The use of orthogonal tRNA-synthetase pairs for stop codon reassignment has enabled the insertion of a huge diversity of noncanonical amino acids into proteins. However, expression levels of these non-natural proteins in cells remain disappointingly low in most cases, which is thought to be a consequence of the multiple checkpoints in translational fidelity and competing release factors. Wang et al. now identify two ways that bacteria actively repress the non-natural system. The authors created a plasmid containing genes for the commonly used Methanococcus jannaschii tyrosyl tRNA and its synthetase as well as a GFP containing an internal TAG codon. After a small number of passages, nonfluorescent bacteria emerged as dominant over the fluorescent cells owing to the insertion of transposons within the synthetase gene, disabling it. When only fluorescent cells were passaged, the authors observed 16-fold increases in expression of YdiI, a putative thioesterase, which the authors demonstrate plays an active part in expelling plasmids from the cell. To bypass bacterial pushback, Zeng et al. co-opt the rarely used AGG codon to insert their residues of interest. Indeed, using a plasmid encoding the pyrrolysine tRNA and synthetase along with GFP as a test protein, the authors were able to incorporate three amino acids at yields up to 92% of the total GFP produced, with no obvious toxicity to the cells.
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Goodman, C. Plasmids out, codons in. Nat Chem Biol 10, 695 (2014). https://doi.org/10.1038/nchembio.1621