Transfer RNA (tRNA) is the most highly and diversely modified class of RNA in all domains of life. However, we still have only a limited understanding of the concerted action of the many enzymes that modify tRNA during tRNA maturation and the synergistic functions of tRNA modifications for protein synthesis. Here, we describe the preparation of in vitro transcribed tRNAs with a partial set of defined modifications and the use of partially modified tRNAs in biochemical assays. By comparing the affinity and activity of tRNA modification enzymes for partially modified and unmodified tRNAs, we gain insight into the preferred pathways of tRNA maturation. Additionally, partially modified tRNAs will be highly useful to investigate the importance of tRNA modifications for tRNA function during translation including the interaction with aminoacyl-tRNA synthases, translation factors and the ribosome. Thereby, the methods described here lay the foundation for understanding the mechanistic function of tRNA modifications.
Keywords: Aminoacylation; Methylation; Modification circuits; Modification order; Pseudouridine; RNA binding; RNA modification; Ribosome; Translation.
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