NF‐kappa B contacts DNA by a heterodimer of the p50 and p65 subunit.

We recently reported that the apparently non‐DNA‐binding 65 kd subunit (p65) of the NF‐kappa B transcription factor can modulate the DNA‐binding specificity of the 50 kd subunit (p50) of NF‐kappa B. In this study we provide an explanation for this property of p65. In electrophoretic mobility shift assays and upon UV cross‐linking to DNA, gel‐purified p65 is shown to be a kappa B‐specific DNA‐binding protein on its own. The binding activity was only detectable if high amounts of p65 were used for the analyses and after the application of a modified renaturation protocol. DNA‐binding of the p65 dimer, in contrast to that of p50, was inhibited by I kappa B‐alpha and ‐beta. This finding is consistent with a receptor function of p65 for both inhibitory subunits. Direct UV cross‐linking of NF‐kappa B to DNA probes which were photoreactive within only one half‐site and a binding competition analysis with p65 showed that p65 has a strong preference for binding to the less conserved half site of kappa B motifs whereas p50 has a moderate preference for the more highly conserved half site. In electrophoretic mobility shift assays and upon sedimentation through glycerol gradients, NF‐kappa B appears to exist as a heterodimer composed of one p50 and one p65 subunit whereas data from gel filtration suggest a higher order complex.