Project Summary/Abstract The ubiquitin-proteasome system is the major pathway for regulatory protein degradation, exhibiting a similar level of target specificity as translation/translation underlying most biological functions. Ubiquitin patterns on substrate molecules determine the specificity, the rate and the outcome of proteolysis by the proteasome. Which features of ubiquitin configurations are important for the selectivity of proteasomal degradation and how the 26S proteasome, with a multitude of ubiquitin receptors, recognizes these features is still poorly understood. Proteasomal degradation involves a sequence of steps. Our published and preliminary studies recorded a strong dependence of the degradation rate on the length, linkage, copy-number and position of conjugated ubiquitin chains. How this selectivity is achieved is still unclear. Here, I propose a systematic investigation to identify how the features of ubiquitin configurations control the kinetics and modes of substrates’ engagement with the ubiquitin receptors on proteasome to determine the rate of degradation. We will employ a single-molecule method I developed previously to differentiate multiple limiting steps in the degradation process and to measure their rate constants. To circumvent the difficulty with preparing protein substrates with defined ubiquitylation, I propose a novel method of using DNA scaffolds to engineer arbitrary yet defined ubiquitin configurations, and systematically vary the features of ubiquitin configuration to determine how they affect substrate’s interaction with proteasome and degradation. Dysregulation of the ubiquitin- proteasome system is implicated in numerous diseases, including cancers, neurodegenerative disorders, autoimmunity and diabetes. The long-term goal of our lab is to elucidate how the proteasome recognizes its targets and selectively engages them into the processive degradation process, and to understand how misregulation of protein degradation leads to the formation of pathogenic inclusions. Our proposed research will provide insights into how mutations in the ubiquitin conjugation pathways lead to human diseases, and will generate valuable information for developing novel therapeutic strategies.