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Bacterial translation machinery for deliberate mistranslation of the genetic code

Significance Aminoacyl-transfer RNA (tRNA) synthetases (aaRSs) are essential enzymes that mediate accurate expression of the genetic code. More than 96% of analyzed species possess duplicated aaRSs genes, which are presumed to encode canonical aaRSs. However, in this study, we show that some of these seemingly canonical aaRSs are in fact repurposed for deliberate mistranslation of the genetic code, enabling organisms to encode two amino acids using a single sense codon. This finding, along with the abundance of duplicated aaRSs genes, suggests that the deliberate mistranslation of sense codons may be more common than previously thought. More broadly, our work illustrates that extant genomes have many exciting functionalities that are currently hidden under a disguise of canonical tRNA synthetases and tRNAs. Inaccurate expression of the genetic code, also known as mistranslation, is an emerging paradigm in microbial studies. Growing evidence suggests that many microbial pathogens can deliberately mistranslate their genetic code to help invade a host or evade host immune responses. However, discovering different capacities for deliberate mistranslation remains a challenge because each group of pathogens typically employs a unique mistranslation mechanism. In this study, we address this problem by studying duplicated genes of aminoacyl-transfer RNA (tRNA) synthetases. Using bacterial prolyl-tRNA synthetase (ProRS) genes as an example, we identify an anomalous ProRS isoform, ProRSx, and a corresponding tRNA, tRNAProA, that are predominately found in plant pathogens from Streptomyces species. We then show that tRNAProA has an unusual hybrid structure that allows this tRNA to mistranslate alanine codons as proline. Finally, we provide biochemical, genetic, and mass spectrometric evidence that cells which express ProRSx and tRNAProA can translate GCU alanine codons as both alanine and proline. This dual use of alanine codons creates a hidden proteome diversity due to stochastic Ala→Pro mutations in protein sequences. Thus, we show that important plant pathogens are equipped with a tool to alter the identity of their sense codons. This finding reveals the initial example of a natural tRNA synthetase/tRNA pair for dedicated mistranslation of sense codons.