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In higher eukaryotes, tRNA methyltransferase 10A (TRMT10A) is responsible for N1-methylguanosine modification at position nine of various cytoplasmic tRNAs. Pathogenic mutations in TRMT10A cause intellectual disability, microcephaly, diabetes, and short stature in humans, and generate cytotoxic tRNA fragments in cultured cells; however, it is not clear how TRMT10A supports codon translation or brain functions. Here, we generated Trmt10a null mice and showed that tRNAGln(CUG) and initiator methionine tRNA levels were universally decreased in various tissues; the same was true in a human cell line lacking TRMT10A. Ribosome profiling of mouse brain revealed that dysfunction of TRMT10A causes ribosome slowdown at the Gln(CAG) codon and increases translation of Atf4 due to higher frequency of leaky scanning of its upstream open reading frames. Broadly speaking, translation of a subset of mRNAs, especially those for neuronal structures, is perturbed in the mutant brain. Despite not showing discernable defects in the pancreas, liver, or kidney, Trmt10a null mice showed lower body weight and smaller hippocampal postsynaptic densities, which is associated with defective synaptic plasticity and memory. Taken together, our study provides mechanistic insight into the roles of TRMT10A in the brain, and exemplifies the importance of universal tRNA modification during translation of specific codons., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The heterotrimeric eIF2 complex consists of a core eIF2γ subunit to which binds eIF2α and eIF2β subunits and plays an important role in delivering the Met-tRNAiMet to the 40S ribosome and start codon selection. The intricacies of eIF2β-γ interaction in promoting Met-tRNAiMet binding are not clearly understood. Previously, the zinc-binding domain (ZBD) eIF2βS264Y mutation was reported to cause Met-tRNAiMet binding defect due to the intrinsic GTPase activity. We showed that the eIF2βS264Y mutation has eIF2β-γ interaction defect. Consistently, the eIF2βT238A intragenic suppressor mutation restored the eIF2β-γ and Met-tRNAiMet binding. The eIF2β-ZBD residues Asn252Asp and Arg253Ala mutation caused Met-tRNAiMet binding defect that was partially rescued by the eIF2βT238A mutation, suggesting the eIF2β-ZBD modulates Met-tRNAiMet binding. The suppressor mutation rescued the translation initiation fidelity defect of the eIF2γN135D SW-I mutation and eIF2βF217A/Q221A double mutation in the HTH domain. The eIF2βT238A suppressor mutation could not rescue the eIF2β binding defect of the eIF2γV281K mutation; however, combining the eIF2βS264Y mutation with the eIF2γV281K mutation was lethal. In addition to the previously known interaction of eIF2β with the eIF2γ subunit via its α1-helix, the eIF2β-ZBD also interacts with the eIF2γ subunit via guanine nucleotide-binding interface; thus, the eIF2β-γ interacts via two distinct binding sites., (© 2024 The Author(s).)

RsgA (small ribosomal subunit, 30S, GTPase), a late-stage biogenesis factor, releases RbfA from 30S-RbfA complex. Escherichia coli Δ rsgA (deleted for rsgA ) shows a slow growth phenotype and an increased accumulation of 17S rRNA (precursor of 16S rRNA) and the ribosomal subunits. Here, we show that the rescue of the Δ rsgA strain by multicopy infB (IF2) is enhanced by simultaneous overexpression of initiator tRNA (i-tRNA), suggesting a role of initiation complex formation in growth rescue. The synergistic effect of IF2/i-tRNA is accompanied by increased processing of 17S rRNA (to 16S), and protection of the 16S rRNA 3'-minor domain. Importantly, we show that an IF2-binding anticonvulsant drug, lamotrigine (Ltg), also rescues the Δ rsgA strain growth. The rescue is accompanied by increased processing of 17S rRNA, protection of the 3'-minor domain of 16S rRNA, and increased 70S ribosomes in polysome profiles. However, Ltg becomes inhibitory to the ΔrsgA strain whose growth was already rescued by an L83R mutation in rbfA . Interestingly, like wild-type infB , overproduction of Ltg R infB alleles (having indel mutations in their domain II) also rescues the Δ rsgA strain (independent of Ltg). Our observations suggest the dual role of IF2 in rescuing the Δ rsgA strain. First, together with i-tRNA, IF2 facilitates the final steps of processing of 17S rRNA. Second, a conformer of IF2 functionally compensates for RsgA, albeit poorly, during 30S biogenesis., Importance: RsgA is a late-stage ribosome biogenesis factor. Earlier, infB (IF2) was isolated as a multicopy suppressor of the Escherichia coli Δ rsgA strain. How IF2 rescued the strain growth remained unclear. This study reveals that (i) the multicopy infB -mediated growth rescue of E. coli Δ rsgA and the processing of 17S precursor to 16S rRNA in the strain are enhanced upon simultaneous overexpression of initiator tRNA and (ii) a conformer of IF2, whose occurrence increases when IF2 is overproduced or when E. coli Δ rsgA is treated with Ltg (an anticonvulsant drug that binds to domain II of IF2), compensates for the function of RsgA. Thus, this study reveals yet another role of IF2 in ribosome biogenesis., Competing Interests: The authors declare no conflict of interest.

Eukaryotic and archaeal translation initiation factor 2 in complex with GTP delivers the initiator methionyl-tRNA to the small ribosomal subunit. Over the past 20 years, thanks to the efforts of various research groups, including ours, this factor from the archaeon Sulfolobus solfataricus and its individual subunits have been crystallized in ten different space groups. Analysis of the molecular packing in these crystals makes it possible to better understand the roles of functionally significant switches and other elements of the nucleotide-binding pocket during the function of the factor as well as the influence of external effects on its transition between active and inactive states.

Methionyl-tRNA synthetase (MetRS) catalyzes the attachment of l-methionine (l-Met) to tRNA Met to generate methionyl-tRNA Met , an essential substrate for protein translation within ribosome. Owing to its indispensable biological function and the structural discrepancies with human counterpart, bacterial MetRS is considered an ideal target for developing antibacterials. Herein, chlorhexidine (CHX) was identified as a potent binder of Staphylococcus aureus MetRS (SaMetRS) through an ATP-aided affinity screening. The co-crystal structure showed that CHX simultaneously occupies the enlarged l-Met pocket (EMP) and the auxiliary pocket (AP) of SaMetRS with its two chlorophenyl groups, while its central hexyl linker swings upwards to interact with some conserved hydrophobic residues. ATP adopts alternative conformations in the active site cavity, and forms ionic bonds and water-mediated hydrogen bonds with CHX. Consistent with this synergistic binding mode, ATP concentration-dependently enhanced the binding affinity of CHX to SaMetRS from 10.2 μM (no ATP) to 0.45 μM (1 mM ATP). While it selectively inhibited two representative type 1 MetRSs from S. aureus and Enterococcus faecalis, CHX did not show significant interactions with three tested type 2 MetRSs, including human cytoplasmic MetRS, in the enzyme inhibition and biophysical binding assays, probably due to the conformational differences between two types of MetRSs at their EMP and AP. Our findings on CHX may inspire the design of MetRS-directed antimicrobials in future., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Masson SAS. All rights reserved.)

Fluorescent initiator tRNAs (tRNAi) play a crucial role in studying protein synthesis, yet generating highly fluorescent tRNAi complexes remains challenging. We present an optimized strategy to effectively generate highly fluorescent initiator-tRNA complexes in living cells. Our strategy allows the generation of Fluo-Met-tRNAi Met complexes. These complexes can have highly chromogenic N-terminal labeling. For generating such complexes, we use either purified fluorescent methionine (PFM) or non-purified fluorescently labeled methionine (NPFM). Furthermore, PFM promotes the active generation of endogenous tRNAi in cells, leading to highly efficient Fluo-Met-tRNAi Met complexes. Finally, PFM-tRNAi Met complexes also facilitate the visualization of native fluorescently labeled Tat binding to beads. This demonstrates the potential of our approach to advance precision protein engineering and biotechnology applications., (© 2024 The Authors. Biotechnology Journal published by Wiley‐VCH GmbH.)

As essential components of the protein synthesis machinery, tRNAs undergo a tightly controlled biogenesis process, which include the incorporation of numerous posttranscriptional modifications. Defects in these tRNA maturation steps may lead to the degradation of hypomodified tRNAs by the rapid tRNA decay (RTD) and nuclear surveillance pathways. We previously identified m1A58 as a late modification introduced after modifications Ψ55 and T54 in yeast elongator tRNAPhe. However, previous reports suggested that m1A58 is introduced early during the tRNA modification process, in particular on primary transcripts of initiator tRNAiMet, which prevents its degradation by RNA decay pathways. Here, aiming to reconcile this apparent inconsistency on the temporality of m1A58 incorporation, we examined its introduction into yeast elongator and initiator tRNAs. We used specifically modified tRNAs to report on the molecular aspects controlling the Ψ55 → T54 → m1A58 modification circuit in elongator tRNAs. We also show that m1A58 is efficiently introduced on unmodified tRNAiMet, and does not depend on prior modifications. Finally, we show that m1A58 has major effects on the structural properties of initiator tRNAiMet, so that the tRNA elbow structure is only properly assembled when this modification is present. This observation provides a structural explanation for the degradation of hypomodified tRNAiMet lacking m1A58 by the nuclear surveillance and RTD pathways., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)

eIF2A was the first eukaryotic initiator tRNA carrier discovered but its exact function has remained enigmatic. Uncharacteristic of translation initiation factors, eIF2A is reported to be non-cytosolic in multiple human cancer cell lines. Attempts to study eIF2A mechanistically have been limited by the inability to achieve high yield of soluble recombinant protein. Here, we developed a purification paradigm that yields ∼360-fold and ∼6000-fold more recombinant human eIF2A from Escherichia coli and insect cells, respectively, than previous reports. Using a mammalian in vitro translation system, we found that increased levels of recombinant human eIF2A inhibit translation of multiple reporter mRNAs, including those that are translated by cognate and near-cognate start codons, and does so prior to start codon recognition. eIF2A also inhibited translation directed by all four types of cap-independent viral IRESs, including the CrPV IGR IRES that does not require initiation factors or initiator tRNA, suggesting excess eIF2A sequesters 40S subunits. Supplementation with additional 40S subunits prevented eIF2A-mediated inhibition and pull-down assays demonstrated direct binding between recombinant eIF2A and purified 40S subunits. These data support a model that eIF2A must be kept away from the translation machinery to avoid sequestering 40S ribosomal subunits., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Translation is a key determinant of gene expression and an important biotechnological engineering target. In bacteria, 5'-untranslated region (5'-UTR) and coding sequence (CDS) are well-known mRNA parts controlling translation and thus cellular protein levels. However, the complex interaction of 5'-UTR and CDS has so far only been studied for few sequences leading to non-generalisable and partly contradictory conclusions. Herein, we systematically assess the dynamic translation from over 1.2 million 5'-UTR-CDS pairs in Escherichia coli to investigate their collective effect using a new method for ultradeep sequence-function mapping. This allows us to disentangle and precisely quantify effects of various sequence determinants of translation. We find that 5'-UTR and CDS individually account for 53% and 20% of variance in translation, respectively, and show conclusively that, contrary to a common hypothesis, tRNA abundance does not explain expression changes between CDSs with different synonymous codons. Moreover, the obtained large-scale data provide clear experimental evidence for a base-pairing interaction between initiator tRNA and mRNA beyond the anticodon-codon interaction, an effect that is often masked for individual sequences and therefore inaccessible to low-throughput approaches. Our study highlights the indispensability of ultradeep sequence-function mapping to accurately determine the contribution of parts and phenomena involved in gene regulation., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Multiple noncanonical amino acids can be installed into proteins in E. coli using mutually orthogonal aminoacyl-tRNA synthetase and tRNA pairs. Here we describe a protocol for simultaneously installing three distinct noncanonical amino acids into proteins for site-specific bioconjugation at three sites. This method relies on an engineered, UAU-suppressing, initiator tRNA, which is aminoacylated with a noncanonical amino acid by Methanocaldococcus jannaschii tyrosyl-tRNA synthetase. Using this initiator tRNA/aminoacyl-tRNA synthetase pair, together with the pyrrolysyl-tRNA synthetase/tRNA Pyl pairs from Methanosarcina mazei and Ca. Methanomethylophilus alvus, three noncanonical amino acids can be installed into proteins in response to the UAU, UAG, and UAA codons., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Initiation factor 3 (IF3) regulates the fidelity of bacterial translation initiation by debarring the use of non-canonical start codons or non-initiator tRNAs and prevents premature docking of the 50S ribosomal subunit to the 30S pre-initiation complex (PIC). The C-terminal domain (CTD) of IF3 can carry out most of the known functions of IF3 and sustain Escherichia coli growth. However, the roles of the N-terminal domain (NTD) have remained unclear. We hypothesized that the interaction between NTD and initiator tRNAfMet (i-tRNA) is essential to coordinate the movement of the two domains during the initiation pathway to ensure fidelity of the process. Here, using atomistic molecular dynamics (MD) simulation, we show that R25A/Q33A/R66A mutations do not impact NTD structure but disrupt its interaction with i-tRNA. These NTD residues modulate the fidelity of translation initiation and are crucial for bacterial growth. Our observations also implicate the role of these interactions in the subunit dissociation activity of CTD of IF3. Overall, the study shows that the interactions between NTD of IF3 and i-tRNA are crucial for coupling the movements of NTD and CTD of IF3 during the initiation pathway and in imparting growth fitness to E. coli., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

All tRNAs have numerous modifications, lack of which often results in growth defects in the budding yeast Saccharomyces cerevisiae and neurological or other disorders in humans. In S. cerevisiae, lack of tRNA body modifications can lead to impaired tRNA stability and decay of a subset of the hypomodified tRNAs. Mutants lacking 7-methylguanosine at G46 (m7G46), N2,N2-dimethylguanosine (m2,2G26), or 4-acetylcytidine (ac4C12), in combination with other body modification mutants, target certain mature hypomodified tRNAs to the rapid tRNA decay (RTD) pathway, catalyzed by 5'-3' exonucleases Xrn1 and Rat1, and regulated by Met22. The RTD pathway is conserved in the phylogenetically distant fission yeast Schizosaccharomyces pombe for mutants lacking m7G46. In contrast, S. cerevisiae trm6/gcd10 mutants with reduced 1-methyladenosine (m1A58) specifically target pre-tRNAiMet(CAU) to the nuclear surveillance pathway for 3'-5' exonucleolytic decay by the TRAMP complex and nuclear exosome. We show here that the RTD pathway has an unexpected major role in the biology of m1A58 and tRNAiMet(CAU) in both S. pombe and S. cerevisiae. We find that S. pombe trm6Δ mutants lacking m1A58 are temperature sensitive due to decay of tRNAiMet(CAU) by the RTD pathway. Thus, trm6Δ mutants had reduced levels of tRNAiMet(CAU) and not of eight other tested tRNAs, overexpression of tRNAiMet(CAU) restored growth, and spontaneous suppressors that restored tRNAiMet(CAU) levels had mutations in dhp1/RAT1 or tol1/MET22. In addition, deletion of cid14/TRF4 in the nuclear surveillance pathway did not restore growth. Furthermore, re-examination of S. cerevisiae trm6 mutants revealed a major role of the RTD pathway in maintaining tRNAiMet(CAU) levels, in addition to the known role of the nuclear surveillance pathway. These findings provide evidence for the importance of m1A58 in the biology of tRNAiMet(CAU) throughout eukaryotes, and fuel speculation that the RTD pathway has a major role in quality control of body modification mutants throughout fungi and other eukaryotes., Competing Interests: The authors have declared that no competing interests exist.

Translation initiation defines the identity and quantity of a synthesized protein. The process is dysregulated in many human diseases 1,2 . A key commitment step is when the ribosomal subunits join at a translation start site on a messenger RNA to form a functional ribosome. Here, we combined single-molecule spectroscopy and structural methods using an in vitro reconstituted system to examine how the human ribosomal subunits join. Single-molecule fluorescence revealed when the universally conserved eukaryotic initiation factors eIF1A and eIF5B associate with and depart from initiation complexes. Guided by single-molecule dynamics, we visualized initiation complexes that contained both eIF1A and eIF5B using single-particle cryo-electron microscopy. The resulting structure revealed how eukaryote-specific contacts between the two proteins remodel the initiation complex to orient the initiator aminoacyl-tRNA in a conformation compatible with ribosomal subunit joining. Collectively, our findings provide a quantitative and architectural framework for the molecular choreography orchestrated by eIF1A and eIF5B during translation initiation in humans., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Aggressive and metastatic cancers show enhanced metabolic plasticity 1 , but the precise underlying mechanisms of this remain unclear. Here we show how two NOP2/Sun RNA methyltransferase 3 (NSUN3)-dependent RNA modifications-5-methylcytosine (m 5 C) and its derivative 5-formylcytosine (f 5 C) (refs. 2-4 )-drive the translation of mitochondrial mRNA to power metastasis. Translation of mitochondrially encoded subunits of the oxidative phosphorylation complex depends on the formation of m 5 C at position 34 in mitochondrial tRNA Met . m 5 C-deficient human oral cancer cells exhibit increased levels of glycolysis and changes in their mitochondrial function that do not affect cell viability or primary tumour growth in vivo; however, metabolic plasticity is severely impaired as mitochondrial m 5 C-deficient tumours do not metastasize efficiently. We discovered that CD36-dependent non-dividing, metastasis-initiating tumour cells require mitochondrial m 5 C to activate invasion and dissemination. Moreover, a mitochondria-driven gene signature in patients with head and neck cancer is predictive for metastasis and disease progression. Finally, we confirm that this metabolic switch that allows the metastasis of tumour cells can be pharmacologically targeted through the inhibition of mitochondrial mRNA translation in vivo. Together, our results reveal that site-specific mitochondrial RNA modifications could be therapeutic targets to combat metastasis., (© 2022. The Author(s).)

The fidelity of initiator tRNA (i-tRNA) selection in the ribosomal P-site is a key step in translation initiation. The highly conserved three consecutive G:C base pairs (3GC pairs) in the i-tRNA anticodon stem play a crucial role in its selective binding in the P-site. Mutations in the 3GC pairs (3GC mutant) render the i-tRNA inactive in initiation. Here, we show that a mutation (E265K) in the unique C-terminal tail domain of RluD, a large ribosomal subunit pseudouridine synthase, results in compromised fidelity of initiation and allows initiation with the 3GC mutant i-tRNA. RluD modifies the uridine residues in H69 to pseudouridines. However, the role of its C-terminal tail domain remained unknown. The E265K mutation does not diminish the pseudouridine synthase activity of RluD, or the growth phenotype of Escherichia coli, or cause any detectable defects in the ribosomal assembly in our assays. However, in our in vivo analyses, we observed that the E265K mutation resulted in increased retention of the ribosome binding factor A (RbfA) on 30S suggesting a new role of RluD in contributing to RbfA release, a function which may be attributed to its (RluD) C-terminal tail domain. The studies also reveal that deficiency of RbfA release from 30S compromises the fidelity of i-tRNA selection in the ribosomal P-site., Competing Interests: Conflicts of interest The authors declare that there are no conflicts of interest., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

In eukaryotes and in archaea late steps of translation initiation involve the two initiation factors e/aIF5B and e/aIF1A. In eukaryotes, the role of eIF5B in ribosomal subunit joining is established and structural data showing eIF5B bound to the full ribosome were obtained. To achieve its function, eIF5B collaborates with eIF1A. However, structural data illustrating how these two factors interact on the small ribosomal subunit have long been awaited. The role of the archaeal counterparts, aIF5B and aIF1A, remains to be extensively addressed. Here, we study the late steps of Pyrococcus abyssi translation initiation. Using in vitro reconstituted initiation complexes and light scattering, we show that aIF5B bound to GTP accelerates subunit joining without the need for GTP hydrolysis. We report the crystallographic structures of aIF5B bound to GDP and GTP and analyze domain movements associated to these two nucleotide states. Finally, we present the cryo-EM structure of an initiation complex containing 30S bound to mRNA, Met-tRNAiMet, aIF5B and aIF1A at 2.7 Å resolution. Structural data shows how archaeal 5B and 1A factors cooperate to induce a conformation of the initiator tRNA favorable to subunit joining. Archaeal and eukaryotic features of late steps of translation initiation are discussed., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

During translation initiation, initiation factor 2 (IF2) holds initiator transfer RNA (fMet-tRNA i fMet ) in a specific orientation in the peptidyl (P) site of the ribosome. Upon subunit joining IF2 hydrolyzes GTP and, concomitant with inorganic phosphate (P i ) release, changes conformation facilitating fMet-tRNA i fMet accommodation into the P site and transition of the 70 S ribosome initiation complex (70S-IC) to an elongation-competent ribosome. The mechanism by which IF2 separates from initiator tRNA at the end of translation initiation remains elusive. Here, we report cryo-electron microscopy (cryo-EM) structures of the 70S-IC from Pseudomonas aeruginosa bound to compact IF2-GDP and initiator tRNA. Relative to GTP-bound IF2, rotation of the switch 2 α-helix in the G-domain bound to GDP unlocks a cascade of large-domain movements in IF2 that propagate to the distal tRNA-binding domain C2. The C2-domain relocates 35 angstroms away from tRNA, explaining how IF2 makes way for fMet-tRNA i fMet accommodation into the P site. Our findings provide the basis by which IF2 gates the ribosome to the elongation phase., (© 2022. The Author(s).)

The Mycobacterium tuberculosis genome contains an abundance of toxin-antitoxin (TA) systems, 50 of which belong to the VapBC family. The activity of VapC toxins is controlled by dynamic association with their cognate antitoxins-the toxin is inactive when complexed with VapB antitoxin but active when freed. Here, we determined the cellular target of two phylogenetically related VapC toxins and demonstrate how their properties can be harnessed for drug development. First, we used a specialized RNA sequencing (RNA-seq) approach, 5' RNA-seq, to accurately identify the in vivo RNA target of M. tuberculosis VapC2 and VapC21 toxins. Both toxins exclusively disable initiator tRNA fMet through cleavage at a single, identical site within their anticodon loop. Consistent with the essential role and global requirement for initiator tRNA fMet in bacteria, expression of each VapC toxin resulted in potent translation inhibition followed by growth arrest and cell death. Guided by previous structural studies, we then mutated two conserved amino acids in the antitoxin (WR→AA) that resided in the toxin-antitoxin interface and were predicted to inhibit toxin activity. Both mutants were markedly less efficient in rescuing growth over time, suggesting that screens for high-affinity small-molecule inhibitors against this or other crucial VapB-VapC interaction sites could drive constitutive inactivation of tRNA fMet by these VapC toxins. Collectively, the properties of the VapBC2 and VapBC21 TA systems provide a framework for development of bactericidal antitubercular agents with high specificity for M. tuberculosis cells.

The eukaryotic translation initiation factor 2 (eIF2) has key functions in the initiation step of protein synthesis. eIF2 guides the initiator tRNA to the ribosome, participates in scanning of the mRNA molecule, supports selection of the start codon, and modulates the translation of mRNAs in response to stress. eIF2 comprises a heterotrimeric complex whose assembly depends on the ATP-grasp protein Cdc123. Mutations of the eIF2γ subunit that compromise eIF2 complex formation cause severe neurological disease in humans. To this date, however, details about the assembly mechanism, step order, and the individual functions of eIF2 subunits remain unclear. Here, we quantified assembly intermediates and studied the behavior of various binding site mutants in budding yeast. Based on these data, we present a model in which a Cdc123-mediated conformational change in eIF2γ exposes binding sites for eIF2α and eIF2β subunits. Contrary to an earlier hypothesis, we found that the associations of eIF2α and eIF2β with the γ-subunit are independent of each other, but the resulting heterodimers are nonfunctional and fail to bind the guanosine exchange factor eIF2B. In addition, levels of eIF2α influence the rate of eIF2 assembly. By binding to eIF2γ, eIF2α displaces Cdc123 and thereby completes the assembly process. Experiments in human cell culture indicate that the mechanism of eIF2 assembly is conserved between yeast and humans. This study sheds light on an essential step in eukaryotic translation initiation, the dysfunction of which is linked to human disease., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

The anticodon stem of initiator tRNA (i-tRNA) possesses the characteristic three consecutive GC base pairs (G29:C41, G30:C40, and G31:C39 abbreviated as GC/GC/GC or 3GC pairs) crucial to commencing translation. To understand the importance of this highly conserved element, we isolated two fast-growing suppressors of Escherichia coli sustained solely on an unconventional i-tRNA (i-tRNA cg/GC/cg ) having cg/GC/cg sequence instead of the conventional GC/GC/GC. Both suppressors have the common mutation of V93A in initiation factor 3 (IF3), and additional mutations of either V32L (Sup-1) or H76L (Sup-2) in small subunit ribosomal protein 12 (uS12). The V93A mutation in IF3 was necessary for relaxed fidelity of i-tRNA selection to sustain on i-tRNA cg/GC/cg though with a retarded growth. Subsequent mutations in uS12 salvaged the retarded growth by enhancing the fidelity of translation. The H76L mutation in uS12 showed better fidelity of i-tRNA selection. However, the V32L mutation compensated for the deficient fidelity of i-tRNA selection by ensuring an efficient fidelity check by ribosome recycling factor (RRF). We reveal unique genetic networks between uS12, IF3 and i-tRNA in initiation and between uS12, elongation factor-G (EF-G), RRF, and Pth (peptidyl-tRNA hydrolase) which, taken together, govern the fidelity of translation in bacteria., (© 2021 John Wiley & Sons Ltd.)

The translation initiation factor eIF2 is critical for protein synthesis initiation, and its regulation is central to the integrated stress response (ISR). eIF2 is a G protein, and the activity is regulated by its GDP or GTP-binding status, such that only GTP-bound eIF2 has high affinity for initiator methionyl tRNA. In the ISR, regulatory signaling reduces the availability of eIF2-GTP and so downregulates protein synthesis initiation in cells. Fluorescence spectroscopy can be used as an analytical tool to study protein-ligand interactions in vitro. Here we describe methods to purify eIF2 and assays of its activity, employing analogs of GDP, GTP, and methionyl initiator tRNA ligands to accurately measure their binding affinities., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Substitution of the conserved Histidine 448 present in one of the three consensus elements characterizing the guanosine nucleotide binding domain (IF2 G2) of Escherichia coli translation initiation factor IF2 resulted in impaired ribosome-dependent GTPase activity which prevented IF2 dissociation from the ribosome, caused a severe protein synthesis inhibition, and yielded a dominant lethal phenotype. A reduced IF2 affinity for the ribosome was previously shown to suppress this lethality. Here, we demonstrate that also a reduced IF2 affinity for fMet-tRNA can suppress this dominant lethal phenotype and allows IF2 to support faithful translation in the complete absence of GTP hydrolysis. These results strengthen the premise that the conformational changes of ribosome, IF2, and fMet-tRNA occurring during the late stages of translation initiation are thermally driven and that the energy generated by IF2-dependent GTP hydrolysis is not required for successful translation initiation and that the dissociation of the interaction between IF2 C2 and the acceptor end of fMet-tRNA, which represents the last tie anchoring the factor to the ribosome before the formation of an elongation-competent 70S complex, is rate limiting for both the adjustment of fMet-tRNA in a productive P site and the IF2 release from the ribosome.

Toxin-antitoxin (TA) systems are genetic modules composed of a toxin interfering with cellular processes and its cognate antitoxin, which counteracts the activity of the toxin. TA modules are widespread in bacterial and archaeal genomes. It has been suggested that TA modules participate in the adaptation of prokaryotes to unfavorable conditions. The Bosea sp. PAMC 26642 used in this study was isolated from the Arctic lichen Stereocaulon sp. There are 12 putative type II TA loci in the genome of Bosea sp. PAMC 26642. Of these, nine functional TA systems have been shown to be toxic in Escherichia coli The toxin inhibits growth, but this inhibition is reversed when the cognate antitoxin genes are coexpressed, indicating that these putative TA loci were bona fide TA modules. Only the BoVapC1 (AXW83_01405) toxin, a homolog of VapC, showed growth inhibition specific to low temperatures, which was recovered by the coexpression of BoVapB1 (AXW83_01400). Microscopic observation and growth monitoring revealed that the BoVapC1 toxin had bacteriostatic effects on the growth of E. coli and induced morphological changes. Quantitative real time polymerase chain reaction and northern blotting analyses showed that the BoVapC1 toxin had a ribonuclease activity on the initiator tRNA fMet , implying that degradation of tRNA fMet might trigger growth arrest in E. coli Furthermore, the BoVapBC1 system was found to contribute to survival against prolonged exposure at 4°C. This is the first study to identify the function of TA systems in cold adaptation., (© 2021 Jeon et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

Initiation factor IF3 is an essential protein that enhances the fidelity and speed of bacterial mRNA translation initiation. Here, we describe the dynamic interplay between IF3 domains and their alternative binding sites using pre-steady state kinetics combined with molecular modelling of available structures of initiation complexes. Our results show that IF3 accommodates its domains at velocities ranging over two orders of magnitude, responding to the binding of each 30S ligand. IF1 and IF2 promote IF3 compaction and the movement of the C-terminal domain (IF3C) towards the P site. Concomitantly, the N-terminal domain (IF3N) creates a pocket ready to accept the initiator tRNA. Selection of the initiator tRNA is accompanied by a transient accommodation of IF3N towards the 30S platform. Decoding of the mRNA start codon displaces IF3C away from the P site and rate limits translation initiation. 70S initiation complex formation brings IF3 domains in close proximity to each other prior to dissociation and recycling of the factor for a new round of translation initiation. Altogether, our results describe the kinetic spectrum of IF3 movements and highlight functional transitions of the factor that ensure accurate mRNA translation initiation., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Macrolide antibiotics, such as erythromycin, bind to the nascent peptide exit tunnel (NPET) of the bacterial ribosome and modulate protein synthesis depending on the nascent peptide sequence. Whereas in vitro biochemical and structural methods have been instrumental in dissecting and explaining the molecular details of macrolide-induced peptidyl-tRNA drop-off and ribosome stalling, the dynamic effects of the drugs on ongoing protein synthesis inside live bacterial cells are far less explored. In the present study, we used single-particle tracking of dye-labeled tRNAs to study the kinetics of mRNA translation in the presence of erythromycin, directly inside live Escherichia coli cells. In erythromycin-treated cells, we find that the dwells of elongator tRNA Phe on ribosomes extend significantly, but they occur much more seldom. In contrast, the drug barely affects the ribosome binding events of the initiator tRNA fMet . By overexpressing specific short peptides, we further find context-specific ribosome binding dynamics of tRNA Phe , underscoring the complexity of erythromycin's effect on protein synthesis in bacterial cells., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

The conserved Gcn2 protein kinase mediates cellular adaptations to amino acid limitation through translational control of gene expression that is exclusively executed by phosphorylation of the α-subunit of the eukaryotic translation initiation factor 2 (eIF2α). Using quantitative phosphoproteomics, however, we discovered that Gcn2 targets auxiliary effectors to modulate translation. Accordingly, Gcn2 also phosphorylates the β-subunit of the trimeric eIF2 G protein complex to promote its association with eIF5, which prevents spontaneous nucleotide exchange on eIF2 and thereby restricts the recycling of the initiator methionyl-tRNA-bound eIF2-GDP ternary complex in amino-acid-starved cells. This mechanism contributes to the inhibition of translation initiation in parallel to the sequestration of the nucleotide exchange factor eIF2B by phosphorylated eIF2α. Gcn2 further phosphorylates Gcn20 to antagonize, in an inhibitory feedback loop, the formation of the Gcn2-stimulatory Gcn1-Gcn20 complex. Thus, Gcn2 plays a substantially more intricate role in controlling translation initiation than hitherto appreciated., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Long terminal repeat (LTR)-retrotransposons constitute a significant part of eukaryotic genomes and influence their function and evolution. Like other RNA viruses, LTR-retrotransposons efficiently utilize their RNA genome to interact with host cell machinery during replication. Here, we provide the first genome-wide RNA secondary structure model for a LTR-retrotransposon in living cells. Using SHAPE probing, we explore the secondary structure of the yeast Ty1 retrotransposon RNA genome in its native in vivo state and under defined in vitro conditions. Comparative analyses reveal the strong impact of the cellular environment on folding of Ty1 RNA. In vivo, Ty1 genome RNA is significantly less structured and more dynamic but retains specific well-structured regions harboring functional cis-acting sequences. Ribosomes participate in the unfolding and remodeling of Ty1 RNA, and inhibition of translation initiation stabilizes Ty1 RNA structure. Together, our findings support the dual role of Ty1 genomic RNA as a template for protein synthesis and reverse transcription. This study also contributes to understanding how a complex multifunctional RNA genome folds in vivo, and strengthens the need for studying RNA structure in its natural cellular context., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

To better understand the interaction between SARS-CoV-2 and human host and find potential ways to block the pandemic, one of the unresolved questions is that how the virus economically utilizes the resources of the hosts. Particularly, the tRNA pool has been adapted to the host genes. If the virus intends to translate its own RNA, then it has to compete with the abundant host mRNAs for the tRNA molecules. Translation initiation is the rate-limiting step during protein synthesis. The tRNAs carrying the initiation Methionine (iMet) recognize the start codon termed initiation ATG (iATG). Other normal Met-carrying tRNAs recognize the internal ATGs. The tAI of virus genes is significantly lower than the tAI of human genes. This disadvantage in translation elongation of viral RNAs must be compensated by more efficient initiation rates. In the human genome, the abundance of iMet-tRNAs to Met-tRNAs is five times higher than the iATG to ATG ratio. However, when SARS-CoV-2 infects human cells, the iMet has an 8.5-time enrichment to iATG. We collected 58 virus species and found that the enrichment of iMet is higher in all viruses compared to human. Our study indicates that the genome sequences of viruses like SARS-CoV-2 have the advantage of competing for the iMet-tRNAs with host mRNAs. The capture of iMet-tRNAs allows the fast translation initiation and the reproduction of virus itself, which compensates the lower tAI of viral genes. This might explain why the virus could rapidly translate its own RNA and reproduce itself from the sea of host mRNAs. Meanwhile, our study reminds the researchers not to ignore the mutations related to ATGs.

Most mammalian cytoplasmic tRNAs contain ribothymidine (T) and pseudouridine (Ψ) at positions 54 and 55, respectively. However, some tRNAs contain Ψ at both positions. Several Ψ54-containing tRNAs function as primers in retroviral DNA synthesis. The Ψ54 of these tRNAs is produced by PUS10, which can also synthesize Ψ55. Two other enzymes, TRUB1 and TRUB2, can also produce Ψ55. By nearest-neighbor analyses of tRNAs treated with recombinant proteins and subcellular extracts of wild-type and specific Ψ55 synthase knockdown cells, we determined that while TRUB1, PUS10, and TRUB2 all have tRNA Ψ55 synthase activities, they have different tRNA structural requirements. Moreover, these activities are primarily present in the nucleus, cytoplasm, and mitochondria, respectively, suggesting a compartmentalization of Ψ55 synthase activity. TRUB1 produces the Ψ55 of most elongator tRNAs, but cytoplasmic PUS10 produces both Ψs of the tRNAs with Ψ54Ψ55. The nuclear isoform of PUS10 is catalytically inactive and specifically binds the unmodified U54U55 versions of Ψ54Ψ55-containing tRNAs, as well as the A54U55-containing tRNA iMet This binding inhibits TRUB1-mediated U55 to Ψ55 conversion in the nucleus. Consequently, the U54U55 of Ψ54Ψ55-containing tRNAs are modified by the cytoplasmic PUS10. Nuclear PUS10 does not bind the U55 versions of T54Ψ55- and A54Ψ55-containing elongator tRNAs. Therefore, TRUB1 is able to produce Ψ55 in these tRNAs. In summary, the tRNA Ψ55 synthase activities of TRUB1 and PUS10 are not redundant but rather are compartmentalized and act on different sets of tRNAs. The significance of this compartmentalization needs further study., (© 2021 Mukhopadhyay et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

Recognition of a start codon by the initiator aminoacyl-tRNA determines the reading frame of messenger RNA (mRNA) translation by the ribosome. In eukaryotes, the GTPase eIF5B collaborates in the correct positioning of the initiator Met-tRNA i Met on the ribosome in the later stages of translation initiation, gating entrance into elongation. Leveraging the long residence time of eIF5B on the ribosome recently identified by single-molecule fluorescence measurements, we determine the cryoEM structure of the naturally long-lived ribosome complex with eIF5B and Met-tRNA i Met immediately before transition into elongation. The structure uncovers an unexpected, eukaryotic specific and dynamic fidelity checkpoint implemented by eIF5B in concert with components of the large ribosomal subunit.

One-carbon metabolism produces methionine and N 10 -formyl-tetrahydrofolate (N 10 -fTHF) required for aminoacylation and formylation of initiator tRNA (i-tRNA), respectively. In Escherichia coli, N 10 -fTHF is made from 5, 10-methylene-THF by a two-step reaction using 5,10-methylene-THF dehydrogenase/cyclohydrolase (FolD). The i-tRNAs from all domains of life possess a highly conserved sequence of three consecutive G-C base pairs (3GC pairs) in their anticodon stem. A 3GC mutant i-tRNA (wherein the 3GC pairs are mutated to those found in elongator tRNA Met ) is incompetent in initiation in E. coli (even though it is efficiently aminoacylated and formylated). Here, we show that E. coli strains having mutations in FolD (G122D or C58Y or P140L) allow a plasmid encoded 3GC mutant i-tRNA to participate in initiation. In vitro, the FolD mutants are highly compromised in their dehydrogenase/cyclohydrolase activities leading to reduced production of N 10 -fTHF and decreased rates of i-tRNA formylation. The perturbation of one-carbon metabolism by trimethoprim (inhibitor of dihydrofolate reductase) phenocopies FolD deficiency and allows initiation with the 3GC mutant i-tRNA. This study reveals an important crosstalk between one-carbon metabolism and the fidelity of translation initiation via formylation of i-tRNA, and suggests that augmentation of the age old sulfa drugs with FolD inhibitors could be an important antibacterial strategy., Competing Interests: Conflicts of Interest The authors declare that there are no conflicts of interest., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

All known riboswitches use their aptamer to senese one metabolite signal and their expression platform to regulate gene expression. Here, we characterize a SAM-I riboswitch (SAM-I Xcc ) from the Xanthomonas campestris that regulates methionine synthesis via the met operon. In vitro and in vivo experiments show that SAM-I Xcc controls the met operon primarily at the translational level in response to cellular S-adenosylmethionine (SAM) levels. Biochemical and genetic data demonstrate that SAM-I Xcc expression platform not only can repress gene expression in response to SAM binding to SAM-I Xcc aptamer but also can sense and bind uncharged initiator Met tRNA, resulting in the sequestering of the anti-Shine-Dalgarno (SD) sequence and freeing the SD for translation initiation. These findings identify a SAM-I riboswitch with a dual functioning expression platform that regulates methionine synthesis through a previously unrecognized mechanism and discover a natural tRNA-sensing RNA element. This SAM-I riboswitch appears to be highly conserved in Xanthomonas species.

tRNA molecules, which contain the most abundant post-transcriptional modifications, are crucial for proper gene expression and protein biosynthesis. Methylation at N1 of adenosine 58 (A58) is critical for maintaining the stability of initiator methionyl-tRNA (tRNAiMet) in bacterial, archaeal, and eukaryotic tRNAs. However, although research has been conducted in yeast and mammals, it remains unclear how A58 in plant tRNAs is modified and involved in development. In this study, we identify the nucleus-localized complex AtTRM61/AtTRM6 in Arabidopsis as tRNA m1A58 methyltransferase. Deficiency or a lack of either AtTRM61 or AtTRM6 leads to embryo arrest and seed abortion. The tRNA m1A level decreases in conditionally complemented Attrm61/LEC1pro::AtTRM61 plants and this is accompanied by reduced levels of tRNAiMet, indicating the importance of the tRNA m1A modification for tRNAiMet stability. Taken together, our results demonstrate that tRNA m1A58 modification is necessary for tRNAiMet stability and is required for embryo development in Arabidopsis., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Mammalian mitochondrial ribosomes are unique molecular machines that translate 11 leaderless mRNAs; however, it is not clear how mitoribosomes initiate translation, since mitochondrial mRNAs lack untranslated regions. Mitochondrial translation initiation shares similarities with prokaryotes, such as the formation of a ternary complex of fMet-tRNA Met , mRNA and the 28 S subunit, but differs in the requirements for initiation factors. Mitochondria have two initiation factors: MTIF2, which closes the decoding center and stabilizes the binding of the fMet-tRNA Met to the leaderless mRNAs, and MTIF3, whose role is not clear. We show that MTIF3 is essential for survival and that heart- and skeletal muscle-specific loss of MTIF3 causes cardiomyopathy. We identify increased but uncoordinated mitochondrial protein synthesis in mice lacking MTIF3, resulting in loss of specific respiratory complexes. Ribosome profiling shows that MTIF3 is required for recognition and regulation of translation initiation of mitochondrial mRNAs and for coordinated assembly of OXPHOS complexes in vivo., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).)

In bacteria, the assembly factors tightly orchestrate the maturation of ribosomes whose competency for protein synthesis is validated by translation machinery at various stages of translation cycle. However, what transpires to the quality control measures when the ribosomes are produced with assembly defects remains enigmatic. In Escherichia coli, we show that 30S ribosomes that harbour assembly defects due to the lack of assembly factors such as RbfA and KsgA display suboptimal initiation codon recognition and bypass the critical codon-anticodon proofreading steps during translation initiation. These premature ribosomes on entering the translation cycle compromise the fidelity of decoding that gives rise to errors during initiation and elongation. We show that the assembly defects compromise the binding of initiation factor 3 (IF3), which in turn appears to license the rapid transition of 30S (pre) initiation complex to 70S initiation complex by tempering the validation of codon-anticodon interaction during translation initiation. This suggests that the premature ribosomes harbouring the assembly defects subvert the IF3 mediated proofreading of cognate initiation codon to enter the translation cycle., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Mitochondrial DNA variants in the MT-TM (mt-tRNA Met ) gene are rare, typically associated with myopathic phenotypes. We identified a novel MT-TM variant resulting in prolonged seizures with childhood-onset myopathy, retinopathy, short stature and elevated CSF lactate associated with bilateral basal ganglia changes on neuroimaging. Muscle biopsy confirmed multiple respiratory chain deficiencies and focal cytochrome c oxidase (COX) histochemical abnormalities. Next-generation sequencing of the mitochondrial genome revealed a novel m.4412G>A variant at high heteroplasmy levels in muscle that fulfils all accepted criteria for pathogenicity including segregation within single muscle fibres, thus broadening the genotypic and phenotypic landscape of mitochondrial tRNA-related disease., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

One of the responses to stress by eukaryotic cells is the down-regulation of protein synthesis by phosphorylation of translation initiation factor eIF2. Phosphorylation results in low availability of the eIF2 ternary complex (eIF2-GTP-tRNAi) by affecting the interaction of eIF2 with its GTP-GDP exchange factor eIF2B. We have determined the cryo-EM structure of yeast eIF2B in complex with phosphorylated eIF2 at an overall resolution of 4.2 Å. Two eIF2 molecules bind opposite sides of an eIF2B hetero-decamer through eIF2α-D1, which contains the phosphorylated Ser51. eIF2α-D1 is mainly inserted between the N-terminal helix bundle domains of δ and α subunits of eIF2B. Phosphorylation of Ser51 enhances binding to eIF2B through direct interactions of phosphate groups with residues in eIF2Bα and indirectly by inducing contacts of eIF2α helix 58-63 with eIF2Bδ leading to a competition with Met-tRNA i .

Protein synthesis in eukaryotes is controlled by signals and stresses via a common pathway, called the integrated stress response (ISR). Phosphorylation of the translation initiation factor eIF2 alpha at a conserved serine residue mediates translational control at the ISR core. To provide insight into the mechanism of translational control we have determined the structures of eIF2 both in phosphorylated and unphosphorylated forms bound with its nucleotide exchange factor eIF2B by electron cryomicroscopy. The structures reveal that eIF2 undergoes large rearrangements to promote binding of eIF2α to the regulatory core of eIF2B comprised of the eIF2B alpha, beta and delta subunits. Only minor differences are observed between eIF2 and eIF2αP binding to eIF2B, suggesting that the higher affinity of eIF2αP for eIF2B drives translational control. We present a model for controlled nucleotide exchange and initiator tRNA binding to the eIF2/eIF2B complex.

Oxazolidinones are synthetic antibiotics used for treatment of infections caused by Gram-positive bacteria. They target the bacterial protein synthesis machinery by binding to the peptidyl transferase centre (PTC) of the ribosome and interfering with the peptidyl transferase reaction. Cadazolid is the first member of quinoxolidinone antibiotics, which are characterized by combining the pharmacophores of oxazolidinones and fluoroquinolones, and it is evaluated for treatment of Clostridium difficile gastrointestinal infections that frequently occur in hospitalized patients. In vitro protein synthesis inhibition by cadazolid was shown in Escherichia coli and Staphylococcus aureus, including an isolate resistant against linezolid, the prototypical oxazolidinone antibiotic. To better understand the mechanism of inhibition, we determined a 3.0 Å cryo-electron microscopy structure of cadazolid bound to the E. coli ribosome in complex with mRNA and initiator tRNA. Here we show that cadazolid binds with its oxazolidinone moiety in a binding pocket in close vicinity of the PTC as observed previously for linezolid, and that it extends its unique fluoroquinolone moiety towards the A-site of the PTC. In this position, the drug inhibits protein synthesis by interfering with the binding of tRNA to the A-site, suggesting that its chemical features also can enable the inhibition of linezolid-resistant strains.

The heterotrimeric eukaryotic translation initiation factor (eIF) 2 plays critical roles in delivering initiator Met-tRNAiMet to the 40S ribosomal subunit and in selecting the translation initiation site. Genetic analyses of patients with MEHMO syndrome, an X-linked intellectual disability syndrome, have identified several unique mutations in the EIF2S3 gene that encodes the γ subunit of eIF2. To gain insights into the molecular consequences of MEHMO syndrome mutations on eIF2 function, we generated a yeast model of the human eIF2γ-I259M mutant, previously identified in a patient with MEHMO syndrome. The corresponding eIF2γ-I318M mutation impaired yeast cell growth and derepressed GCN4 expression, an indicator of defective eIF2-GTP-Met-tRNAiMet complex formation, and, likewise, overexpression of human eIF2γ-I259M derepressed ATF4 messenger RNA translation in human cells. The yeast eIF2γ-I318M mutation also increased initiation from near-cognate start codons. Biochemical analyses revealed a defect in Met-tRNAiMet binding to the mutant yeast eIF2 complexes in vivo and in vitro. Overexpression of tRNAiMet restored Met-tRNAiMet binding to eIF2 in vivo and rescued the growth defect in the eIF2γ-I318M strain. Based on these findings and the structure of eIF2, we propose that the I259M mutation impairs Met-tRNAiMet binding, causing altered control of protein synthesis that underlies MEHMO syndrome.

The initiator tRNA (Met-tRNA i Met ) at the P site of the small ribosomal subunit plays an important role in the recognition of an mRNA start codon. In bacteria, the initiator tRNA carrier, IF2, facilitates the positioning of Met-tRNA i Met on the small ribosomal subunit. Eukarya contain the Met-tRNA i Met carrier, eIF2 (unrelated to IF2), whose carrier activity is inhibited under stress conditions by the phosphorylation of its α-subunit by stress-activated eIF2α kinases. The stress-resistant initiator tRNA carrier, eIF2A, was recently uncovered and shown to load Met-tRNA i Met on the 40S ribosomal subunit associated with a stress-resistant mRNA under stress conditions. Here, we report that eIF2A interacts and functionally cooperates with eIF5B (a homolog of IF2), and we describe the functional domains of eIF2A that are required for its binding of Met-tRNA i Met , eIF5B, and a stress-resistant mRNA. The results indicate that the eukaryotic eIF5B-eIF2A complex functionally mimics the bacterial IF2 containing ribosome-, GTP-, and initiator tRNA-binding domains in a single polypeptide.

In archaeal translation initiation, a preinitiation complex (PIC) made up of aIF1, aIF1A, the ternary complex (TC, e/aIF2-GTP-Met-tRNAiMet) and mRNA bound to the small ribosomal subunit is responsible for start codon selection. Many archaeal mRNAs contain a Shine-Dalgarno (SD) sequence allowing the PIC to be prepositioned in the vicinity of the start codon. Nevertheless, cryo-EM studies have suggested local scanning to definitely establish base pairing of the start codon with the tRNA anticodon. Here, using fluorescence anisotropy, we show that aIF1 and mRNA have synergistic binding to the Pyrococcus abyssi 30S. Stability of 30S:mRNA:aIF1 strongly depends on the SD sequence. Further, toeprinting experiments show that aIF1-containing PICs display a dynamic conformation with the tRNA not firmly accommodated in the P site. AIF1-induced destabilization of the PIC is favorable for proofreading erroneous initiation complexes. After aIF1 departure, the stability of the PIC increases reflecting initiator tRNA fully base-paired to the start codon. Altogether, our data support the idea that some of the main events governing start codon selection in eukaryotes and archaea occur within a common structural and functional core. However, idiosyncratic features in loop 1 sequence involved in 30S:mRNA binding suggest adjustments of e/aIF1 functioning in the two domains.

Prokaryotic toxin-antitoxin (TA) modules are highly abundant and are involved in stress response and drug tolerance. The most common type II TA modules consist of two interacting proteins. The type II toxins are diverse enzymes targeting various essential intracellular targets. The antitoxin binds to cognate toxin and inhibits its function. Recently, TA modules whose toxins are GNAT-family acetyltransferases were described. For two such systems, the target of acetylation was shown to be aminoacyl-tRNA: the TacT toxin targets aminoacylated elongator tRNAs, while AtaT targets the amino acid moiety of initiating tRNAMet. We show that the itaRT gene pair from Escherichia coli encodes a TA module with acetyltransferase toxin ItaT that specifically and exclusively acetylates Ile-tRNAIle thereby blocking translation and inhibiting cell growth. ItaT forms a tight complex with the ItaR antitoxin, which represses the transcription of itaRT operon. A comprehensive bioinformatics survey of GNAT acetyltransferases reveals that enzymes encoded by validated or putative TA modules are common and form a distinct branch of the GNAT family tree. We speculate that further functional analysis of such TA modules will result in identification of enzymes capable of specifically targeting many, perhaps all, aminoacyl tRNAs.

Mitochondria maintain their own specialized protein synthesis machinery, which in mammals is used exclusively for the synthesis of the membrane proteins responsible for oxidative phosphorylation 1,2 . The initiation of protein synthesis in mitochondria differs substantially from bacterial or cytosolic translation systems. Mitochondrial translation initiation lacks initiation factor 1, which is essential in all other translation systems from bacteria to mammals 3,4 . Furthermore, only one type of methionyl transfer RNA (tRNA Met ) is used for both initiation and elongation 4,5 , necessitating that the initiation factor specifically recognizes the formylated version of tRNA Met (fMet-tRNA Met ). Lastly, most mitochondrial mRNAs do not possess 5' leader sequences to promote mRNA binding to the ribosome 2 . There is currently little mechanistic insight into mammalian mitochondrial translation initiation, and it is not clear how mRNA engagement, initiator-tRNA recruitment and start-codon selection occur. Here we determine the cryo-EM structure of the complete translation initiation complex from mammalian mitochondria at 3.2 Å. We describe the function of an additional domain insertion that is present in the mammalian mitochondrial initiation factor 2 (mtIF2). By closing the decoding centre, this insertion stabilizes the binding of leaderless mRNAs and induces conformational changes in the rRNA nucleotides involved in decoding. We identify unique features of mtIF2 that are required for specific recognition of fMet-tRNA Met and regulation of its GTPase activity. Finally, we observe that the ribosomal tunnel in the initiating ribosome is blocked by insertion of the N-terminal portion of mitochondrial protein mL45, which becomes exposed as the ribosome switches to elongation mode and may have an additional role in targeting of mitochondrial ribosomes to the protein-conducting pore in the inner mitochondrial membrane.

Biallelic missense mutations in MARS are responsible for rare but severe cases of pulmonary alveolar proteinosis (PAP) prevalent on the island of La Réunion. MARS encodes cytosolic methionyl-tRNA synthetase (MetRS), an essential translation factor. The multisystemic effects observed in patients with this form of PAP are consistent with a loss-of-function defect in an ubiquitously expressed enzyme. The pathophysiological mechanisms involved in MARS-related PAP are currently unknown. In this work, we analyzed the effect of the PAP-related mutations in MARS on the thermal stability and on the catalytic parameters of the MetRS mutants, relative to wild-type. The effect of these mutations on the structural integrity of the enzyme as a member of the cytosolic multisynthetase complex was also investigated. Our results establish that the PAP-related substitutions in MetRS impact the tRNA Met -aminoacylation reaction especially at the level of methionine recognition, and suggest a direct link between the loss of activity of the enzyme and the pathological disorders in PAP., (© 2018 Federation of European Biochemical Societies.)

Our ability to directly relate results from test-tube biochemical experiments to the kinetics in living cells is very limited. Here we present experimental and analytical tools to directly study the kinetics of fast biochemical reactions in live cells. Dye-labeled molecules are electroporated into bacterial cells and tracked using super-resolved single-molecule microscopy. Trajectories are analyzed by machine-learning algorithms to directly monitor transitions between bound and free states. In particular, we measure the dwell time of tRNAs on ribosomes, and hence achieve direct measurements of translation rates inside living cells at codon resolution. We find elongation rates with tRNA Phe that are in perfect agreement with previous indirect estimates, and once fMet-tRNA fMet has bound to the 30S ribosomal subunit, initiation of translation is surprisingly fast and does not limit the overall rate of protein synthesis. The experimental and analytical tools for direct kinetics measurements in live cells have applications far beyond bacterial protein synthesis.

Chemical modifications of RNA have been attracting increasing interest because of their impact on RNA fate and function. Therefore, the characterization of enzymes catalyzing such modifications is of great importance. The RNA cytosine methyltransferase NSUN3 was recently shown to generate 5-methylcytosine in the anticodon loop of mitochondrial tRNA Met . Further oxidation of this position is required for normal mitochondrial translation and function in human somatic cells. Because embryonic stem cells (ESCs) are less dependent on oxidative phosphorylation than somatic cells, we examined the effects of catalytic inactivation of Nsun3 on self-renewal and differentiation potential of murine ESCs. We demonstrate that Nsun3-mutant cells show strongly reduced mt-tRNA Met methylation and formylation as well as reduced mitochondrial translation and respiration. Despite the lower dependence of ESCs on mitochondrial activity, proliferation of mutant cells was reduced, while pluripotency marker gene expression was not affected. By contrast, ESC differentiation was skewed towards the meso- and endoderm lineages at the expense of neuroectoderm. Wnt3 was overexpressed in early differentiating mutant embryoid bodies and in ESCs, suggesting that impaired mitochondrial function disturbs normal differentiation programs by interfering with cellular signalling pathways. Interestingly, basal levels of reactive oxygen species (ROS) were not altered in ESCs, but Nsun3 inactivation attenuated induction of mitochondrial ROS upon stress, which may affect gene expression programs upon differentiation. Our findings not only characterize Nsun3 as an important regulator of stem cell fate but also provide a model system to study the still incompletely understood interplay of mitochondrial function with stem cell pluripotency and differentiation.

Upon glucose restriction, eukaryotic cells upregulate oxidative metabolism to maintain homeostasis. Using genetic screens, we find that the mitochondrial serine hydroxymethyltransferase (SHMT2) is required for robust mitochondrial oxygen consumption and low glucose proliferation. SHMT2 catalyzes the first step in mitochondrial one-carbon metabolism, which, particularly in proliferating cells, produces tetrahydrofolate (THF)-conjugated one-carbon units used in cytoplasmic reactions despite the presence of a parallel cytoplasmic pathway. Impairing cytoplasmic one-carbon metabolism or blocking efflux of one-carbon units from mitochondria does not phenocopy SHMT2 loss, indicating that a mitochondrial THF cofactor is responsible for the observed phenotype. The enzyme MTFMT utilizes one such cofactor, 10-formyl THF, producing formylmethionyl-tRNAs, specialized initiator tRNAs necessary for proper translation of mitochondrially encoded proteins. Accordingly, SHMT2 null cells specifically fail to maintain formylmethionyl-tRNA pools and mitochondrially encoded proteins, phenotypes similar to those observed in MTFMT-deficient patients. These findings provide a rationale for maintaining a compartmentalized one-carbon pathway in mitochondria., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Defective nucleotide modifications of mitochondrial tRNAs have been associated with several human diseases, but their pathophysiology remains poorly understood. In this report, we investigated the pathogenic molecular mechanism underlying a hypertension-associated 4435A→G mutation in mitochondrial tRNA Met The m.4435A→G mutation affected a highly conserved adenosine at position 37, 3' adjacent to the tRNA's anticodon, which is important for the fidelity of codon recognition and stabilization. We hypothesized that the m.4435A→G mutation introduced an m 1 G37 modification of tRNA Met , altering its structure and function. Primer extension and methylation activity assays indeed confirmed that the m.4435A→G mutation created a tRNA methyltransferase 5 (TRMT5)-catalyzed m 1 G37 modification of tRNA Met We found that this mutation altered the tRNA Met structure, indicated by an increased melting temperature and electrophoretic mobility of the mutated tRNA compared with the wildtype molecule. We demonstrated that cybrid cell lines carrying the m.4435A→G mutation exhibited significantly decreased efficiency in aminoacylation and steady-state levels of tRNA Met , as compared with those of control cybrids. The aberrant tRNA Met metabolism resulted in variable decreases in mitochondrial DNA (mtDNA)-encoded polypeptides in the mutant cybrids. Furthermore, we found that the m.4435A→G mutation caused respiratory deficiency, markedly diminished mitochondrial ATP levels and membrane potential, and increased the production of reactive oxygen species in mutant cybrids. These results demonstrated that an aberrant m 1 G37 modification of mitochondrial tRNA Met affected the structure and function of its tRNA and consequently altered mitochondrial function. Our findings provide critical insights into the pathophysiology of maternally inherited hypertension, which is manifested by the deficient tRNA nucleotide modification., (© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.)

Type II toxin-antitoxin (TA) systems play a critical role in the establishment and maintenance of bacterial dormancy. They are composed of a protein toxin and its cognate protein antitoxin. They function to regulate growth under conditions of stress, such as starvation or antibiotic treatment. As cellular proteases degrade the antitoxin, which normally binds and neutralizes the toxin, this frees the toxin to act on its cellular targets and arrest bacterial growth. TA systems are of particular concern in regard to pathogenic organisms, such as nontypeable Haemophilus influenzae (NTHi), as dormancy may lead to chronic infections and failure of antibiotic treatment. Many targets of VapC toxins have not been identified, to date, and this knowledge is crucial to understanding how toxins control the establishment and maintenance of bacterial dormancy. Accordingly, we characterized the target specificity of the VapC toxins from the two paralogous NTHi vapBC TA systems. RNA sequencing and Northern blot analysis revealed that VapC1 and VapC2 cleave tRNA fMet in the anticodon loop. Overexpression of tRNA fMet suppresses VapC toxicity, suggesting that translation inhibition results from the depletion of tRNA fMet These experiments also identified base pairs in the tRNA fMet anticodon stem that play a key role in VapC-specific cleavage of the tRNA. Together these findings suggest the potential for NTHi VapC1 and VapC2 to induce dormancy by sequence-specific cleavage of tRNA fMet IMPORTANCE Bacterial persistence is a significant concern in regard to pathogenic organisms, such as nontypeable Haemophilus influenzae , as it can result in recurrent and chronic infections. Toxin-antitoxin systems can lead to persistence by causing bacteria to enter a slow-growing state that renders them antibiotic tolerant. Type II toxin components affect a wide variety of bacterial targets in order to elicit dormancy, and for many toxin-antitoxin systems, these mechanisms are not well understood. Thus, in order to understand how vapBC toxin-antitoxin systems cause dormancy, it is crucial to investigate the substrate specificity of VapC toxins. This study identifies the target of the VapC1 and VapC2 toxins from NTHi and takes important steps toward understanding the specificity of these toxins for their tRNA target., (Copyright © 2018 American Society for Microbiology.)