Summary/Abstract The mechanism by which transcription factors assemble active transcription complexes on specific DNA sequences does not appear to follow a simple recognition code. Direct readout, wherein specific residues in the transcription factor “read” the specific DNA sequence through direct interactions is most often assumed to apply due to an oversimplified view of DNA as a rigid molecule. However, subtle, and not-so-subtle, structural changes occur when DNA binds to transcription factors. In addition, the DNA binding domains of transcription factors exhibit a large range of flexibility and often contain intrinsically disordered regions. These elements of flexibility endow the problem of transcription factor-DNA molecular recognition with many of the features of the protein folding problem. Our overall hypothesis is that transcription factor-DNA binding would instead be better described by similar principles as have been elucidated for the protein folding problem. Here, we will focus on the stress-response transcription factor, nuclear factor κB (NFκB), which activates hundreds of genes involved in growth regulation and the immune response. We will combine rigorous theory with molecular biophysical experiments to study the assembly kinetics of NFκB transcriptosome complexes. We will investigate coupling between DNA and NFκB as it relates to tandem κB sites, nucleosomal DNA, and the DNA-binding co-activator, RPS3. We predict that NFκB and additional nuclear proteins assemble into specific NFκB transcriptosomes on κB-DNA sites via a cooperative assembly process. We will test this hypothesis with the following aims: Aim 1 Determine the role of DNA context in NFκB binding. We will test the hypothesis that DNA context plays a key role in determining which NFκB binding events result in transcription activation by studying the binding of NFκB to a series of bona fide NFκB promoter and enhancer sequences both theoretically and experimentally. Aim 2 Explore how NFκB interacts with nucleosomal DNA and can invade or unwind nucleosomal DNA. We will test the hypothesis that NFκB is capable of disrupting nucleosome stability in a manner dependent on NFκB concentration and the sequence of DNA that is wrapped by the nucleosome, thereby exposing DNA for the initiation of transcription. Atomic force microscopy and computational modeling of the NFκB interaction with nucleosomes will be pursued. Aim 3 Determine how the ternary interaction between DNA, NFκB and the transcription co-activator, RPS3 forms. The NFκB coactivator, RPS3, associates with, and activates, subsets of NFκB transcription activation sites forming higher-order NFκB transcriptosome complexes. We will use the AWSEM-Suite code to predict the structures of these larger protein complexes and will verify the predicted long-range contacts between proteins and domains by NMR paramagnetic relaxation, SAXS, and HDX-MS experiments.