Natural homolog of tRNA synthetase editing domain rescues conditional lethality caused by mistranslation

AlaXp is a widely distributed (from bacteria to humans) genome-encoded homolog of the editing domain of alanyl-tRNA synthetases. Editing repairs the confusion of serine and glycine for alanine through clearance of mischarged (with Ser or Gly) tRNA(Ala). Because genome-encoded fragments of editing domains of other synthetases are scarce, the AlaXp redundancy of the editing domain of alanyl-tRNA synthetase is thought to reflect an unusual sensitivity of cells to mistranslation at codons for Ala. Indeed, a small defect in the editing activity of alanyl-tRNA synthetase is causally linked to neurodegeneration in the mouse. Although limited earlier studies demonstrated that AlaXp deacylated mischarged tRNA(Ala) in vitro, the significance of this activity in vivo has not been clear. Here we describe a bacterial system specifically designed to investigate activity of AlaXp in vivo. Serine toxicity, experienced by a strain harboring an editing-defective alanyl-tRNA synthetase, was rescued by an AlaXp-encoding transgene. Rescue was dependent on amino acid residues in AlaXp that are needed for its in vitro catalytic activity. Thus, the editing activity per se of AlaXp was essential for suppressing mistranslation. The results support the idea that the unique widespread distribution of AlaXp arises from the singular difficulties, for translation, poised by alanine.     

Mistranslation in a eucaryotic organism.

Previous work from our laboratory has demonstrated that a subclass of the aminoglycoside antibiotics, those containing the drug fragment paromamine, stimulates mistranslation in cell-free protein-synthesizing systems derived from eucaryotic cells. We report here experiments which show that the ciliate Tetrahymena thermophila (formerly T. pyriformis, syngen 1) is sensitive to the paromamine-containing aminoglycoside antibiotics. The drugs are active with respect to growth inhibition, inhibition of protein synthesis in the whole organism, inhibition of protein synthesis in vitro and the stimulation of mistranslation in cell-free protein-synthesizing systems. Because of their misreading properties, these drugs may be useful in isolating and propagating strains carrying mutations which can be translationally suppressed (that is, missense and nonsense mutations).     

Temperature dependent mistranslation in a hyperthermophile adapts proteins to lower temperatures

All organisms universally encode, synthesize and utilize proteins that function optimally within a subset of growth conditions. While healthy cells are thought to maintain high translational fidelity within their natural habitats, natural environments can easily fluctuate outside the optimal functional range of genetically encoded proteins. The hyperthermophilic archaeon Aeropyrum pernix (A. pernix) can grow throughout temperature variations ranging from 70 to 100°C, although the specific factors facilitating such adaptability are unknown. Here, we show that A. pernix undergoes constitutive leucine to methionine mistranslation at low growth temperatures. Low-temperature mistranslation is facilitated by the misacylation of tRNA^Leu with methionine by the methionyl-tRNA synthetase (MetRS). At low growth temperatures, the A. pernix MetRS undergoes a temperature dependent shift in tRNA charging fidelity, allowing the enzyme to conditionally charge tRNA^Leu with methionine. We demonstrate enhanced low-temperature activity for A. pernix citrate synthase that is synthesized during leucine to methionine mistranslation at low-temperature growth compared to its high-fidelity counterpart synthesized at high-temperature. Our results show that conditional leucine to methionine mistranslation can make protein adjustments capable of improving the low-temperature activity of hyperthermophilic proteins, likely by facilitating the increasing flexibility required for greater protein function at lower physiological temperatures.     

Reduced Amino Acid Specificity of Mammalian Tyrosyl-tRNA Synthetase Is Associated with Elevated Mistranslation of Tyr Codons

Quality control operates at different steps in translation to limit errors to approximately one mistranslated codon per 10,000 codons during mRNA-directed protein synthesis. Recent studies have suggested that error rates may actually vary considerably during translation under different growth conditions. Here we examined the misincorporation of Phe at Tyr codons during synthesis of a recombinant antibody produced in tyrosine-limited Chinese hamster ovary (CHO) cells. Tyr to Phe replacements were previously found to occur throughout the antibody at a rate of up to 0.7% irrespective of the identity or context of the Tyr codon translated. Despite this comparatively high mistranslation rate, no significant change in cellular viability was observed. Monitoring of Phe and Tyr levels revealed that changes in error rates correlated with changes in amino acid pools, suggesting that mischarging of tRNA^Tyr with noncognate Phe by tyrosyl-tRNA synthetase was responsible for mistranslation. Steady-state kinetic analyses of CHO cytoplasmic tyrosyl-tRNA synthetase revealed a 25-fold lower specificity for Tyr over Phe as compared with previously characterized bacterial enzymes, consistent with the observed increase in translation error rates during tyrosine limitation. Functional comparisons of mammalian and bacterial tyrosyl-tRNA synthetase revealed key differences at residues responsible for amino acid recognition, highlighting differences in evolutionary constraints for translation quality control.     

Function and origin of mistranslation in distinct cellular contexts

Mistranslation describes errors during protein synthesis that prevent the amino acid sequences specified in the genetic code from being reflected within proteins. For a long time, mistranslation has largely been considered an aberrant cellular process that cells actively avoid at all times. However, recent evidence has demonstrated that cells from all three domains of life not only tolerate certain levels and forms of mistranslation, but actively induce mistranslation under certain circumstances. To this end, dedicated biological mechanisms have recently been found to reduce translational fidelity, which indicates that mistranslation is not exclusively an erroneous process and can even benefit cells in particular cellular contexts. There currently exists a spectrum of mistranslational processes that differ not only in their origins, but also in their molecular and cellular effects. These findings suggest that the optimal degree of translational fidelity largely depends on a specific cellular context. This review aims to conceptualize the basis and functional consequence of the diverse types of mistranslation that have been described so far.     

Protein mistranslation protects bacteria against oxidative stress

Accurate flow of genetic information from DNA to protein requires faithful translation. An increased level of translational errors (mistranslation) has therefore been widely considered harmful to cells. Here we demonstrate that surprisingly, moderate levels of mistranslation indeed increase tolerance to oxidative stress in Escherichia coli. Our RNA sequencing analyses revealed that two antioxidant genes katE and osmC, both controlled by the general stress response activator RpoS, were upregulated by a ribosomal error-prone mutation. Mistranslation-induced tolerance to hydrogen peroxide required rpoS, katE and osmC. We further show that both translational and post-translational regulation of RpoS contribute to peroxide tolerance in the error-prone strain, and a small RNA DsrA, which controls translation of RpoS, is critical for the improved tolerance to oxidative stress through mistranslation. Our work thus challenges the prevailing view that mistranslation is always detrimental, and provides a mechanism by which mistranslation benefits bacteria under stress conditions.     

Mistranslation and its control by tRNA synthetases

Aminoacyl tRNA synthetases are ancient proteins that interpret the genetic material in all life forms. They are thought to have appeared during the transition from the RNA world to the theatre of proteins. During translation, they establish the rules of the genetic code, whereby each amino acid is attached to a tRNA that is cognate to the amino acid. Mistranslation occurs when an amino acid is attached to the wrong tRNA and subsequently is misplaced in a nascent protein. Mistranslation can be toxic to bacteria and mammalian cells, and can lead to heritable mutations. The great challenge for nature appears to be serine-for-alanine mistranslation, where even small amounts of this mistranslation cause severe neuropathologies in the mouse. To minimize serine-for-alanine mistranslation, powerful selective pressures developed to prevent mistranslation through a special editing activity imbedded within alanyl-tRNA synthetases (AlaRSs). However, serine-for-alanine mistranslation is so challenging that a separate, genome-encoded fragment of the editing domain of AlaRS is distributed throughout the Tree of Life to redundantly prevent serine-to-alanine mistranslation. Detailed X-ray structural and functional analysis shed light on why serine-for-alanine mistranslation is a universal problem, and on the selective pressures that engendered the appearance of AlaXps at the base of the Tree of Life.     

Evolution of Robustness to Protein Mistranslation by Accelerated Protein Turnover

Translational errors occur at high rates, and they influence organism viability and the onset of genetic diseases. To investigate how organisms mitigate the deleterious effects of protein synthesis errors during evolution, a mutant yeast strain was engineered to translate a codon ambiguously (mistranslation). It thereby overloads the protein quality-control pathways and disrupts cellular protein homeostasis. This strain was used to study the capacity of the yeast genome to compensate the deleterious effects of protein mistranslation. Laboratory evolutionary experiments revealed that fitness loss due to mistranslation can rapidly be mitigated. Genomic analysis demonstrated that adaptation was primarily mediated by large-scale chromosomal duplication and deletion events, suggesting that errors during protein synthesis promote the evolution of genome architecture. By altering the dosages of numerous, functionally related proteins simultaneously, these genetic changes introduced large phenotypic leaps that enabled rapid adaptation to mistranslation. Evolution increased the level of tolerance to mistranslation through acceleration of ubiquitin-proteasome–mediated protein degradation and protein synthesis. As a consequence of rapid elimination of erroneous protein products, evolution reduced the extent of toxic protein aggregation in mistranslating cells. However, there was a strong evolutionary trade-off between adaptation to mistranslation and survival upon starvation: the evolved lines showed fitness defects and impaired capacity to degrade mature ribosomes upon nutrient limitation. Moreover, as a response to an enhanced energy demand of accelerated protein turnover, the evolved lines exhibited increased glucose uptake by selective duplication of hexose transporter genes. We conclude that adjustment of proteome homeostasis to mistranslation evolves rapidly, but this adaptation has several side effects on cellular physiology. Our work also indicates that translational fidelity and the ubiquitin-proteasome system are functionally linked to each other and may, therefore, co-evolve in nature.     

Mistranslation in E. coli.

Flagellin, the protomeric subunit of bacterial flagella, contains no cysteine. We have detected the incorporation of trace quantities of 35S-cysteine into flagellin, highly purified and then resolved by SDS polyacrylamide gel electrophoresis, to measure mistranslation in vivo. Under normal conditions, this value is about 6 X 10(-4) pmoles cysteine per pmole flagellin. This value is greatly increased during growth in low concentrations of streptomycin and neomycin, antibiotics which are known to stimulate misreading in vitro. Of the specific types of misreading which streptomycin stimulates in vitro, only misreading of the CGU and CGC arginine codons could give rise to illegitimate incorporation of cysteine. In agreement, partial arginine starvation increases the incorporation of 35S-cysteine into flagellin in a relA- mutant, with or without streptomycin, but has no such effect in its isogenic relA+ partner- Assuming from these results that 35S-cysteine incorporation into flagellin reflects misreading of CGU/C coda, we deduce a misreading probability per codon in the range of 10(-4).     

Measurement of translational accuracy in vivo: missense reporting using inactive enzyme mutants

The measurement and potential technological significance of in vivo missense errors are briefly reviewed. A recently developed approach is described in which reporter enzyme activity is generated by mistranslation of a gene coding for an inactive mutant form of the enzyme. Initial results obtained using the alpha subunit of E coli tryptophan synthetase and bacterial luciferase are discussed, as well as the prospects for further development of this method.     

Mistranslation of the genetic code

During mRNA decoding at the ribosome, deviations from stringent codon identity, or “mistranslation,” are generally deleterious and infrequent. Observations of organisms that decode some codons ambiguously, and the discovery of a compensatory increase in mistranslation frequency to combat environmental stress have changed the way we view “errors” in decoding. Modern tools for the study of the frequency and phenotypic effects of mistranslation can provide quantitative and sensitive measurements of decoding errors that were previously inaccessible. Mistranslation with non-protein amino acids, in particular, is an enticing prospect for new drug therapies and the study of molecular evolution.     

Global tRNA misacylation induced by anaerobiosis and antibiotic exposure broadly increases stress resistance in Escherichia coli

High translational fidelity is commonly considered a requirement for optimal cellular health and protein function. However, recent findings have shown that inducible mistranslation specifically with methionine engendered at the tRNA charging level occurs in mammalian cells, yeast and archaea, yet it was unknown whether bacteria were capable of mounting a similar response. Here, we demonstrate that Escherichia coli misacylates non-methionyl-tRNAs with methionine in response to anaerobiosis and antibiotic exposure via the methionyl–tRNA synthetase (MetRS). Two MetRS succinyl-lysine modifications independently confer high tRNA charging fidelity to the otherwise promiscuous, unmodified enzyme. Strains incapable of tRNA mismethionylation are less adept at growth in the presence of antibiotics and stressors. The presence of tRNA mismethionylation and its potential role in mistranslation within the bacterial domain establishes this response as a pervasive biological mechanism and connects it to diverse cellular functions and modes of fitness.     

High-Fidelity Translation of Recombinant Human Hemoglobin in Escherichia coli

Coexpression of di-α-globin and β-globin in Escherichia coli in the presence of exogenous heme yielded high levels of soluble, functional recombinant human hemoglobin (rHb1.1). High-level expression of rHb1.1 provides a good model for measuring mistranslation in heterologous proteins. rHb1.1 does not contain isoleucine; therefore, any isoleucine present could be attributed to mistranslation, most likely mistranslation of one or more of the 200 codons that differ from an isoleucine codon by 1 bp. Sensitive amino acid analysis of highly purified rHb1.1 typically revealed ≤0.2 mol of isoleucine per mol of hemoglobin. This corresponds to a translation error rate of ≤0.001, which is not different from typical translation error rates found for E. coli proteins. Two different expression systems that resulted in accumulation of globin proteins to levels equivalent to ~20% of the level of E. coli soluble proteins also resulted in equivalent translational fidelity.     

In vivo translational inaccuracy in Escherichia coli: missense reporting using extremely low activity mutants of Vibrio harveyi luciferase

A convenient, sensitive assay for measurement of in vivo missense translational errors is reported that uses luciferase activity generated by mistranslation of a gene encoding an inactive mutant alpha chain of the Vibrio harveyi enzyme. Mutations were introduced at alpha45 His, a position known to be highly intolerant of amino acids other than histidine. To normalize for any variations in expression level, the concentration of wild-type luciferase alphabeta dimer was determined by a novel assay using co-refolding of active/wild-type beta enzyme subunits with inactive alpha subunits in lysate with an excess of exogenously added active alpha subunits. Four His alpha45 missense mutants of luciferase encoded by leucine codons (CUC, CUU, CUG, and UUG) had histidine misincorporation rates of 2.0 x 10(-6), 1.3 x 10(-6), 9.0 x 10(-8), and 1.5 x 10(-8) respectively, a variation of over 133-fold among synonymous codons. Any substantial contribution of mutation was ruled out by a Luria-Delbrück fluctuation test. The two leucine codons with the highest rates, CUU and CUC, have a single central-mismatch to the histidyl-tRNAQUG anticodon. Aminoglycoside antibiotics known to enhance mistranslation increased the error rate of the CUC codon more than those of the CUU and CUG codons, consistent with the hypothesis that CUC codon mistranslation arises primarily from miscoding events such as the selection of noncognate histidyl-tRNAQUG at the central position of the codon.     

Methionine Mistranslation Bypasses the Restraint of the Genetic Code to Generate Mutant Proteins with Distinct Activities

Although mistranslation is commonly believed to be deleterious, recent evidence indicates that mistranslation can be actively regulated and be beneficial in stress response. Methionine mistranslation in mammalian cells is regulated by reactive oxygen species where cells deliberately alter the proteome through incorporating Met at non-Met positions to enhance oxidative stress response. However, it was not known whether specific, mistranslated mutant proteins have distinct activities from the wild-type protein whose sequence is restrained by the genetic code. Here, we show that Met mistranslation with and without Ca²⁺ overload generates specific mutant Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) proteins substituting non-Met with Met at multiple locations. Compared to the genetically encoded wild-type CaMKII, specific mutant CaMKIIs can have distinct activation profiles, intracellular localization and enhanced phenotypes. Our results demonstrate that Met-mistranslation, or “Met-scan” can indeed generate mutant proteins in cells that expand the activity profile of the wild-type protein, and provide a molecular mechanism for the role of regulated mistranslation.     

Streptomycin-induced, third-position misreading of the genetic code

Streptomycin was used to increase the frequency of errors in protein synthesis in vivo. In the system under study two misreading errors were observed. Both involved the erroneous insertion of lysine at asparagine codons, because of misreading of a pyrimidine as a purine at the 3' position of the codon. Streptomycin increased the errors at the two codons AAU and AAC to the same extent, thereby maintaining the error ratio found for basal level mistranslation.     

Mistranslation Reduces Mutation Load in Evolving Proteins through Negative Epistasis with DNA Mutations

Translational errors during protein synthesis cause phenotypic mutations that are several orders of magnitude more frequent than DNA mutations. Such phenotypic mutations may affect adaptive evolution through their interactions with DNA mutations. To study how mistranslation may affect the adaptive evolution of evolving proteins, we evolved populations of green fluorescent protein (GFP) in either high-mistranslation or low-mistranslation Escherichia coli hosts. In both hosts, we first evolved GFP under purifying selection for the ancestral phenotype green fluorescence, and then under directional selection toward the new phenotype yellow fluorescence. High-mistranslation populations evolved modestly higher yellow fluorescence during each generation of evolution than low-mistranslation populations. We demonstrate by high-throughput sequencing that elevated mistranslation reduced the accumulation of deleterious DNA mutations under both purifying and directional selection. It did so by amplifying the fitness effects of deleterious DNA mutations through negative epistasis with phenotypic mutations. In contrast, mistranslation did not affect the incidence of beneficial mutations. Our findings show that phenotypic mutations interact epistatically with DNA mutations. By reducing a population’s mutation load, mistranslation can affect an important determinant of evolvability.     

Selection of tRNA charging quality control mechanisms that increase mistranslation of the genetic code

Mistranslation can follow two events during protein synthesis: production of non-cognate amino acid:transfer RNA (tRNA) pairs by aminoacyl-tRNA synthetases (aaRSs) and inaccurate selection of aminoacyl-tRNAs by the ribosome. Many aaRSs actively edit non-cognate amino acids, but editing mechanisms are not evolutionarily conserved, and their physiological significance remains unclear. To address the connection between aaRSs and mistranslation, the evolutionary divergence of tyrosine editing by phenylalanyl-tRNA synthetase (PheRS) was used as a model. Certain PheRSs are naturally error prone, most notably a Mycoplasma example that displayed a low level of specificity consistent with elevated mistranslation of the proteome. Mycoplasma PheRS was found to lack canonical editing activity, relying instead on discrimination against the non-cognate amino acid by kinetic proofreading. This mechanism of discrimination is inadequate for organisms where translation is more accurate, as Mycoplasma PheRS failed to support Escherichia coli growth. However, minor changes in the defunct editing domain of the Mycoplasma enzyme were sufficient to restore E. coli growth, indicating that translational accuracy is an evolutionarily selectable trait.