| The Rel/NF-κB family proteins are important transcription factors that regulate the expression of genes involved in growth, development, stress response, and innate and adaptive immunity. There are five members of the family, and they function as homo- and heterodimers. In resting cells they are held inactive in the cytoplasm by a family of proteins known as the IκBs (Inhibitors of κB). Upon stimulation of appropriate upstream receptors or sensors, IκB proteins are degraded and NFκB dimers are freed to enter the nucleus and bind to specific DNA targets in gene enhancers, referred to as κB-sites. DNA-bound NFκB interacts with other transcriptional activators to form a large multi-protein/DNA complex known as the enhanceosome. Functional enhanceosomes recruit the basal transcriptional machinery, activating and sustaining gene transcription.; Using a combination of in vitro biochemistry and structural biology I attempted to quantitate and visualize the interactions between NFκB dimers and their partners in transcriptional regulation: IκB proteins, DNA, and other transcriptional activators.; The IκBα/NFκB interface was probed using protein:protein gel mobility shift assays and deletional analysis to determine the contribution to binding of the independently flexible domains/regions of NFκB and IκB. These revealed that the nuclear localization sequence containing polypeptide of p65 was particularly important in recognition by IκBα and that C-terminal phosphorylation of IκBα is necessary for full binding to NFκB dimers.; DNA binding assayed by fluorescence anisotropy revealed that NFκB dimers bind their κB-DNA targets through a cooperative, dimerization-dependent mechanism. Analysis of the effects of changes in temperature, pH, and ionic strength further indicated that the binding is entropically driven through the dehydration of pre-ordered protein and DNA interfaces. DNA recognition depends on the composition of the NFκB dimer, and only the oncogenic NFκB vRel is capable of binding tightly to multiple classes of κB DNA.; Structural studies by X-ray crystallography were also undertaken on a complex of an NFκB dimer and the DNA-binding domain of another transcriptional activator, Sp1. The Sp1 DNA binding domain interfered with NFκB-DNA binding and the two proteins show overlapping DNA specificity. This prevented the growth of crystals of stable ternary complexes. |
