an oligodeoxyribo nucleotide probe. DNA sequence analysis showed that the cDNA for the p haseolin G-box-binding protein (PGI) encodes a basic region/helix-loop -helix (bHLH) domain near the carboxyl terminus. Sequence comparison w ith other known plant bHLH proteins such as maize R/B and Antirrhinum DEL suggests that PGI represents a new member of bHLH protein family i n plants, Electrophoresis mobility-shift and DNase I footprinting assa ys demonstrated that PG1 protein binds preferentially to the G-box (CA CGTG) and not to other two phaseolin E-box motifs (CACCTG and CATATG). Expression of PG1 RNA was detectable in all stages of seed developmen t as well as in flower, root, and leaf. We discuss the implications of the bHLH protein for transcriptional regulation of the beta-phaseolin gene.