Collaborative Proposal: Role of tRNA base modifications in genetic code accuracy and cellular fitness

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Organisms are presumed to maintain a high accuracy of translation to ensure that little variation occurs in the sequences of proteins involved in essential cellular functions. However, a relaxed translational stringency has recently been observed across all branches of life, suggesting that under certain circumstances organisms may tolerate higher translation error rates than previously estimated. Indeed, a decrease in translation stringency has the potential to yield new proteins that provide short- or long-term survival benefits. The overall objective of this project is to understand the connections between tRNA base modifications, changes in translation accuracy and cellular fitness. Graduate and undergraduate students will receive interdisciplinary training through their work on this project. An evolution-in-action module (EvolvingSTEM) has been implemented in New Hampshire and Pennsylvania high schools and will be introduced to biology students in two Winston-Salem, North Carolina public high schools.This project will test the hypothesis that modification of the tRNA wobble base is a tunable component of translation rather than a toggle switch that when turned off leads to error catastrophe. This proposal has three objectives. Molecular dynamics and enzyme kinetics approaches will be used to characterize evolved BcTilS:tRNAIle2 variants in vitro and in silico in order to understand the catalytic and conformational changes that occur in these variants. The in vivo effects of TilS:tRNAIle2 dysfunction such as which amino acid is inserted at AUA codons, which amino acid is present at the 3'-end of tRNAIle2, and what is the metabolic mechanism of enhanced fitness will be investigated. Finally, noncanonical cellular partners of TilS and tRNAIle2 that may play alternative or additional roles in enhanced bacterial fitness will be identified.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.