Fig. 1. FUS3 and LEC2 activate the At2S3 promoter. (A) Comparison of nucleotide sequences of cis-elements present on At2S3 (upper sequence) and Brassica napus napA (bottom sequence) promoters. The B-box element contains DistB and ProxB, the RY-G-box complex contains a G-box surrounded by 2 RY motifs. (B) FUS3 and LEC2 bind to the RY-G-box complex in the At2S3 promoter. Yeast reporter strains B::HIS3 and RY-G::HIS3 carrying either the control plasmid pCV70 or expressing FUS3 or LEC2 were streaked (as depicted on the right) on medium containing histidine (+His) or restrictive medium (–His, + 1 mM 3-AT). (C) FUS3 protein forms a gel retardation complex (arrow) with both RY elements. FUS3 protein was incubated with a radiolabelled fragment of the At2S3 promoter (–98 to –48 relative to transcription start) containing both RY elements and the G-box. The reactions contain in vitro transcribed and translated control plasmids (lane 1) or FUS3 expression plasmid (lanes 2-17). Non-labelled competitor DNA (see Materials and methods) was added in 5-fold molar excess (lanes 3, 6, 9, 12, 15), 10-fold molar excess (lanes 4, 7, 10, 13, 16) and 20-fold (lanes 5, 8, 11, 14, 17). Competitor DNA used was wild type (lanes 3-5), mutant RY1 (lanes 6-8), mutant RY2 (lanes 9-11), mutant RYs (lanes 12-14) and mutant G-box (lanes 15-17).