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.
Urban
M B
MB
Laboratorium für Molekulare Biologie, Ludwig-Maximilians-Universität München, Martinsried, FRG.
Schreck
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R
Baeuerle
P A
PA
D001483:Base Sequence; D001667:Binding, Competitive; D002499:Centrifugation, Density Gradient; D003432:Cross-Linking Reagents; D003600:Cytosol / Q000737:chemistry; D015342:DNA Probes; D004591:Electrophoresis, Polyacrylamide Gel; D005990:Glycerol; D006801:Humans; D008969:Molecular Sequence Data; D016328:NF-kappa B / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D011257:Pregnancy Proteins / Q000302:isolation & purification; D013329:Structure-Activity Relationship; D014466:Ultraviolet Rays
D003432:Cross-Linking Reagents; D015342:DNA Probes; D016328:NF-kappa B; D011257:Pregnancy Proteins; D005990:Glycerol