Fig. 9.

Gel-shift assays of various tandem TGAC core motifs (bold, upper-case nucleotides) in three putative target genes of StBEL5 and POTH1 with a range of linker sequence (lower-case nucleotides) between motifs. Upstream sequences of StBEL6 contain the tandem motifs in a tail-to-tail orientation on opposite DNA strands and StBEL22 motifs exhibit a head-to-head orientation, whereas StBEL34 contains the tandem motifs in a tail-to-head orientation on the same (+) DNA strand. The StBEL5 and POTH1 proteins were expressed and purified with a C-terminal GST fusion tag. Each DNA bait was tested for binding with StBEL5–GST, POTH1–GST, or GST alone or with StBEL5–GST and POTH1–GST together. Ten femtomoles of synthesized DNA probes of 30 (StBEL6) or 50 nt labelled with biotin were used in the binding reaction. The amounts of StBEL5 and POTH1 proteins used in these assays were adjusted to achieve equivalent molarity. Unlabelled DNA bait at 100×, 200×, and 500× concentrations relative to the labelled probe was used in the competition assays.