The absence of the queuosine tRNA modification leads to pleiotropic phenotypes revealing perturbations of metal and oxidative stress homeostasis in Escherichia coli K12.

Queuosine (Q) is a conserved hypermodification of the wobble base of tRNA containing GUN anticodons but the physiological consequences of Q deficiency are poorly understood in bacteria. This work combines transcriptomic, proteomic and physiological studies to characterize a Q-deficient Escherichia coli K12 MG1655 mutant. The absence of Q led to an increased resistance to nickel and cobalt, and to an increased sensitivity to cadmium, compared to the wild-type (WT) strain. Transcriptomic analysis of the WT and Q-deficient strains, grown in the presence and absence of nickel, revealed that the nickel transporter genes (nikABCDE) are downregulated in the Q- mutant, even when nickel is not added. This mutant is therefore primed to resist to high nickel levels. Downstream analysis of the transcriptomic data suggested that the absence of Q triggers an atypical oxidative stress response, confirmed by the detection of slightly elevated reactive oxygen species (ROS) levels in the mutant, increased sensitivity to hydrogen peroxide and paraquat, and a subtle growth phenotype in a strain prone to accumulation of ROS.

tRNA-modifying enzyme mutations induce codon-specific mistranslation and protein aggregation in yeast.

Protein synthesis rate and accuracy are tightly controlled by the cell and are essential for proteome homoeostasis (proteostasis); however, the full picture of how mRNA translational factors maintain protein synthesis accuracy and co-translational protein folding are far from being fully understood. To address this question, we evaluated the role of 70 yeast tRNA-modifying enzyme genes on protein aggregation and used mass spectrometry to identify the aggregated proteins. We show that modification of uridine at anticodon position 34 (U34) by the tRNA-modifying enzymes Elp1, Elp3, Sml3 and Trm9 is critical for proteostasis, the mitochondrial tRNA-modifying enzyme Slm3 plays a fundamental role in general proteostasis and that stress response proteins whose genes are enriched in codons decoded by tRNAs lacking mcm5U34, mcm5s2U34, ncm5U34, ncm5Um34, modifications are overrepresented in protein aggregates of the ELP1, SLM3 and TRM9 KO strains. Increased rates of amino acid misincorporation were also detected in these strains at protein sites that specifically mapped to the codons sites that are decoded by the hypomodified tRNAs, demonstrating that U34 tRNA modifications safeguard the proteome from translational errors, protein misfolding and proteotoxic stress.