From an analysis of many TF-ChIP data sets, TF-TF interactions were predicted on the basis of regular DNA sequence motif arrangements. 27 predicted TF-TF interactions were tested using a pull down assay that measures the \'Luminescence Intensity Ratio\'. A cut-off of 7 was used, well above values for six negative controls.
Interaction in vitro; bait produced by coupled in vitro translation; prey produced by coupled in vitro translation.
hkb protein was incubated with biotin labeled DNA containing two copies of its cognate DNA binding motif. 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 TF.
Unlabeled competitors containing only one motif were far less effective at competing for binding than oligos containing two nearby motifs. Binding of hkb to its cognate motif is cooperative.
Interaction in vitro; protein produced by in vitro translation.