Knowledge Bank

In recent years, numerous defects in tRNA biogenesis and related processes, including tRNA aminoacylation, have been linked to human disease. As direct mediators of translational fidelity, aminoacyl-tRNA synthetases (aaRS) are responsible for the correct pairing of an amino acid with its cognate tRNA. Mispaired aminoacyl-tRNA (aa-tRNA) species occur due to a lack of amino acid discrimination within the aaRS active site. These misacylation events make tRNA proofreading and editing mechanisms essential to maintaining the accuracy of translation. Historically, misincorporation of Tyr into the proteome at Phe codons has been considered a major contributor to an overall decrease in cell viability. Looking at the accumulation of mutations over time is useful for identifying factors that contribute to disease progression, but does not provide much insight into the source of mutations. It is possible that mutation rates are affected by factors other than proteome fidelity in this model. To address this question, a yeast-based mutation rate analysis will be performed, thereby precluding contributing factors of the tumor microenvironment. We hypothesize that changes in proteome fidelity caused by aaRS mediated mistranslation lead to dramatic genomic alterations through an increase in mutation rate. A mutation fluctuation assay to monitor cell survival, which reflects the frequency of inactivation of the CAN1 locus, will be performed. CAN1 is responsible for importing canavanine, a toxic analog of arginine. Data obtained from the mutation rate analysis will provide insight into the direct contribution of aaRS mediated mistranslation to genome stability. If these initial experiments are successful, our results could be further applied to human cell lines using the endogenous yeast enzyme.