Noncognate tRNAs Research Articles

Structural basis for the selective methylation of 5-carboxymethoxyuridine in tRNA modification.

Posttranscriptional modifications of tRNA are widely conserved in all domains of life. Especially, those occurring within the anticodon often modulate translational efficiency. Derivatives of 5-hydroxyuridine are specifically found in bacterial tRNA, where 5-methoxyuridine and 5-carboxymethoxyuridine are the major species in Gram-positive and Gram-negative bacteria, respectively. In certain tRNA species, 5-carboxymethoxyuridine can be further methylated by CmoM to form the methyl ester. In this report, we present the X-ray crystal structure of Escherichia coli CmoM complexed with tRNASer1, which contains 5-carboxymethoxyuridine at the 5'-end of anticodon (the 34th position of tRNA). The 2.22 Å resolution structure of the enzyme-tRNA complex reveals that both the protein and tRNA undergo local conformational changes around the binding interface. Especially, the hypomodified uracil base is flipped out from the canonical stacked conformation enabling the specific molecular interactions with the enzyme. Moreover, the structure illustrates that the enzyme senses exclusively the anticodon arm region of the substrate tRNA and examines the presence of key determinants, 5-carboxymethoxyuridine at position 34 and guanosine at position 35, offering molecular basis for the discriminatory mechanism against non-cognate tRNAs.

Investigating the effects of the Geobacillus kaustophilus TilS lysidine modification on accurate mRNA decoding and aminoacylation.

Accurate protein biosynthesis requires translational fidelity and decoding efficiency. Mistranslated proteins are susceptible to misfolding, aggregation, or degradation; often resulting in disease. Accurate protein biosynthesis is expedited by attaching the correct amino acid to the 3′ end of the corresponding tRNA. Aminoacyl-tRNA synthetase (aaRS) enzymes catalyze the covalent linkage of amino acids to the cognate tRNA during translation. Due to the importance of accuracy, these enzymes must be specific in which tRNA substrates they utilize. Wobble-base pairing can occur between tRNAIle2UAU and the AUG methionine codon. Thus, evolution has dictated tRNAIle2 minor isoacceptors that are transcribed with a CAU codon. Typically, tRNAIle2 is encoded with the CAU anticodon; however, the same codon is utilized by tRNAMet when decoding the AUG codon.In bacterial systems, a post-transcriptional modification of C34 of tRNAIle2CAU facilitates accurate protein biosynthesis as aaRS's use the lysidine modification as an identity element for recognition and proper charging. The tRNA isoleucine lysidine synthetase enzyme (TilS) modifies the wobble position of tRNAIle2 precursors generating a LAU codon. This prevents methionyl tRNA synthetase (MetRS) mischarging via steric hindrances and modified tRNAIle2LAU is ultimately charged by IleRS. This work attempts to further characterize Geobacillus kaustophilus TilS by exploring the effects of distal residue mutations on lysidinylation function and investigating the recognition capacity of GkMetRS and GkIleRS for both cognate and noncognate tRNA substrates. Exploring the impact of the lysidine modification on charging efficiency will provide an increased understanding of aaRS discrimination and codon recognition mechanisms.

Composition of human-specific slow codons and slow di-codons in SARS-CoV and 2019-nCoV are lower than other coronaviruses suggesting a faster protein synthesis rate of SARS-CoV and 2019-nCoV.

Translation of a genetic codon without a cognate tRNA gene is affected by both the cognate tRNA availability and the interaction with non-cognate isoacceptor tRNAs. Moreover, two consecutive slow codons (slow di-codons) lead to a much slower translation rate. Calculating the composition of host specific slow codons and slow di-codons in the viral protein coding sequences can predict the order of viral protein synthesis rates between different virus strains.Comparison of human-specific slow codon and slow di-codon compositions in the genomes of 590 coronaviruses infect humans revealed that the protein synthetic rates of 2019 novel coronavirus (2019-nCoV) and severe acute respiratory syndrome-related coronavirus (SARS-CoV) may be much faster than other coronaviruses infect humans. Analysis of host-specific slow codon and di-codon compositions provides links between viral genomic sequences and capability of virus replication in host cells that may be useful for surveillance of the transmission potential of novel viruses.

Molecular dynamics simulations of cognate and non-cognate AspRS-tRNAAsp complexes

Aspartyl tRNA synthetase (AspRS), one of the 20 aminoacyl-tRNA synthetases, plays an important role in protein synthesis by catalyzing the aminoacylation reaction and synthesises Aspartyl-tRNA (tRNAAsp). A typical three-dimensional structure of AspRS comprises three distinct domains for the recognition of cognate tRNA and catalysis, namely, anti-codon binding domain/N-terminal domain, hinge domain and catalytic domain through their interactions with anti-codon loop, D-stem and acceptor arm of cognate tRNA, respectively. In this work, we have studied the structural characteristics of each domain of AspRS to understand the recognition mechanism of tRNAAsp using molecular dynamics simulations. The dynamics of AspRS-tRNAAsp complexes from E.coli (cognate and non-cognate), S.cerevisiae (cognate) and T.thermophilus (non-cognate) were compared to understand the differences in recognition of cognate and non-cognate tRNAs. Our results explain that the conformational changes associated with the recognition of tRNA occur only in the cognate complexes. Among the cognate complexes, the conformational changes in yeast AspRS are highly controlled during tRNAAsp recognition than that of in the E. coli AspRS. Moreover, the functional motions required for the tRNA recognition are observed only in the cognate complexes, and the conformational changes in AspRS and their recognition of tRNAAsp are organism specific.Communicated by Ramaswamy H. Sarma

Overexpression of mitochondrial histidyl-tRNA synthetase restores mitochondrial dysfunction caused by a deafness-associated tRNAHis mutation.

The deafness-associated m.12201T>C mutation affects the A5-U68 base-pairing within the acceptor stem of mitochondrial tRNAHis The primary defect in this mutation is an alteration in tRNAHis aminoacylation. Here, we further investigate the molecular mechanism of the deafness-associated tRNAHis 12201T>C mutation and test whether the overexpression of the human mitochondrial histidyl-tRNA synthetase gene (HARS2) in cytoplasmic hybrid (cybrid) cells carrying the m.12201T>C mutation reverses mitochondrial dysfunctions. Using molecular dynamics simulations, we demonstrate that the m.12201T>C mutation perturbs the tRNAHis structure and function, supported by decreased melting temperature, conformational changes, and instability of mutated tRNA. We show that the m.12201T>C mutation-induced alteration of aminoacylation tRNAHis causes mitochondrial translational defects and respiratory deficiency. We found that the transfer of HARS2 into the cybrids carrying the m.12201T>C mutation raises the levels of aminoacylated tRNAHis from 56.3 to 75.0% but does not change the aminoacylation of other tRNAs. Strikingly, HARS2 overexpression increased the steady-state levels of tRNAHis and of noncognate tRNAs, including tRNAAla, tRNAGln, tRNAGlu, tRNALeu(UUR), tRNALys, and tRNAMet, in cells bearing the m.12201T>C mutation. This improved tRNA metabolism elevated the efficiency of mitochondrial translation, activities of oxidative phosphorylation complexes, and respiration capacity. Furthermore, HARS2 overexpression markedly increased mitochondrial ATP levels and membrane potential and reduced production of reactive oxygen species in cells carrying the m.12201T>C mutation. These results indicate that HARS2 overexpression corrects the mitochondrial dysfunction caused by the tRNAHis mutation. These findings provide critical insights into the pathophysiology of mitochondrial disease and represent a step toward improved therapeutic interventions for mitochondrial disorders.

Stereospecificity control in aminoacyl-tRNA-synthetases: new evidence of d-amino acids activation and editing.

The homochirality of amino acids is vital for the functioning of the translation apparatus. l-Amino acids predominate in proteins and d-amino acids usually represent diverse regulatory functional physiological roles in both pro- and eukaryotes. Aminoacyl-tRNA-synthetases (aaRSs) ensure activation of proteinogenic or nonproteinogenic amino acids and attach them to cognate or noncognate tRNAs. Although many editing mechanisms by aaRSs have been described, data about the protective role of aaRSs in d-amino acids incorporation remained unknown. Tyrosyl- and alanyl-tRNA-synthetases were represented as distinct members of this enzyme family. To study the potential to bind and edit noncognate substrates, Thermus thermophilus alanyl-tRNA-synthetase (AlaRS) and tyrosyl-tRNA-synthetase were investigated in the context of d-amino acids recognition. Here, we showed that d-alanine was effectively activated by AlaRS and d-Ala-tRNAAla, formed during the erroneous aminoacylation, was edited by AlaRS. On the other hand, it turned out that d-aminoacyl-tRNA-deacylase (DTD), which usually hydrolyzes d-aminoacyl-tRNAs, was inactive against d-Ala-tRNAAla. To support the finding about DTD, computational docking and molecular dynamics simulations were run. Overall, our work illustrates the novel function of the AlaRS editing domain in stereospecificity control during translation together with trans-editing factor DTD. Thus, we propose different evolutionary strategies for the maintenance of chiral selectivity during translation.

Naturally occurring dual recognition of tRNAHis substrates with and without a universal identity element

ABSTRACTThe extra 5ʹ guanine nucleotide (G-1) on tRNAHis is a nearly universal feature that specifies tRNAHis identity. The G-1 residue is either genome encoded or post-transcriptionally added by tRNAHis guanylyltransferase (Thg1). Despite Caenorhabditis elegans being a Thg1-independent organism, its cytoplasmic tRNAHis (CetRNAnHis) retains a genome-encoded G-1. Our study showed that this eukaryote possesses a histidyl-tRNA synthetase (CeHisRS) gene encoding two distinct HisRS isoforms that differ only at their N-termini. Most interestingly, its mitochondrial tRNAHis (CetRNAmHis) lacks G-1, a scenario never observed in any organelle. This tRNA, while lacking the canonical identity element, can still be efficiently aminoacylated in vivo. Even so, addition of G-1 to CetRNAmHis strongly enhanced its aminoacylation efficiency in vitro. Overexpression of CeHisRS successfully bypassed the requirement for yeast THG1 in the presence of CetRNAnHis without G-1. Mutagenesis assays showed that the anticodon takes a primary role in CetRNAHis identity recognition, being comparable to the universal identity element. Consequently, simultaneous introduction of both G-1 and the anticodon of tRNAHis effectively converted a non-cognate tRNA to a tRNAHis-like substrate. Our study suggests that a new balance between identity elements of tRNAHis relieves HisRS from the absolute requirement for G-1.

Determining the fidelity of tRNA aminoacylation via microarrays

The fidelity of tRNA aminoacylation is a critical determinant for the ultimate accuracy of protein synthesis. Although aminoacyl-tRNA synthetases are assumed to consistently maintain high tRNA charging fidelity, recent evidence has demonstrated that the fidelity of the aminoacylation reaction can be actively regulated and liable to change. Accordingly, the ability to conveniently assay the fidelity of tRNA charging is becoming increasingly relevant for studying mistranslation. Here we describe a combined radioactivity and microarray based method that can quantitatively elucidate which individual cognate or noncognate tRNA isoacceptors are charged with amino acid. In this technique, in vitro tRNA charging reactions or in vivo pulse-labeling is performed using a radiolabeled amino acid and tRNA microarrays are used to distinguish tRNA isoacceptors in total tRNA. During the tRNA array hybridization, each tRNA will hybridize to its unique probe and subsequent phosphorimaging of the array can determine which tRNAs were aminoacylated with the radiolabeled amino acid. The method can be used to assess the fidelity of tRNA charging in vivo or in vitro and can be applied to any organism with annotated tRNA genes.

A dual fluorescent reporter for the investigation of methionine mistranslation in live cells.

In mammalian cells under oxidative stress, the methionyl-tRNA synthetase (MetRS) misacylates noncognate tRNAs at frequencies as high as 10% distributed among up to 28 tRNA species. Instead of being detrimental for the cell, misincorporation of methionine residues in the proteome reduces the risk of oxidative damage to proteins, which aids the oxidative stress response. tRNA microarrays have been essential for the detection of the full pattern of misacylated tRNAs, but have limited capacity to investigate the misacylation and mistranslation mechanisms in live cells. Here we develop a dual-fluorescence reporter to specifically measure methionine misincorporation at glutamic acid codons GAA and GAG via tRNAGlu mismethionylation in human cells. Our method relies on mutating a specific Met codon in the active site of the fluorescent protein mCherry to a Glu codon that renders mCherry nonfluorescent when translation follows the genetic code. Mistranslation utilizing mismethionylated tRNAGlu restores fluorescence in proportion to the amount of misacylated tRNAGlu. This cellular approach works well for both transient transfection and established stable HEK293 lines. It is rapid, straightforward, and well suited for high-throughput activity analysis under a wide range of physiological conditions. As a proof of concept, we apply this method to characterize the effect of human tRNAGlu isodecoders on mistranslation and discuss the implications of our findings.

MIST, a Novel Approach to Reveal Hidden Substrate Specificity in Aminoacyl-tRNA Synthetases.

Aminoacyl-tRNA synthetases (AARSs) constitute a family of RNA-binding proteins, that participate in the translation of the genetic code, by covalently linking amino acids to appropriate tRNAs. Due to their fundamental importance for cell life, AARSs are likely to be one of the most ancient families of enzymes and have therefore been characterized extensively. Paradoxically, little is known about their capacity to discriminate tRNAs mainly because of the practical challenges that represent precise and systematic tRNA identification. This work describes a new technical and conceptual approach named MIST (Microarray Identification of Shifted tRNAs) designed to study the formation of tRNA/AARS complexes independently from the aminoacylation reaction. MIST combines electrophoretic mobility shift assays with microarray analyses. Although MIST is a non-cellular assay, it fully integrates the notion of tRNA competition. In this study we focus on yeast cytoplasmic Arginyl-tRNA synthetase (yArgRS) and investigate in depth its ability to discriminate cellular tRNAs. We report that yArgRS in submicromolar concentrations binds cognate and non-cognate tRNAs with a wide range of apparent affinities. In particular, we demonstrate that yArgRS binds preferentially to type II tRNAs but does not support their misaminoacylation. Our results reveal important new trends in tRNA/AARS complex formation and potential deep physiological implications.

Deducing the Kinetics of Protein Synthesis In Vivo from the Transition Rates Measured In Vitro

The molecular machinery of life relies on complex multistep processes that involve numerous individual transitions, such as molecular association and dissociation steps, chemical reactions, and mechanical movements. The corresponding transition rates can be typically measured in vitro but not in vivo. Here, we develop a general method to deduce the in-vivo rates from their in-vitro values. The method has two basic components. First, we introduce the kinetic distance, a new concept by which we can quantitatively compare the kinetics of a multistep process in different environments. The kinetic distance depends logarithmically on the transition rates and can be interpreted in terms of the underlying free energy barriers. Second, we minimize the kinetic distance between the in-vitro and the in-vivo process, imposing the constraint that the deduced rates reproduce a known global property such as the overall in-vivo speed. In order to demonstrate the predictive power of our method, we apply it to protein synthesis by ribosomes, a key process of gene expression. We describe the latter process by a codon-specific Markov model with three reaction pathways, corresponding to the initial binding of cognate, near-cognate, and non-cognate tRNA, for which we determine all individual transition rates in vitro. We then predict the in-vivo rates by the constrained minimization procedure and validate these rates by three independent sets of in-vivo data, obtained for codon-dependent translation speeds, codon-specific translation dynamics, and missense error frequencies. In all cases, we find good agreement between theory and experiment without adjusting any fit parameter. The deduced in-vivo rates lead to smaller error frequencies than the known in-vitro rates, primarily by an improved initial selection of tRNA. The method introduced here is relatively simple from a computational point of view and can be applied to any biomolecular process, for which we have detailed information about the in-vitro kinetics.

The SmpB C-terminal tail helps tmRNA to recognize and enter stalled ribosomes.

In bacteria, transfer-messenger RNA (tmRNA) and SmpB comprise the most common and effective system for rescuing stalled ribosomes. Ribosomes stall on mRNA transcripts lacking stop codons and are rescued as the defective mRNA is swapped for the tmRNA template in a process known as trans-translation. The tmRNA–SmpB complex is recruited to the ribosome independent of a codon–anticodon interaction. Given that the ribosome uses robust discriminatory mechanisms to select against non-cognate tRNAs during canonical decoding, it has been hard to explain how this can happen. Recent structural and biochemical studies show that SmpB licenses tmRNA entry through its interactions with the decoding center and mRNA channel. In particular, the C-terminal tail of SmpB promotes both EFTu activation and accommodation of tmRNA, the former through interactions with 16S rRNA nucleotide G530 and the latter through interactions with the mRNA channel downstream of the A site. Here we present a detailed model of the earliest steps in trans-translation, and in light of these mechanistic considerations, revisit the question of how tmRNA preferentially reacts with stalled, non-translating ribosomes.

Coevolution of Specificity Determinants in Eukaryotic Glutamyl- and Glutaminyl-tRNA Synthetases

The glutaminyl-tRNA synthetase (GlnRS) enzyme, which pairs glutamine with tRNAGln for protein synthesis, evolved by gene duplication in early eukaryotes from a nondiscriminating glutamyl-tRNA synthetase (GluRS) that aminoacylates both tRNAGln and tRNAGlu with glutamate. This ancient GluRS also separately differentiated to exclude tRNAGln as a substrate, and the resulting discriminating GluRS and GlnRS further acquired additional protein domains assisting function in cis (the GlnRS N-terminal Yqey domain) or in trans (the Arc1p protein associating with GluRS). These added domains are absent in contemporary bacterial GlnRS and GluRS. Here, using Saccharomyces cerevisiae enzymes as models, we find that the eukaryote-specific protein domains substantially influence amino acid binding, tRNA binding and aminoacylation efficiency, but they play no role in either specific nucleotide readout or discrimination against noncognate tRNA. Eukaryotic tRNAGln and tRNAGlu recognition determinants are found in equivalent positions and are mutually exclusive to a significant degree, with key nucleotides located adjacent to portions of the protein structure that differentiated during the evolution of archaeal nondiscriminating GluRS to GlnRS. These findings provide important corroboration for the evolutionary model and suggest that the added eukaryotic domains arose in response to distinctive selective pressures associated with the greater complexity of the eukaryotic translational apparatus. We also find that the affinity of GluRS for glutamate is significantly increased when Arc1p is not associated with the enzyme. This is consistent with the lower concentration of intracellular glutamate and the dissociation of the Arc1p:GluRS complex upon the diauxic shift to respiratory conditions.

Flipping of the Ribosomal A-Site Adenines Provides a Basis for tRNA Selection

Ribosomes control the missense error rate of ~10−4 during translation though quantitative contributions of individual mechanistic steps of the conformational changes yet to be fully determined. Biochemical and biophysical studies led to a qualitative tRNA selection model in which ribosomal A-site residues A1492 and A1493 (A1492/3) flip out in response to cognate tRNA binding, promoting the subsequent reactions, but not in the case of near-cognate or non-cognate tRNA. However, this model was recently questioned by X-ray structures revealing conformations of extrahelical A1492/3 and domain closure of the decoding center in both cognate and near-cognate tRNA bound ribosome complexes, suggesting that the non-specific flipping of A1492/3 has no active role in tRNA selection. We explore this question by carrying out molecular dynamics simulations, aided with fluorescence and NMR experiments, to probe the free energy cost of extrahelical flipping of 1492/3 and the strain energy associated with domain conformational change. Our rigorous calculations demonstrate that the A1492/3 flipping is indeed a specific response to the binding of cognate tRNA, contributing 3kcal/mol to the specificity of tRNA selection. Furthermore, the different A-minor interactions in cognate and near-cognate complexes propagate into the conformational strain and contribute another 4kcal/mol in domain closure. The recent structure of ribosome with features of extrahelical A1492/3 and closed domain in near-cognate complex is reconciled by possible tautomerization of the wobble base pair in mRNA–tRNA. These results quantitatively rationalize other independent experimental observations and explain the ribosomal discrimination mechanism of selecting cognate versus near-cognate tRNA.

Promiscuous methionyl-tRNA synthetase mediates adaptive mistranslation to protect cells against oxidative stress.

ABSTRACTAminoacyl-tRNA synthetases (ARSs) acylate transfer (t)RNAs with amino acids. Charging tRNAs with the right amino acids is the first step in translation; therefore, the accurate and error-free functioning of ARSs is an essential prerequisite for translational fidelity. A recent study found that methionine (Met) can be incorporated into non-Met residues of proteins through methionylation of non-cognate tRNAs under conditions of oxidative stress. However, it was not understood how this mis-methionylation is achieved. Here, we report that methionyl-tRNA synthetase (MRS) is phosphorylated at Ser209 and Ser825 by extracellular signal-related kinase (ERK1/2) under conditions of stress caused by reactive oxygen species (ROS), and that this phosphorylated MRS shows increased affinity for non-cognate tRNAs with lower affinity for tRNAMet, leading to an increase in Met residues in cellular proteins. The expression of a mutant MRS containing the substitutions S209D and S825D, mimicking dual phosphorylation, reduced ROS levels and cell death. This controlled inaccuracy of MRS seems to serve as a defense mechanism against ROS-mediated damage at the cost of translational fidelity.

Thermodynamic properties distinguish human mitochondrial aspartyl-tRNA synthetase from bacterial homolog with same 3D architecture

In the mammalian mitochondrial translation apparatus, the proteins and their partner RNAs are coded by two genomes. The proteins are nuclear-encoded and resemble their homologs, whereas the RNAs coming from the rapidly evolving mitochondrial genome have lost critical structural information. This raises the question of molecular adaptation of these proteins to their peculiar partner RNAs. The crystal structure of the homodimeric bacterial-type human mitochondrial aspartyl-tRNA synthetase (DRS) confirmed a 3D architecture close to that of Escherichia coli DRS. However, the mitochondrial enzyme distinguishes by an enlarged catalytic groove, a more electropositive surface potential and an alternate interaction network at the subunits interface. It also presented a thermal stability reduced by as much as 12°C. Isothermal titration calorimetry analyses revealed that the affinity of the mitochondrial enzyme for cognate and non-cognate tRNAs is one order of magnitude higher, but with different enthalpy and entropy contributions. They further indicated that both enzymes bind an adenylate analog by a cooperative allosteric mechanism with different thermodynamic contributions. The larger flexibility of the mitochondrial synthetase with respect to the bacterial enzyme, in combination with a preserved architecture, may represent an evolutionary process, allowing nuclear-encoded proteins to cooperate with degenerated organelle RNAs.

Structure Analysis of Entamoeba histolytica DNMT2 (EhMeth)

In eukaryotes, DNA methylation is an important epigenetic modification that is generally involved in gene regulation. Methyltransferases (MTases) of the DNMT2 family have been shown to have a dual substrate specificity acting on DNA as well as on three specific tRNAs (tRNAAsp, tRNAVal, tRNAGly). Entamoeba histolytica is a major human pathogen, and expresses a single DNA MTase (EhMeth) that belongs to the DNMT2 family and shows high homology to the human enzyme as well as to the bacterial DNA MTase M.HhaI. The molecular basis for the recognition of the substrate tRNAs and discrimination of non-cognate tRNAs is unknown. Here we present the crystal structure of the cytosine-5-methyltransferase EhMeth at a resolution of 2.15 Å, in complex with its reaction product S-adenosyl-L-homocysteine, revealing all parts of a DNMT2 MTase, including the active site loop. Mobility shift assays show that in vitro the full length tRNA is required for stable complex formation with EhMeth.

A tyrosyl‐tRNA synthetase variant that is responsible for Dominant Intermediate Charcot‐Marie‐Tooth disorder Type C displays decreased fidelity

Charcot‐Marie‐Tooth disorder (CMT) is the most common inherited peripheral neuropathy, affecting 1 in 2,500 people in the U.S. Mutations in over 30 different genes have been found to give rise to CMT, including the genes encoding the glycyl‐, alanyl‐, and tyrosyl‐tRNA synthetases. There is currently no cure for CMT.Three mutations in the gene encoding the human cytoplasmic tyrosyl‐tRNA synthetase, G41R, Δ(153–156), and E196K, have been found to give rise to Dominant Intermediate Charcot‐Marie‐Tooth disorder, Type C (DI‐CMTC). Based on modeling studies, we postulated that the E196K substitution affects the fidelity of tyrosyl‐tRNA synthestase. To test this hypothesis, the ability of the E196K variant to catalyze the misacylation of noncognate tRNAs was investigated. We find that the E196K variant is ~100‐fold more efficient at aminoacylating tRNATyr variants containing a noncognate G1:C72 base pair than is the wild type enzyme. Replacing Glu 196 with alanine also decreases the fidelity of the enzyme, indicating that Glu 196 acts as an anti‐determinant to prevent the misacylation of tRNAs containing a G1:C72 base pair. This observation suggests that DI‐CMTC is due to misincorporation of tyrosine into proteins, leading to defects in either their stability or ability to fold correctly. This research is supported by MDA 4268 to EAF.

Quality control by the ribosome following peptide bond formation

The overall fidelity of protein synthesis has been thought to rely on the combined accuracy of two basic processes: the aminoacylation of transfer RNAs with their cognate amino acid by the aminoacyl-tRNA synthetases, and the selection of cognate aminoacyl-tRNAs by the ribosome in cooperation with the GTPase elongation factor EF-Tu. These two processes, which together ensure the specific acceptance of a correctly charged cognate tRNA into the aminoacyl (A) site, operate before peptide bond formation. Here we report the identification of an additional mechanism that contributes to high fidelity protein synthesis after peptidyl transfer, using a well-defined in vitro bacterial translation system. In this retrospective quality control step, the incorporation of an amino acid from a non-cognate tRNA into the growing polypeptide chain leads to a general loss of specificity in the A site of the ribosome, and thus to a propagation of errors that results in abortive termination of protein synthesis.

Crystal Structure of Glutamyl-Queuosine tRNA Asp Synthetase Complexed with l-Glutamate: Structural Elements Mediating tRNA-Independent Activation of Glutamate and Glutamylation of tRNA Asp Anticodon

Glutamyl-queuosine tRNA Asp synthetase (Glu-Q-RS) from Escherichia coli is a paralog of the catalytic core of glutamyl-tRNA synthetase (GluRS) that catalyzes glutamylation of queuosine in the wobble position of tRNA Asp. Despite important structural similarities, Glu-Q-RS and GluRS diverge strongly by their functional properties. The only feature common to both enzymes consists in the activation of Glu to form Glu-AMP, the intermediate of transfer RNA (tRNA) aminoacylation. However, both enzymes differ by the mechanism of selection of the cognate amino acid and by the mechanism of its activation. Whereas GluRS selects l-Glu and activates it only in the presence of the cognate tRNA Glu, Glu-Q-RS forms Glu-AMP in the absence of tRNA. Moreover, while GluRS transfers the activated Glu to the 3′ accepting end of the cognate tRNA Glu, Glu-Q-RS transfers the activated Glu to Q34 located in the anticodon loop of the noncognate tRNA Asp. In order to gain insight into the structural elements leading to distinct mechanisms of amino acid activation, we solved the three-dimensional structure of Glu-Q-RS complexed to Glu and compared it to the structure of the GluRS·Glu complex. Comparison of the catalytic site of Glu-Q-RS with that of GluRS, combined with binding experiments of amino acids, shows that a restricted number of residues determine distinct catalytic properties of amino acid recognition and activation by the two enzymes. Furthermore, to explore the structural basis of the distinct aminoacylation properties of the two enzymes and to understand why Glu-Q-RS glutamylates only tRNA Asp among the tRNAs possessing queuosine in position 34, we performed a tRNA mutational analysis to search for the elements of tRNA Asp that determine recognition by Glu-Q-RS. The analyses made on tRNA Asp and tRNA Asn show that the presence of a C in position 38 is crucial for glutamylation of Q34. The results are discussed in the context of the evolution and adaptation of the tRNA glutamylation system.