Project Summary The NF- κB dimers bind to specific DNA response elements known as the κB DNA sites located in the promoters and enhancers of thousands of genes, and regulate their expression. Although the roughly 10 bp long κB sequences follow a consensus, hundreds of specific sequences can fit the consensus. Sequence variations can result in differences in NF-κB-DNA binding affinity, kinetics and conformations leading to changes in transcriptional output. Indeed, other and we reported that as few as a single bp change can have severe effect in gene regulation by the NF-κB dimers. Affinities of the NF-κB:DNA complexes derived from in vitro experiments do not always correlate with in vivo binding and gene regulation. These observations suggest that there are modulators present in the cell and without their inclusion in in vitro experiments in vivo and in vitro results will not reconcile. On the other hand, without proper in vitro experimental set up, proper investigation of regulatory mechanisms in vivo is difficult to accomplish. Cellular experiments performed over the past 10 years established the presence of several of such modulators, but their mechanisms of action could not be properly explained without thorough biochemical and biophysical experiments. We term these modulators cofactors. These cofactors alter the DNA binding affinity of NF-κB p50:RelA heterodimer and RelA homodimers in a κB sequence-specific manner. The focus of this proposal is to use new experiments to propose a unifying principle of how the cofactors regulate NF-κB activity. We propose that when the affinity between an NF-κB:κB DNA complex is weak, a cofactor can act positively enhancing the affinity of NF-κB:DNA complexes by directly contacting NF-κB without contacting DNA allowing gene expression to occur. Alternatively, a cofactor can act negatively by removing NF-κB off the DNA (or reduce affinity). Several positive cofactors and few negative cofactors are known. We will investigate the mode of actions of a few of these cofactors in vitro. Specifically, we will identify the site of interaction of both positive and negative cofactors on RelA and how they use allosteric mechanism to alter DNA binding by RelA. Since no cofactor specific to p50 is known, we also plan to identify cofactors specific to the p50 subunit and investigate if and how these new cofactors act together with RelA-specific cofactors. We will generate mutants of RelA defective in cofactor binding and test how gene expression profile and DNA binding in cells alters in response to specific stimulus.