Stress-response transcription factors must be tightly regulated so that they remain completely inactive in the resting state of the cell, are robustly activated in response to the appropriate signals, and are then rapidly and completely inactivated to avoid inappropriate gene expression when the stress condition is mitigated. The stress- response transcription factor nuclear factor ?B (NF?B) regulates cell growth, immune responses, inflammatory viral responses, and apoptotic cell death and is often misregulated in cancer, arthritis, asthma, diabetes, AIDS, and viral infections. Members of the NF?B family of transcription factors are held in the cytoplasm in an inactive state by their bound inhibitors (I?Bs) until the cell receives an external signal. NF?B is activated by phosphorylation and ubiquitination of the I?B?, which is then targeted for proteasomal degradation, releasing the NF?B to translocate into the nucleus. In a classical negative feedback mechanism, NF?B upregulates transcription of I?B? in addition to signal-specific stress-response genes: newly-synthesized I?B? kinetically enhances NF?B dissociation from the DNA in a process we have termed molecular stripping. A transient ternary complex intermediate is formed during the stripping process; we characterized this ternary complex by NMR and cryo-EM at equilibrium, and demonstrated by stopped-flow methods at lower concentrations that the dissociation of NF?B from its cognate DNA is accelerated by I?B?. In an exciting new observation, it has recently been shown that the stability of the resting NF?B-I?B? complex in the cytoplasm is enhanced by interaction with a specific long non-coding RNA (lncRNA), which appears to form a stable ternary complex analogous to the transient NF?B-I?B?-DNA complex formed in the nucleus during molecular stripping.
Specific Aim 1 of this proposal is concerned with the structural characterization of this NF?B-I?B?-RNA complex using a variety of biophysical techniques, including NMR, cryo-electron microscopy, in collaboration with Dr. Gabriel Lander, and small-angle X-ray scattering, in collaboration with Dr. John Tainer.
Specific Aim 2 will probe the structural and dynamic differences between the binary and ternary complexes of NF?B, I?B? and DNA, and the ternary NF?B-I?B?-RNA complex using specifically methyl-labeled proteins. Labeling methods and NMR experiments pioneered by the Kay lab at the University of Toronto enable dynamic information to be obtained even on systems as large as the NF?B-I?B? complexes, and we have demonstrated in previous work that this system is amenable to these approaches. Fulfilment of these specific aims will enable us to describe the structures and dynamics of these large dynamic complexes in unprecedented detail, and we anticipate that the experimental design utilized in this project will provide an important precedent for the study of the many other dynamic macromolecular complexes that have remained difficult to characterize.
The nuclear factor ?B (NF?B) signaling system regulates cell growth, immune responses, inflammatory viral responses, and apoptotic death and is often misregulated in cancer, arthritis, asthma, diabetes, AIDS, and viral infections. We propose studies aimed towards a detailed understanding of the structural and dynamic mechanisms by which NF?B proteins interact with their partners, using a biophysical approach combining a range of different techniques. Such an understanding is an important prerequisite for the design of drugs directed towards these targets.
