Mechanism and function of tRNA modification and folding

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Protein synthesis is a fundamentally critical process to sustain all forms of life. In addition, protein synthesis is also an important part of bioengineering, specifically the production of large quantities of proteins outside living cells for commercial applications. Also, defects in protein synthesis lead to a plethora of different diseases. During protein synthesis, the genetic information is decoded and translated into a protein sequence by critical adaptor molecules called transfer ribonucleic acids or tRNAs. Despite more than 50 years of research, we still cannot define the design principles common to all tRNA. Notably, tRNAs carry numerous chemical modifications, but it remains poorly understood how tRNA modifying enzymes enhance the functionality and structure of tRNAs. This gap in our understanding of tRNA design hampers our abilities in protein bioengineering. To incorporate non-natural building blocks in engineered proteins, we require novel, designer tRNAs that are efficient in protein synthesis; but to-date we are unable to utilize chemical modifications to enhance the functionality of tRNAs. Therefore, my current research program focuses on understanding the formation and function of natural tRNAs, specifically the optimization of tRNAs by modifying enzymes. The proposed research builds on our breakthrough discovery that a prototypic tRNA modifying enzyme also assists in generating the correct three-dimensional shape of tRNAs. Here, we will systematically study the function of multiple tRNA modifying enzymes for tRNA structure and function in protein synthesis. Specifically, we will investigate how tRNAs are chemically modified by different enzymes, how they fold into a correct three-dimensional shape, and how tRNA modification and folding affects their function during protein synthesis. By increasing our fundamental knowledge on tRNA biology, we are laying the foundation to engineer novel tRNAs for the efficient production of proteins with novel properties for commercial applications. At the same time, our research may improve our understanding of inherited diseases resulting from tRNA modification defects.