Project Abstract This proposal seeks to understand how biochemical networks organize specific reactions in space and time, with a focus on regulatory mechanisms in cell signaling and gene expression. We aim to develop biochemical model systems to understand how cells direct kinase activity in complex, interconnected signaling networks and to engineer tools to study structure-function relationships in eukaryotic genomes. These efforts will help us to dissect the mechanistic features that enable precise spatial and temporal control over biochemical reactions, provide new strategies to engineer cellular functions, and inform the design of more effective therapeutics that target specific cellular processes. In cell signaling, scaffold proteins coordinate the formation of physical complexes that assemble multiple signaling proteins. Scaffold proteins are widely assumed to act by tethering proteins together to accelerate specific reactions. Recent mechanistic and structural studies, however, have revealed unexpectedly complex biochemical mechanisms that regulate signaling through conformational changes and allosteric effects. This complexity is likely magnified in cellular environments, where multiple pathways compete for shared components and a plethora of different regulatory factors perform functions that are poorly understood at the molecular level. In particular, we currently lack a mechanistic framework to understand how scaffold-mediated kinase reactions are affected by competing scaffolds, phosphatases, and accessory proteins that assemble higher-order complexes. Addressing this knowledge gap with quantitative kinetic frameworks, functional models and structural studies is critical for understanding how cells precisely process and integrate different signaling inputs to achieve specific outcomes. Conceptually-related questions arise in genome regulation, where spatial organization also plays a central role. Eukaryotic genomes adopt 3D structures that appear to promote specific biochemical processes by positioning genes in proximity to remote DNA regulatory sites or to localized proteins, but this model has not been rigorously tested. A major challenge is the lack of robust tools to systematically perturb genome structures and assess effects on function. Recent CRISPR-Cas methods show promise but have not yet been widely adopted, possibly due to limited efficacy and generalizability. Developing more robust tools to systematically perturb genome structure will enable us to address critical knowledge gaps in the relationship between genome structure and function.