DNA binding of purified transcription factor NF-kappa B. Affinity, specificity, Zn2+ dependence, and differential half-site recognition.

U Zabel,R Schreck,P A Baeuerle

doi:10.1016/s0021-9258(18)52428-5

U Zabel, R Schreck + Show 1 more

Open Access

https://doi.org/10.1016/s0021-9258(18)52428-5

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Abstract

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.

Highlights

From the Laboratory for Molecular Biology, Gene Center, LudwigMaximilians- University Munich, Am Klopferspitz, 0-8033 Martinsried, FederalRepublic of Germany
Addition of increasing amounts of polynucleotides showed that poly(&-dT) had a much higher efficiency than poly(d1-dC) to compete for binding of NF-KB to the radioactive DNA probe (Fig. 4 B ) .In Fig. 4C, we show the quantitation of titrationexperimentswiththree different polynucleotides
DNA probe, the equilibrium of binding was reached after 1 h sites are arranged such that the half-site with the and only 20% of the amount of radioactive complex could three subsequent G residues is on the left (Fig. 6)

Summary

RESULTS

We subjected the NF-KBwhich was activated during thepurification of IKB(Zabel and Baeuerle, 1990) to sequence-specific DNA affinity chromatography and examined whether chloroquine. Among the determined using EMSAs as described (Meisterernst et aL, 1988).A few polypeptides that eluted a t 300 mM KC1 from the column, double-stranded oligonucleotidewasusedwhich containedinits p50 and p65 were the major species (Fig. lB, lane 6 ). These center the KBmotif from the mouse K light chain enhancer (Sen and Baltimore, 1986) and 4 base pairs which flank the site in situ.

Total protein

GGGAT TTCCC GGGAAATCCC GGGAC TTTCC GGGGC TTTCC

Findings

DISCUSSION