Proteome-wide measurement of non-canonical bacterial mistranslation by quantitative mass spectrometry of protein modifications.

The genetic code is virtually universal in biology and was likely established before the advent of cellular life. The extent to which mistranslation occurs is poorly understood and presents a fundamental question in basic research and production of recombinant proteins. Here we used shotgun proteomics combined with unbiased protein modification analysis to quantitatively analyze in vivo mistranslation in an E. coli strain with a defect in the editing mechanism of leucyl-tRNA synthetase. We detected the misincorporation of a non-proteinogenic amino acid norvaline on 10% of all measured leucine residues under microaerobic conditions and revealed preferential deployment of a tRNA(Leu)(CAG) isoacceptor during norvaline misincorporation. The strain with the norvalylated proteome demonstrated a substantial reduction in cell fitness under both prolonged aerobic and microaerobic cultivation. Unlike norvaline, isoleucine did not substitute for leucine even under harsh error-prone conditions. Our study introduces shotgun proteomics as a powerful tool in quantitative analysis of mistranslation

Naturally Occurring Isoleucyl-tRNA Synthetase without tRNA-dependent Pre-transfer Editing

Isoleucyl-tRNA synthetase (IleRS) is unusual among aminoacyl-tRNA synthetases in having a tRNA-dependent pre-transfer editing activity. Alongside the typical bacterial IleRS (such as Escherichia coli IleRS), some bacteria also have the enzymes (eukaryote-like) that cluster with eukaryotic IleRSs and exhibit low sensitivity to the antibiotic mupirocin. Our phylogenetic analysis suggests that the ileS1 and ileS2 genes of contemporary bacteria are the descendants of genes that might have arisen by an ancient duplication event before the separation of bacteria and archaea. We present the analysis of evolutionary constraints of the synthetic and editing reactions in eukaryotic/eukaryote-like IleRSs, which share a common origin but diverged through adaptation to different cell environments. The enzyme from the yeast cytosol exhibits tRNA-dependent pre-transfer editing analogous to E. coli IleRS. This argues for the presence of this proofreading in the common ancestor of both IleRS types and an ancient origin of the synthetic site-based quality control step. Yet surprisingly, the eukaryote-like enzyme from Streptomyces griseus IleRS lacks this capacity; at the same time, its synthetic site displays the 10(3)-fold drop in sensitivity to antibiotic mupirocin relative to the yeast enzyme. The discovery that pre-transfer editing is optional in IleRSs lends support to the notion that the conserved post-transfer editing domain is the main checkpoint in these enzymes. We substantiated this by showing that under error-prone conditions S. griseus IleRS is able to rescue the growth of an E. coli lacking functional IleRS, providing the first evidence that tRNA-dependent pre-transfer editing in IleRS is not essential for cell viability

Točnost sinteze seril-tRNA

The high level of translational fidelity is ensured by various types of quality control mechanisms, which are adapted to prevent or correct naturally occurring mistakes. Accurate aminoacyl-tRNA synthesis is mostly dependent on the specificity of the aminoacyl-tRNA synthetases (aaRS), i.e. their ability to choose among competing structurally similar substrates. Our studies have revealed that accurate seryl-tRNA synthesis in yeast and plants is accomplished via tRNA-assisted optimization of amino acid binding to the active site of seryl-tRNA synthetase (SerRS). Based on our recent kinetic data, a mechanism is proposed by which transient protein : RNA complex activates the cognate amino acid more efficiently and more specifically than the apoenzyme alone. This may proceed via a tRNA induced conformational change in the enzyme’s active site. The influence of tRNASer, on the activation of serine by SerRS variants mutated in the active site, is much less pronounced. Although SerRS misactivates structurally similar threonine in vitro, the formation of such erroneous threonyl-adenylate is reduced in the presence of nonchargeable tRNASer analog. Thus, the sequence-specific tRNA : SerRS interactions enhance the accuracy of amino acid recognition. Another type of quality control mechanism in tRNA serylation is assumed to be based on the complex formation between SerRS and a nonsynthetase protein. Using in vivo interaction screen, yeast peroxin Pex21p was identified as SerRS interacting protein. This was confirmed by an in vitro binding assay. Kinetic experiments performed in the presence of Pex21p revealed that this peroxin acts as an activator of seryl-tRNA synthetase in the aminoacylation reaction.Točnost biosinteze proteina nadziru različiti kontrolni mehanizmi koji sprečavaju ili ispravljaju gre{ke u translaciji. Specifičnost aminoacil tRNA-sintetaza (aaRS) pri izboru i kovalentnom povezivanju pripadnih aminokiselina i tRNA ključna je u ovom procesu. Istraživanja u našem laboratoriju pokazala su da se specifičnost i učinkovitost sinteze seril-tRNA u kvascu i biljkama povećavaju tRNA-ovisnim prilagođavanjem veznog mjesta za serin u aktivnom mjestu seril tRNA-sintetaze (SerRS). Dakle, makromolekularni kompleksi tRNA i enzima imaju bolja katalitička svojstva od apoenzima. Naši rezultati kinetike pokazuju da se vezanjem tRNA bitno mijenja konformacija veznog mjesta za serin u enzimu divljeg tipa, dok je konformacijska promjena slabija kod enzima s mutacijama u aktivnom mjestu. Iako SerRS može aktivirati i serinu sličan treonin, stvaranje treonil adenilata smanjeno je u prisutnosti aminoacilacijski inaktivnog analoga tRNA. Time je pokazano da su interakcije između pripadne tRNA i SerRS bitne za točan izbor aminokiseline. Djelotvornost serilacije povećava se i interakcijom SerRS s nesintetaznim proteinom, peroksinom Pex21p. Ta neočekivana interakcija uočena je prvo in vivo, pretragom kvaščeve biblioteke u sustavu dvaju hibrida sa SerRS kao interakcijskim proteinom. Interakcija je potvrđena in vitro. Kinetički eksperimenti pokazali su da Pex21p djeluje kao aktivator SerRS, što ovu neobičnu interakciju čini biološki značajnom jer povećava učinkovitost aminoaciliranja

The physiological target for LeuRS translational quality control is norvaline

The fidelity of protein synthesis depends on the capacity of aminoacyl-tRNA synthetases (AARSs) to couple only cognate amino acid-tRNA pairs. If amino acid selectivity is compromised, fidelity can be ensured by an inherent AARS editing activity that hydrolyses mischarged tRNAs. Here we show that the editing activity of Escherichia coli leucyl-tRNA synthetase (EcLeuRS) is not required to prevent incorrect isoleucine incorporation. Rather, as shown by kinetic, structural and in vivo approaches, the prime biological function of LeuRS editing is to prevent mis-incorporation of the non-standard amino acid norvaline. This conclusion follows from a reassessment of the discriminatory power of LeuRS against isoleucine and the demonstration that a LeuRS editing- deficient E. coli strain grows normally in high concentrations of isoleucine but not under oxygen deprivation conditions when norvaline accumulates to substantial levels. Thus, AARS- based translational quality control is a key feature for bacterial adaptive response to oxygen deprivation. The non-essential role for editing under normal bacterial growth has important implications for the development of resistance to antimicrobial agents targeting the LeuRS editing site

Modular pathways for editing non-cognate amino acids by human cytoplasmic leucyl-tRNA synthetase

To prevent potential errors in protein synthesis, some aminoacyl-transfer RNA (tRNA) synthetases have evolved editing mechanisms to hydrolyze misactivated amino acids (pre-transfer editing) or misacylated tRNAs (post-transfer editing). Class Ia leucyl-tRNA synthetase (LeuRS) may misactivate various natural and non-protein amino acids and then mischarge tRNALeu. It is known that the fidelity of prokaryotic LeuRS depends on multiple editing pathways to clear the incorrect intermediates and products in the every step of aminoacylation reaction. Here, we obtained human cytoplasmic LeuRS (hcLeuRS) and tRNALeu (hctRNALeu) with high activity from Escherichia coli overproducing strains to study the synthetic and editing properties of the enzyme. We revealed that hcLeuRS could adjust its editing strategy against different non-cognate amino acids. HcLeuRS edits norvaline predominantly by post-transfer editing; however, it uses mainly pre-transfer editing to edit α-amino butyrate, although both amino acids can be charged to tRNALeu. Post-transfer editing as a final checkpoint of the reaction was very important to prevent mis-incorporation in vitro. These results provide insight into the modular editing pathways created to prevent genetic code ambiguity by evolution

Mapping Hidden Potential Identity Elements by Computing the Average Discriminating Power of Individual tRNA Positions

The recently published discrete mathematical method, extended consensus partition (ECP), identifies nucleotide types at each position that are strictly absent from a given sequence set, while occur in other sets. These are defined as discriminating elements (DEs). In this study using the ECP approach, we mapped potential hidden identity elements that discriminate the 20 different tRNA identities. We filtered the tDNA data set for the obligatory presence of well-established tRNA features, and then separately for each identity set, the presence of already experimentally identified strictly present identity elements. The analysis was performed on the three kingdoms of life. We determined the number of DE, e.g. the number of sets discriminated by the given position, for each tRNA position of each tRNA identity set. Then, from the positional DE numbers obtained from the 380 pairwise comparisons of the 20 identity sets, we calculated the average excluding value (AEV) for each tRNA position. The AEV provides a measure on the overall discriminating power of each position. Using a statistical analysis, we show that positional AEVs correlate with the number of already identified identity elements. Positions having high AEV but lacking published identity elements predict hitherto undiscovered tRNA identity elements

Točnost sinteze seril-tRNA

The high level of translational fidelity is ensured by various types of quality control mechanisms, which are adapted to prevent or correct naturally occurring mistakes. Accurate aminoacyl-tRNA synthesis is mostly dependent on the specificity of the aminoacyl-tRNA synthetases (aaRS), i.e. their ability to choose among competing structurally similar substrates. Our studies have revealed that accurate seryl-tRNA synthesis in yeast and plants is accomplished via tRNA-assisted optimization of amino acid binding to the active site of seryl-tRNA synthetase (SerRS). Based on our recent kinetic data, a mechanism is proposed by which transient protein : RNA complex activates the cognate amino acid more efficiently and more specifically than the apoenzyme alone. This may proceed via a tRNA induced conformational change in the enzyme’s active site. The influence of tRNASer, on the activation of serine by SerRS variants mutated in the active site, is much less pronounced. Although SerRS misactivates structurally similar threonine in vitro, the formation of such erroneous threonyl-adenylate is reduced in the presence of nonchargeable tRNASer analog. Thus, the sequence-specific tRNA : SerRS interactions enhance the accuracy of amino acid recognition. Another type of quality control mechanism in tRNA serylation is assumed to be based on the complex formation between SerRS and a nonsynthetase protein. Using in vivo interaction screen, yeast peroxin Pex21p was identified as SerRS interacting protein. This was confirmed by an in vitro binding assay. Kinetic experiments performed in the presence of Pex21p revealed that this peroxin acts as an activator of seryl-tRNA synthetase in the aminoacylation reaction.Točnost biosinteze proteina nadziru različiti kontrolni mehanizmi koji sprečavaju ili ispravljaju gre{ke u translaciji. Specifičnost aminoacil tRNA-sintetaza (aaRS) pri izboru i kovalentnom povezivanju pripadnih aminokiselina i tRNA ključna je u ovom procesu. Istraživanja u našem laboratoriju pokazala su da se specifičnost i učinkovitost sinteze seril-tRNA u kvascu i biljkama povećavaju tRNA-ovisnim prilagođavanjem veznog mjesta za serin u aktivnom mjestu seril tRNA-sintetaze (SerRS). Dakle, makromolekularni kompleksi tRNA i enzima imaju bolja katalitička svojstva od apoenzima. Naši rezultati kinetike pokazuju da se vezanjem tRNA bitno mijenja konformacija veznog mjesta za serin u enzimu divljeg tipa, dok je konformacijska promjena slabija kod enzima s mutacijama u aktivnom mjestu. Iako SerRS može aktivirati i serinu sličan treonin, stvaranje treonil adenilata smanjeno je u prisutnosti aminoacilacijski inaktivnog analoga tRNA. Time je pokazano da su interakcije između pripadne tRNA i SerRS bitne za točan izbor aminokiseline. Djelotvornost serilacije povećava se i interakcijom SerRS s nesintetaznim proteinom, peroksinom Pex21p. Ta neočekivana interakcija uočena je prvo in vivo, pretragom kvaščeve biblioteke u sustavu dvaju hibrida sa SerRS kao interakcijskim proteinom. Interakcija je potvrđena in vitro. Kinetički eksperimenti pokazali su da Pex21p djeluje kao aktivator SerRS, što ovu neobičnu interakciju čini biološki značajnom jer povećava učinkovitost aminoaciliranja

The Accuracy of Seryl-tRNA Synthesis

The high level of translational fidelity is ensured by various types of quality control mechanisms, which are adapted to prevent or correct naturally occurring mistakes. Accurate aminoacyl-tRNA synthesis is mostly dependent on the specificity of the aminoacyl-tRNA synthetases (aaRS), i.e. their ability to choose among competing structurally similar substrates. Our studies have revealed that accurate seryl-tRNA synthesis in yeast and plants is accomplished via tRNA-assisted optimization of amino acid binding to the active site of seryl-tRNA synthetase (SerRS). Based on our recent kinetic data, a mechanism is proposed by which transient protein : RNA complex activates the cognate amino acid more efficiently and more specifically than the apoenzyme alone. This may proceed via a tRNA induced conformational change in the enzyme’s active site. The influence of tRNASer, on the activation of serine by SerRS variants mutated in the active site, is much less pronounced. Although SerRS misactivates structurally similar threonine in vitro, the formation of such erroneous threonyl-adenylate is reduced in the presence of nonchargeable tRNASer analog. Thus, the sequence-specific tRNA : SerRS interactions enhance the accuracy of amino acid recognition. Another type of quality control mechanism in tRNA serylation is assumed to be based on the complex formation between SerRS and a nonsynthetase protein. Using in vivo interaction screen, yeast peroxin Pex21p was identified as SerRS interacting protein. This was confirmed by an in vitro binding assay. Kinetic experiments performed in the presence of Pex21p revealed that this peroxin acts as an activator of seryl-tRNA synthetase in the aminoacylation reaction