Fixen, Gretchen M., authorStasevich, Timothy, advisorNishimura, Erin, committee memberChung, Jean, committee member2024-05-272024-05-272024https://hdl.handle.net/10217/238411Genes encoded in our DNA are fundamental to human health and well-being. Their imperative role requires tight regulation throughout their journey to becoming functional proteins. These regulations, when disrupted, have been linked to many neurodegenerative disorders and cancers, stressing the importance of deconvolving their components. Translation is one of the final steps in this journey that has been extensively explored, resulting in a recent technique developed known as nascent chain tracking (NCT) coupled with MS2 stem loop tagging. Using this technique, we are able to track translation dynamics in real-time and in live cells. Despite this, there are still limitations in spatially and temporally tracking the recruitment of translation effectors to translation sites and accurately measuring these dynamics. With the incorporation of optogenetic blue-light-sensitive proteins, we can generate inducible biomolecular condensates that recruit green fluorescent protein (GFP)-tagged proteins and our reporter mRNAs. Using this controlled test-tube-like environment, we can discover the direct effects ribosomal quality control proteins have on translation dynamics. A main quality control pathway involves ZNF598, GIGYF2, and 4EHP proteins that mediate translation control during ribosome stalling. We discovered that both GIGYF2 and 4EHP can be recruited to these clusters and co-localize with our active translation sites in live cells. Further exploration found that 4EHP alone cannot fully cause translation inhibition with our system. Despite this, we do see translation initiation occurring over time due to complex formation with HIF-2∝. However, GIGYF2 has distinct effects on these kinetics that are variable. This tool, when optimized, will be able to describe different proteins' effects on translation kinetics in an isolated environment in live cells.born digitalmasters thesesengCopyright and other restrictions may apply. User is responsible for compliance with all applicable laws. For information about copyright law, please see https://libguides.colostate.edu/copyright.optogeneticstranslationquality controlnascent chain trackingText