mRNA decoding in human is kinetically and structurally distinct from bacteria.

In all species, ribosomes synthesize proteins by faithfully decoding messenger RNA (mRNA) nucleotide sequences using aminoacyl-tRNA substrates. Current knowledge of the decoding mechanism derives principally from studies on bacterial systems1. Although key features are conserved across evolution2, eukaryotes achieve higher-fidelity mRNA decoding than bacteria3. In human, changes in decoding fidelity are linked to ageing and disease and represent a potential point of therapeutic intervention in both viral and cancer treatment4-6. Here we combine single-molecule imaging and cryogenic electron microscopy methods to examine the molecular basis of human ribosome fidelity to reveal that the decoding mechanism is both kinetically and structurally distinct from that of bacteria. Although decoding is globally analogous in both species, the reaction coordinate of aminoacyl-tRNA movement is altered on the human ribosome and the process is an order of magnitude slower. These distinctions arise from eukaryote-specific structural elements in the human ribosome and in the elongation factor eukaryotic elongation factor 1A (eEF1A) that together coordinate faithful tRNA incorporation at each mRNA codon. The distinct nature and timing of conformational changes within the ribosome and eEF1A rationalize how increased decoding fidelity is achieved and potentially regulated in eukaryotic species.

The mechanism of peptidyl transfer catalysis by the ribosome.

The ribosome catalyzes two fundamental biological reactions: peptidyl transfer, the formation of a peptide bond during protein synthesis, and peptidyl hydrolysis, the release of the complete protein from the peptidyl tRNA upon completion of translation. The ribosome is able to utilize and distinguish the two different nucleophiles for each reaction, the α-amine of the incoming aminoacyl tRNA versus the water molecule. The correct binding of substrates induces structural rearrangements of ribosomal active-site residues and the substrates themselves, resulting in an orientation suitable for catalysis. In addition, active-site residues appear to provide further assistance by ordering active-site water molecules and providing an electrostatic environment via a hydrogen network that stabilizes the reaction intermediates and possibly shuttles protons. Major questions remain concerning the timing, components, and mechanism of the proton transfer steps. This review summarizes the recent progress in structural, biochemical, and computational advances and presents the current mechanistic models for these two reactions.

When Paul Berg meets Donald Crothers: an achiral connection through protein biosynthesis.

Outliers in scientific observations are often ignored and mostly remain unreported. However, presenting them is always beneficial since they could reflect the actual anomalies that might open new avenues. Here, we describe two examples of the above that came out of the laboratories of two of the pioneers of nucleic acid research in the area of protein biosynthesis, Paul Berg and Donald Crothers. Their work on the identification of D-aminoacyl-tRNA deacylase (DTD) and 'Discriminator hypothesis', respectively, were hugely ahead of their time and were partly against the general paradigm at that time. In both of the above works, the smallest and the only achiral amino acid turned out to be an outlier as DTD can act weakly on glycine charged tRNAs with a unique discriminator base of 'Uracil'. This peculiar nature of glycine remained an enigma for nearly half a century. With a load of available information on the subject by the turn of the century, our work on 'chiral proofreading' mechanisms during protein biosynthesis serendipitously led us to revisit these findings. Here, we describe how we uncovered an unexpected connection between them that has implications for evolution of different eukaryotic life forms.

An anticodon-sensing T-boxzyme generates the elongator nonproteinogenic aminoacyl-tRNA in situ of a custom-made translation system for incorporation.

In the hypothetical RNA world, ribozymes could have acted as modern aminoacyl-tRNA synthetases (ARSs) to charge tRNAs, thus giving rise to the peptide synthesis along with the evolution of a primitive translation apparatus. We previously reported a T-boxzyme, Tx2.1, which selectively charges initiator tRNA with N-biotinyl-phenylalanine (BioPhe) in situ in a Flexible In-vitro Translation (FIT) system to produce BioPhe-initiating peptides. Here, we performed in vitro selection of elongation-capable T-boxzymes (elT-boxzymes), using para-azido-l-phenylalanine (PheAZ) as an acyl-donor. We implemented a new strategy to enrich elT-boxzyme-tRNA conjugates that self-aminoacylated on the 3'-terminus selectively. One of them, elT32, can charge PheAZ onto tRNA in trans in response to its cognate anticodon. Further evolution of elT32 resulted in elT49, with enhanced aminoacylation activity. We have demonstrated the translation of a PheAZ-containing peptide in an elT-boxzyme-integrated FIT system, revealing that elT-boxzymes are able to generate the PheAZ-tRNA in response to the cognate anticodon in situ of a custom-made translation system. This study, together with Tx2.1, illustrates a scenario where a series of ribozymes could have overseen aminoacylation and co-evolved with a primitive RNA-based translation system.

Distinct localization of chiral proofreaders resolves organellar translation conflict in plants.

Plants have two endosymbiotic organelles originated from two bacterial ancestors. The transition from an independent bacterium to a successful organelle would have required extensive rewiring of biochemical networks for its integration with archaeal host. Here, using Arabidopsis as a model system, we show that plant D-aminoacyl-tRNA deacylase 1 (DTD1), of bacterial origin, is detrimental to organellar protein synthesis owing to its changed tRNA recognition code. Plants survive this conflict by spatially restricting the conflicted DTD1 to the cytosol. In addition, plants have targeted archaeal DTD2 to both the organelles as it is compatible with their translation machinery due to its strict D-chiral specificity and lack of tRNA determinants. Intriguingly, plants have confined bacterial-derived DTD1 to work in archaeal-derived cytosolic compartment whereas archaeal DTD2 is targeted to bacterial-derived organelles. Overall, the study provides a remarkable example of the criticality of optimization of biochemical networks for survival and evolution of plant mitochondria and chloroplast.

Conditional Switch between Frameshifting Regimes upon Translation of dnaX mRNA.

Ribosome frameshifting during translation of bacterial dnaX can proceed via different routes, generating a variety of distinct polypeptides. Using kinetic experiments, we show that -1 frameshifting predominantly occurs during translocation of two tRNAs bound to the slippery sequence codons. This pathway depends on a stem-loop mRNA structure downstream of the slippery sequence and operates when aminoacyl-tRNAs are abundant. However, when aminoacyl-tRNAs are in short supply, the ribosome switches to an alternative frameshifting pathway that is independent of a stem-loop. Ribosome stalling at a vacant 0-frame A-site codon results in slippage of the P-site peptidyl-tRNA, allowing for -1-frame decoding. When the -1-frame aminoacyl-tRNA is lacking, the ribosomes switch into -2 frame. Quantitative mass spectrometry shows that the -2-frame product is synthesized in vivo. We suggest that switching between frameshifting routes may enrich gene expression at conditions of aminoacyl-tRNA limitation.

Intrinsic Ribosome Destabilization Underlies Translation and Provides an Organism with a Strategy of Environmental Sensing.

Nascent polypeptides can modulate the polypeptide elongation speed on the ribosome. Here, we show that nascent chains can even destabilize the translating Escherichia coli ribosome from within. This phenomenon, termed intrinsic ribosome destabilization (IRD), occurs in response to a special amino acid sequence of the nascent chain, without involving the release or the recycling factors. Typically, a consecutive array of acidic residues and those intermitted by alternating prolines induce IRD. The ribosomal protein bL31, which bridges the two subunits, counteracts IRD, such that only strong destabilizing sequences abort translation in living cells. We found that MgtL, the leader peptide of a Mg2+ transporter (MgtA), contains a translation-aborting sequence, which sensitizes the ribosome to a decline in Mg2+ concentration and thereby triggers the MgtA-upregulating genetic scheme. Translation proceeds at an inherent risk of ribosomal destabilization, and nascent chain-ribosome complexes can function as a Mg2+ sensor by harnessing IRD.

eIF5A Functions Globally in Translation Elongation and Termination.

The eukaryotic translation factor eIF5A, originally identified as an initiation factor, was later shown to promote translation elongation of iterated proline sequences. Using a combination of ribosome profiling and in vitro biochemistry, we report a much broader role for eIF5A in elongation and uncover a critical function for eIF5A in termination. Ribosome profiling of an eIF5A-depleted strain reveals a global elongation defect, with abundant ribosomes stalling at many sequences, not limited to proline stretches. Our data also show ribosome accumulation at stop codons and in the 3' UTR, suggesting a global defect in termination in the absence of eIF5A. Using an in vitro reconstituted translation system, we find that eIF5A strongly promotes the translation of the stalling sequences identified by profiling and increases the rate of peptidyl-tRNA hydrolysis more than 17-fold. We conclude that eIF5A functions broadly in elongation and termination, rationalizing its high cellular abundance and essential nature.

Context-based sensing of orthosomycin antibiotics by the translating ribosome.

Orthosomycin antibiotics inhibit protein synthesis by binding to the large ribosomal subunit in the tRNA accommodation corridor, which is traversed by incoming aminoacyl-tRNAs. Structural and biochemical studies suggested that orthosomycins block accommodation of any aminoacyl-tRNAs in the ribosomal A-site. However, the mode of action of orthosomycins in vivo remained unknown. Here, by carrying out genome-wide analysis of antibiotic action in bacterial cells, we discovered that orthosomycins primarily inhibit the ribosomes engaged in translation of specific amino acid sequences. Our results reveal that the predominant sites of orthosomycin-induced translation arrest are defined by the nature of the incoming aminoacyl-tRNA and likely by the identity of the two C-terminal amino acid residues of the nascent protein. We show that nature exploits this antibiotic-sensing mechanism for directing programmed ribosome stalling within the regulatory open reading frame, which may control expression of an orthosomycin-resistance gene in a variety of bacterial species.

Chloroplast translation factor EF-Tu of Arabidopsis thaliana can be inactivated via oxidation of a specific cysteine residue.

Translational elongation factor EF-Tu, which delivers aminoacyl-tRNA to the ribosome, is susceptible to inactivation by reactive oxygen species (ROS) in the cyanobacterium Synechocystis sp. PCC 6803. However, the sensitivity to ROS of chloroplast-localized EF-Tu (cpEF-Tu) of plants remains to be elucidated. In the present study, we generated a recombinant cpEF-Tu protein of Arabidopsis thaliana and examined its sensitivity to ROS in vitro. In cpEF-Tu that lacked a bound nucleotide, one of the two cysteine residues, Cys149 and Cys451, in the mature protein was sensitive to oxidation by H2O2, with the resultant formation of sulfenic acid. The translational activity of cpEF-Tu, as determined with an in vitro translation system, derived from Escherichia coli, that had been reconstituted without EF-Tu, decreased with the oxidation of a cysteine residue. Replacement of Cys149 with an alanine residue rendered cpEF-Tu insensitive to inactivation by H2O2, indicating that Cys149 might be the target of oxidation. In contrast, cpEF-Tu that had bound either GDP or GTP was less sensitive to oxidation by H2O2 than nucleotide-free cpEF-Tu. The addition of thioredoxin f1, a major thioredoxin in the Arabidopsis chloroplast, to oxidized cpEF-Tu allowed the reduction of Cys149 and the reactivation of cpEF-Tu, suggesting that the oxidation of cpEF-Tu might be a reversible regulatory mechanism that suppresses the chloroplast translation system in a redox-dependent manner.

Drop-off-reinitiation triggered by EF-G-driven mistranslocation and its alleviation by EF-P.

In ribosomal translation, peptidyl transfer occurs between P-site peptidyl-tRNA and A-site aminoacyl-tRNA, followed by translocation of the resulting P-site deacylated-tRNA and A-site peptidyl-tRNA to E and P site, respectively, mediated by EF-G. Here, we report that mistranslocation of P-site peptidyl-tRNA and A-site aminoacyl-tRNA toward E and A site occurs when high concentration of EF-G triggers the migration of two tRNAs prior to completion of peptidyl transfer. Consecutive incorporation of less reactive amino acids, such as Pro and d-Ala, makes peptidyl transfer inefficient and thus induces the mistranslocation event. Consequently, the E-site peptidyl-tRNA drops off from ribosome to give a truncated peptide lacking the C-terminal region. The P-site aminoacyl-tRNA allows for reinitiation of translation upon accommodation of a new aminoacyl-tRNA at A site, leading to synthesis of a truncated peptide lacking the N-terminal region, which we call the 'reinitiated peptide'. We also revealed that such a drop-off-reinitiation event can be alleviated by EF-P that promotes peptidyl transfer of Pro. Moreover, this event takes place both in vitro and in cell, showing that reinitiated peptides during protein synthesis could be accumulated in this pathway in cells.

Negative catalysis by the editing domain of class I aminoacyl-tRNA synthetases.

Aminoacyl-tRNA synthetases (AARS) translate the genetic code by loading tRNAs with the cognate amino acids. The errors in amino acid recognition are cleared at the AARS editing domain through hydrolysis of misaminoacyl-tRNAs. This ensures faithful protein synthesis and cellular fitness. Using Escherichia coli isoleucyl-tRNA synthetase (IleRS) as a model enzyme, we demonstrated that the class I editing domain clears the non-cognate amino acids well-discriminated at the synthetic site with the same rates as the weakly-discriminated fidelity threats. This unveiled low selectivity suggests that evolutionary pressure to optimize the rates against the amino acids that jeopardize translational fidelity did not shape the editing site. Instead, we propose that editing was shaped to safeguard cognate aminoacyl-tRNAs against hydrolysis. Misediting is prevented by the residues that promote negative catalysis through destabilisation of the transition state comprising cognate amino acid. Such powerful design allows broad substrate acceptance of the editing domain along with its exquisite specificity in the cognate aminoacyl-tRNA rejection. Editing proceeds by direct substrate delivery to the editing domain (in cis pathway). However, we found that class I IleRS also releases misaminoacyl-tRNAIle and edits it in trans. This minor editing pathway was up to now recognized only for class II AARSs.

Structure of Escherichia coli heat shock protein Hsp15 in complex with the ribosomal 50S subunit bearing peptidyl-tRNA.

In Escherichia coli, the heat shock protein 15 (Hsp15) is part of the cellular response to elevated temperature. Hsp15 interacts with peptidyl-tRNA-50S complexes that arise upon dissociation of translating 70S ribosomes, and is proposed to facilitate their rescue and recycling. A previous structure of E. coli Hsp15 in complex with peptidyl-tRNA-50S complex reported a binding site located at the central protuberance of the 50S subunit. By contrast, recent structures of RqcP, the Hsp15 homolog in Bacillus subtilis, in complex with peptidyl-tRNA-50S complexes have revealed a distinct site positioned between the anticodon-stem-loop (ASL) of the P-site tRNA and H69 of the 23S rRNA. Here we demonstrate that exposure of E. coli cells to heat shock leads to a decrease in 70S ribosomes and accumulation of 50S subunits, thus identifying a natural substrate for Hsp15 binding. Additionally, we have determined a cryo-EM reconstruction of the Hsp15-50S-peptidyl-tRNA complex isolated from heat shocked E. coli cells, revealing that Hsp15 binds to the 50S-peptidyl-tRNA complex analogously to its B. subtilis homolog RqcP. Collectively, our findings support a model where Hsp15 stabilizes the peptidyl-tRNA in the P-site and thereby promotes access to the A-site for putative rescue factors to release the aberrant nascent polypeptide chain.

A [3Fe-4S] cluster and tRNA-dependent aminoacyltransferase BlsK in the biosynthesis of Blasticidin S.

Blasticidin S is a peptidyl nucleoside antibiotic. Its biosynthesis involves a cryptic leucylation and two leucylated intermediates, LDBS and LBS, have been found in previous studies. Leucylation has been proposed to be a new self-resistance mechanism during blasticidin S biosynthesis, and the leucyl group was found to be important for the methylation of ß-amino group of the arginine side chain. However, the responsible enzyme and its associated mechanism of the leucyl transfer process remain to be elucidated. Here, we report results investigating the leucyl transfer step forming the intermediate LDBS in blasticidin biosynthesis. A hypothetical protein, BlsK, has been characterized by genetic and in vitro biochemical experiments. This enzyme catalyzes the leucyl transfer from leucyl-transfer RNA (leucyl-tRNA) to the ß-amino group on the arginine side chain of DBS. Furthermore, BlsK was found to contain an iron-sulfur cluster that is necessary for activity. These findings provide an example of an iron-sulfur protein that catalyzes an aminoacyl-tRNA (aa-tRNA)-dependent amide bond formation in a natural product biosynthetic pathway.

Structure of Gcn1 bound to stalled and colliding 80S ribosomes.

The Gcn pathway is conserved in all eukaryotes, including mammals such as humans, where it is a crucial part of the integrated stress response (ISR). Gcn1 serves as an essential effector protein for the kinase Gcn2, which in turn is activated by stalled ribosomes, leading to phosphorylation of eIF2 and a subsequent global repression of translation. The fine-tuning of this adaptive response is performed by the Rbg2/Gir2 complex, a negative regulator of Gcn2. Despite the wealth of available biochemical data, information on structures of Gcn proteins on the ribosome has remained elusive. Here we present a cryo-electron microscopy structure of the yeast Gcn1 protein in complex with stalled and colliding 80S ribosomes. Gcn1 interacts with both 80S ribosomes within the disome, such that the Gcn1 HEAT repeats span from the P-stalk region on the colliding ribosome to the P-stalk and the A-site region of the lead ribosome. The lead ribosome is stalled in a nonrotated state with peptidyl-tRNA in the A-site, uncharged tRNA in the P-site, eIF5A in the E-site, and Rbg2/Gir2 in the A-site factor binding region. By contrast, the colliding ribosome adopts a rotated state with peptidyl-tRNA in a hybrid A/P-site, uncharged-tRNA in the P/E-site, and Mbf1 bound adjacent to the mRNA entry channel on the 40S subunit. Collectively, our findings reveal the interaction mode of the Gcn2-activating protein Gcn1 with colliding ribosomes and provide insight into the regulation of Gcn2 activation. The binding of Gcn1 to a disome has important implications not only for the Gcn2-activated ISR, but also for the general ribosome-associated quality control pathways.

Bacterial translation machinery for deliberate mistranslation of the genetic code.

Inaccurate expression of the genetic code, also known as mistranslation, is an emerging paradigm in microbial studies. Growing evidence suggests that many microbial pathogens can deliberately mistranslate their genetic code to help invade a host or evade host immune responses. However, discovering different capacities for deliberate mistranslation remains a challenge because each group of pathogens typically employs a unique mistranslation mechanism. In this study, we address this problem by studying duplicated genes of aminoacyl-transfer RNA (tRNA) synthetases. Using bacterial prolyl-tRNA synthetase (ProRS) genes as an example, we identify an anomalous ProRS isoform, ProRSx, and a corresponding tRNA, tRNAProA, that are predominately found in plant pathogens from Streptomyces species. We then show that tRNAProA has an unusual hybrid structure that allows this tRNA to mistranslate alanine codons as proline. Finally, we provide biochemical, genetic, and mass spectrometric evidence that cells which express ProRSx and tRNAProA can translate GCU alanine codons as both alanine and proline. This dual use of alanine codons creates a hidden proteome diversity due to stochastic Ala→Pro mutations in protein sequences. Thus, we show that important plant pathogens are equipped with a tool to alter the identity of their sense codons. This finding reveals the initial example of a natural tRNA synthetase/tRNA pair for dedicated mistranslation of sense codons.

Ribosomal protein S18 acetyltransferase RimI is responsible for the acetylation of elongation factor Tu.

N-terminal acetylation is widespread in the eukaryotic proteome but in bacteria is restricted to a small number of proteins mainly involved in translation. It was long known that elongation factor Tu (EF-Tu) is N-terminally acetylated, whereas the enzyme responsible for this process was unclear. Here, we report that RimI acetyltransferase, known to modify ribosomal protein S18, is likewise responsible for N-acetylation of the EF-Tu. With the help of inducible tufA expression plasmid, we demonstrated that the acetylation does not alter the stability of EF-Tu. Binding of aminoacyl tRNA to the recombinant EF-Tu in vitro was found to be unaffected by the acetylation. At the same time, with the help of fast kinetics methods, we demonstrate that an acetylated variant of EF-Tu more efficiently accelerates A-site occupation by aminoacyl-tRNA, thus increasing the efficiency of in vitro translation. Finally, we show that a strain devoid of RimI has a reduced growth rate, expanded to an evolutionary timescale, and might potentially promote conservation of the acetylation mechanism of S18 and EF-Tu. This study increased our understanding of the modification of bacterial translation apparatus.

RNA-dependent synthesis of ergosteryl-3ß-O-glycine in Ascomycota expands the diversity of steryl-amino acids.

A wide range of bacteria possess virulence factors such as aminoacyl-tRNA transferases (ATTs) that are capable of rerouting aminoacyl-transfer RNAs away from protein synthesis to conjugate amino acids onto glycerolipids. We recently showed that, although these pathways were thought to be restricted to bacteria, higher fungi also possess ergosteryl-3ß-O-L-aspartate synthases (ErdSs), which transfer the L-Asp moiety of aspartyl-tRNAAsp onto the 3ß-OH group of ergosterol (Erg), yielding ergosteryl-3ß-O-L-aspartate (Erg-Asp). Here, we report the discovery, in fungi, of a second type of fungal sterol-specific ATTs, namely, ergosteryl-3ß-O-glycine (Erg-Gly) synthase (ErgS). ErgS consists of a freestanding DUF2156 domain encoded by a gene distinct from and paralogous to that of ErdS. We show that the enzyme only uses Gly-tRNAGly produced by an independent glycyl-tRNA synthetase (GlyRS) to transfer glycine onto the 3ß-OH of Erg, producing Erg-Gly. Phylogenomics analysis also show that the Erg-Gly synthesis pathway exists only in Ascomycota, including species of biotechnological interest, and more importantly, in human pathogens, such as Aspergillus fumigatus. The discovery of a second type of Erg-aa not only expands the repertoire of this particular class of fungal lipids but suggests that Erg-aa synthases might constitute a genuine subfamily of lipid-modifying ATTs.

Rate-limiting hydrolysis in ribosomal release reactions revealed by ester activation.

Translation terminates by releasing the polypeptide chain in one of two chemical reactions catalyzed by the ribosome. Release is also a target for engineering, as readthrough of a stop codon enables incorporation of unnatural amino acids and treatment of genetic diseases. Hydrolysis of the ester bond of peptidyl-tRNA requires conformational changes of both a class I release factor (RF) protein and the peptidyl transferase center of a large subunit rRNA. The rate-limiting step was proposed to be hydrolysis at physiological pH and an RF conformational change at higher pH, but evidence was indirect. Here, we tested this by activating the ester electrophile at the Escherichia coli ribosomal P site using a trifluorine-substituted amino acid. Quench-flow kinetics revealed that RF1-catalyzed release could be accelerated, but only at pH 6.2-7.7 and not higher pH. This provided direct evidence for rate-limiting hydrolysis at physiological or lower pH and a different rate limitation at higher pH. Additionally, we optimized RF-free release catalyzed by unacylated tRNA or the CCA trinucleotide (in 30% acetone). We determined that these two model release reactions, although very slow, were surprisingly accelerated by the trifluorine analog but to a different extent from each other and from RF-catalyzed release. Hence, hydrolysis was rate limiting in all three reactions. Furthermore, in 20% ethanol, we found that there was significant competition between fMet-ethyl ester formation and release in all three release reactions. We thus favor proposed mechanisms for translation termination that do not require a fully-negatively-charged OH- nucleophile.