Direct observation of a transient ternary complex during I kappa B alpha-mediated dissociation of NF-kappa B from DNA

作者：Alverdi Vera; Hetrick Byron; Joseph Simpson; Komives Elizabeth A^*

来源：Proceedings of the National Academy of Sciences of the United States of America, 2014, 111(1): 225-230.

DOI：10.1073/pnas.1318115111

• transcription activation
• DNA binding
• NF-kappa B transcription
• NF-kappa B post-induction repression
• stopped-flow fluorescence kinetics

We previously demonstrated that I kappa B alpha markedly increases the dissociation rate of DNA from NF-kappa B. The mechanism of this process remained a puzzle because no ternary complex was observed, and structures show that the DNA and I kappa B alpha binding sites on NF-kappa B are overlapping. The kinetics of interaction of I kappa B alpha with NF-kappa B 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 I kappa B alpha were monitored. Rate constants governing the individual steps in the reaction were obtained from analysis of the measured rate vs. concentration profiles. The NF-kappa B association with DNA is extremely rapid with a rate constant of 1.5 x 10(8) M(-1.)s(-1). The NF-kappa B-DNA complex dissociates with a rate constant of 0.41 s(-1), yielding a K-D of 2.8 nM. When I kappa B alpha is added to the NF-kappa B-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 I kappa B alpha-mediated dissociation is nearly equal to the rate constant of association of I kappa B alpha with the NF-kappa B-DNA complex, showing that I kappa B alpha is optimized to repress transcription. The rate constants for the individual steps of a more folded mutant I kappa B alpha were also measured. This mutant associates with NF-kappa B more rapidly than wild-type I kappa B alpha, but it associates with the NF-kappa B-DNA complex more slowly and also is less efficient at mediating dissociation of the NF-kappa B-DNA complex.

• 出版日期2014-1-7