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Translation initiation is a highly regulated, multi-step process that is critical for efficient and accurate protein synthesis. In bacteria, initiation begins when mRNA, initiation factors, and a dedicated initiator fMet-tRNA fMet bind the small (30S) ribosomal subunit. Specific binding of fMet-tRNA fMet in the peptidyl (P) site is mediated by the inspection of the fMet moiety by initiation factor IF2 and of three conserved G-C base pairs in the tRNA anticodon stem by the 30S head domain. Tandem A-minor interactions form between 16S ribosomal RNA nucleotides A1339 and G1338 and tRNA base pairs G30-C40 and G29-C41, respectively. Swapping the G30-C40 pair of tRNA fMet with C-G (called tRNA fMet M1) reduces discrimination against the noncanonical start codon CUG in vitro, suggesting crosstalk between the gripping of the anticodon stem and recognition of the start codon. Here, we solved electron cryomicroscopy structures of Escherichia coli 70S initiation complexes containing the fMet-tRNA fMet M1 variant paired to the noncanonical CUG start codon, in the presence or absence of IF2 and the non-hydrolyzable GTP analog GDPCP, alongside structures of 70S initiation complexes containing this tRNA fMet variant paired to the canonical bacterial start codons AUG, GUG, and UUG. We find that the M1 mutation weakens A-minor interactions between tRNA fMet and 16S nucleotides A1339 and G1338, with IF2 strengthening the interaction of G1338 with the tRNA minor groove. These structures suggest how even slight changes to the recognition of the fMet-tRNA fMet anticodon stem by the ribosome can impact the start codon selection., Competing Interests: Conflicts of interest The authors declare that they have no conflicts of interest regarding the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Ribosomal incorporation of β-amino acids into nascent peptides is much less efficient than that of the canonical α-amino acids. To overcome this, we have engineered a tRNA chimera bearing T-stem of tRNAGlu and D-arm of tRNAPro1, referred to as tRNAPro1E2, which efficiently recruits EF-Tu and EF-P. Using tRNAPro1E2 indeed improved β-amino acid incorporation. However, multiple/consecutive incorporations of β-amino acids are still detrimentally poor. Here, we attempted fine-tuning of the anticodon arm of tRNAPro1E2 aiming at further enhancement of β-amino acid incorporation. By screening various mutations introduced into tRNAPro1E2, C31G39/C28G42 mutation showed an approximately 3-fold enhancement of two consecutive incorporation of β-homophenylglycine (βPhg) at CCG codons. The use of this tRNA made it possible for the first time to elongate up to ten consecutive βPhg's. Since the enhancement effect of anticodon arm mutations differs depending on the codon used for β-amino acid incorporation, we optimized anticodon arm sequences for five codons (CCG, CAU, CAG, ACU and UGG). Combination of the five optimal tRNAs for these codons made it possible to introduce five different kinds of β-amino acids and analogs simultaneously into model peptides, including a macrocyclic scaffold. This strategy would enable ribosomal synthesis of libraries of macrocyclic peptides containing multiple β-amino acids., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Angiogenin, an RNase-A-family protein, promotes angiogenesis and has been implicated in cancer, neurodegenerative diseases and epigenetic inheritance 1-10 . After activation during cellular stress, angiogenin cleaves tRNAs at the anticodon loop, resulting in translation repression 11-15 . However, the catalytic activity of isolated angiogenin is very low, and the mechanisms of the enzyme activation and tRNA specificity have remained a puzzle 3,16-23 . Here we identify these mechanisms using biochemical assays and cryogenic electron microscopy (cryo-EM). Our study reveals that the cytosolic ribosome is the activator of angiogenin. A cryo-EM structure features angiogenin bound in the A site of the 80S ribosome. The C-terminal tail of angiogenin is rearranged by interactions with the ribosome to activate the RNase catalytic centre, making the enzyme several orders of magnitude more efficient in tRNA cleavage. Additional 80S-angiogenin structures capture how tRNA substrate is directed by the ribosome into angiogenin's active site, demonstrating that the ribosome acts as the specificity factor. Our findings therefore suggest that angiogenin is activated by ribosomes with a vacant A site, the abundance of which increases during cellular stress 24-27 . These results may facilitate the development of therapeutics to treat cancer and neurodegenerative diseases., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

The ribosome brings 3'-aminoacyl-tRNA and 3'-peptidyl-tRNAs together to enable peptidyl transfer by binding them in two major ways. First, their anticodon loops are bound to mRNA, itself anchored at the ribosomal subunit interface, by contiguous anticodon:codon pairing augmented by interactions with the decoding center of the small ribosomal subunit. Second, their acceptor stems are bound by the peptidyl transferase center, which aligns the 3'-aminoacyl- and 3'-peptidyl-termini for optimal interaction of the nucleophilic amino group and electrophilic ester carbonyl group. Reasoning that intrinsic codon:anticodon binding might have been a major contributor to bringing tRNA 3'-termini into proximity at an early stage of ribosomal peptide synthesis, we wondered if primordial amino acids might have been assigned to those codons that bind the corresponding anticodon loops most tightly. By measuring the binding of anticodon stem loops to short oligonucleotides, we determined that family-box codon:anticodon pairings are typically tighter than split-box codon:anticodon pairings. Furthermore, we find that two family-box anticodon stem loops can tightly bind a pair of contiguous codons simultaneously, whereas two split-box anticodon stem loops cannot. The amino acids assigned to family boxes correspond to those accessible by what has been termed cyanosulfidic chemistry, supporting the contention that these limited amino acids might have been the first used in primordial coded peptide synthesis.

The anticodon modifications of transfer RNAs (tRNAs) finetune the codon recognition on the ribosome for accurate translation. Bacteria and archaea utilize the modified cytidines, lysidine (L) and agmatidine (agm 2 C), respectively, in the anticodon of tRNA Ile to decipher AUA codon. L and agm 2 C contain long side chains with polar termini, but their functions remain elusive. Here we report the cryogenic electron microscopy structures of tRNAs Ile recognizing the AUA codon on the ribosome. Both modifications interact with the third adenine of the codon via a unique C-A geometry. The side chains extend toward 3' direction of the mRNA, and the polar termini form hydrogen bonds with 2'-OH of the residue 3'-adjacent to the AUA codon. Biochemical analyses demonstrated that AUA decoding is facilitated by the additional interaction between the polar termini of the modified cytidines and 2'-OH of the fourth mRNA residue. We also visualized cyclic N 6 -threonylcarbamoyladenosine (ct 6 A), another tRNA modification, and revealed a molecular basis how ct 6 A contributes to efficient decoding., (© 2024. The Author(s), under exclusive licence to Springer Nature America, Inc.)

The frequency of errors upon decoding of messenger RNA by the bacterial ribosome is low, with one misreading event per 1 × 10 4 codons. In the universal genetic code, the AUN codon box specifies two amino acids, isoleucine and methionine. In bacteria and archaea, decoding specificity of the AUA and AUG codons relies on the wobble avoidance strategy that requires modification of C34 in the anticodon loop of isoleucine transfer RNA Ile CAU (tRNA Ile CAU ). Bacterial tRNA Ile CAU with 2-lysylcytidine (lysidine) at the wobble position deciphers AUA while avoiding AUG. Here we report cryo-electron microscopy structures of the Escherichia coli 70S ribosome complexed with elongation factor thermo unstable (EF-Tu) and isoleucine-tRNA Ile LAU in the process of decoding AUA and AUG. Lysidine in tRNA Ile LAU excludes AUG by promoting the formation of an unusual Hoogsteen purine-pyrimidine nucleobase geometry at the third position of the codon, weakening the interactions with the mRNA and destabilizing the EF-Tu ternary complex. Our findings elucidate the molecular mechanism by which tRNA Ile LAU specifically decodes AUA over AUG., (© 2024. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Rapid and accurate translation is essential in all organisms to produce properly folded and functional proteins. mRNA codons that define the protein-coding sequences are decoded by tRNAs on the ribosome in the aminoacyl (A) binding site. The mRNA codon and the tRNA anticodon interaction is extensively monitored by the ribosome to ensure accuracy in tRNA selection. While other polymerases that synthesize DNA and RNA can correct for misincorporations, the ribosome is unable to correct mistakes. Instead, when a misincorporation occurs, the mismatched tRNA-mRNA pair moves to the peptidyl (P) site and, from this location, causes a reduction in the fidelity at the A site, triggering post-peptidyl transfer quality control. This reduced fidelity allows for additional incorrect tRNAs to be accepted and for release factor 2 (RF2) to recognize sense codons, leading to hydrolysis of the aberrant peptide. Here, we present crystal structures of the ribosome containing a tRNA Lys in the P site with a U•U mismatch with the mRNA codon. We find that when the mismatch occurs in the second position of the P-site codon-anticodon interaction, the first nucleotide of the A-site codon flips from the mRNA path to engage highly conserved 16S rRNA nucleotide A1493 in the decoding center. We propose that this mRNA nucleotide mispositioning leads to reduced fidelity at the A site. Further, this state may provide an opportunity for RF2 to initiate premature termination before erroneous nascent chains disrupt the cellular proteome., Competing Interests: Conflict of 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 © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Cognate tRNAs deliver specific amino acids to translating ribosomes according to the standard genetic code, and three codons with no cognate tRNAs serve as stop codons. Some protists have reassigned all stop codons as sense codons, neglecting this fundamental principle 1-4 . Here we analyse the in-frame stop codons in 7,259 predicted protein-coding genes of a previously undescribed trypanosomatid, Blastocrithidia nonstop. We reveal that in this species in-frame stop codons are underrepresented in genes expressed at high levels and that UAA serves as the only termination codon. Whereas new tRNAs Glu fully cognate to UAG and UAA evolved to reassign these stop codons, the UGA reassignment followed a different path through shortening the anticodon stem of tRNA Trp CCA from five to four base pairs (bp). The canonical 5-bp tRNA Trp recognizes UGG as dictated by the genetic code, whereas its shortened 4-bp variant incorporates tryptophan also into in-frame UGA. Mimicking this evolutionary twist by engineering both variants from B. nonstop, Trypanosoma brucei and Saccharomyces cerevisiae and expressing them in the last two species, we recorded a significantly higher readthrough for all 4-bp variants. Furthermore, a gene encoding B. nonstop release factor 1 acquired a mutation that specifically restricts UGA recognition, robustly potentiating the UGA reassignment. Virtually the same strategy has been adopted by the ciliate Condylostoma magnum. Hence, we describe a previously unknown, universal mechanism that has been exploited in unrelated eukaryotes with reassigned stop codons., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

Modified nucleotides in tRNAs are important determinants of folding, structure and function. Here we identify METTL8 as a mitochondrial matrix protein and active RNA methyltransferase responsible for installing m 3 C 32 in the human mitochondrial (mt-)tRNA Thr and mt-tRNA Ser(UCN) . METTL8 crosslinks to the anticodon stem loop (ASL) of many mt-tRNAs in cells, raising the question of how methylation target specificity is achieved. Dissection of mt-tRNA recognition elements revealed U 34 G 35 and t 6 A 37 /(ms 2 )i 6 A 37 , present concomitantly only in the ASLs of the two substrate mt-tRNAs, as key determinants for METTL8-mediated methylation of C 32 . Several lines of evidence demonstrate the influence of U 34 , G 35 , and the m 3 C 32 and t 6 A 37 /(ms 2 )i 6 A 37 modifications in mt-tRNA Thr/Ser(UCN) on the structure of these mt-tRNAs. Although mt-tRNA Thr/Ser(UCN) lacking METTL8-mediated m 3 C 32 are efficiently aminoacylated and associate with mitochondrial ribosomes, mitochondrial translation is mildly impaired by lack of METTL8. Together these results define the cellular targets of METTL8 and shed new light on the role of m 3 C 32 within mt-tRNAs., (© 2022. The Author(s).)

During protein synthesis on ribosome, tRNA recognizes its cognate codon of mRNA through base-pairing with the anticodon. The 5'-end nucleotide of the anticodon is capable of wobble base-pairing, offering a molecular basis for codon degeneracy. The wobble nucleotide is often targeted for post-transcriptional modification, which affects the specificity and fidelity of the decoding process. Flipping-out of a wobble nucleotide in the anticodon loop has been proposed to be necessary for modifying enzymes to access the target nucleotide, which has been captured in selective structures of protein-bound complexes. Meanwhile, all other structures of free or ribosome-bound tRNA display anticodon bases arranged in stacked conformation. We report the X-ray crystal structure of unbound tRNA Val1 to a 2.04 Å resolution showing two different conformational states of wobble uridine in the anticodon loop, one stacked on the neighboring base and the other swiveled out toward solvent. In addition, the structure reveals a rare magnesium ion coordination to the nitrogen atom of a nucleobase, which has been sampled very rarely among known structures of nucleic acids., (© 2021 Jeong and Kim; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

Human mitochondrial transcripts contain messenger and ribosomal RNAs flanked by transfer RNAs (tRNAs), which are excised by mitochondrial RNase (mtRNase) P and Z to liberate all RNA species. In contrast to nuclear or bacterial RNase P, mtRNase P is not a ribozyme but comprises three protein subunits that carry out RNA cleavage and methylation by unknown mechanisms. Here, we present the cryo-EM structure of human mtRNase P bound to precursor tRNA, which reveals a unique mechanism of substrate recognition and processing. Subunits TRMT10C and SDR5C1 form a subcomplex that binds conserved mitochondrial tRNA elements, including the anticodon loop, and positions the tRNA for methylation. The endonuclease PRORP is recruited and activated through interactions with its PPR and nuclease domains to ensure precise pre-tRNA cleavage. The structure provides the molecular basis for the first step of RNA processing in human mitochondria., (© 2021. The Author(s).)

Colicin D is a plasmid-encoded bacteriocin that specifically cleaves tRNA Arg of sensitive Escherichia coli cells. E. coli has four isoaccepting tRNA Arg s; the cleavage occurs at the 3' end of anticodon-loop, leading to translation impairment in the sensitive cells. tRNAs form a common L-shaped structure and have many conserved nucleotides that limit tRNA identity elements. How colicin D selects tRNA Arg s from the tRNA pool of sensitive E. coli cells is therefore intriguing. Here, we reveal the recognition mechanism of colicin D via biochemical analyses as well as structural modelling. Colicin D recognizes tRNA Arg ICG , the most abundant species of E. coli tRNA Arg s, at its anticodon-loop and D-arm, and selects it as the most preferred substrate by distinguishing its anticodon-loop sequence from that of others. It has been assumed that translation impairment is caused by a decrease in intact tRNA molecules due to cleavage. However, we found that intracellular levels of intact tRNA Arg ICG do not determine the viability of sensitive cells after such cleavage; rather, an accumulation of cleaved ones does. Cleaved tRNA Arg ICG dominant-negatively impairs translation in vitro . Moreover, we revealed that EF-Tu, which is required for the delivery of tRNAs, does not compete with colicin D for binding tRNA Arg ICG , which is consistent with our structural model. Finally, elevation of cleaved tRNA Arg ICG level decreases the viability of sensitive cells. These results suggest that cleaved tRNA Arg ICG transiently occupies ribosomal A-site in an EF-Tu-dependent manner, leading to translation impairment. The strategy should also be applicable to other tRNA-targeting RNases, as they, too, recognize anticodon-loops. Abbreviations: mnm 5 U: 5-methylaminomethyluridine; mcm 5 s 2 U: 5-methoxycarbonylmethyl-2-thiouridine.

Biogenic polyamines are natural aliphatic polycations formed from amino acids by biochemical pathways that are highly conserved from bacteria to humans. Their cellular concentrations are carefully regulated and dysregulation causes severe cell growth defects. Polyamines have high affinity for nucleic acids and are known to interact with mRNA, tRNA and rRNA to stimulate the translational machinery, but the exact molecular mechanism(s) for this stimulus is still unknown. Here we exploit that Escherichia coli is viable in the absence of polyamines, including the universally conserved putrescine and spermidine. Using global macromolecule labelling approaches we find that ribosome efficiency is reduced by 50-70% in the absence of polyamines and this reduction is caused by slow translation elongation speed. The low efficiency causes rRNA and multiple tRNA species to be overproduced in the absence of polyamines, suggesting an impact on the feedback regulation of stable RNA transcription. Importantly, we find that polyamine deficiency affects both tRNA levels and tRNA modification patterns. Specifically, a large fraction of tRNA his , tRNA tyr and tRNA asn lack the queuosine modification in the anticodon "wobble" base, which can be reversed by addition of polyamines to the growth medium. In conclusion, we demonstrate that polyamines are needed for modification of specific tRNA, possibly by facilitating the interaction with modification enzymes., Competing Interests: Conflict of interest statement None declared., (Copyright © 2021 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Specific interaction between the start codon, 5'-AUG-3', and the anticodon, 5'-CAU-3', ensures accurate initiation of translation. Recent studies show that several near-cognate start codons (e.g. GUG and CUG) can play a role in initiating translation in eukaryotes. However, the mechanism allowing initiation through mismatched base-pairs at the ribosomal decoding site is still unclear at an atomic level. In this work, we propose an extended simulation-based method to evaluate free energy profiles, through computing the distance between each base-pair of the triplet interactions involved in recognition of start codons in eukaryotic translation pre-initiation complex. Our method provides not only the free energy penalty for mismatched start codons relative to the AUG start codon, but also the preferred pathways of transitions between bound and unbound states, which has not been described by previous studies. To verify the method, the binding dynamics of cognate (AUG) and near-cognate start codons (CUG and GUG) were simulated. Evaluated free energy profiles agree with experimentally observed changes in initiation frequencies from respective codons. This work proposes for the first time how a G:U mismatch at the first position of codon (GUG)-anticodon base-pairs destabilizes the accommodation in the initiating eukaryotic ribosome and how initiation at a CUG codon is nearly as strong as, or sometimes stronger than, that at a GUG codon. Our method is expected to be applied to study the affinity changes for various mismatched base-pairs., Competing Interests: The authors have declared that no competing interests exist.

Transfer RNA (tRNA) is the most diversely modified RNA. Although the strictly conserved purine position 37 in the anticodon stem-loop undergoes modifications that are phylogenetically distributed, we do not yet fully understand the roles of these modifications. Therefore, molecular dynamics simulations are used to provide molecular-level details for how such modifications impact the structure and function of tRNA. A focus is placed on three hypermodified base families that include the parent i 6 A, t 6 A, and yW modifications, as well as derivatives. Our data reveal that the hypermodifications exhibit significant conformational flexibility in tRNA, which can be modulated by additional chemical functionalization. Although the overall structure of the tRNA anticodon stem remains intact regardless of the modification considered, the anticodon loop must rearrange to accommodate the bulky, dynamic hypermodifications, which includes changes in the nucleotide glycosidic and backbone conformations, and enhanced or completely new nucleobase-nucleobase interactions compared to unmodified tRNA or tRNA containing smaller (m 1 G) modifications at the 37th position. Importantly, the extent of the changes in the anticodon loop is influenced by the addition of small functional groups to parent modifications, implying each substituent can further fine-tune tRNA structure. Although the dominant conformation of the ASL is achieved in different ways for each modification, the molecular features of all modified tRNA drive the ASL domain to adopt the functional open-loop conformation. Importantly, the impact of the hypermodifications is preserved in different sequence contexts. These findings highlight the likely role of regulating mRNA structure and translation., (© 2021 Seelam Prabhakar et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

A recent crystal structure of a ribosome complex undergoing partial translocation in the absence of elongation factor EF-G showed disruption of codon-anticodon pairing and slippage of the reading frame by -1, directly implicating EF-G in preservation of the translational reading frame. Among mutations identified in a random screen for dominant-lethal mutations of EF-G were a cluster of six that map to the tip of domain IV, which has been shown to contact the codon-anticodon duplex in trapped translocation intermediates. In vitro synthesis of a full-length protein using these mutant EF-Gs revealed dramatically increased -1 frameshifting, providing new evidence for a role for domain IV of EF-G in maintaining the reading frame. These mutations also caused decreased rates of mRNA translocation and rotational movement of the head and body domains of the 30S ribosomal subunit during translocation. Our results are in general agreement with recent findings from Rodnina and coworkers based on in vitro translation of an oligopeptide using EF-Gs containing mutations at two positions in domain IV, who found an inverse correlation between the degree of frameshifting and rates of translocation. Four of our six mutations are substitutions at positions that interact with the translocating tRNA, in each case contacting the RNA backbone of the anticodon loop. We suggest that EF-G helps to preserve the translational reading frame by preventing uncoupled movement of the tRNA through these contacts; a further possibility is that these interactions may stabilize a conformation of the anticodon that favors base-pairing with its codon., (© 2021 Niblett et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

The translational accuracy in protein synthesis is contributed to by several mechanisms in the ribosome, generally called kinetic proofreading. This process in the ribosome inhibits the non-cognate codon-anticodon interaction. However, it is not sufficient for fidelity of protein synthesis since a wrong amino acid can easily be added to the growing polypeptide chain if a tRNA while cognate to the mRNA, carries a non-cognate amino acid. Therefore, additional to the kinetic proofreading, there must be some hitherto unknown characteristic in misacylated-tRNAs to stop the process of protein synthesis if such misacylated-tRNA is accommodated in the ribosomal A-site. In order to understand this characteristic, we have performed computational quantum chemistry analysis on five different tRNA molecules, each one attached to five different amino acids with one being cognate to the tRNA and the other four non-cognate. This study shows the importance of aminoacyl-tRNA binding energy in ensuring fidelity of protein synthesis.

Viruses commonly use specifically folded RNA elements that interact with both host and viral proteins to perform functions important for diverse viral processes. Examples are found at the 3' termini of certain positive-sense ssRNA virus genomes where they partially mimic tRNAs, including being aminoacylated by host cell enzymes. Valine-accepting tRNA-like structures (TLS Val ) are an example that share some clear homology with canonical tRNAs but have several important structural differences. Although many examples of TLS Val have been identified, we lacked a full understanding of their structural diversity and phylogenetic distribution. To address this, we undertook an in-depth bioinformatic and biochemical investigation of these RNAs, guided by recent high-resolution structures of a TLS Val We cataloged many new examples in plant-infecting viruses but also in unrelated insect-specific viruses. Using biochemical and structural approaches, we verified the secondary structure of representative TLS Val substrates and tested their ability to be valylated, confirming previous observations of structural heterogeneity within this class. In a few cases, large stem-loop structures are inserted within variable regions located in an area of the TLS distal to known host cell factor binding sites. In addition, we identified one virus whose TLS has switched its anticodon away from valine, causing a loss of valylation activity; the implications of this remain unclear. These results refine our understanding of the structural and functional mechanistic details of tRNA mimicry and how this may be used in viral infection., (© 2021 Sherlock et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

Because ambient temperature affects biochemical reactions, organisms living in extreme temperature conditions adapt protein composition and structure to maintain biochemical functions. While it is not feasible to experimentally determine optimal growth temperature (OGT) for every known microbial species, organisms adapted to different temperatures have measurable differences in DNA, RNA and protein composition that allow OGT prediction from genome sequence alone. In this study, we built a 'tRNA thermometer' model using tRNA sequence to predict OGT. We used sequences from 100 archaea and 683 bacteria species as input to train two Convolutional Neural Network models. The first pairs individual tRNA sequences from different species to predict which comes from a more thermophilic organism, with accuracy ranging from 0.538 to 0.992. The second uses the complete set of tRNAs in a species to predict optimal growth temperature, achieving a maximum ${r^2}$ of 0.86; comparable with other prediction accuracies in the literature despite a significant reduction in the quantity of input data. This model improves on previous OGT prediction models by providing a model with minimum input data requirements, removing laborious feature extraction and data preprocessing steps and widening the scope of valid downstream analyses., (Published by Oxford University Press on behalf of Nucleic Acids Research 2020.)

Various pathogenic variants in both mitochondrial tRNA Phe and Phenylalanyl-tRNA synthetase mitochondrial protein coding gene (FARS2) gene encoding for the human mitochondrial PheRS have been identified and associated with neurological and/or muscle-related pathologies. An important Guanine-34 (G34)A anticodon mutation associated with myoclonic epilepsy with ragged red fibers (MERRF) syndrome has been reported in hmit-tRNA Phe . The majority of G34 contacts in available aaRSs-tRNAs complexes specifically use that base as an important tRNA identity element. The network of intermolecular interactions providing its specific recognition also largely conserved. However, their conservation depends also on the invariance of the residues in the anticodon binding domain (ABD) of human mitochondrial Phenylalanyl-tRNA synthetase (hmit-PheRS). A defect in recognition of the anticodon of tRNA Phe may happen not only because of G34A mutation, but also due to mutations in the ABD. Indeed, a pathogenic mutation in FARS2 has been recently reported in a 9-year-old female patient harboring a p.Asp364Gly mutation. Asp364 is hydrogen bonded (HB) to G34 in WT hmit-PheRS. Thus, there are two pathogenic variants disrupting HB between G34 and Asp364: one is associated with G34A mutation, and the other with Asp364Gly mutation. We have measured the rates of tRNA Phe aminoacylation catalyzed by WT hmit-PheRS and mutant enzymes. These data ranked the residues making a HB with G34 according to their contribution to activity and the signal transduction pathway in the hmit-PheRS-tRNA Phe complex. Furthermore, we carried out extensive MD simulations to reveal the interdomain contact topology on the dynamic trajectories of the complex, and gaining insight into the structural and dynamic integrity effects of hmit-PheRS complexed with tRNA Phe . DATABASE: Structural data are available in PDB database under the accession number(s): 3CMQ, 3TUP, 5MGH, 5MGV., (© 2020 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Queuosine (Q) is a conserved tRNA modification in bacteria and eukaryotes. Eukaryotic Q-tRNA modification occurs through replacing the guanine base with the scavenged metabolite queuine at the wobble position of tRNAs with G34 U 35 N 36 anticodon (Tyr, His, Asn, Asp) by the QTRT1/QTRT2 heterodimeric enzyme encoded in the genome. In humans, Q-modification in tRNA Tyr and tRNA Asp are further glycosylated with galactose and mannose, respectively. Although galactosyl-Q (galQ) and mannosyl-Q (manQ) can be measured by LC/MS approaches, the difficulty of detecting and quantifying these modifications with low sample inputs has hindered their biological investigations. Here we describe a simple acid denaturing gel and nonradioactive northern blot method to detect and quantify the fraction of galQ/manQ-modified tRNA using just microgram amounts of total RNA. Our method relies on the secondary amine group of galQ/manQ becoming positively charged to slow their migration in acid denaturing gels commonly used for tRNA charging studies. We apply this method to determine the Q and galQ/manQ modification kinetics in three human cells lines. For Q-modification, tRNA Asp is modified the fastest, followed by tRNA His , tRNA Tyr , and tRNA Asn Compared to Q-modification, glycosylation occurs at a much slower rate for tRNA Asp , but at a similar rate for tRNA Tyr Our method enables easy access to study the function of these enigmatic tRNA modifications., (© 2020 Zhang et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

In the Elongator-dependent modification pathway, chemical modifications are introduced at the wobble uridines at position 34 in transfer RNAs (tRNAs), which serve to optimize codon translation rates. Here, we show that this three-step modification pathway exists in Dictyostelium discoideum, model of the evolutionary superfamily Amoebozoa. Not only are previously established modifications observable by mass spectrometry in strains with the most conserved genes of each step deleted, but also additional modifications are detected, indicating a certain plasticity of the pathway in the amoeba. Unlike described for yeast, D. discoideum allows for an unconditional deletion of the single tQCUG gene, as long as the Elongator-dependent modification pathway is intact. In gene deletion strains of the modification pathway, protein amounts are significantly reduced as shown by flow cytometry and Western blotting, using strains expressing different glutamine leader constructs fused to GFP. Most dramatic are these effects, when the tQCUG gene is deleted, or Elp3, the catalytic component of the Elongator complex is missing. In addition, Elp3 is the most strongly conserved protein of the modification pathway, as our phylogenetic analysis reveals. The implications of this observation are discussed with respect to the evolutionary age of the components acting in the Elongator-dependent modification pathway., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Bacterial transfer RNAs (tRNAs) contain evolutionarily conserved sequences and modifications that ensure uniform binding to the ribosome and optimal translational accuracy despite differences in their aminoacyl attachments and anticodon nucleotide sequences. In the tRNA anticodon stem-loop, the anticodon sequence is correlated with a base pair in the anticodon loop (nucleotides 32 and 38) to tune the binding of each tRNA to the decoding center in the ribosome. Disruption of this correlation renders the ribosome unable to distinguish correct from incorrect tRNAs. The molecular basis for how these two tRNA features combine to ensure accurate decoding is unclear. Here, we solved structures of the bacterial ribosome containing either wild-type [Formula: see text] or [Formula: see text] containing a reversed 32-38 pair on cognate and near-cognate codons. Structures of wild-type [Formula: see text] bound to the ribosome reveal 23S ribosomal RNA (rRNA) nucleotide A1913 positional changes that are dependent on whether the codon-anticodon interaction is cognate or near cognate. Further, the 32-38 pair is destabilized in the context of a near-cognate codon-anticodon pair. Reversal of the pairing in [Formula: see text] ablates A1913 movement regardless of whether the interaction is cognate or near cognate. These results demonstrate that disrupting 32-38 and anticodon sequences alters interactions with the ribosome that directly contribute to misreading., Competing Interests: The authors declare no competing interest.

Translation fidelity relies essentially on the ability of ribosomes to accurately recognize triplet interactions between codons on mRNAs and anticodons of tRNAs. To determine the codon-anticodon pairs that are efficiently accepted by the eukaryotic ribosome, we took advantage of the IRES from the intergenic region (IGR) of the Cricket Paralysis Virus. It contains an essential pseudoknot PKI that structurally and functionally mimics a codon-anticodon helix. We screened the entire set of 4096 possible combinations using ultrahigh-throughput screenings combining coupled transcription/translation and droplet-based microfluidics. Only 97 combinations are efficiently accepted and accommodated for translocation and further elongation: 38 combinations involve cognate recognition with Watson-Crick pairs and 59 involve near-cognate recognition pairs with at least one mismatch. More than half of the near-cognate combinations (36/59) contain a G at the first position of the anticodon (numbered 34 of tRNA). G34-containing tRNAs decoding 4-codon boxes are almost absent from eukaryotic genomes in contrast to bacterial genomes. We reconstructed these missing tRNAs and could demonstrate that these tRNAs are toxic to cells due to their miscoding capacity in eukaryotic translation systems. We also show that the nature of the purine at position 34 is correlated with the nucleotides present at 32 and 38., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Deaminations C->T and A->G are frequent mutations producing nucleotide content gradients across genomes proportional to singlestrandedness during replication/transcription. Hence, within single codons, deamination risks increase from first to third codon positions, while second codon positions are functionally most crucial. Here genetic codes are analyzed assuming that after anticodons protected codons from deaminations, first and second codon positions swapped (N2N1N3->N1N2N3), with lowest deamination risks for N2 in presumed primitive N2N1N3 codons. N2N1N3, not standard N1N2N3, codon structure minimizes deaminations inversely proportionally to cognate amino acid hydrophobicity and parallel betasheet conformational preference. For N1N2N3, deamination minimization increases with genetic code integration order of cognate amino acids: during the presumed N2N1N3->N1N2N3 codon structure transition, protein synthesis combined direct codon-amino acid interactions for late amino acids and tRNA-based translation for early amino acids. Hence N2N1N3 codons would correspond to tRNA-free translation by spontaneous codon-amino acid affinities, and tRNA-mediated translation presumably caused N2N1N3->N1N2N3 swaps. Results show that rational, not arbitrary rules link codon and amino acid structures. Some analyses detect mitochondrial RNAs and peptides in public data corresponding to systematic position swaps, suggesting occasional swapping polymerase activity., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Ubiquitous across all domains of life, tRNAs constitute an essential component of cellular physiology, carry out an indispensable role in protein synthesis, and have been historically the subject of a wide range of biochemical and biophysical studies as prototypical folded RNA molecules. Although conformational flexibility is a well-established characteristic of tRNA structure, it is typically regarded as an adaptive property exhibited in response to an inducing event, such as the binding of a tRNA synthetase or the accommodation of an aminoacyl-tRNA into the ribosome. In this study, we present crystallographic data of a tRNA molecule to expand on this paradigm by showing that structural flexibility and plasticity are intrinsic properties of tRNAs, apparent even in the absence of other factors. Based on two closely related conformations observed within the same crystal, we posit that unbound tRNAs by themselves are flexible and dynamic molecules. Furthermore, we demonstrate that the formation of the T-loop conformation by the tRNA TΨC stem-loop, a well-characterized and classic RNA structural motif, is possible even in the absence of important interactions observed in fully folded tRNAs., (© 2020 Chan et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

Expanding the chemical diversity of aptamers remains an important thrust in the field in order to increase their functional potential. Previously, our group developed LOOPER, which enables the incorporation of up to 16 unique modifications throughout a ssDNA sequence, and applied it to the in vitro evolution of thrombin binders. As LOOPER-derived highly modified nucleic acids polymers are governed by two interrelated evolutionary variables, namely, functional modifications and sequence, the evolution of this polymer contrasts with that of canonical DNA. Herein we provide in-depth analysis of the evolution, including structure-activity relationships, mapping of evolutionary pressures on the library, and analysis of plausible evolutionary pathways that resulted in the first LOOPER-derived aptamer, TBL1. A detailed picture of how TBL1 interacts with thrombin and how it may mimic known peptide binders of thrombin is also proposed. Structural modeling and folding studies afford insights into how the aptamer displays critical modifications and also how modifications enhance the structural stability of the aptamer. A discussion of benefits and potential limitations of LOOPER during in vitro evolution is provided, which will serve to guide future evolutions of this highly modified class of aptamers.

Of the four bases, guanine is the most susceptible to oxidation, which results in the formation of 8-oxoguanine (8-oxoG). In protein-free DNA, 8-oxodG adopts the syn conformation more frequently than the anti one. In the syn conformation, 8-oxodG base pairs with dA. The equilibrium between the anti and syn conformations of the adduct are known to be altered by the enzyme recognizing 8-oxodG. We previously showed that 8-oxoG in mRNA severely disrupts tRNA selection, but the underlying mechanism for these effects was not addressed. Here, we use miscoding antibiotics and ribosome mutants to probe how 8-oxoG interacts with the tRNA anticodon in the decoding center. Addition of antibiotics and introduction of error-inducing mutations partially suppressed the effects of 8-oxoG. Under these conditions, rates and/or endpoints of peptide-bond formation for the cognate (8-oxoG•C) and near-cognate (8-oxoG•A) aminoacyl-tRNAs increased. In contrast, the antibiotics had little effect on other mismatches, suggesting that the lesion restricts the nucleotide from forming other interactions. Our findings suggest that 8-oxoG predominantly adopts the syn conformation in the A site. However, its ability to base pair with adenosine in this conformation is not sufficient to promote the necessary structural changes for tRNA selection to proceed., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Aminoacyl tRNA synthetases ligate tRNAs specifically with their cognate amino acid. These synthetases are among life's earliest proteins, class II tRNA synthetases (cognates A, D, F, G, H, K, N, P, S, and T) presumably preceding class I tRNA synthetases (cognates C, E, I, L, M, Q, R, V, W, and Y). Classification of codons into palindromic (structure XYX), 5'-dominant (YXX), and 3'-dominant (XXY) (Codon Directional Asymmetry [CDA]) shows that class II tRNA synthetases aminoacylate amino acids associated with XXY. Our working hypothesis expects bias for XXY codons in primordial RNAs, such as theoretical minimal RNA rings, designed in silico to mimic life's earliest RNAs. Twenty-five RNA rings have been computed, which code over a minimal length (22 nucleotides) for a start codon, stop codon, and one and only one codon for each of the 20 amino acids, and form stem-loop hairpins preventing degradation; these 25 minimal RNAs are the only ones matching these constraints and they seem homologous to consensus tRNA sequences. This similarity defined candidate RNA ring anticodons and corresponding cognate amino acids. Here, analyses of RNA ring codon contents confirm bias for XXY codons in 13 among 14 RNA rings with unequal XXY and YXX codon numbers. This bias increases with the genetic code integration order of the RNA ring's cognate amino acid across and within tRNA synthetase classes, suggesting that evolutionary processes, and not physicochemical constraints, produced the association between CDA and tRNA synthetase classes. The self-referential hypothesis for genetic code origin, a very complete genetic code evolutionary hypothesis integrating many translational machinery components, predicts best among genetic code evolutionary hypotheses CDA biases in RNA rings. The RNA rings' simple design inadvertently reproduces CDAs predicted by the genetic code's structure, confirming theoretical minimal RNA rings as good proxies for life's earliest RNAs.

The genetic code likely arose when a bidirectional gene replicating as a quasi-species began to produce ancestral aminoacyl-tRNA synthetases (aaRS) capable of distinguishing between two distinct sets of amino acids. The synthetase class division therefore necessarily implies a mechanism by which the two ancestral synthetases could also discriminate between two different kinds of tRNA substrates. We used regression methods to uncover the possible patterns of base sequences capable of such discrimination and find that they appear to be related to thermodynamic differences in the relative stabilities of a hairpin necessary for recognition of tRNA substrates by Class I aaRS. The thermodynamic differences appear to be exploited by secondary structural differences between models for the ancestral aaRS called synthetase Urzymes and reinforced by packing of aromatic amino acid side chains against the nonpolar face of the ribose of A76 if and only if the tRNA CCA sequence forms a hairpin. The patterns of bases 1, 2, and 73 and stabilization of the hairpin by structural complementarity with Class I, but not Class II, aaRS Urzymes appear to be necessary and sufficient to have enabled the generation of the first two aaRS-tRNA cognate pairs, and the launch of a rudimentary binary genetic coding related recognizably to contemporary cognate pairs. As a consequence, it seems likely that nonrandom aminoacylation of tRNAs preceded the advent of the tRNA anticodon stem-loop. Consistent with this suggestion, coding rules in the acceptor-stem bases also reveal a palimpsest of the codon-anticodon interaction, as previously proposed. © 2019 IUBMB Life, 2019 © 2019 IUBMB Life, 71(8):1088-1098, 2019., (© 2019 International Union of Biochemistry and Molecular Biology.)

Transfer RNAs require essential maturation steps to become functional. Among them, RNase P removes 5' leader sequences of pre-tRNAs. Although RNase P was long thought to occur universally as ribonucleoproteins, different types of protein-only RNase P enzymes were discovered in both eukaryotes and prokaryotes. Interestingly, all these enzymes belong to the super-group of PilT N-terminal-like nucleases (PIN)-like ribonucleases. This wide family of enzymes can be subdivided into major subgroups. Here, we review recent studies at both functional and mechanistic levels on three PIN-like ribonucleases groups containing enzymes connected to tRNA maturation and/or translation regulation. The evolutive distribution of these proteins containing PIN-like domains as well as their organization and fusion with various functional domains is discussed and put in perspective with the diversity of functions they acquired during evolution, for the maturation and homeostasis of tRNA and a wider array of RNA substrates. © 2019 IUBMB Life, 2019 © 2019 IUBMB Life, 71(8):1117-1125, 2019., (© 2019 International Union of Biochemistry and Molecular Biology.)

The highly conserved Elongator complex modifies transfer RNAs (tRNAs) in their wobble base position, thereby regulating protein synthesis and ensuring proteome stability. The precise mechanisms of tRNA recognition and its modification reaction remain elusive. Here, we show cryo-electron microscopy structures of the catalytic subcomplex of Elongator and its tRNA-bound state at resolutions of 3.3 and 4.4 Å. The structures resolve details of the catalytic site, including the substrate tRNA, the iron-sulfur cluster, and a SAM molecule, which are all validated by mutational analyses in vitro and in vivo. tRNA binding induces conformational rearrangements, which precisely position the targeted anticodon base in the active site. Our results provide the molecular basis for substrate recognition of Elongator, essential to understand its cellular function and role in neurodegenerative diseases and cancer.

tRNA His guanylyltransferase (Thg1) adds a single guanine to the -1 position of tRNA His as part of its maturation. This seemingly modest addition of one nucleotide to tRNA His ensures translational fidelity by providing a critical identity element for the histidyl aminoacyl tRNA synthetase (HisRS). Like HisRS, Thg1 utilizes the GUG anticodon for selective tRNA His recognition, and Thg1-tRNA complex structures have revealed conserved residues that interact with anticodon nucleotides. Separately, kinetic analysis of alanine variants has demonstrated that many of these same residues are required for catalytic activity. A model in which loss of activity with the variants was attributed directly to loss of the critical anticodon interaction has been proposed to explain the combined biochemical and structural results. Here we used RNA chemical footprinting and binding assays to test this model and further probe the molecular basis for the requirement for two critical tRNA-interacting residues, His-152 and Lys-187, in the context of human Thg1 (hThg1). Surprisingly, we found that His-152 and Lys-187 alanine-substituted variants maintain a similar overall interaction with the anticodon region, arguing against the sufficiency of this interaction for driving catalysis. Instead, conservative mutagenesis revealed a new direct function for these residues in recognition of a non-Watson-Crick G -1 :A 73 bp, which had not been described previously. These results have important implications for the evolution of eukaryotic Thg1 from a family of ancestral promiscuous RNA repair enzymes to the highly selective enzymes needed for their essential function in tRNA His maturation., (© 2019 Matlock et al.)

1-Methyladenosine (m1A) is a modified nucleoside found at positions 9, 14, 22 and 58 of tRNAs, which arises from the transfer of a methyl group onto the N1-atom of adenosine. The yqfN gene of Bacillus subtilis encodes the methyltransferase TrmK (BsTrmK) responsible for the formation of m1A22 in tRNA. Here, we show that BsTrmK displays a broad substrate specificity, and methylates seven out of eight tRNA isoacceptor families of B. subtilis bearing an A22. In addition to a non-Watson-Crick base-pair between the target A22 and a purine at position 13, the formation of m1A22 by BsTrmK requires a full-length tRNA with intact tRNA elbow and anticodon stem. We solved the crystal structure of BsTrmK showing an N-terminal catalytic domain harbouring the typical Rossmann-like fold of Class-I methyltransferases and a C-terminal coiled-coil domain. We used NMR chemical shift mapping to drive the docking of BstRNASer to BsTrmK in complex with its methyl-donor cofactor S-adenosyl-L-methionine (SAM). In this model, validated by methyltransferase activity assays on BsTrmK mutants, both domains of BsTrmK participate in tRNA binding. BsTrmK recognises tRNA with very few structural changes in both partner, the non-Watson-Crick R13-A22 base-pair positioning the A22 N1-atom close to the SAM methyl group., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Adenosine deaminase acting on transfer RNA (ADAT) is an essential eukaryotic enzyme that catalyzes the deamination of adenosine to inosine at the first position of tRNA anticodons. Mammalian ADATs modify eight different tRNAs, having increased their substrate range from a bacterial ancestor that likely deaminated exclusively tRNA Arg Here we investigate the recognition mechanisms of tRNA Arg and tRNA Ala by human ADAT to shed light on the process of substrate expansion that took place during the evolution of the enzyme. We show that tRNA recognition by human ADAT does not depend on conserved identity elements, but on the overall structural features of tRNA. We find that ancestral-like interactions are conserved for tRNA Arg , while eukaryote-specific substrates use alternative mechanisms. These recognition studies show that human ADAT can be inhibited by tRNA fragments in vitro, including naturally occurring fragments involved in important regulatory pathways., (© 2019 Roura Frigolé et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

Modification of anticodon nucleotides allows tRNAs to decode multiple codons, expanding the genetic code. Additionally, modifications located in the anticodon loop, outside the anticodon itself, stabilize tRNA–codon interactions, increasing decoding fidelity. Anticodon loop nucleotide 37 is 3′ to the anticodon and, in tRNA CGG Pro , is methylated at the N1 position in its nucleobase (m 1 G37). The m 1 G37 modification in tRNA CGG Pro stabilizes its interaction with the codon and maintains the mRNA frame. However, it is unclear how m 1 G37 affects binding at the decoding center to both cognate and +1 slippery codons. Here, we show that the tRNA CGG Pro m 1 G37 modification is important for the association step during binding to a cognate CCG codon. In contrast, m 1 G37 prevented association with a slippery CCC-U or +1 codon. Similar analyses of frameshift suppressor tRNA SufA6 , a tRNA CGG Pro derivative containing an extra nucleotide in its anticodon loop that undergoes +1 frameshifting, reveal that m 1 G37 destabilizes interactions with both the cognate CCG and slippery codons. One reason for this destabilization is the disruption of a conserved U32·A38 nucleotide pairing in the anticodon stem through insertion of G37.5. Restoring the tRNA SufA6 U32·A37.5 pairing results in a high-affinity association on the slippery CCC-U codon. Further, an X-ray crystal structure of the 70S ribosome bound to tRNA SufA6 U32·A37.5 at 3.6 Å resolution shows a reordering of the anticodon loop consistent with the findings from the high-affinity measurements. Our results reveal how the tRNA modification at nucleotide 37 stabilizes interactions with the mRNA codon to preserve the mRNA frame., (© 2019 Nguyen et al.)

All types of cellular RNAs are post-transcriptionally modified, constituting the so called 'epitranscriptome'. In particular, tRNAs and their anticodon stem loops represent major modification hotspots. The attachment of small chemical groups at the heart of the ribosomal decoding machinery can directly affect translational rates, reading frame maintenance, co-translational folding dynamics and overall proteome stability. The variety of tRNA modification patterns is driven by the activity of specialized tRNA modifiers and large modification complexes. Notably, the absence or dysfunction of these cellular machines is correlated with several human pathophysiologies. In this review, we aim to highlight the most recent scientific progress and summarize currently available structural information of the most prominent eukaryotic tRNA modifiers., (Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Initiator tRNAs (i-tRNAs) possess highly conserved three consecutive GC base pairs (GC/GC/GC, 3GC pairs) in their anticodon stems. Additionally, in bacteria and eukaryotic organelles, the amino acid attached to i-tRNA is formylated by Fmt to facilitate its targeting to 30S ribosomes. Mutations in GC/GC/GC to UA/CG/AU in i-tRNACUA/3GC do not affect its formylation. However, the i-tRNACUA/3GC is non-functional in initiation. Here, we characterised an Escherichia coli strain possessing an amber mutation in its fmt gene (fmtam274), which affords initiation with i-tRNACUA/3GC. Replacement of fmt with fmtam274 in the parent strain results in production of truncated Fmt, accumulation of unformylated i-tRNA, and a slow growth phenotype. Introduction of i-tRNACUA/3GC into the fmtam274 strain restores accumulation of formylated i-tRNAs and rescues the growth defect of the strain. We show that i-tRNACUA/3GC causes a low level suppression of am274 in fmtam274. Low levels of cellular Fmt lead to compromised efficiency of formylation of i-tRNAs, which in turn results in distribution of the charged i-tRNAs between IF2 and EF-Tu allowing the plasmid borne i-tRNACUA/3GC to function at both the initiation and elongation steps. We show that a speedy formylation of i-tRNA population is crucial for its preferential binding (and preventing other tRNAs) into the P-site., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Decoding is thought to be governed by a conformational transition in the ribosome-open (off) to closed (on)-that occurs upon codon-anticodon pairing in the A site. Ribosomal ambiguity (ram) mutations increase miscoding and map to disparate regions, consistent with a role for ribosome dynamics in decoding, yet precisely how these mutations act has been unclear. Here, we solved crystal structures of 70S ribosomes harboring 16S ram mutations G299A and G347U in the absence A-site tRNA (A-tRNA) and in the presence of a near-cognate anticodon stem-loop (ASL). In the absence of an A-tRNA, each of the mutant ribosomes exhibits a partially closed (on) state. In the 70S-G347U structure, the 30S shoulder is rotated inward and intersubunit bridge B8 is disrupted. In the 70S-G299A structure, the 30S shoulder is rotated inward and decoding nucleotide G530 flips into the anti conformation. Both of these mutant ribosomes adopt the fully closed (on) conformation in the presence of near-cognate A-tRNA, just as they do with cognate A-tRNA. Thus, these ram mutations act by promoting the open (off) to closed (on) transition, albeit in somewhat distinct ways. This work reveals the functional importance of 30S shoulder rotation for productive aminoacylated-tRNA incorporation., (© The Author(s) 2018. Published by Oxford University Press on behalf of Nucleic Acids Research.)

tRNAs are the central adaptor molecules in translation. Their decoding properties are influenced by post-transcriptional modifications, particularly in the critical anticodon-stem-loop (ASL) region. Synonymous codon choice, also called codon usage bias, affects both translation efficiency and accuracy, and ASL modifications play key roles in both of these processes. In combination with a handful of historical examples, recent studies integrating ribosome profiling, proteomics, codon-usage analyses, and modification quantifications show that levels of tRNA modifications can change under stress, during development, or under specific metabolic conditions and can modulate the expression of specific genes. Deconvoluting the different responses (global or specific) to tRNA modification deficiencies can be difficult because of pleiotropic effects, but, as more cases emerge, it does seem that tRNA modification changes could add another layer of regulation in the transfer of information from DNA to protein.

The precise interplay between the mRNA codon and the tRNA anticodon is crucial for ensuring efficient and accurate translation by the ribosome. The insertion of RNA nucleobase derivatives in the mRNA allowed us to modulate the stability of the codon-anticodon interaction in the decoding site of bacterial and eukaryotic ribosomes, allowing an in-depth analysis of codon recognition. We found the hydrogen bond between the N 1 of purines and the N 3 of pyrimidines to be sufficient for decoding of the first two codon nucleotides, whereas adequate stacking between the RNA bases is critical at the wobble position. Inosine, found in eukaryotic mRNAs, is an important example of destabilization of the codon-anticodon interaction. Whereas single inosines are efficiently translated, multiple inosines, e.g., in the serotonin receptor 5-HT 2C mRNA, inhibit translation. Thus, our results indicate that despite the robustness of the decoding process, its tolerance toward the weakening of codon-anticodon interactions is limited.

Class I and II aaRS recognition of opposite grooves was likely among the earliest determinants fixed in the tRNA acceptor stem bases. A new regression model identifies those determinants in bacterial tRNAs. Integral coefficients relate digital dependent to independent variables with perfect agreement between observed and calculated grooves for all twenty isoaccepting tRNAs. Recognition is mediated by the Discriminator base 73, the first base pair, and base 2 of the acceptor stem. Subsets of these coefficients also identically compute grooves recognized by smaller numbers of aaRS. Thus, the model is hierarchical, suggesting that new rules were added to pre-existing ones as new amino acids joined the coding alphabet. A thermodynamic rationale for the simplest model implies that Class-dependent aaRS secondary structures exploited differential tendencies of the acceptor stem to form the hairpin observed in Class I aaRS•tRNA complexes, enabling the earliest groove discrimination. Curiously, groove recognition also depends explicitly on the identity of base 2 in a manner consistent with the middle bases of the codon table, confirming a hidden ancestry of codon-anticodon pairing in the acceptor stem. That, and the lack of correlation with anticodon bases support prior productive coding interaction of tRNA minihelices with proto-mRNA.

Alanine is encoded by the four codons of the GC box (GCA, GCG, GCU, and GCC). Known alanine anticodons include the UGC, IGC, and VGC triplets (I = inosine; V = uridine-5-oxyacetic acid). The energy-minimized structures of all possible codon-anticodon combinations involving all the alanine codons GCA, GCG, GCU, and GCC with the alanine anticodons UGC, IGC, and VGC are studied using the AMBER software. Fifteen H-bonded duplex structures arising out of these combinations are studied here, all having Watson-Crick-type base pairs at the first and second codon positions, and a variety of base pairing possibilities at the third (or wobble) position. Structural and stability considerations suggest that some codon-anticodon duplexes would be more favored than others for accommodation during the translation process. The UGC anticodon is predicted to favor the GCA codon for reading, while the GCC codon is least favored. The IGC anticodon would prefer to read the GCC codon, the GCG codon being least favored, while a syn conformer for A in the GCA codon could allow for it to be read. For the VGC anticodon, the GCA codon is predicted to be read most favorably, and the GCC codon least favorably, while a syn conformer for V in the anticodon would allow for the codon GCU to be read through a wobble pair which involves the exocyclic 5-oxyacetate group of V in H-bonding.

The decipherment of the tRNA's operational code, known as the identity problem, requires the location of the sites in the tRNA structure that are involved in their correct recognition by the corresponding aminoacyl-tRNA synthetase. In this work, we determine the identity elements of each tRNA isoacceptor by means of the variation of information measure from information theory. We show that all isoacceptors exhibit sites associated with some bases of the anticodon. These sites form clusters that are scattered along the tRNA structure. The clusters determine the identity elements of each tRNA. We derive a catalogue of clustered sites for each tRNA that expands previously reported elements.

Hypermodified bases present at 3'-adjacent (37 th ) position in anticodon loop of tRNA Phe are well known for their contribution in modulating codon-anticodon interactions. Peroxywybutosine (o2yW), a wyosine family member, is one of such tricyclic modified bases observed at the 37 th position in tRNA Phe . Conformational preferences and three-dimensional structural analysis of peroxywybutosine have not been investigated in detail at atomic level. Hence, in the present study quantum chemical semi-empirical RM1 and multiple molecular dynamics (MD) simulations have been used to study structural significance of peroxywybutosine in tRNA Phe . Full geometry optimizations over the peroxywybutosine base have also been performed using ab-initio HF-SCF (6-31G**), DFT (B3LYP/6-31G**) and semi-empirical PM6 method to compare the salient properties. RM1 predicted most stable structure shows that the amino-carboxy-propyl side chain of o2yW remains 'distal' to the five membered imidazole ring of tricyclic guanosine. MD simulation trajectory of the isolated peroxy base showed restricted periodical fluctuations of peroxywybutosine side chain which might be helpful to maintain proper anticodon loop structure and mRNA reading frame during protein biosynthesis process. Another comparative MD simulation study of the anticodon stem loop with codon UUC showed various properties, which justify the functional implications of peroxywybutosine at 37 th position along with other modified bases present in ASL of tRNA Phe . Thus, this study presents an atomic view into the structural properties of peroxywybutosine, which can be useful to determine its role in the anticodon stem loop in context of codon-anticodon interactions and frame shift mutations., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Endoribonuclease toxins (ribotoxins) are produced by bacteria and fungi to respond to stress, eliminate non-self competitor species, or interdict virus infection. PrrC is a bacterial ribotoxin that targets and cleaves tRNA Lys UUU in the anticodon loop. In vitro studies suggested that the post-transcriptional modification threonylcarbamoyl adenosine (t 6 A) is required for PrrC activity but this prediction had never been validated in vivo. Here, by using t 6 A-deficient yeast derivatives, it is shown that t 6 A is a positive determinant for PrrC proteins from various bacterial species. Streptococcus mutans is one of the few bacteria where the t 6 A synthesis gene tsaE (brpB) is dispensable and its genome encodes a PrrC toxin. We had previously shown using an HPLC-based assay that the S. mutans tsaE mutant was devoid of t 6 A. However, we describe here a novel and a more sensitive hybridization-based t 6 A detection method (compared to HPLC) that showed t 6 A was still present in the S. mutans ΔtsaE, albeit at greatly reduced levels (93% reduced compared with WT). Moreover, mutants in 2 other S. mutans t 6 A synthesis genes (tsaB and tsaC) were shown to be totally devoid of the modification thus confirming its dispensability in this organism. Furthermore, analysis of t 6 A modification ratios and of t 6 A synthesis genes mRNA levels in S. mutans suggest they may be regulated by growth phase.

High-fidelity translation and a strictly accurate proteome were originally assumed as essential to life and cellular viability. Yet recent studies in bacteria and eukaryotic model organisms suggest that proteome-wide mistranslation can provide selective advantages and is tolerated in the cell at higher levels than previously thought (one error in 6.9 × 10 -4 in yeast) with a limited impact on phenotype. Previously, we selected a tRNA Pro containing a single mutation that induces mistranslation with alanine at proline codons in yeast. Yeast tolerate the mistranslation by inducing a heat-shock response and through the action of the proteasome. Here we found a homologous human tRNA Pro (G3:U70) mutant that is not aminoacylated with proline, but is an efficient alanine acceptor. In live human cells, we visualized mistranslation using a green fluorescent protein reporter that fluoresces in response to mistranslation at proline codons. In agreement with measurements in yeast, quantitation based on the GFP reporter suggested a mistranslation rate of up to 2-5% in HEK 293 cells. Our findings suggest a stress-dependent phenomenon where mistranslation levels increased during nutrient starvation. Human cells did not mount a detectable heat-shock response and tolerated this level of mistranslation without apparent impact on cell viability. Because humans encode ∼600 tRNA genes and the natural population has greater tRNA sequence diversity than previously appreciated, our data also demonstrate a cell-based screen with the potential to elucidate mutations in tRNAs that may contribute to or alleviate disease.

The modification of adenosine to inosine at position 34 of tRNA anticodons has a profound impact upon codon-anticodon recognition. In bacteria, I34 is thought to exist only in tRNA Arg , while in eukaryotes the modification is present in eight different tRNAs. In eukaryotes, the widespread use of I34 strongly influenced the evolution of genomes in terms of tRNA gene abundance and codon usage. In humans, codon usage indicates that I34 modified tRNAs are preferred for the translation of highly repetitive coding sequences, suggesting that I34 is an important modification for the synthesis of proteins of highly skewed amino acid composition. Here we extend the analysis of distribution of codons that are recognized by I34 containing tRNAs to all phyla known to use this modification. We find that the preference for codons recognized by such tRNAs in genes with highly biased codon compositions is universal among eukaryotes, and we report that, unexpectedly, some bacterial phyla show a similar preference. We demonstrate that the genomes of these bacterial species contain previously undescribed tRNA genes that are potential substrates for deamination at position 34.

The currently available structural information is insufficient for a detailed analysis of interactions between human glycyl-tRNA synthetase (GARS) and enterovirus IRESs. At the same time, this information is required in order to understand how this IRES trans-acting factor (ITAF) functions during viral mRNA translation, which is in turn crucial for the development of direct-action antiviral agents. In this paper, a theoretical model of the complex between a cadicivirus A IRES fragment and the anticodon-binding domain of human GARS is constructed using molecular dynamics simulation based on all of the available structural and biochemical data. The proposed model enables the structural interpretation of the previously obtained biochemical data.

Horizontal gene transfer is crucial for the adaptation of microorganisms to environmental cues. The acidophilic, bioleaching bacterium Acidithiobacillus ferrooxidans encodes an integrative-conjugative genetic element (ICEAfe1) inserted in the gene encoding a tRNA Ala . This genetic element is actively excised from the chromosome upon induction of DNA damage. A similar genetic element (ICEAca TY .2) is also found in an equivalent position in the genome of Acidithiobacillus caldus. The local genomic context of both mobile genetic elements is highly syntenous and the cognate integrases are well conserved. By means of site directed mutagenesis, target site deletions and in vivo integrations assays in the heterologous model Escherichia coli, we assessed the target sequence requirements for site-specific recombination to be catalyzed by these integrases. We determined that each enzyme recognizes a specific small DNA segment encoding the anticodon stem/loop of the tRNA as target site and that specific positions in these regions are well conserved in the target attB sites of orthologous integrases. Also, we demonstrate that the local genetic context of the target sequence is not relevant for the integration to take place. These findings shed new light on the mechanism of site-specific integration of integrative-conjugative elements in members of Acidithiobacillus genus.