Redox Status Affects the Catalytic Activity of Glutamyl-tRNA Synthetase

Glutamyl-tRNA synthetases (GluRS) provide Glu-tRNA for different processes including protein synthesis, glutamine transamidation and tetrapyrrole biosynthesis. Many organisms contain multiple GluRSs, but whether these duplications solely broaden tRNA specificity or also play additional roles in tetrapyrrole biosynthesis is not known. Previous studies have shown that GluRS1, one of two GluRSs from the extremophile Acidithiobacillus ferrooxidans, is inactivated when intracellular heme is elevated suggesting a specific role for GluRS1 in the regulation of tetrapyrrole biosynthesis. We now show that, in vitro, GluRS1 activity is reversibly inactivated upon oxidation by hemin and hydrogen peroxide. The targets for oxidation-based inhibition were found to be cysteines from a SWIM zinc-binding motif located in the tRNA acceptor helix-binding domain. tRNAGlu was able to protect GluRS1 against oxidative inactivation by hemin plus hydrogen peroxide. The sensitivity to oxidation of A. ferrooxidans GluRS1 might provide a means to regulate tetrapyrrole and protein biosynthesis in response to extreme changes in both the redox and heme status of the cell via a single enzyme

tRNAs: Cellular Barcodes for Amino Acids

The role of tRNA in translating the genetic code has received considerable attention over the last 50 years, and we now know in great detail how particular amino acids are specifically selected and brought to the ribosome in response to the corresponding mRNA codon. Over the same period, it has also become increasingly clear that the ribosome is not the only destination to which tRNAs deliver amino acids, with processes ranging from lipid modification to antibiotic biosynthesis all using aminoacyl‐tRNAs as substrates. Here we review examples of alternative functions for tRNA beyond translation, which together suggest that the role of tRNA is to deliver amino acids for a variety of processes that includes, but is not limited to, protein synthesis

Cell-Specific Differences in the Requirements for Translational Quality Control

Protein synthesis has an overall error rate of approximately 10-4 for each mRNA codon translated. The fidelity of translation is mainly determined by two events: synthesis of cognate amino acid:tRNA pairs by aminoacyl-tRNA synthetases (aaRSs) and accurate selection of aminoacyl-tRNAs (aa-tRNAs) by the ribosome. To ensure faithful aa-tRNA synthesis, many aaRSs employ a proofreading (“editing”) activity, such as phenylalanyl-tRNA synthetases (PheRS) that hydrolyze mischarged Tyr-tRNAPhe. Eukaryotes maintain two distinct PheRS enzymes, a cytoplasmic (ctPheRS) and an organellar form. CtPheRS is similar to bacterial enzymes in that it consists of a heterotetramer in which the α-subunits contain the active site and the β-subunits harbor the editing site. In contrast, mitochondrial PheRS (mtPheRS) is an α-subunit monomer that does not edit Tyr-tRNAPhe, and a comparable transacting activity does not exist in organelles. Although mtPheRS does not edit, it is extremely specific as only one Tyr-tRNAPhe is synthesized for every ∼7,300 Phe-tRNAPhe, compatible with an error rate in translation of ∼10-4. When the error rate of mtPheRS was increased 17-fold, the corresponding strain could not grow on respiratory media and the mitochondrial genome was rapidly lost. In contrast, error-prone mtPheRS, editing-deficient ctPheRS, and their wild-type counterparts all supported cytoplasmic protein synthesis and cell growth. These striking differences reveal unexpectedly divergent requirements for quality control in different cell compartments and suggest that the limits of translational accuracy may be largely determined by cellular physiology

Mechanisms of Resistance to an Amino Acid Antibiotic That Targets Translation

Structural and functional diversity among the aminoacyl-tRNA synthetases prevent infiltration of the genetic code by noncognate amino acids. To explore whether these same features distinguish the synthetases as potential sources of resistance against antibiotic amino acid analogues, we investigated bacterial growth inhibition by S-(2-aminoethyl)-l-cysteine (AEC). Wild-type lysyl-tRNA synthetase (LysRS) and a series of active site variants were screened for their ability to restore growth of an Escherichia coli LysRS null strain at increasing concentrations of AEC. While wild-type E. coli growth is completely inhibited at 5 µM AEC, two LysRS variants, Y280F and F426W, provided substantial resistance and allowed E. coli to grow in the presence of up to 1 mM AEC. Elevated resistance did not reflect changes in the kinetics of amino acid activation or tRNALys aminoacylation, which showed at best 4−6-fold improvements, but instead correlated with the binding affinity for AEC, which was decreased ∼50-fold in the LysRS variants. In addition to changes in LysRS, AEC resistance has also been attributed to mutations in the L box riboswitch, which regulates expression of the lysC gene, encoding aspartokinase. The Y280F and F426W LysRS mutants contained wild-type L box riboswitches that responded normally to AEC in vitro, indicating that LysRS is the primary cellular target of this antibiotic. These findings suggest that the AEC resistance conferred by L box mutations is an indirect effect resulting from derepression of lysC expression and increased cellular pools of lysine, which results in more effective competition with AEC for binding to LysRS