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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.

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.)

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spreads all over the world and brings great harm to humans in many countries. Many new SARS-CoV-2 variants appeared during its transmission. In the present study, the Delta variants (B.1.617.2) of SARS-CoV-2, which have appeared in many countries, were considered for analysis. In order to evaluate the evolutionary divergence of the Delta variants(B.1.617.2), the codon usage divergence in Delta variants (B.1.617.2) of SARS-CoV-2 was compared to that of the SARS-CoV-2 genomes emerged before June 2020. All Delta variants (B.1.617.2) and 350 early genomes of SARS-CoV-2 in the NCBI database were downloaded. Codon usage pattern including the basic composition, the GC ratio of the third position (GC3) and the first two positions (GC12) in codons, overall GC contents, the effective number of codons (ENC), the codon bias index (CBI), the relative synonymous codon usage (RSCU) values, etc., of all concerned important gene sequences were all calculated. Codon usage divergence of them was calculated via summing their standard deviations. The results suggested that base compositions in both Delta variants (B.1.617.2) of SARS-CoV-2 and the early SARS-CoV-2 genomes were similar to each other. However, the internal codon usage divergence for most genes in Delta variants (B.1.617.2) was significantly wider than that of SARS-CoV-2. The RSCU values were further used to explore the synonymous and non-synonymous mutations in the sequences of the Delta variants (B.1.617.2), and the results showed the synonymous mutations are more obvious than the non-synonymous in the concerned sequences. The related codon usage divergence analysis is helpful for further study on the adaptability and disease prognosis of the SARS-CoV-2 variants., (Copyright © 2021. Published by Elsevier B.V.)

A key characteristic of cancer cells is their increased proliferative capacity, which requires elevated levels of protein synthesis. The process of protein synthesis involves the translation of codons within the mRNA coding sequence into a string of amino acids to form a polypeptide chain. As most amino acids are encoded by multiple codons, the nucleotide sequence of a coding region can vary dramatically without altering the polypeptide sequence of the encoded protein. Although mutations that do not alter the final amino acid sequence are often thought of as silent/synonymous, these can still have dramatic effects on protein output. Because each codon has a distinct translation elongation rate and can differentially impact mRNA stability, each codon has a different degree of 'optimality' for protein synthesis. Recent data demonstrates that the codon preference of a transcriptome matches the abundance of tRNAs within the cell and that this supply and demand between tRNAs and mRNAs varies between different cell types. The largest observed distinction is between mRNAs encoding proteins associated with proliferation or differentiation. Nevertheless, precisely how codon optimality and tRNA expression levels regulate cell fate decisions and their role in malignancy is not fully understood. This review describes the current mechanistic understanding on codon optimality, its role in malignancy and discusses the potential to target codon optimality therapeutically in the context of cancer., (© 2021. The Author(s).)

MHC-I associated peptides (MAPs) play a central role in the elimination of virus-infected and neoplastic cells by CD8 T cells. However, accurately predicting the MAP repertoire remains difficult, because only a fraction of the transcriptome generates MAPs. In this study, we investigated whether codon arrangement (usage and placement) regulates MAP biogenesis. We developed an artificial neural network called Codon Arrangement MAP Predictor (CAMAP), predicting MAP presentation solely from mRNA sequences flanking the MAP-coding codons (MCCs), while excluding the MCC per se. CAMAP predictions were significantly more accurate when using original codon sequences than shuffled codon sequences which reflect amino acid usage. Furthermore, predictions were independent of mRNA expression and MAP binding affinity to MHC-I molecules and applied to several cell types and species. Combining MAP ligand scores, transcript expression level and CAMAP scores was particularly useful to increase MAP prediction accuracy. Using an in vitro assay, we showed that varying the synonymous codons in the regions flanking the MCCs (without changing the amino acid sequence) resulted in significant modulation of MAP presentation at the cell surface. Taken together, our results demonstrate the role of codon arrangement in the regulation of MAP presentation and support integration of both translational and post-translational events in predictive algorithms to ameliorate modeling of the immunopeptidome., Competing Interests: The authors have declared that no competing interests exist.

Understanding the origins of mutations in tumor suppressor genes and oncogenes associated with cancers in different tissues is critical to the development of potential prevention strategies. Analysis of >10,000 nonsense mutations in 63 tumor suppressor genes based on the ratio of the number of nonsense mutations per codon type is reported for each gene. The ratio for C•G→T•A nonsense mutations at Arg CGA codons to the number of CGA codons in all cancers is 23 (3088 total nonsense mutations for 134 CGA codons in the 63 suppressor genes). The ratio for this codon, which is attributed to hydrolytic deamination of 5-methylcytosine at CpG sites based on the sequence context, is 6-fold higher than the next highest ratio that involves a C•G→T•A transition at Trp TGG codons. C•G→A•T transversions at Glu, Ser, Tyr, Gly and Cys codons account for 25 % of the total nonsense mutations but the mutation per codon ratio for these codons is 1.0. Analysis of the bases 5' of the mutated CGA codons in the 63 tumor suppressor genes in all cancers shows a preference of 5'-G > C ∼ T ∼ A, which is not indicative of a role for enzymatic deamination by deaminases. Overall C•G→T•A mutations account for 61 % of all of the nonsense mutations in the collection of tumor suppressor genes. It is demonstrated that the ratio of C•G→T•A deamination-associated nonsense mutations at CGA codons (hydrolytic deamination) to the number of frame shift insertion/deletion mutations (i.e., replication based) for 5 major tumor suppressors genes are very similar in 3 different tissues that undergo a wide range of stem cell divisions. Therefore, the frequency of deamination mutations parallels the number of stem cell replications. This may reflect the generation of more solvent accessible single-stranded DNA regions during polymerization that are kinetically more prone to deamination., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Synonymous codon substitutions are not always selectively neutral as revealed by several types of analyses, including studies of codon usage patterns among genes. We analyzed codon usage in 13 bacterial genomes sampled from across a large order of bacteria, Enterobacterales, and identified presumptively neutral and selected classes of synonymous substitutions. To estimate substitution rates, given a neutral/selected classification of synonymous substitutions, we developed a flexible [Formula: see text] substitution model that allows multiple classes of synonymous substitutions. Under this multiclass synonymous substitution (MSS) model, the denominator of [Formula: see text] includes only the strictly neutral class of synonymous substitutions. On average, the value of [Formula: see text] under the MSS model was 80% of that under the standard codon model in which all synonymous substitutions are assumed to be neutral. The indication is that conventional [Formula: see text] analyses overestimate these values and thus overestimate the frequency of positive diversifying selection and underestimate the strength of purifying selection. To quantify the strength of selection necessary to explain this reduction, we developed a model of selected compensatory codon substitutions. The reduction in synonymous substitution rate, and thus the contribution that selection makes to codon bias variation among genes, can be adequately explained by very weak selection, with a mean product of population size and selection coefficient, [Formula: see text]., Competing Interests: The authors declare no competing interest.

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.)

Ribosome profiling, also known as Ribo-seq, has become a popular approach to investigate regulatory mechanisms of translation in a wide variety of biological contexts. Ribo-seq not only provides a measurement of translation efficiency based on the relative abundance of ribosomes bound to transcripts, but also has the capacity to reveal dynamic and local regulation at different stages of translation based on positional information of footprints across individual transcripts. While many computational tools exist for the analysis of Ribo-seq data, no method is currently available for rigorous testing of the pattern differences in ribosome footprints. In this work, we develop a novel approach together with an R package, RiboDiPA, for Differential Pattern Analysis of Ribo-seq data. RiboDiPA allows for quick identification of genes with statistically significant differences in ribosome occupancy patterns for model organisms ranging from yeast to mammals. We show that differential pattern analysis reveals information that is distinct and complimentary to existing methods that focus on translational efficiency analysis. Using both simulated Ribo-seq footprint data and three benchmark data sets, we illustrate that RiboDiPA can uncover meaningful pattern differences across multiple biological conditions on a global scale, and pinpoint characteristic ribosome occupancy patterns at single codon resolution., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The residue 2-thiouridine (s 2 U) provides a remarkable example for the "modified wobble" hypothesis, which postulates that some post-transcriptional modifications at the wobble position of tRNAs restrict recognition of degenerate codons. Through extensive molecular dynamics simulations using our χ IDRP force field parameters, we demonstrate how this modification shifts the conformational ensemble from a predominantly disordered, as in the case of an RNA pentamer (GUUUC), to a substantially ordered population in Gs 2 UUUC. Our simulations clearly showed that the van der Waals interaction of sulfur played a major role in driving the disorder-to-order transition. The conformational redistribution and the slowing down of the transition between the clusters within the population in the presence of s 2 U suggest ensemble allostery to be a key mechanism that may play a general role in the functioning of the wobble modifications of tRNAs.

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.)

Two optimization strategies, codon usage modification and glycine supplementation, were adopted to improve the extracellular production of Bacillus sp. NR5 UPM β-cyclodextrin glycosyltransferase (CGT-BS) in recombinant Escherichia coli . Several rare codons were eliminated and replaced with the ones favored by E. coli cells, resulting in an increased codon adaptation index (CAI) from 0.67 to 0.78. The cultivation of the codon modified recombinant E. coli following optimization of glycine supplementation enhanced the secretion of β-CGTase activity up to 2.2-fold at 12 h of cultivation as compared to the control. β-CGTase secreted into the culture medium by the transformant reached 65.524 U/mL at post-induction temperature of 37 °C with addition of 1.2 mM glycine and induced at 2 h of cultivation. A 20.1-fold purity of the recombinant β-CGTase was obtained when purified through a combination of diafiltration and nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography. This combined strategy doubled the extracellular β-CGTase production when compared to the single approach, hence offering the potential of enhancing the expression of extracellular enzymes, particularly β-CGTase by the recombinant E. coli .

Stereochemical nucleotide-amino acid interactions, in the form of noncovalent nucleotide-amino acid interactions, potentially produced the genetic code's codon-amino acid assignments. Empirical estimates of single nucleotide-amino acid affinities on surfaces and in solution are used to test whether trinucleotide-amino acid affinities determined genetic code assignments pending the principle "first arrived, first served": presumed early amino acids have greater codon-amino acid affinities than ulterior ones. Here, these single nucleotide affinities are used to approximate all 64 × 20 trinucleotide-amino acid affinities. Analyses show that (1) on surfaces, genetic code codon-amino acid assignments tend to match high affinities for the amino acids that integrated earliest the genetic code (according to Wong's metabolic coevolution hypothesis between nucleotides and amino acids) and (2) in solution, the same principle holds for the anticodon-amino acid assignments. Affinity analyses match best genetic code assignments when assuming that trinucleotides competed for amino acids, rather than amino acids for trinucleotides. Codon-amino acid affinities stick better to genetic code assignments than anticodon-amino acid affinities. Presumably, two independent coding systems, on surfaces and in solution, converged, and formed the current translation system. Proto-translation on surfaces by direct codon-amino acid interactions without tRNA-like adaptors coadapted with a system emerging in solution by proto-tRNA anticodon-amino acid interactions. These systems assigned identical or similar cognates to codons on surfaces and to anticodons in solution. Results indicate that a prebiotic metabolism predated genetic code self-organization.

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.)

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.

Protein-coding nucleic acids exhibit composition and codon biases between sequences coding for intrinsically disordered regions (IDRs) and those coding for structured regions. IDRs are regions of proteins that are folding self-insufficient and which function without the prerequisite of folded structure. Several authors have investigated composition bias or codon selection in regions encoding for IDRs, primarily in Eukaryota, and concluded that elevated GC content is the result of the biased amino acid composition of IDRs. We substantively extend previous work by examining GC content in regions encoding IDRs, from 44 species in Eukaryota, Archaea, and Bacteria, spanning a wide range of GC content. We confirm that regions coding for IDRs show a significantly elevated GC content, even across all domains of life. Although this is largely attributable to the amino acid composition bias of IDRs, we show that this bias is independent of the overall GC content and, most importantly, we are the first to observe that GC content bias in IDRs is significantly different than expected from IDR amino acid composition alone. We empirically find compensatory codon selection that reduces the observed GC content bias in IDRs. This selection is dependent on the overall GC content of the organism. The codon selection bias manifests as use of infrequent, AT-rich codons in encoding IDRs. Further, we find these relationships to be independent of the intrinsic disorder prediction method used, and independent of estimated translation efficiency. These observations are consistent with the previous work, and we speculate on whether the observed biases are causal or symptomatic of other driving forces.

The family of giant viruses is still expanding, and evidence of a translational machinery is emerging in the virosphere. The Klosneuvirinae group of giant viruses was first reconstructed from in silico studies, and then a unique member was isolated, Bodo saltans virus. Here we describe the isolation of a new member in this group using coculture with the free-living amoeba Vermamoeba vermiformis This giant virus, called Yasminevirus, has a 2.1-Mb linear double-stranded DNA genome encoding 1,541 candidate proteins, with a GC content estimated at 40.2%. Yasminevirus possesses a nearly complete translational machinery, with a set of 70 tRNAs associated with 45 codons and recognizing 20 amino acids (aa), 20 aminoacyl-tRNA synthetases (aaRSs) recognizing 20 aa, as well as several translation factors and elongation factors. At the genome scale, evolutionary analyses placed this virus in the Klosneuvirinae group of giant viruses. Rhizome analysis demonstrated that the genome of Yasminevirus is mosaic, with ∼34% of genes having their closest homologues in other viruses, followed by ∼13.2% in Eukaryota , ∼7.2% in Bacteria , and less than 1% in Archaea Among giant virus sequences, Yasminevirus shared 87% of viral hits with Klosneuvirinae. This description of Yasminevirus sheds light on the Klosneuvirinae group in a captivating quest to understand the evolution and diversity of giant viruses. IMPORTANCE Yasminevirus is an icosahedral double-stranded DNA virus isolated from sewage water by amoeba coculture. Here its structure and replicative cycle in the amoeba Vermamoeba vermiformis are described and genomic and evolutionary studies are reported. This virus belongs to the Klosneuvirinae group of giant viruses, representing the second isolated and cultivated giant virus in this group, and is the first isolated using a coculture procedure. Extended translational machinery pointed to Yasminevirus among the quasiautonomous giant viruses with the most complete translational apparatus of the known virosphere., (Copyright © 2019 American Society for Microbiology.)

Translation initiation is a critical step in the regulation of protein synthesis, and it is subjected to different control mechanisms, such as 5' UTR secondary structure and initiation codon context, that can influence the rates at which initiation and consequentially translation occur. For some genes, translation elongation also affects the rate of protein synthesis. With a GFP library containing nearly all possible combinations of nucleotides from the 3 rd to the 5 th codon positions in the protein coding region of the mRNA, it was previously demonstrated that some nucleotide combinations increased GFP expression up to four orders of magnitude. While it is clear that the codon region from positions 3 to 5 can influence protein expression levels of artificial constructs, its impact on endogenous proteins is still unknown. Through bioinformatics analysis, we identified the nucleotide combinations of the GFP library in Escherichia coli genes and examined the correlation between the expected levels of translation according to the GFP data with the experimental measures of protein expression. We observed that E. coli genes were enriched with the nucleotide compositions that enhanced protein expression in the GFP library, but surprisingly, it seemed to affect the translation efficiency only marginally. Nevertheless, our data indicate that different enterobacteria present similar nucleotide composition enrichment as E. coli , suggesting an evolutionary pressure towards the conservation of short translational enhancer sequences.

In-frame decoding in the ribosome occurs through canonical or wobble Watson-Crick pairing of three mRNA codon bases (a triplet) with a triplet of anticodon bases in tRNA. Departures from the triplet-triplet interaction can result in frameshifting, meaning downstream mRNA codons are then read in a different register. There are many mechanisms to induce frameshifting, and most are insufficiently understood. One previously proposed mechanism is doublet decoding, in which only codon bases 1 and 2 are read by anticodon bases 34 and 35, which would lead to -1 frameshifting. In E. coli, tRNA Ser3 GCU can induce -1 frameshifting at alanine (GCA) codons. The logic of the doublet decoding model is that the Ala codon's GC could pair with the tRNA Ser3' s GC, leaving the third anticodon residue U36 making no interactions with mRNA. Under that model, a U36C mutation would still induce -1 frameshifting, but experiments refute this. We perform all-atom simulations of wild-type tRNA Ser3 , as well as a U36C mutant. Our simulations revealed a hydrogen bond between U36 of the anticodon and G1 of the codon. The U36C mutant cannot make this interaction, as it lacks the hydrogen-bond-donating H3. The simulation thus suggests a novel, non-doublet decoding mechanism for -1 frameshifting by tRNA Ser3 at Ala codons., Competing Interests: The authors declare no conflict of interest.

Chromosomally-encoded toxin-antitoxin complexes are ubiquitous in bacteria and regulate growth through the release of the toxin component typically in a stress-dependent manner. Type II ribosome-dependent toxins adopt a RelE-family RNase fold and inhibit translation by degrading mRNAs while bound to the ribosome. Here, we present biochemical and structural studies of the Escherichia coli YoeB toxin interacting with both a UAA stop and an AAU sense codon in pre- and post-mRNA cleavage states to provide insights into possible mRNA substrate selection. Both mRNAs undergo minimal changes during the cleavage event in contrast to type II ribosome-dependent RelE toxin. Further, the 16S rRNA decoding site nucleotides that monitor the mRNA in the aminoacyl(A) site adopt different orientations depending upon which toxin is present. Although YoeB is a RelE family member, it is the sole ribosome-dependent toxin that is dimeric. We show that engineered monomeric YoeB is active against mRNAs bound to both the small and large subunit. However, the stability of monomeric YoeB is reduced ∼20°C, consistent with potential YoeB activation during heat shock in E. coli as previously demonstrated. These data provide a molecular basis for the ability of YoeB to function in response to thermal stress., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Translation is controlled by numerous accessory proteins and translation factors. In the yeast Saccharomyces cerevisiae, translation elongation requires an essential elongation factor, the ABCF ATPase eEF3. A closely related protein, New1, is encoded by a non-essential gene with cold sensitivity and ribosome assembly defect knock-out phenotypes. Since the exact molecular function of New1 is unknown, it is unclear if the ribosome assembly defect is direct, i.e. New1 is a bona fide assembly factor, or indirect, for instance due to a defect in protein synthesis. To investigate this, we employed yeast genetics, cryo-electron microscopy (cryo-EM) and ribosome profiling (Ribo-Seq) to interrogate the molecular function of New1. Overexpression of New1 rescues the inviability of a yeast strain lacking the otherwise strictly essential translation factor eEF3. The structure of the ATPase-deficient (EQ2) New1 mutant locked on the 80S ribosome reveals that New1 binds analogously to the ribosome as eEF3. Finally, Ribo-Seq analysis revealed that loss of New1 leads to ribosome queuing upstream of 3'-terminal lysine and arginine codons, including those genes encoding proteins of the cytoplasmic translational machinery. Our results suggest that New1 is a translation factor that fine-tunes the efficiency of translation termination or ribosome recycling., (© 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.)

Although protein synthesis dynamics has been studied both with theoretical models and by profiling ribosome footprints, the determinants of ribosome flux along open reading frames (ORFs) are not fully understood. Combining measurements of protein synthesis rate with ribosome footprinting data, we here inferred translation initiation and elongation rates for over a 1,000 ORFs in exponentially growing wild-type yeast cells. We found that the amino acid composition of synthesized proteins is as important a determinant of translation elongation rate as parameters related to codon and transfer RNA (tRNA) adaptation. We did not find evidence of ribosome collisions curbing the protein output of yeast transcripts, either in high translation conditions associated with exponential growth, or in strains in which deletion of individual ribosomal protein (RP) genes leads to globally increased or decreased translation. Slow translation elongation is characteristic of RP-encoding transcripts, which have markedly lower protein output compared with other transcripts with equally high ribosome densities., Competing Interests: The authors declare no conflict of interest., (Copyright © 2019 the Author(s). Published by PNAS.)

Deaminations (A->G, C->T) increase with DNA singlestrandedness during replication, presumably creating spontaneous genomic mutational and nucleotide frequency gradients. Alternatively, genes are positioned to avoid deaminations. Deamination gradients affect directly mitogene third codon positions; conserved vertebrate mitochondrial tRNA and protein coding gene arrangements minimize deaminations in anticodons, and first and second codon positions in mitogenes. Here we describe deamination gradients across theoretical minimal RNA rings, 22 nucleotide-long RNAs designed to simulate prebiotic RNAs. These RNA rings code for a start/stop codon and a single codon for each amino acid, and form stem-loop hairpins slowing degradation. They resemble consensus tRNAs, defining potential anticodons and cognate amino acids. Theoretical minimal RNA rings are not designed to include deamination gradients, yet deamination gradients occur in RNA rings. tRNA homology produces stronger evidence for deamination gradients than RNA ring homology defined by coding properties. Deamination gradients start at predicted RNA ring anticodons, corresponding to known homologies between mitochondrial tRNAs and replication origins, and between bacterial tRNA synthetases and mitochondrial DNA polymerase gamma. Deamination gradients are strongest for RNA rings with predicted anticodons matching cognate amino acids that integrated early the genetic code. Presumably protections against deaminations evolved while amino acids integrated the genetic code. Results confirm tRNA-RNA ring homologies. Coding constraints defining RNA rings presumably produce deamination gradients starting at predicted anticodons. Hence, the universal genetic code determines nucleotide deamination gradients in theoretical minimal RNA rings, suggesting adaptation to prevent consequences of deamination mutations. Results also indicate that the genetic code's structure determined evolution of tRNAs, their cognates, tRNA synthetases, and polymerases.

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.

Functional paralogous ABO, GBGT1, A3GALT2, and GGTA1 genes encode blood group A and B transferases (AT and BT), Forssman glycolipid synthase (FS), isoglobotriaosylceramide synthase (iGb3S), and α1,3-galactosyltransferase (GT), respectively. These glycosyltransferases transfer N-acetyl-d-galactosamine (GalNAc) or d-galactose forming an α1,3-glycosidic linkage. However, their acceptor substrates are diverse. Previously, we demonstrated that the amino acids at codons 266 and 268 of human AT/BT are crucial to their distinct sugar specificities, elucidating the molecular genetic basis of the ABO glycosylation polymorphism of clinical importance in transfusion and transplantation medicine. We also prepared in vitro mutagenized ATs/BTs having any of 20 possible amino acids at those codons, and showed that those codons determine the transferase activity and sugar specificity. We have expanded structural analysis to include evolutionarily related α1,3-Gal(NAc) transferases. Eukaryotic expression constructs were prepared of AT, FS, iGb3S, and GT, possessing selected tripeptides of AT-specific AlaGlyGly or LeuGlyGly, BT-specific MetGlyAla, FS-specific GlyGlyAla, or iGb3S and GT-specific HisAlaAla, at the codons corresponding to 266-268 of human AT/BT. DNA transfection was performed using appropriate recipient cells existing and newly created, and the appearance of cell surface oligosaccharide antigens was immunologically examined. The results have shown that several tripeptides other than the originals also bestowed transferase activity. However, the repertoire of functional amino acids varied among those transferases, suggesting that structures around those codons differentially affected the interactions between donor nucleotide-sugar and acceptor substrates. It was concluded that different tripeptide sequences at the substrate-binding pocket have contributed to the generation of α1,3-Gal(NAc) transferases with diversified specificities.

Gene prediction, also known as gene identification, gene finding, gene recognition, or gene discovery, is among one of the important problems of molecular biology and is receiving increasing attention due to the advent of large-scale genome sequencing projects. We designed an ab initio model (called ChemGenome) for gene prediction in prokaryotic genomes based on physicochemical characteristics of codons. In this chapter, we present the methodology of the latest version of this model ChemGenome2.1 (CG2.1). The first module of the protocol builds a three-dimensional vector from three calculated quantities for each codon-the double-helical trinucleotide base pairing energy, the base pair stacking energy, and an index of the propensity of a codon for protein-nucleic acid interactions. As this three-dimensional vector moves along any genome, the net orientation of the resultant vector should differ significantly for gene and non-genic regions to make a distinction feasible. The predicted putative protein-coding genes from above parameters are passed through a second module of the protocol which reduces the number of false positives by utilizing a filter based on stereochemical properties of protein sequences. The chemical properties of amino acid side chains taken into consideration are the presence of sp3 hybridized γ carbon atom, hydrogen bond donor ability, short/absence of δ carbon and linearity of the side chains/non-occurrence of bi-dentate forks with terminal hydrogen atoms in the side chain. The final prediction of the potential protein-coding genes is based on the frequency of occurrence of amino acids in the predicted protein sequences and their deviation from the frequency values of Swissprot protein sequences, both at monomer and tripeptide levels. The final screening is based on Z-score. Though CG2.1 is a gene finding tool for prokaryotes, considering the underlying similarity in the chemical and physical properties of DNA among prokaryotes and eukaryotes, we attempted to evaluate its applicability for gene finding in the lower eukaryotes. The results give a hope that the concept of gene finding based on physicochemical model of codons is a viable idea for eukaryotes as well, though, undoubtedly, improvements are needed.

HLA-C*07:639 differs from HLA-C*07:19 by one nucleotide substitution at codon 324 in exon 6., (© 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

One nucleotide substitution at codon 152 of HLA-B*58:01:01:01 results in a novel allele, HLA-B*58:19., (© 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

HLA-DPB1*612:01 differs from HLA-DPB1*13:01:01:01 by a single nucleotide substitution at codon 37 (CGC- > TGC)., (© 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Pacific Bioscience's SMRT sequencing was used to confirm and extend HLA-DPB1*64:01N and 701:01., (© 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

HLA-DPA1*02:01:01:06 differs from HLA-DPA1*02:01:01:01 by a single nucleotide substitution (T → G) at position 948., (© 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

The new HLA alleles HLA-B*56:55:01:02, -C*03:374 and -DPB1*13:01:03 were characterized by NGS methodology., (© 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

HLA-B*13:02:21 has one nucleotide substitution, C > T(GAC to GAT) compared with B*13:02:01., (© 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

HLA-A*23:91N differs from HLA-A*23:01:01 by a single-nucleotide substitution (G → A) at position 369., (© 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

HLA-DRB3*02:02:11 differs from HLA-DRB3*02:02:01:01 by a single synonymous substitution in exon 3'., (© 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

HLA-DPA1*01:03:01:12 differs from HLA-DPA1*01:03:01:01 by a single nucleotide substitution (G → C) at position 4239., (© 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

HLA-B*15:476 differs from HLA-B*15:01:01:01 by one nucleotide substitution at codon 299 in exon 5., (© 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

The novel HLA-B allele, HLA-B*50:31, was identified by sequencing genomic DNA., (© 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Pacific Bioscience's SMRT DNA sequencing was used to identify two novel intronic variants of HLA-A*32:01:01:01., (© 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

HLA-DPB1*763:01 differs from HLA-DPB1*105:01:01:01 by one nucleotide substitution at codon 128 in exon 3., (© 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Novel HLA-B alleles HLA-B*53:01:17 and HLA-B*58:83 are described in two patients from Guinea-Bissau., (© 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

One nucleotide replacement at residue 369 of HLA-B*46:01:01 results in a new allele, HLA-B*46:01:19., (© 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

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.

During protein synthesis genetic instructions are passed from DNA via mRNA to the ribosome to assemble a protein chain. Occasionally, stop codons in the mRNA are bypassed and translation continues into the untranslated region (3'-UTR). This process, called translational readthrough (TR), yields a protein chain that becomes longer than would be predicted from the DNA sequence alone. Protein sequences vary in propensity for translational errors, which may yield evolutionary constraints by limiting evolutionary paths. Here we investigated TR in Saccharomyces cerevisiae by analysing ribosome profiling data. We clustered proteins as either prone or non-prone to TR, and conducted comparative analyses. We find that a relatively high frequency (5%) of genes undergo TR, including ribosomal subunit proteins. Our main finding is that proteins undergoing TR are highly expressed and have intrinsically disordered C-termini. We suggest that highly expressed proteins may compensate for the deleterious effects of TR by having intrinsically disordered C-termini, which may provide conformational flexibility but without distorting native function. Moreover, we discuss whether minimizing deleterious effects of TR is also enabling exploration of the phenotypic landscape of protein isoforms.

Perturbations play an important role both in engineered systems and cellular processes. Thus, understanding their effect on protein synthesis should contribute to all biomedical disciplines. Here we describe the first genome-scale analysis of perturbations in translation-related factors in S. cerevisiae. To this end, we used simulations based on a computational model that takes into consideration the fundamental stochastic and bio-physical nature of translation. We found that the initiation rate has a key role in determining the sensitivity to perturbations. For low initiation rates, the first codons of the coding region dominate the sensitivity, which is highly correlated with the ratio between initiation rate and mean elongation rate (r = -0.95), with the open reading frame (ORF) length (r = 0.6) and with protein abundance (r = 0.45). For high initiation rates (that may rise, for example, due to cellular growth), the sensitivity of a gene is dominated by all internal codons and is correlated with the decoding rate. We found that various central intracellular functions are associated with the sensitivity: for example, both genes that are sensitive and genes that are robust to perturbations are over-represented in the group of genes related to translation regulation; this may suggest that robustness to perturbations is a trait that undergoes evolutionary selection in relation to the function of the encoded protein. We believe that the reported results, due to their quantitative value and genome-wide perspective, should contribute to disciplines such as synthetic biology, functional genomics, comparative genomics and molecular evolution.

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.

In all translation systems, the genetic code assigns codons to amino acids as building blocks of polypeptides, defining their chemical, structural and physiological properties. The canonical genetic code, however, utilizes only 20 proteinogenic amino acids redundantly encoded in 61 codons. In order to expand the building block repertoire, this redundancy was reduced by tuning composition of the transfer RNA (tRNA) mixture in vitro. Depletion of particular tRNAs from the total tRNA mixture or its reconstitution with in vitro-transcribed tRNA SNN s (S = C or G, N = U, C, A or G) divided a codon box to encode two amino acids, expanding the repertoire to 23. The expanded genetic codes may benefit analysis of cellular regulatory pathways and drug screening., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

It is well known that sequences of bases in DNA are translated into sequences of amino acids in cells via the genetic code. More recently, it has been discovered that the sequence of DNA bases also influences the geometry and deformability of the DNA. These two correspondences represent a naturally arising example of duplexed codes, providing two different ways of interpreting the same DNA sequence. This paper will set up the notation and basic results necessary to mathematically investigate the relationship between these two natural DNA codes. It then undertakes two very different such investigations: one graphical approach based only on expected values and another analytic approach incorporating the deformability of the DNA molecule and approximating the mutual information of the two codes. Special emphasis is paid to whether there is evidence that pressure to maximize the duplexing efficiency influenced the evolution of the genetic code. Disappointingly, the results fail to support the hypothesis that the genetic code was influenced in this way. In fact, applying both methods to samples of realistic alternative genetic codes shows that the duplexing of the genetic code found in nature is just slightly less efficient than average. The implications of this negative result are considered in the final section of the paper.