Figure 5

Specific interaction of NF-κB dimers with canonical and non-canonical sequences. (a) Interaction of four NF-κB dimers, p50p50, RELARELA, RELAp50 and RELAp52, with canonical sequences containing either a H-2 binding site (lanes 1 to 5), or a HIV recognition site (lanes 6 to 10). These were profiled using EMSA (top panel), UV laser (middle panel) and DNAse I (bottom panel) footprinting techniques (with interactor regions demarcated with vertical black lines). For example, RELA dimer-DNA complexes were detected with EMSA (lanes 3 and 8; red arrows). Furthermore, a 'UV footprint' in the form of lower intensity banding observed within the interactor region (relative to controls in lanes 1 and 6) indicates specific interactions of varying affinities between the dimer and DNA. (b) Interaction of RELARELA with the non-canonical sequences was non-specific. With both sequences, distinct dimer-DNA complexes were observed by EMSA with all dimers except RELARELA, for which a smear was obtained (lane 4: RELARELA). No footprint was observed with RELARELA, whilst for the other dimers a stronger footprint was obtained with AGGGGAAGTTA compared to CTGGGGATTTA. (c) Median enrichment of 11-mers bound by the three RELA-containing dimers in EMSA-Seq. Five groupings of sequences were formed on the basis of MATCH similarity (Grp1 ≤ 0.20, 0.201 ≥ Grp2 ≤ 0.40, 0.401 ≥ Grp3 ≤ 0.60, 0.601 ≥ Grp4 ≤ 0.80 and Grp5 ≥ 0.801). There is a trend of enrichment increasing alongside MATCH similarity. Also shown are the average enrichment values and corresponding similarities to the reference for the six 11-mer sequences that were footprinted (crosses with sequence indicated).