DNA binding of purified transcription factor NF-kappa B. Affinity, specificity, Zn2+ dependence, and differential half-site recognition.
A rapid purification procedure for the NF-kappa B transcription factor from the cytosol of human placenta is demonstrated which exploits the insensitivity of the NF-kappa B.DNA complex toward the intercalating agent chloroquine. Purified NF-kappa B required 100 mM KCl or NaCl and a pH of 7.5 to optimally bind to DNA. Equilibrium of binding was reached within less than 5 min in the absence of competitor DNA and after 1 h in the presence of 0.1 mg/ml poly(dI-dC). DNA binding of NF-kappa B was specifically blocked by the chelating agent 1,10-orthophenantroline and could only be reconstituted by addition of Zn2+. Under optimal binding conditions, the dissociation constant for the complex of the purified NF-kappa B with its most frequent cognate DNA motif 5'-GGGACTTTCC-3' was in the range of 10(-12) to 10(-13) M. Various other cis-acting kappa B motifs were recognized by NF-kappa B with lower affinities. A comparative analysis of known NF-kappa B-binding sites and competition experiments with synthetic polynucleotides and oligonucleotides encompassing only one half-site or single-stranded kappa B motifs suggested that the two DNA-binding monomers in the NF-kappa B protein complex can interact differentially with the half-sites of the decameric cognate motif.
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Laboratory for Molecular Biology, Ludwig-Maximilians-University Munich, Martinsried, Federal Republic of Germany.
Schreck
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Baeuerle
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PA
D001483:Base Sequence; D001665:Binding Sites; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D004247:DNA / Q000378:metabolism*; D015342:DNA Probes; D005260:Female; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D016328:NF-kappa B / Q000302:isolation & purification / Q000378:metabolism*; D015345:Oligonucleotide Probes; D010920:Placenta / Q000378:metabolism; D011089:Polydeoxyribonucleotides / Q000494:pharmacology; D011247:Pregnancy; D013816:Thermodynamics; D015032:Zinc / Q000494:pharmacology*
D015342:DNA Probes; D016328:NF-kappa B; D015345:Oligonucleotide Probes; D011089:Polydeoxyribonucleotides; D004247:DNA; D015032:Zinc