Oxidative stress strongly restricts the effect of codon choice on the efficiency of protein synthesis in Escherichia coli.

Introduction: The response of enterobacteria to oxidative stress is usually considered to be regulated by transcription factors such as OxyR and SoxR. Nevertheless, several reports have shown that under oxidative stress the levels, modification and aminoacylation of tRNAs may be altered suggesting a role of codon bias in regulation of gene expression under this condition. Methods: In order to characterize the effects of oxidative stress on translation elongation we constructed a library of 61 plasmids, each coding for the green fluorescent protein (GFP) translationally fused to a different set of four identical codons. Results: Using these reporters, we observed that GFP production levels vary widely (~15 fold) when Escherichia coli K-12 is cultured in minimal media as a consequence of codon choice variations. When bacteria are cultured under oxidative stress caused by paraquat the levels of GFP produced by most clones is reduced and, in contrast to control conditions, the range of GFP levels is restricted to a ~2 fold range. Restricting elongation of particular sequences does not increase the range of GFP production under oxidative stress, but altering translation initiation rates leads to an increase in this range. Discussion: Altogether, our results suggest that under normal conditions the speed of translation elongation is in the range of the speed of initiation and, consequently, codon choice impacts the speed of protein synthesis. In contrast, under oxidative stress translation initiation becomes much slower than elongation, limiting the speed of translation such that codon choice has at most only subtle effects on the overall output of translation.

Modulation of Escherichia coli Translation by the Specific Inactivation of tRNAGly Under Oxidative Stress.

Bacterial oxidative stress responses are generally controlled by transcription factors that modulate the synthesis of RNAs with the aid of some sRNAs that control the stability, and in some cases the translation, of specific mRNAs. Here, we report that oxidative stress additionally leads to inactivation of tRNAGly in Escherichia coli, inducing a series of physiological changes. The observed inactivation of tRNAGly correlated with altered efficiency of translation of Gly codons, suggesting a possible mechanism of translational control of gene expression under oxidative stress. Changes in translation also depended on the availability of glycine, revealing a mechanism whereby bacteria modulate the response to oxidative stress according to the prevailing metabolic state of the cells.

Codon usage revisited: Lack of correlation between codon usage and the number of tRNA genes in enterobacteria.

It is widely believed that if a high number of genes are found for any tRNA in a rapidly replicating bacteria, then the cytoplasmic levels of that tRNA will be high and an open reading frame containing a higher frequency of the complementary codon will be translated faster. This idea is based on correlations between the number of tRNA genes, tRNA concentration and the frequency of codon usage observed in a limited number of strains as well as from the fact that artificially changing the number of tRNA genes alters translation efficiency and consequently the amount of properly folded protein synthesized. tRNA gene number may greatly vary in a genome due to duplications, deletions and lateral transfer which in turn would alter the levels and functionality of many proteins. Such changes are potentially deleterious for fitness and as a result it is expected that changes in tRNA gene numbers should be accompanied by a modification of the frequency of codon usage. In contrast to this model, when comparing the number of tRNA genes and the frequency of codon usage of several Salmonella enterica and Escherichia coli strains we found that changes in the number of tRNA genes are not correlated to changes in codon usage. Furthermore, these changes are not correlated with a change in the efficiency of codon translation. These results suggest that once a genome gains or loses tRNA genes, it responds by modulating the concentrations of tRNAs rather than modifying its frequency of codon usage.

Non-canonical roles of tRNAs and tRNA mimics in bacterial cell biology.

Transfer RNAs (tRNAs) are the macromolecules that transfer activated amino acids from aminoacyl-tRNA synthetases to the ribosome, where they are used for the mRNA guided synthesis of proteins. Transfer RNAs are ancient molecules, perhaps even predating the existence of the translation machinery. Albeit old, these molecules are tremendously conserved, a characteristic that is well illustrated by the fact that some bacterial tRNAs are efficient and specific substrates of eukaryotic aminoacyl-tRNA synthetases and ribosomes. Considering their ancient origin and high structural conservation, it is not surprising that tRNAs have been hijacked during evolution for functions outside of translation. These roles beyond translation include synthetic, regulatory and information functions within the cell. Here we provide an overview of the non-canonical roles of tRNAs and their mimics in bacteria, and discuss some of the common themes that arise when comparing these different functions.

Effect of hydrogen peroxide on the biosynthesis of heme and proteins: potential implications for the partitioning of Glu-tRNA(Glu) between these pathways.

Glutamyl-tRNA (Glu-tRNA(Glu)) is the common substrate for both protein translation and heme biosynthesis via the C5 pathway. Under normal conditions, an adequate supply of this aminoacyl-tRNA is available to both pathways. However, under certain circumstances, Glu-tRNA(Glu) can become scarce, resulting in competition between the two pathways for this aminoacyl-tRNA. In Acidithiobacillus ferrooxidans, glutamyl-tRNA synthetase 1 (GluRS1) is the main enzyme that synthesizes Glu-tRNA(Glu). Previous studies have shown that GluRS1 is inactivated in vitro by hydrogen peroxide (H2O2). This raises the question as to whether H2O2 negatively affects in vivo GluRS1 activity in A. ferrooxidans and whether Glu-tRNA(Glu) distribution between the heme and protein biosynthesis processes may be affected by these conditions. To address this issue, we measured GluRS1 activity. We determined that GluRS1 is inactivated when cells are exposed to H2O2, with a concomitant reduction in intracellular heme level. The effects of H2O2 on the activity of purified glutamyl-tRNA reductase (GluTR), the key enzyme for heme biosynthesis, and on the elongation factor Tu (EF-Tu) were also measured. While exposing purified GluTR, the first enzyme of heme biosynthesis, to H2O2 resulted in its inactivation, the binding of glutamyl-tRNA to EF-Tu was not affected. Taken together, these data suggest that in A. ferrooxidans, the flow of glutamyl-tRNA is diverted from heme biosynthesis towards protein synthesis under oxidative stress conditions.

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. tRNA(Glu) 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.