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¿Ba, 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¿Ba in addition to signal-specific stress-response genes: newly-synthesized I¿Ba 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¿Ba. In an exciting new observation, it has recently been shown
that the stability of the resting NF¿B-I¿Ba 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¿Ba-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¿Ba-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¿Ba and DNA, and the ternary NF¿B-I¿Ba-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¿Ba 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.