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Mutations that introduce premature termination codons (PTCs) within protein-coding genes are associated with incurable and severe genetic diseases. Many PTC-associated disorders are life-threatening and have no approved medical treatment options. Suppressor transfer RNAs (sup-tRNAs) with the capacity to translate PTCs represent a promising therapeutic strategy to treat these conditions; however, developing novel sup-tRNAs with high efficiency and specificity often requires extensive engineering and screening. Moreover, these efforts are not always successful at producing more efficient sup-tRNAs. Here we show that a pyrrolysine (Pyl) tRNA (tRNAPyl), which naturally translates the UAG stop codon, offers a favorable scaffold for developing sup-tRNAs that restore protein synthesis from PTC-containing genes. We created a series of rationally designed Pyl tRNAScaffold Suppressor-tRNAs (PASS-tRNAs) that are substrates of bacterial and human alanyl-tRNA synthetase. Using a PTC-containing fluorescent reporter gene, PASS-tRNAs restore protein synthesis to wild-type levels in bacterial cells. In human cells, PASS-tRNAs display robust and consistent PTC suppression in multiple reporter genes, including pathogenic mutations in the tumor suppressor gene BRCA1 associated with breast and ovarian cancer. Moreover, PTC suppression occurred with high codon specificity and no observed cellular dysregulation. Collectively, these results unveil a new class of sup-tRNAs with encouraging potential for tRNA-based therapeutic applications., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Transfer RNAs (tRNA) are essential small non-coding RNAs that enable the translation of genomic information into proteins in all life forms. The principal function of tRNAs is to bring amino acid building blocks to the ribosomes for protein synthesis. In the ribosome, tRNAs interact with messenger RNA (mRNA) to mediate the incorporation of amino acids into a growing polypeptide chain following the rules of the genetic code. Accurate interpretation of the genetic code requires tRNAs to carry amino acids matching their anticodon identity and decode the correct codon on mRNAs. Errors in these steps cause the translation of codons with the wrong amino acids (mistranslation), compromising the accurate flow of information from DNA to proteins. Accumulation of mutant proteins due to mistranslation jeopardizes proteostasis and cellular viability. However, the concept of mistranslation is evolving, with increasing evidence indicating that mistranslation can be used as a mechanism for survival and acclimatization to environmental conditions. In this review, we discuss the central role of tRNAs in modulating translational fidelity through their dynamic and complex interplay with translation factors. We summarize recent discoveries of mistranslating tRNAs and describe the underlying molecular mechanisms and the specific conditions and environments that enable and promote mistranslation., Competing Interests: Conflicts of interest The authors declare no conflict of interest., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The rules of the genetic code are implemented by the unique features that define the amino acid identity of each transfer RNA (tRNA). These features, known as "identity elements," mark tRNAs for recognition by aminoacyl-tRNA synthetases (ARSs), the enzymes responsible for ligating amino acids to tRNAs. While tRNA identity elements enable stringent substrate selectivity of ARSs, these enzymes are prone to errors during amino acid selection, leading to the synthesis of incorrect aminoacyl-tRNAs that jeopardize the fidelity of protein synthesis. Many error-prone ARSs have evolved specialized domains that hydrolyze incorrectly synthesized aminoacyl-tRNAs. These domains, known as editing domains, also exist as free-standing enzymes and, together with ARSs, safeguard protein synthesis fidelity. Here, we discuss how the same identity elements that define tRNA aminoacylation play an integral role in aminoacyl-tRNA editing, synergistically ensuring the correct translation of genetic information into proteins. Moreover, we review the distinct strategies of tRNA selection used by editing enzymes and ARSs to avoid undesired hydrolysis of correctly aminoacylated tRNAs., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Cruz and Vargas-Rodriguez.)

Suppressor transfer RNAs (sup-tRNAs) are receiving renewed attention for their promising therapeutic properties in treating genetic diseases caused by nonsense mutations. Traditionally, sup-tRNAs have been created by replacing the anticodon sequence of native tRNAs with a suppressor sequence. However, due to their complex interactome, considering other structural and functional tRNA features for design and engineering can yield more effective sup-tRNA therapies. For over 2 decades, the field of genetic code expansion (GCE) has created a wealth of knowledge, resources, and tools to engineer sup-tRNAs. In this Mini Review, we aim to shed light on how existing knowledge and strategies to develop sup-tRNAs for GCE can be adopted to accelerate the discovery of efficient and specific sup-tRNAs for medical treatment options. We highlight methods and milestones and discuss how these approaches may enlighten the research and development of tRNA medicines., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Awawdeh, Radecki and Vargas-Rodriguez.)

Ribosomes are often used in synthetic biology as a tool to produce desired proteins with enhanced properties or entirely new functions. However, repurposing ribosomes for producing designer proteins is challenging due to the limited number of engineering solutions available to alter the natural activity of these enzymes. In this study, we advance ribosome engineering by describing a novel strategy based on functional fusions of ribosomal RNA (rRNA) with messenger RNA (mRNA). Specifically, we create an mRNA-ribosome fusion called RiboU, where the 16S rRNA is covalently attached to selenocysteine insertion sequence (SECIS), a regulatory RNA element found in mRNAs encoding selenoproteins. When SECIS sequences are present in natural mRNAs, they instruct ribosomes to decode UGA codons as selenocysteine (Sec, U) codons instead of interpreting them as stop codons. This enables ribosomes to insert Sec into the growing polypeptide chain at the appropriate site. Our work demonstrates that the SECIS sequence maintains its functionality even when inserted into the ribosome structure. As a result, the engineered ribosomes RiboU interpret UAG codons as Sec codons, allowing easy and site-specific insertion of Sec in a protein of interest with no further modification to the natural machinery of protein synthesis. To validate this approach, we use RiboU ribosomes to produce three functional target selenoproteins in Escherichia coli by site-specifically inserting Sec into the proteins' active sites. Overall, our work demonstrates the feasibility of creating functional mRNA-rRNA fusions as a strategy for ribosome engineering, providing a novel tool for producing Sec-containing proteins in live bacterial cells., Competing Interests: Competing interests statement:The authors declare no competing interest.

The correct coupling of amino acids with transfer RNAs (tRNAs) is vital for translating genetic information into functional proteins. Errors during this process lead to mistranslation, where a codon is translated using the wrong amino acid. While unregulated and prolonged mistranslation is often toxic, growing evidence suggests that organisms, from bacteria to humans, can induce and use mistranslation as a mechanism to overcome unfavorable environmental conditions. Most known cases of mistranslation are caused by translation factors with poor substrate specificity or when substrate discrimination is sensitive to molecular changes such as mutations or posttranslational modifications. Here we report two novel families of tRNAs, encoded by bacteria from the Streptomyces and Kitasatospora genera, that adopted dual identities by integrating the anticodons AUU (for Asn) or AGU (for Thr) into the structure of a distinct proline tRNA. These tRNAs are typically encoded next to a full-length or truncated version of a distinct isoform of bacterial-type prolyl-tRNA synthetase. Using two protein reporters, we showed that these tRNAs translate asparagine and threonine codons with proline. Moreover, when expressed in Escherichia coli, the tRNAs cause varying growth defects due to global Asn-to-Pro and Thr-to-Pro mutations. Yet, proteome-wide substitutions of Asn with Pro induced by tRNA expression increased cell tolerance to the antibiotic carbenicillin, indicating that Pro mistranslation can be beneficial under certain conditions. Collectively, our results significantly expand the catalog of organisms known to possess dedicated mistranslation machinery and support the concept that mistranslation is a mechanism for cellular resiliency against environmental stress., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Post-translational modifications (PTMs) can occur on almost all amino acids in eukaryotes as a key mechanism for regulating protein function. The ability to study the role of these modifications in various biological processes requires techniques to modify proteins site-specifically. One strategy for this is genetic code expansion (GCE) in bacteria. The low frequency of post-translational modifications in bacteria makes it a preferred host to study whether the presence of a post-translational modification influences a protein's function. Genetic code expansion employs orthogonal translation systems engineered to incorporate a modified amino acid at a designated protein position. Selenoproteins, proteins containing selenocysteine, are also known to be post-translationally modified. Selenoproteins have essential roles in oxidative stress, immune response, cell maintenance, and skeletal muscle regeneration. Their complicated biosynthesis mechanism has been a hurdle in our understanding of selenoprotein functions. As technologies for selenocysteine insertion have recently improved, we wanted to create a genetic system that would allow the study of post-translational modifications in selenoproteins. By combining genetic code expansion techniques and selenocysteine insertion technologies, we were able to recode stop codons for insertion of N ε -acetyl-l-lysine and selenocysteine, respectively, into multiple proteins. The specificity of these amino acids for their assigned position and the simplicity of reverting the modified amino acid via mutagenesis of the codon sequence demonstrates the capacity of this method to study selenoproteins and the role of their post-translational modifications. Moreover, the evidence that Sec insertion technology can be combined with genetic code expansion tools further expands the chemical biology applications., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Morosky, Comyns, Nunes, Chung, Hoffmann, Söll, Vargas-Rodriguez and Krahn.)

Ribosomes are remarkable in their malleability to accept diverse aminoacyl-tRNA substrates from both the same organism and other organisms or domains of life. This is a critical feature of the ribosome that allows the use of orthogonal translation systems for genetic code expansion. Optimization of these orthogonal translation systems generally involves focusing on the compatibility of the tRNA, aminoacyl-tRNA synthetase, and a non-canonical amino acid with each other. As we expand the diversity of tRNAs used to include non-canonical structures, the question arises as to the tRNA suitability on the ribosome. Specifically, we investigated the ribosomal translation of allo-tRNAUTu1, a uniquely shaped (9/3) tRNA exploited for site-specific selenocysteine insertion, using single-molecule fluorescence. With this technique we identified ribosomal disassembly occurring from translocation of allo-tRNAUTu1 from the A to the P site. Using cryo-EM to capture the tRNA on the ribosome, we pinpointed a distinct tertiary interaction preventing fluid translocation. Through a single nucleotide mutation, we disrupted this tertiary interaction and relieved the translation roadblock. With the continued diversification of genetic code expansion, our work highlights a targeted approach to optimize translation by distinct tRNAs as they move through the ribosome., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Intricate evolutionary events enabled the emergence of the full set of aminoacyl-tRNA synthetase (aaRS) families that define the genetic code. The diversification of aaRSs has continued in organisms from all domains of life, yielding aaRSs with unique characteristics as well as aaRS-like proteins with innovative functions outside translation. Recent bioinformatic analyses have revealed the extensive occurrence and phylogenetic diversity of aaRS gene duplication involving every synthetase family. However, only a fraction of these duplicated genes has been characterized, leaving many with biological functions yet to be discovered. Here we discuss how genomic duplication is associated with the occurrence of novel aaRSs and aaRS-like proteins that provide adaptive advantages to their hosts. We illustrate the variety of activities that have evolved from the primordial aaRS catalytic sites. This precedent underscores the need to investigate currently unexplored aaRS genomic duplications as they may hold a key to the discovery of exciting biological processes, new drug targets, important bioactive molecules, and tools for synthetic biology applications., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Krahn, Söll and Vargas-Rodriguez.)

Site-specific incorporation of distinct non-canonical amino acids into proteins via genetic code expansion requires mutually orthogonal aminoacyl-tRNA synthetase/tRNA pairs. Pyrrolysyl-tRNA synthetase (PylRS)/tRNAPyl pairs are ideal for genetic code expansion and have been extensively engineered for developing mutually orthogonal pairs. Here, we identify two novel wild-type PylRS/tRNAPyl pairs simultaneously present in the deep-rooted extremely halophilic euryarchaeal methanogen Candidatus Methanohalarchaeum thermophilum HMET1, and show that both pairs are functional in the model halophilic archaeon Haloferax volcanii. These pairs consist of two different PylRS enzymes and two distinct tRNAs with dissimilar discriminator bases. Surprisingly, these two PylRS/tRNAPyl pairs display mutual orthogonality enabled by two unique features, the A73 discriminator base of tRNAPyl2 and a shorter motif 2 loop in PylRS2. In vivo translation experiments show that tRNAPyl2 charging by PylRS2 is defined by the enzyme's shortened motif 2 loop. Finally, we demonstrate that the two HMET1 PylRS/tRNAPyl pairs can simultaneously decode UAG and UAA codons for incorporation of two distinct noncanonical amino acids into protein. This example of a single base change in a tRNA leading to additional coding capacity suggests that the growth of the genetic code is not yet limited by the number of identity elements fitting into the tRNA structure., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Inaccurate expression of the genetic code, also known as mistranslation, is an emerging paradigm in microbial studies. Growing evidence suggests that many microbial pathogens can deliberately mistranslate their genetic code to help invade a host or evade host immune responses. However, discovering different capacities for deliberate mistranslation remains a challenge because each group of pathogens typically employs a unique mistranslation mechanism. In this study, we address this problem by studying duplicated genes of aminoacyl-transfer RNA (tRNA) synthetases. Using bacterial prolyl-tRNA synthetase (ProRS) genes as an example, we identify an anomalous ProRS isoform, ProRSx, and a corresponding tRNA, tRNA ProA , that are predominately found in plant pathogens from Streptomyces species. We then show that tRNA ProA has an unusual hybrid structure that allows this tRNA to mistranslate alanine codons as proline. Finally, we provide biochemical, genetic, and mass spectrometric evidence that cells which express ProRSx and tRNA ProA can translate GCU alanine codons as both alanine and proline. This dual use of alanine codons creates a hidden proteome diversity due to stochastic Ala→Pro mutations in protein sequences. Thus, we show that important plant pathogens are equipped with a tool to alter the identity of their sense codons. This finding reveals the initial example of a natural tRNA synthetase/tRNA pair for dedicated mistranslation of sense codons., Competing Interests: The authors declare no competing interest.

Accurate translation of genetic information into proteins is vital for cell sustainability. ProXp-ala prevents proteome-wide Pro-to-Ala mutations by hydrolyzing misacylated Ala-tRNA Pro , which is synthesized by prolyl-tRNA synthetase. Bacterial ProXp-ala was previously shown to combine a size-based exclusion mechanism with conformational and chemical selection for the recognition of the alanyl moiety, whereas tRNA Pro is selected via recognition of tRNA acceptor-stem elements G72 and A73. The identity of these critical bases changed during evolution with eukaryotic cytosolic tRNA Pro possessing a cytosine at the corresponding positions. The mechanism by which eukaryotic ProXp-ala adapted to these changes remains unknown. In this work, recognition of the aminoacyl moiety and tRNA acceptor stem by human ( Homo sapiens , or Hs ) ProXp-ala was examined. Enzymatic assays revealed that Hs ProXp-ala requires C72 and C73 in the context of Hs cytosolic tRNA Pro for efficient deacylation of mischarged Ala-tRNA Pro The strong dependence on these bases prevents cross-species deacylation of bacterial Ala-tRNA Pro or of Hs mitochondrial Ala-tRNA Pro by the human enzyme. Similar to the bacterial enzyme, Hs ProXp-ala showed strong tRNA acceptor-stem recognition but differed in its amino acid specificity profile relative to bacterial ProXp-ala. Changes at conserved residues in both the Hs and bacterial ProXp-ala substrate-binding pockets modulated this specificity. These results illustrate how the mechanism of substrate selection diverged during the evolution of the ProXp-ala family, providing the first example of a trans -editing domain whose specificity evolved to adapt to changes in its tRNA substrate., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Vargas-Rodriguez et al.)

Selenocysteine (Sec) is the 21st genetically encoded amino acid in organisms across all domains of life. Although structurally similar to cysteine (Cys), the Sec selenol group has unique properties that are attractive for protein engineering and biotechnology applications. Production of designer proteins with Sec (selenoproteins) at desired positions is now possible with engineered translation systems in Escherichia coli However, obtaining pure selenoproteins at high yields is limited by the accumulation of free Sec in cells, causing undesired incorporation of Sec at Cys codons due to the inability of cysteinyl-tRNA synthetase (CysRS) to discriminate against Sec. Sec misincorporation is toxic to cells and causes protein aggregation in yeast. To overcome this limitation, here we investigated a CysRS from the selenium accumulator plant Astragalus bisulcatus that is reported to reject Sec in vitro Sequence analysis revealed a rare His → Asn variation adjacent to the CysRS catalytic pocket. Introducing this variation into E. coli and Saccharomyces cerevisiae CysRS increased resistance to the toxic effects of selenite and selenomethionine (SeMet), respectively. Although the CysRS variant could still use Sec as a substrate in vitro , we observed a reduction in the frequency of Sec misincorporation at Cys codons in vivo We surmise that the His → Asn variation can be introduced into any CysRS to provide a fitness advantage for strains burdened by Sec misincorporation and selenium toxicity. Our results also support the notion that the CysRS variant provides higher specificity for Cys as a mechanism for plants to grow in selenium-rich soils., (© 2019 Hoffman et al.)

Genetic incorporation of noncanonical amino acids (ncAAs) has become a powerful tool to enhance existing functions or introduce new ones into proteins through expanded chemistry. This technology relies on the process of nonsense suppression, which is made possible by directing aminoacyl-tRNA synthetases (aaRSs) to attach an ncAA onto a cognate suppressor tRNA. However, different mechanisms govern aaRS specificity toward its natural amino acid (AA) substrate and hinder the engineering of aaRSs for applications beyond the incorporation of a single l-α-AA. Directed evolution of aaRSs therefore faces two interlinked challenges: the removal of the affinity for cognate AA and improvement of ncAA acylation. Here we review aspects of AA recognition that directly influence the feasibility and success of aaRS engineering toward d- and β-AAs incorporation into proteins in vivo. Emerging directed evolution methods are described and evaluated on the basis of aaRS active site plasticity and its inherent constraints.

During protein synthesis, ribosomes discriminate chirality of amino acids and prevent incorporation of D-amino acids into nascent proteins by slowing down the rate of peptide bond formation. Despite this phenomenon being known for nearly forty years, no structures have ever been reported that would explain the poor reactivity of D-amino acids. Here we report a 3.7Å-resolution crystal structure of a bacterial ribosome in complex with a D-aminoacyl-tRNA analog bound to the A site. Although at this resolution we could not observe individual chemical groups, we could unambiguously define the positions of the D-amino acid side chain and the amino group based on chemical restraints. The structure reveals that similarly to L-amino acids, the D-amino acid binds the ribosome by inserting its side chain into the ribosomal A-site cleft. This binding mode does not allow optimal nucleophilic attack of the peptidyl-tRNA by the reactive α-amino group of a D-amino acid. Also, our structure suggests that the D-amino acid cannot participate in hydrogen-bonding with the P-site tRNA that is required for the efficient proton transfer during peptide bond formation. Overall, our work provides the first mechanistic insight into the ancient mechanism that helps living cells ensure the stereochemistry of protein synthesis., (© The Author(s) 2018. Published by Oxford University Press on behalf of Nucleic Acids Research.)

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Synthesis of proteins with noncanonical amino acids (ncAAs) enables the creation of protein-based biomaterials with diverse new chemical properties that may be attractive for material science. Current methods for large-scale production of ncAA-containing proteins, frequently carried out in Escherichia coli , involve the use of orthogonal aminoacyl-tRNA synthetases (o-aaRSs) and tRNAs (o-tRNAs). Although o-tRNAs are designed to be orthogonal to endogenous aaRSs, their orthogonality to the components of the E. coli metabolism remains largely unexplored. We systematically investigated how the E. coli tRNA modification machinery affects the efficiency and orthogonality of o-tRNA Sep used for production of proteins with the ncAA O- phosphoserine (Sep). The incorporation of Sep into a green fluorescent protein (GFP) in 42 E. coli strains carrying deletions of single tRNA modification genes identified several genes that affect the o-tRNA activity. Deletion of cysteine desulfurase ( iscS ) increased the yield of Sep-containing GFP more than eightfold, while overexpression of dimethylallyltransferase MiaA and pseudouridine synthase TruB improved the specificity of Sep incorporation. These results highlight the importance of tRNA modifications for the biosynthesis of proteins containing ncAAs, and provide a novel framework for optimization of o-tRNAs., Competing Interests: The authors declare no conflict of interest.

Incorporation of nonstandard amino acids (nsAAs) leads to chemical diversification of proteins, which is an important tool for the investigation and engineering of biological processes. However, the aminoacyl-tRNA synthetases crucial for this process are polyspecific in regard to nsAAs and standard amino acids. Here, we develop a quality control system called "posttranslational proofreading" to more accurately and rapidly evaluate nsAA incorporation. We achieve this proofreading by hijacking a natural pathway of protein degradation known as the N-end rule, which regulates the lifespan of a protein based on its amino-terminal residue. We find that proteins containing certain desired N-terminal nsAAs have much longer half-lives compared with those proteins containing undesired amino acids. We use the posttranslational proofreading system to further evolve a Methanocaldococcus jannaschii tyrosyl-tRNA synthetase (TyrRS) variant and a tRNA Tyr species for improved specificity of the nsAA biphenylalanine in vitro and in vivo. Our newly evolved biphenylalanine incorporation machinery enhances the biocontainment and growth of genetically engineered Escherichia coli strains that depend on biphenylalanine incorporation. Finally, we show that our posttranslational proofreading system can be designed for incorporation of other nsAAs by rational engineering of the ClpS protein, which mediates the N-end rule. Taken together, our posttranslational proofreading system for in vivo protein sequence verification presents an alternative paradigm for molecular recognition of amino acids and is a major advance in our ability to accurately expand the genetic code., Competing Interests: Conflict of interest statement: G.M.C. has related financial interests in ReadCoor, EnEvolv, and GRO Biosciences. A.M.K. and G.M.C. have filed a provisional patent on posttranslational proofreading, and A.M.K., D.S., E.K., and G.M.C. have filed a provisional patent on evolved BipA OTS variants. For a complete list of G.M.C.’s financial interests, please visit arep.med.harvard.edu/gmc/tech.html., (Copyright © 2018 the Author(s). Published by PNAS.)

In many organisms, the UGA stop codon is recoded to insert selenocysteine (Sec) into proteins. Sec incorporation in bacteria is directed by an mRNA element, known as the Sec-insertion sequence (SECIS), located downstream of the Sec codon. Unlike other aminoacyl-tRNAs, Sec-tRNA Sec is delivered to the ribosome by a dedicated elongation factor, SelB. We recently identified a series of tRNA Sec -like tRNA genes distributed across Bacteria that also encode a canonical tRNA Sec . These tRNAs contain sequence elements generally recognized by cysteinyl-tRNA synthetase (CysRS). While some of these tRNAs contain a UCA Sec anticodon, most have a GCA Cys anticodon. tRNA Sec with GCA anticodons are known to recode UGA codons. Here we investigate the clostridial Desulfotomaculum nigrificans tRNA Sec -like tRNA Cys , and show that this tRNA is acylated by CysRS, recognized by SelB, and capable of UGA recoding with Cys in Escherichia coli. We named this non-canonical group of tRNA Cys as 'tRNA ReC ' (Recoding with Cys). We performed a comprehensive survey of tRNA ReC genes to establish their phylogenetic distribution, and found that, in a particular lineage of clostridial Pelotomaculum, the Cys identity elements of tRNA ReC had mutated. This novel tRNA, which contains a UCA anticodon, is capable of Sec incorporation in E. coli, albeit with lower efficiency relative to Pelotomaculum tRNA Sec . We renamed this unusual tRNA Sec derived from tRNA ReC as 'tRNA ReU ' (Recoding with Sec). Together, our results suggest that tRNA ReC and tRNA ReU may serve as safeguards in the production of selenoproteins and - to our knowledge - they provide the first example of programmed codon-anticodon mispairing in bacteria.

Hundreds of non-proteinogenic (np) amino acids (AA) are found in plants and can in principle enter human protein synthesis through foods. While aminoacyl-tRNA synthetase (AARS) editing potentially provides a mechanism to reject np AAs, some have pathological associations. Co-crystal structures show that vegetable-sourced azetidine-2-carboxylic acid (Aze), a dual mimic of proline and alanine, is activated by both human prolyl- and alanyl-tRNA synthetases. However, it inserts into proteins as proline, with toxic consequences in vivo. Thus, dual mimicry increases odds for mistranslation through evasion of one but not both tRNA synthetase editing systems.

Molecular sieves ensure proper pairing of tRNAs and amino acids during aminoacyl-tRNA biosynthesis, thereby avoiding detrimental effects of mistranslation on cell growth and viability. Mischarging errors are often corrected through the activity of specialized editing domains present in some aminoacyl-tRNA synthetases or via single-domain trans -editing proteins. ProXp-ala is a ubiquitous trans -editing enzyme that edits Ala-tRNA Pro , the product of Ala mischarging by prolyl-tRNA synthetase, although the structural basis for discrimination between correctly charged Pro-tRNA Pro and mischarged Ala-tRNA Ala is unclear. Deacylation assays using substrate analogs reveal that size discrimination is only one component of selectivity. We used NMR spectroscopy and sequence conservation to guide extensive site-directed mutagenesis of Caulobacter crescentus ProXp-ala, along with binding and deacylation assays to map specificity determinants. Chemical shift perturbations induced by an uncharged tRNA Pro acceptor stem mimic, microhelix Pro , or a nonhydrolyzable mischarged Ala-microhelix Pro substrate analog identified residues important for binding and deacylation. Backbone 15 N NMR relaxation experiments revealed dynamics for a helix flanking the substrate binding site in free ProXp-ala, likely reflecting sampling of open and closed conformations. Dynamics persist on binding to the uncharged microhelix, but are attenuated when the stably mischarged analog is bound. Computational docking and molecular dynamics simulations provide structural context for these findings and predict a role for the substrate primary α-amine group in substrate recognition. Overall, our results illuminate strategies used by a trans -editing domain to ensure acceptance of only mischarged Ala-tRNA Pro , including conformational selection by a dynamic helix, size-based exclusion, and optimal positioning of substrate chemical groups., Competing Interests: The authors declare no conflict of interest.

We report the identification of novel tRNA species with 12-base pair amino-acid acceptor branches composed of longer acceptor stem and shorter T-stem. While canonical tRNAs have a 7/5 configuration of the branch, the novel tRNAs have either 8/4 or 9/3 structure. They were found during the search for selenocysteine tRNAs in terabytes of genome, metagenome and metatranscriptome sequences. Certain bacteria and their phages employ the 8/4 structure for serine and histidine tRNAs, while minor cysteine and selenocysteine tRNA species may have a modified 8/4 structure with one bulge nucleotide. In Acidobacteria, tRNAs with 8/4 and 9/3 structures may function as missense and nonsense suppressor tRNAs and/or regulatory noncoding RNAs. In δ-proteobacteria, an additional cysteine tRNA with an 8/4 structure mimics selenocysteine tRNA and may function as opal suppressor. We examined the potential translation function of suppressor tRNA species in Escherichia coli; tRNAs with 8/4 or 9/3 structures efficiently inserted serine, alanine and cysteine in response to stop and sense codons, depending on the identity element and anticodon sequence of the tRNA. These findings expand our view of how tRNA, and possibly the genetic code, is diversified in nature., (© The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The degeneracy of the genetic code allows nucleic acids to encode amino acid identity as well as noncoding information for gene regulation and genome maintenance. The rare arginine codons AGA and AGG (AGR) present a case study in codon choice, with AGRs encoding important transcriptional and translational properties distinct from the other synonymous alternatives (CGN). We created a strain of Escherichia coli with all 123 instances of AGR codons removed from all essential genes. We readily replaced 110 AGR codons with the synonymous CGU codons, but the remaining 13 "recalcitrant" AGRs required diversification to identify viable alternatives. Successful replacement codons tended to conserve local ribosomal binding site-like motifs and local mRNA secondary structure, sometimes at the expense of amino acid identity. Based on these observations, we empirically defined metrics for a multidimensional "safe replacement zone" (SRZ) within which alternative codons are more likely to be viable. To evaluate synonymous and nonsynonymous alternatives to essential AGRs further, we implemented a CRISPR/Cas9-based method to deplete a diversified population of a wild-type allele, allowing us to evaluate exhaustively the fitness impact of all 64 codon alternatives. Using this method, we confirmed the relevance of the SRZ by tracking codon fitness over time in 14 different genes, finding that codons that fall outside the SRZ are rapidly depleted from a growing population. Our unbiased and systematic strategy for identifying unpredicted design flaws in synthetic genomes and for elucidating rules governing codon choice will be crucial for designing genomes exhibiting radically altered genetic codes., Competing Interests: G.K., M.J.L., M.L., M.G.N., D.B.G., and G.M.C. are inventors on patent application #62350468 submitted by the President and Fellows of Harvard College. G.M.C. is a founder of Enevolv Inc. and Gen9bio. Other potentially relevant financial interests are listed at arep.med.harvard.edu/gmc/tech.html.

Aminoacyl-tRNA synthetases (ARSs) establish the rules of the genetic code, whereby each amino acid is attached to a cognate tRNA. Errors in this process lead to mistranslation, which can be toxic to cells. The selective forces exerted by species-specific requirements and environmental conditions potentially shape quality-control mechanisms that serve to prevent mistranslation. A family of editing factors that are homologous to the editing domain of bacterial prolyl-tRNA synthetase includes the previously characterized trans-editing factors ProXp-ala and YbaK, which clear Ala-tRNA(Pro) and Cys-tRNA(Pro), respectively, and three additional homologs of unknown function, ProXp-x, ProXp-y, and ProXp-z. We performed an in vivo screen of 230 conditions in which an Escherichia coli proXp-y deletion strain was grown in the presence of elevated levels of amino acids and specific ARSs. This screen, together with the results of in vitro deacylation assays, revealed Ser- and Thr-tRNA deacylase function for this homolog. A similar activity was demonstrated for Bordetella parapertussis ProXp-z in vitro. These proteins, now renamed "ProXp-ST1" and "ProXp-ST2," respectively, recognize multiple tRNAs as substrates. Taken together, our data suggest that these free-standing editing domains have the ability to prevent mistranslation errors caused by a number of ARSs, including lysyl-tRNA synthetase, threonyl-tRNA synthetase, seryl-tRNA synthetase, and alanyl-tRNA synthetase. The expression of these multifunctional enzymes is likely to provide a selective growth advantage to organisms subjected to environmental stresses and other conditions that alter the amino acid pool.

Accurate protein synthesis requires the hydrolytic editing of tRNAs incorrectly aminoacylated by aminoacyl-tRNA synthetases (ARSs). Recognition of cognate tRNAs by ARS is less error-prone than amino acid recognition, and, consequently, editing domains are generally believed to act only on the tRNAs cognate to their related ARSs. For example, the AlaX family of editing domains, including the editing domain of alanyl-tRNA synthetase and the related free-standing trans-editing AlaX enzymes, are thought to specifically act on tRNA(Ala), whereas the editing domains of threonyl-tRNA synthetases are specific for tRNA(Thr). Here we show that, contrary to this belief, AlaX-S, the smallest of the extant AlaX enzymes, deacylates Ser-tRNA(Thr) in addition to Ser-tRNA(Ala) and that a single residue is important to determine this behavior. Our data indicate that promiscuous forms of AlaX are ancestral to tRNA-specific AlaXs. We propose that former AlaX domains were used to maintain translational fidelity in earlier stages of genetic code evolution when mis-serylation of several tRNAs was possible., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Errors in protein synthesis due to mispairing of amino acids with tRNAs jeopardize cell viability. Several checkpoints to prevent formation of Ala- and Cys-tRNA(Pro) have been described, including the Ala-specific editing domain (INS) of most bacterial prolyl-tRNA synthetases (ProRSs) and an autonomous single-domain INS homolog, YbaK, which clears Cys-tRNA(Pro) in trans. In many species where ProRS lacks an INS domain, ProXp-ala, another single-domain INS-like protein, is responsible for editing Ala-tRNA(Pro). Although the amino acid specificity of these editing domains has been established, the role of tRNA sequence elements in substrate selection has not been investigated in detail. Critical recognition elements for aminoacylation by bacterial ProRS include acceptor stem elements G72/A73 and anticodon bases G35/G36. Here, we show that ProXp-ala and INS require these same acceptor stem and anticodon elements, respectively, whereas YbaK lacks inherent tRNA specificity. Thus, these three related domains use divergent approaches to recognize tRNAs and prevent mistranslation. Whereas some editing domains have borrowed aspects of tRNA recognition from the parent aminoacyl-tRNA synthetase, relaxed tRNA specificity leading to semi-promiscuous editing may offer advantages to cells.

Aminoacyl-tRNA synthetases (ARSs) catalyze the attachment of specific amino acids to cognate tRNAs. Although the accuracy of this process is critical for overall translational fidelity, similar sizes of many amino acids provide a challenge to ARSs. For example, prolyl-tRNA synthetases (ProRSs) mischarge alanine and cysteine onto tRNA(Pro). Many bacterial ProRSs possess an alanine-specific proofreading domain (INS) but lack the capability to edit Cys-tRNA(Pro). Instead, Cys-tRNA(Pro) is cleared by a single-domain homolog of INS, the trans-editing YbaK protein. A global bioinformatics analysis revealed that there are six types of "INS-like" proteins. In addition to INS and YbaK, four additional single-domain homologs are widely distributed throughout bacteria: ProXp-ala (formerly named PrdX), ProXp-x (annotated as ProX), ProXp-y (annotated as YeaK), and ProXp-z (annotated as PA2301). The last three are domains of unknown function. Whereas many bacteria encode a ProRS containing an INS domain in addition to YbaK, many other combinations of INS-like proteins exist throughout the bacterial kingdom. Here, we focus on Caulobacter crescentus, which encodes a ProRS with a truncated INS domain that lacks catalytic activity, as well as YbaK and ProXp-ala. We show that C. crescentus ProRS can readily form Cys- and Ala-tRNA(Pro), and deacylation studies confirmed that these species are cleared by C. crescentus YbaK and ProXp-ala, respectively. Substrate specificity of C. crescentus ProXp-ala is determined, in part, by elements in the acceptor stem of tRNA(Pro) and further ensured through collaboration with elongation factor Tu. These results highlight the diversity of approaches used to prevent proline mistranslation and reveal a novel triple-sieve mechanism of editing that relies exclusively on trans-editing factors.