ovo and Kr proteins were incubated with biotin labeled DNA containing both cognate DNA binding motifs. DNA was pulled down with streptavidin beads and the amount of luciferase activity pulled down was measured. Unlabeled competitor oligos lacking one or both DNA binding motifs were added in excess to assess the cooperativity of DNA binding by the two TFs.
Unlabeled competitors containing both ovo and Kr DNA motifs within the same oligo efficiently compete binding. When ovo and Kr motifs are provided on separate DNA molecules, they are not able to compete. This suggests that binding of ovo and Kr to oligos containing both cognate sites is cooperative.
Interaction in vitro; proteins produced by coupled in vitro translation; prey produced by coupled in vitro translation.