Inducible orthogonal aminoacylation demonstrates that charging is required for mitochondrial tRNA import in Trypanosoma brucei

Huot, Jonathan; Shikha, Shikha; Schneider, André (2019). Inducible orthogonal aminoacylation demonstrates that charging is required for mitochondrial tRNA import in Trypanosoma brucei. Scientific Reports, 9(1) Nature Publishing Group 10.1038/s41598-019-47268-4

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Orthogonal aminoacyl-tRNA synthetase/tRNA pairs have emerged as powerful means of site-specifically introducing non-standard amino acids into proteins in vivo. Using amino acids with crosslinking moieties this method allows the identification of transient protein-protein interactions. Here we have introduced a previously characterized evolved tyrosyl-tRNA synthetase/suppressor tRNATyr pair from E. coli into the parasitic protozoan Trypanosoma brucei. Upon addition of a suitable non-standard amino acid the suppressor tRNATyr was charged and allowed translation of a green fluorescent protein whose gene contained a nonsense mutation. - T. brucei is unusual in that its mitochondrion lacks tRNA genes indicating that all its organellar tRNAs are imported from the cytosol. Expression of the bacterial tyrosyl-tRNA synthetase in our system is tetracycline-inducible. We have therefore used it to demonstrate that cytosolic aminoacylation of the suppressor tRNATyr induces its import into the mitochondrion.

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