During protein synthesis, aminoacyl-tRNA synthetases (aaRSs) catalyse the ligation of amino acids to transfer RNAs (tRNAs) to form the aminoacyl-tRNAs. As tRNAs recognise the codons on the messenger RNA (mRNA) through their anticodons in the ribosome, the amino acids are linked to the growing polypeptide chain. The correct coupling of amino acids with tRNAs and the binding of anticodons to the correct codons are vital for translating genetic information into functional proteins. However, growing evidence suggests that organisms, from bacteria to human cells, can induce and use mistranslation as a mechanism to respond to environmental stress. In this work, the researchers reported two novel families of non-canonical tRNAs, encoded by bacteria from the Streptomyces and Kitasatospora genera, that can induce mistranslation of the genetic code. It was found that a dual identity was adapted by these tRNAs to replace proline anticodon with AUU asparagine anticodon or AGU threonine anticodon. Using a previously developed superfolder green fluorescent protein (stGFP) and β-lactamase reporters respectively, these tRNAs were shown to translate threonine and asparagine codons with proline. Additionally, liquid-chromatography tandem mass spectrometry (LC-MS/MS) was used for the detection of mistranslation events of Asn with Pro in the stGFP reporter. Asn-to-Pro mistranslation was detected at three different positions within sfGFP, and these substitutions were not observed in the sfGFP purified without tRNAs. Moreover, when expressed in Escherichia coli, mistranslation by these tRNAs caused cell growth defects under normal growth conditions. However, under specific stress conditions, such as in the presence of antibiotics, mistranslation of Asn and Thr with Pro increased cell tolerance to the environment. Interestingly, in the absence of cognate bacterial prolyl-tRNA synthetase (ProRS), tRNAs were still shown to incorporate Pro in response to Asn and Thr codons with the aids of endogenous E. coli ProRS. Taken together, these results expand the catalogue of organisms known to possess dedicated mistranslation and support the concept that mistranslation is a mechanism for increased cellular resiliency against environmental stress.
How was PEAKS used?
MS raw files were processed using PEAKS XPro (v10.6, Bioinformatics Solutions Inc., Ontario, Canada). The data was searched against a custom database containing the sfGFP sequence in the E. coli K12 Uniprot-reviewed database. A precursor ion mass tolerance of 10 ppm and a fragment ion mass tolerance was set to 0.02 Da. Semi-specific cleavage with trypsin or chymotrypsin was selected with a maximum of 3 missed cleavages. A fixed modification of carbamidomethylation (C) and variable modifications of deamidation (NQ) and oxidation (M) were added, and a maximum of two variable modifications were allowed per peptide. The peptide false discovery rate was set to 1% for the database search. Only confident mutations with an A-Score of 20 or higher and minimum mutation ion intensities of 1% were considered. To ensure the accuracy in identifying mutations, a false discovery rate (FDR) filter of <1% for peptide identification and minimum fragment ion intensity of 1% for each mutation were applied.
Schuntermann, Dominik B., et al. “Mistranslation of the genetic code by a new family of bacterial transfer RNAs.” Journal of Biological Chemistry 299.7 (2023). https://doi.org/10.1016/j.jbc.2023.104852
The correct coupling of amino acids with transfer RNAs (tRNAs) is vital for translating genetic information into functional proteins. Errors during this process lead to mistranslation, where a codon is translated using the wrong amino acid. While unregulated and prolonged mistranslation is often toxic, growing evidence suggests that organisms, from bacteria to humans, can induce and use mistranslation as a mechanism to overcome unfavorable environmental conditions. Most known cases of mistranslation are caused by translation factors with poor substrate specificity or when substrate discrimination is sensitive to molecular changes such as mutations or posttranslational modifications. Here we report two novel families of tRNAs, encoded by bacteria from the Streptomyces and Kitasatospora genera, that adopted dual identities by integrating the anticodons AUU (for Asn) or AGU (for Thr) into the structure of a distinct proline tRNA. These tRNAs are typically encoded next to a full-length or truncated version of a distinct isoform of bacterial-type prolyl-tRNA synthetase. Using two protein reporters, we showed that these tRNAs translate asparagine and threonine codons with proline. Moreover, when expressed in Escherichia coli, the tRNAs cause varying growth defects due to global Asn-to-Pro and Thr-to-Pro mutations. Yet, proteome-wide substitutions of Asn with Pro induced by tRNA expression increased cell tolerance to the antibiotic carbenicillin, indicating that Pro mistranslation can be beneficial under certain conditions. Collectively, our results significantly expand the catalog of organisms known to possess dedicated mistranslation machinery and support the concept that mistranslation is a mechanism for cellular resiliency against environmental stress.