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Proc Natl Acad Sci U S A. 2014 Jan 7;111(1):225-30. doi: 10.1073/pnas.1318115111. Epub 2013 Dec 23.

Direct observation of a transient ternary complex during IκBα-mediated dissociation of NF-κB from DNA.

Author information

1
Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA 92092-0378.

Abstract

We previously demonstrated that IκBα markedly increases the dissociation rate of DNA from NF-κB. The mechanism of this process remained a puzzle because no ternary complex was observed, and structures show that the DNA and IκBα binding sites on NF-κB are overlapping. The kinetics of interaction of IκBα with NF-κB and its complex with DNA were analyzed by using stopped-flow experiments in which fluorescence changes in pyrene-labeled DNA or the native tryptophan in IκBα were monitored. Rate constants governing the individual steps in the reaction were obtained from analysis of the measured rate vs. concentration profiles. The NF-κB association with DNA is extremely rapid with a rate constant of 1.5 × 10(8) M(-1)⋅s(-1). The NF-κB-DNA complex dissociates with a rate constant of 0.41 s(-1), yielding a KD of 2.8 nM. When IκBα is added to the NF-κB-DNA complex, we observe the formation of a transient ternary complex in the first few milliseconds of the fluorescence trace, which rapidly rearranges to release DNA. The rate constant of this IκBα-mediated dissociation is nearly equal to the rate constant of association of IκBα with the NF-κB-DNA complex, showing that IκBα is optimized to repress transcription. The rate constants for the individual steps of a more folded mutant IκBα were also measured. This mutant associates with NF-κB more rapidly than wild-type IκBα, but it associates with the NF-κB-DNA complex more slowly and also is less efficient at mediating dissociation of the NF-κB-DNA complex.

KEYWORDS:

DNA binding; NF-κB post-induction repression; NF-κB transcription; stopped-flow fluorescence kinetics; transcription activation

PMID:
24367071
PMCID:
PMC3890772
DOI:
10.1073/pnas.1318115111
[Indexed for MEDLINE]
Free PMC Article

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