The threonyl-tRNA synthetase is an enzyme that catalyzes the attachment of the amino acid threonine to the threonyl-tRNA so that protein synthesis at the ribosome can occur. In order to maintain translational fidelity, this tRNA synthetase must employ an editing function that is structurally and functionally separate from this enzyme's aminoacylation capacity. Here, we studied the importance editing function of the Bacillus subtilis threonyl synthetase, ThrRS, by making a single amino acid substitution in the separate editing domain. The data in this study suggest that disrupting editing function in the major B. subtilis ThrRS, thrS, is toxic to the cells under normal growth conditions. A future goal in this project is to study the effect of knocking out the editing function of the minor B. subtilis ThrRS, thrZ.

Faithful translation of the genetic code into amino acid sequences is important for the viability of organisms. One source of error in translation is the mischarging of tRNAs with the incorrect amino acid due to structural similarities between the cognate and non-cognate amino acids. If gone unchecked, these mischarged tRNAs would provide an amino acid to the ribosome that does not match its codon, thereby causing mistranslation of the mRNA sequence. The proteins that are responsible for charging tRNAs with the correct amino acids are called aminoacyl-tRNA synthetases (aaRS). Some of these aaRSs have evolved an editing mechanism that allows them to cleave off a non-cognate amino acid from the mischarged tRNA, which is broadly conserved across all domains of life. This editing activity seems like it would be essential for life, however there are many examples of organisms who have lost their editing function to no ill effect. Moreover, there are examples of organisms that have conserved their editing function, but do not show a growth defect when it is eliminated, such as E. coli and its phenylalanine aaRS (PheRS).

We chose to study E. coli’s PheRS to understand why its editing function is evolutionarily conserved. We discovered that the non-protein amino acid meta-Tyrosine (m-Tyr) is toxic to PheRS editing-defective (PheRS edit-) E. coli. We then sought to understand why m-Tyr is so toxic to PheRS edit- cells. We used chemical mutagenesis to find m-Tyr resistant mutants and then performed whole genome sequencing to find mutated genes that could contribute to the resistance. We found that mutations in uptake and efflux transport could provide resistance by keeping or getting m-Tyr out of the cell. We also identified a resistance mutation that likely elevated Phe production, which provided resistance by most likely increasing competitive inhibition of the m-Tyr. We also observed PheRS edit- E. coli after m-Tyr exposure directly via light and electron microscopy. We observed large protein aggregates forming in the cells, which indicated that the m-Tyr destabilized a large fraction of the proteome. We also performed transcriptomic analysis of PheRS edit- E. coli after m-Tyr exposure to see what stress responses they used to deal with m-Tyr toxicity. We found a strong induction of the unfolded protein stress response, as well as oxidative stress, DNA damage stress, and indications of lost ion homeostasis. Based on these findings, we proposed a model of m-Tyr toxicity that involves a cascading and self-reinforcing chain reaction of cellular stresses that ultimately leads to cell death.

Cells maintain fidelity in protein synthesis through various mechanisms including aminoacyl-tRNA synthetases (aaRSs) that are able to discriminate tRNAs and their cognate amino acids. Substrate specificity along with editing functions of aaRSs act to prevent or remove incorrect amino acids charged onto tRNAs. Editing domains are highly conserved and found in both prokaryotes and eukaryotes; knocking out the editing function of an aaRS does not seem to affect cell viability. To determine the biological relevance of the editing domains found on aaRSs, it is essential to determine the contribution of editing to protein fidelity in vivo. Bacillus subtilis serves as an ideal model to study this phenomenon because it possesses a small gene-enocoded, 10 amino acid pheromone peptide, ComX. ComX is processed, modified, and secreted by B. subtilis as a signaling molecule that can easily be purified. The amino acid sequence of ComX can be altered to some degree while maintaining cellular activity enabling different strains that produce variants of ComX pheromone to be compared for levels of mistranslation. Ultimately future experiments will reveal the impact that editing by an aaRS has on the fidelity of protein synthesis in vivo by quantifying the rates of mistranslation in strains of B. subtilis that express an editing-defective tRNA synthetase and comparing it to levels of mistranslation to wild type strains.