Mechanism of kappa B DNA binding by Rel/NF-kappa B dimers.
The DNA binding of three different NF-kappaB dimers, the p50 and p65 homodimers and the p50/p65 heterodimer, has been examined using a combination of gel mobility shift and fluorescence anisotropy assays. The NF-kappaB p50/p65 heterodimer is shown here to bind the kappaB DNA target site of the immunoglobulin kappa enhancer (Ig-kappaB) with an affinity of approximately 10 nm. The p50 and p65 homodimers bind to the same site with roughly 5- and 15-fold lower affinity, respectively. The nature of the binding isotherms indicates a cooperative mode of binding for all three dimers to the DNA targets. We have further characterized the role of pH, salt, and temperature on the formation of the p50/p65 heterodimer-Ig-kappaB complex. The heterodimer binds to the Ig-kappaB DNA target in a pH-dependent manner, with the highest affinity between pH 7.0 and 7.5. A strong salt-dependent interaction between Ig-kappaB and the p50/p65 heterodimer is observed, with optimum binding occurring at monovalent salt concentrations below 75 mm, with binding becoming virtually nonspecific at a salt concentration of 200 mm. Binding of the heterodimer to DNA was unchanged across a temperature range between 4 degrees C and 42 degrees C. The sensitivity to ionic environment and insensitivity to temperature indicate that NF-kappaB p50/p65 heterodimers form complexes with specific DNA in an entropically driven manner.
Phelps
C B
CB
Department of Chemistry and Biochemistry, University of California San Diego, La Jolla, California 92093, USA.
Sengchanthalangsy
L L
LL
Malek
S
S
Ghosh
G
G
D019943:Amino Acid Substitution; D001665:Binding Sites; D004247:DNA / Q000737:chemistry* / Q000378:metabolism*; D019281:Dimerization; D016325:HIV Long Terminal Repeat; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008956:Models, Chemical; D016297:Mutagenesis, Site-Directed; D016328:NF-kappa B / Q000737:chemistry* / Q000378:metabolism*; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000378:metabolism*; D055503:Protein Multimerization; D020569:Proto-Oncogene Proteins c-rel / Q000737:chemistry* / Q000378:metabolism*; D013816:Thermodynamics
D016328:NF-kappa B; D009838:Oligodeoxyribonucleotides; D020569:Proto-Oncogene Proteins c-rel; D004247:DNA