The role of the p50 and p65 subunits of NF-kappa B in the recognition of cognate sequences.
An important regulatory element for expression of the MHC class I gene H-2Kb is a palindromic 11-bp enhancer sequence, 5'-GGGGATTCCCC-3', which was shown to interact with the constitutive factor(s) KBF1/H2TF1 and the inducible nuclear factor NF-kappa B, a multisubunit transcription activator. Recent data from molecular cloning and biochemical analyses of KBF1 and NF-kappa B have shown that KBF1 corresponds to the dimerized DNA-binding subunit of NF-kappa B (p50) and that NF-kappa B forms a DNA-binding heterodimer of one p50 and one p65 subunit. In this study, we investigated differences in the binding specificity of the p50 dimer and p50-p65 by using mutants of the H-2Kb motif in binding competition assays. Both forms of NF-kappa B bound with high affinity to a wild-type H-2Kb motif. In contrast to the p50 dimer, p50-p65 had a strongly reduced requirement for an 11th base pair to efficiently recognize the motif. Only in the presence of spermine could the p50 dimer also efficiently bind to motifs altered in positions 1 or 11, suggesting that DNA bending was important for protein-DNA recognition. An H-2Kb motif mutated in one half site such that the frequently found kappa B motif 5'-GGGACTTTCC-3' is created was efficiently recognized only by p50-p65. The decameric kappa B motifs of reduced symmetry, which are most frequently found as cis-acting elements in promoter and enhancer sequences, seem to be bound preferentially by the p50-p65 heterodimer. It thus appears that p65 extends the DNA-binding specificity of NF-kappa B and thereby allows efficient recognition of a wider spectrum of binding motifs.
Urban
M B
MB
Laboratorium für molekulare Biologie, Ludwig-Maximilians-Universität München, Germany.
Baeuerle
P A
PA
D001483:Base Sequence; D001667:Binding, Competitive; D004247:DNA / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004742:Enhancer Elements, Genetic; D005805:Genes, MHC Class I; D006183:H-2 Antigens / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D016328:NF-kappa B / Q000302:isolation & purification / Q000378:metabolism*; D009841:Oligonucleotides; D013096:Spermine / Q000494:pharmacology
D006183:H-2 Antigens; C032876:H-2Kb protein, mouse; D016328:NF-kappa B; D009841:Oligonucleotides; D013096:Spermine; D004247:DNA