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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.

The nucleotide binding protein 35 (Nbp35)/cytosolic Fe-S cluster deficient 1 (Cfd1)/alternative pyrimidine biosynthetic protein C (ApbC) protein homologs have been identified in all three domains of life. In eukaryotes, the Nbp35/Cfd1 heterocomplex is an essential Fe-S cluster assembly scaffold required for the maturation of Fe-S proteins in the cytosol and nucleus, whereas the bacterial ApbC is an Fe-S cluster transfer protein only involved in the maturation of a specific target protein. Here, we show that the Nbp35/ApbC homolog MMP0704 purified from its native archaeal host Methanococcus maripaludis contains a [4Fe-4S] cluster that can be transferred to a [4Fe-4S] apoprotein. Deletion of mmp0704 from M. maripaludis does not cause growth deficiency under our tested conditions. Our data indicate that Nbp35/ApbC is a nonessential [4Fe-4S] cluster transfer protein in methanogenic archaea.

Here, we report that wild type Escherichia coli ribosomes accept and elongate precharged initiator tRNAs acylated with multiple benzoic acids, including aramid precursors, as well as malonyl (1,3-dicarbonyl) substrates to generate a diverse set of aramid-peptide and polyketide-peptide hybrid molecules. This work expands the scope of ribozyme- and ribosome-catalyzed chemical transformations, provides a starting point for in vivo translation engineering efforts, and offers an alternative strategy for the biosynthesis of polyketide-peptide natural products.

SummaryProtein translation using four-base codons occurs in both natural and synthetic systems. What constraints contributed to the universal adoption of a triplet-codon, rather than quadruplet-codon, genetic code? Here, we investigate the tolerance of the E. coli genetic code to tRNA mutations that increase codon size. We found that tRNAs from all twenty canonical isoacceptor classes can be converted to functional quadruplet tRNAs (qtRNAs), many of which selectively incorporate a single amino acid in response to a specified four-base codon. However, efficient quadruplet codon translation often requires multiple tRNA mutations, potentially constraining evolution. Moreover, while tRNAs were largely amenable to quadruplet conversion, only nine of the twenty aminoacyl tRNA synthetases tolerate quadruplet anticodons. These constitute a functional and mutually orthogonal set, but one that sharply limits the chemical alphabet available to a nascent all-quadruplet code. Our results illuminate factors that led to selection and maintenance of triplet codons in primordial Earth and provide a blueprint for synthetic biologists to deliberately engineer an all-quadruplet expanded genetic code.

Genetic code expansion technologies supplement the natural codon repertoire with assignable variants in vivo, but are often limited by heterologous translational components and low suppression efficiencies. Here, we explore engineered Escherichia coli tRNAs supporting quadruplet codon translation by first developing a library-cross-library selection to nominate quadruplet codon–anticodon pairs. We extend our findings using a phage-assisted continuous evolution strategy for quadruplet-decoding tRNA evolution (qtRNA-PACE) that improved quadruplet codon translation efficiencies up to 80-fold. Evolved qtRNAs appear to maintain codon-anticodon base pairing, are typically aminoacylated by their cognate tRNA synthetases, and enable processive translation of adjacent quadruplet codons. Using these components, we showcase the multiplexed decoding of up to four unique quadruplet codons by their corresponding qtRNAs in a single reporter. Cumulatively, our findings highlight how E. coli tRNAs can be engineered, evolved, and combined to decode quadruplet codons, portending future developments towards an exclusively quadruplet codon translation system., Genetic code expansion strategies are limited to specific codons that can be reassigned to new amino acids. Here the authors show that quadruplet-decoding tRNAs (qtRNAs) can be rapidly discovered and evolved to decode new quadruplet codons, enabling four independent decoding events in a single protein in living cells.

Antibiotic resistance frequently evolves through fitness trade-offs in which the genetic alterations that confer resistance to a drug can also cause growth defects in resistant cells. Here, through experimental evolution in a microfluidics-based turbidostat, we demonstrate that antibiotic-resistant cells can be efficiently inhibited by amplifying the fitness costs associated with drug-resistance evolution. Using tavaborole-resistant Escherichia coli as a model, we show that genetic mutations in leucyl-tRNA synthetase (that underlie tavaborole resistance) make resistant cells intolerant to norvaline, a chemical analog of leucine that is mistakenly used by tavaborole-resistant cells for protein synthesis. We then show that tavaborole-sensitive cells quickly outcompete tavaborole-resistant cells in the presence of norvaline due to the amplified cost of the molecular defect of tavaborole resistance. This finding illustrates that understanding molecular mechanisms of drug resistance allows us to effectively amplify even small evolutionary vulnerabilities of resistant cells to potentially enhance or enable adaptive therapies by accelerating posttreatment competition between resistant and susceptible cells.

Antibiotic resistance frequently evolves through fitness trade-offs in which the genetic alterations that confer resistance to a drug can also cause growth defects in resistant cells. Here, through experimental evolution in a microfluidics-based turbidostat, we demonstrate that antibiotic-resistant cells can be efficiently inhibited by amplifying the fitness costs associated with drug-resistance evolution. Using tavaborole-resistant E. coli as a model, we show that genetic mutations in leucyl-tRNA synthetase (that underlie tavaborole resistance) make resistant cells intolerant to norvaline, a chemical analog of leucine that is mistakenly used by tavaborole-resistant cells for protein synthesis. We then show that tavaborole-sensitive cells quickly outcompete tavaborole-resistant cells in the presence of norvaline due to the amplified cost of the molecular defect of tavaborole resistance. This finding illustrates a potentially generalizable approach for combating therapeutic resistance, prolonging the effectiveness of drugs and enabling the use of drugs that are no longer effective due to the rapid evolution of resistance.

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.

Transfer RNA (tRNA) has a long-established role in protein synthesis. The tRNA molecule serves as an adaptor [1] between the genetic instructions written in nucleic acid sequences and the protein p...

Selenocysteine (Sec), a rare genetically encoded amino acid with unusual chemical properties, is of great interest for protein engineering. Sec is synthesized on its cognate tRNA (tRNA(Sec)) by the concerted action of several enzymes. While all other aminoacyl-tRNAs are delivered to the ribosome by the elongation factor Tu (EF-Tu), Sec-tRNA(Sec) requires a dedicated factor, SelB. Incorporation of Sec into protein requires recoding of the stop codon UGA aided by a specific mRNA structure, the SECIS element. This unusual biogenesis restricts the use of Sec in recombinant proteins, limiting our ability to study the properties of selenoproteins. Several methods are currently available for the synthesis selenoproteins. Here we focus on strategies for in vivo Sec insertion at any position(s) within a recombinant protein in a SECIS-independent manner: (i) engineering of tRNA(Sec) for use by EF-Tu without the SECIS requirement, and (ii) design of a SECIS-independent SelB route.

Ribosomal proteins are indispensable components of a living cell, and yet their structures are remarkably diverse in different species. Here we use manually curated structural alignments to provide a comprehensive catalog of structural variations in homologous ribosomal proteins from bacteria, archaea, eukaryotes, and eukaryotic organelles. By resolving numerous ambiguities and errors of automated structural and sequence alignments, we uncover a whole new class of structural variations that reside within seemingly conserved segments of ribosomal proteins. We then illustrate that these variations reflect an apparent adaptation of ribosomal proteins to the specific environments and lifestyles of living species. Finally, we show that most of these structural variations reside within nonglobular extensions of ribosomal proteins—protein segments that are thought to promote ribosome biogenesis by stabilizing the proper folding of ribosomal RNA. We show that although the extensions are thought to be the most ancient peptides on our planet, they are in fact the most rapidly evolving and most structurally and functionally diverse segments of ribosomal proteins. Overall, our work illustrates that, despite being long considered as slowly evolving and highly conserved, ribosomal proteins are more complex and more specialized than is generally recognized.

Development of new aminoacyl-tRNA synthetase (aaRS)•tRNA pairs is central for incorporation of novel non-canonical amino acids (ncAAs) into proteins via genetic code expansion (GCE). The Escherichia coli and Caulobacter crescentus histidyl-tRNA synthetases (HisRS) evolved divergent mechanisms of tRNAE. coli was genetically engineered to use a C. crescentus HisRS•tRNAA genomically modified E. coli strain (named MEOV1) was created. MEVO1 requires an active C. crescentus HisRS•tRNAThe C. crescentus HisRS•tRNAWe developed a platform that allows protein engineering of E. coli HisRS that should facilitate GCE in E. coli. This article is part of a Special Issue entitled "Biochemistry of Synthetic Biology - Recent Developments" Guest Editor: Dr. Ilka Heinemann and Dr. Patrick O'Donoghue.

Pyrrolysyl-tRNA synthetase (PylRS) is a major tool in genetic code expansion with non-canonical amino acids, yet understanding of its structure and activity is incomplete. Here we describe the crystal structure of the previously uncharacterized essential N-terminal domain of this unique enzyme in complex with tRNAPyl. This structure explains why PylRS remains orthogonal in a broad range of organisms, from bacteria to humans. The structure also illustrates why tRNAPyl recognition by PylRS is anticodon-independent; the anticodon does not contact the enzyme. Using standard microbiological culture equipment, we then established a new method for laboratory evolution – a non-continuous counterpart of the previously developed phage-assisted continuous evolution. With this method, we evolved novel PylRS variants with enhanced activity and amino acid specificity. We finally employed an evolved PylRS variant to determine its N-terminal domain structure and show how its mutations improve PylRS activity in the genetic encoding of a non-canonical amino acid.

By transplanting identity elements into E. coli tRNAfMet , we have engineered an orthogonal initiator tRNA (itRNATy2 ) that is a substrate for Methanocaldococcus jannaschii TyrRS. We demonstrate that itRNATy2 can initiate translation in vivo with aromatic non-canonical amino acids (ncAAs) bearing diverse sidechains. Although the initial system suffered from low yields, deleting redundant copies of tRNAfMet from the genome afforded an E. coli strain in which the efficiency of non-canonical initiation equals elongation. With this improved system we produced a protein containing two distinct ncAAs at the first and second positions, an initial step towards producing completely unnatural polypeptides in vivo. This work provides a valuable tool to synthetic biology and demonstrates remarkable versatility of the E. coli translational machinery for initiation with ncAAs in vivo.

Eukaryotic cells are divided into the nucleus and the cytosol, and, to enter the nucleus, proteins typically possess short signal sequences, known as nuclear localization signals (NLSs). Although NLSs have long been considered as features unique to eukaryotic proteins, we show here that similar or identical protein segments are present in ribosomal proteins from the Archaea. Specifically, the ribosomal proteins uL3, uL15, uL18, and uS12 possess NLS-type motifs that are conserved across all major branches of the Archaea, including the most ancient groups Microarchaeota and Diapherotrites, pointing to the ancient origin of NLS-type motifs in the Archaea. Furthermore, by using fluorescence microscopy, we show that the archaeal NLS-type motifs can functionally substitute eukaryotic NLSs and direct the transport of ribosomal proteins into the nuclei of human cells. Collectively, these findings illustrate that the origin of NLSs preceded the origin of the cell nucleus, suggesting that the initial function of NLSs was not related to intracellular trafficking, but possibly was to improve recognition of nucleic acids by cellular proteins. Overall, our study reveals rare evolutionary intermediates among archaeal cells that can help elucidate the sequence of events that led to the origin of the eukaryotic cell.

In the past two decades, tRNA molecules and their corresponding aminoacyl-tRNA synthetases (aaRS) have been extensively used in synthetic biology to genetically encode post-translationally modified and unnatural amino acids. In this review, we briefly examine one fundamental requirement for the successful application of tRNA/aaRS pairs for expanding the genetic code. This requirement is known as “orthogonality„—the ability of a tRNA and its corresponding aaRS to interact exclusively with each other and avoid cross-reactions with additional types of tRNAs and aaRSs in a given organism.

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.

The sulfur-containing nucleosides in transfer RNA (tRNAs) are present in all three domains of life; they have critical functions for accurate and efficient translation, such as tRNA structure stabilization and proper codon recognition. The tRNA modification enzymes ThiI (in bacteria and archaea) and Ncs6 (in archaea and eukaryotic cytosols) catalyze the formation of 4-thiouridine (s4U) and 2-thiouridine (s2U), respectively. The ThiI homologs were proposed to transfer sulfur via cysteine persulfide enzyme adducts, whereas the reaction mechanism of Ncs6 remains unknown. Here we show that ThiI from the archaeon Methanococcus maripaludis contains a [3Fe-4S] cluster that is essential for its tRNA thiolation activity. Furthermore, the archaeal and eukaryotic Ncs6 homologs as well as phosphoseryl-tRNA (Sep-tRNA):Cys-tRNA synthase (SepCysS), which catalyzes the Sep-tRNA to Cys-tRNA conversion in methanogens, also possess a [3Fe-4S] cluster similar to the methanogenic archaeal ThiI. These results suggest that the diverse tRNA thiolation processes in archaea and eukaryotic cytosols share a common mechanism dependent on a [3Fe-4S] cluster for sulfur transfer.

Selenocysteine is the only proteinogenic amino acid encoded by a recoded in-frame UGA codon that does not operate as the canonical opal stop codon. A specialized translation elongation factor, eEFSec in eukaryotes and SelB in prokaryotes, promotes selenocysteine incorporation into selenoproteins by a still poorly understood mechanism. Our structural and biochemical results reveal that four domains of human eEFSec fold into a chalice-like structure that has similar binding affinities for GDP, GTP and other guanine nucleotides. Surprisingly, unlike in eEF1A and EF-Tu, the guanine nucleotide exchange does not cause a major conformational change in domain 1 of eEFSec, but instead induces a swing of domain 4. We propose that eEFSec employs a non-canonical mechanism involving the distinct C-terminal domain 4 for the release of the selenocysteinyl-tRNA during decoding on the ribosome., Specialized translation elongation factors (eEFSec and SelB) promote selenocysteine incorporation into proteins. Here, the authors report the structure of human eEFSec, examine its interactions with guanine nucleotides, and propose a non-canonical mechanism for decoding selenocysteine.

Protein phosphorylation is one of the most important post-translational modifications in nature. However, the site-specific incorporation of O-phosphotyrosine into proteins in vivo has not yet been reported. Endogenous phosphatases present in cells can dephosphorylate phosphotyrosine as a free amino acid or as a protein residue. Therefore, we deleted the genes of five phosphatases from the genome of Escherichia coli with the aim of stabilizing phosphotyrosine. Together with an engineered aminoacyl-tRNA synthetase (derived from Methanocaldococcus jannaschii tyrosyl-tRNA synthetase) and an elongation factor Tu variant, we were able to co-translationally incorporate O-phosphotyrosine into the super-folder green fluorescent protein at a desired position in vivo. This system will facilitate future studies of tyrosine phosphorylation.

Cisplatin is a widely prescribed anticancer drug, which triggers cell death by covalent binding to a broad range of biological molecules. Among cisplatin targets, cellular RNAs remain the most poorly characterized molecules. Although cisplatin was shown to inactivate essential RNAs, including ribosomal, spliceosomal and telomeric RNAs, cisplatin binding sites in most RNA molecules are unknown, and therefore it remains challenging to study how modifications of RNA by cisplatin contributes to its toxicity. Here we report a 2.6Å-resolution X-ray structure of cisplatin-modified 70S ribosome, which describes cisplatin binding to the ribosome and provides the first nearly atomic model of cisplatin–RNA complex. We observe nine cisplatin molecules bound to the ribosome and reveal consensus structural features of the cisplatin-binding sites. Two of the cisplatin molecules modify conserved functional centers of the ribosome—the mRNA-channel and the GTPase center. In the mRNA-channel, cisplatin intercalates between the ribosome and the messenger RNA, suggesting that the observed inhibition of protein synthesis by cisplatin is caused by impaired mRNA-translocation. Our structure provides an insight into RNA targeting and inhibition by cisplatin, which can help predict cisplatin-binding sites in other cellular RNAs and design studies to elucidate a link between RNA modifications by cisplatin and cisplatin toxicity.

Withstanding 3.5 billion years of genetic drift, the canonical genetic code remains such a fundamental foundation for the complexity of life that it is highly conserved across all three phylogenetic domains. Genome engineering technologies are now making it possible to rationally change the genetic code, offering resistance to viruses, genetic isolation from horizontal gene transfer, and prevention of environmental escape by genetically modified organisms. We discuss the biochemical, genetic, and technological challenges that must be overcome in order to engineer the genetic code.

Microsporidia are fungi-like parasites that have the smallest known eukaryotic genome, and for that reason they are used as a model to study the phenomenon of genome decay in parasitic forms of life. Similar to other intracellular parasites that reproduce asexually in an environment with alleviated natural selection, Microsporidia experience continuous genome decay that is driven by Muller&rsquo, s ratchet&mdash, an evolutionary process of irreversible accumulation of deleterious mutations that lead to gene loss and the miniaturization of cellular components. Particularly, Microsporidia have remarkably small ribosomes in which the rRNA is reduced to the minimal enzymatic core. In this study, we analyzed microsporidian ribosomes to study an apparent impact of Muller&rsquo, s ratchet on structure of RNA and protein molecules in parasitic forms of life. Through mass spectrometry of microsporidian proteome and analysis of microsporidian genomes, we found that massive rRNA reduction in microsporidian ribosomes appears to annihilate the binding sites for ribosomal proteins eL8, eL27, and eS31, suggesting that these proteins are no longer bound to the ribosome in microsporidian species. We then provided an evidence that protein eS31 is retained in Microsporidia due to its non-ribosomal function in ubiquitin biogenesis. Our study illustrates that, while Microsporidia carry the same set of ribosomal proteins as non-parasitic eukaryotes, some ribosomal proteins are no longer participating in protein synthesis in Microsporidia and they are preserved from genome decay by having extra-ribosomal functions. More generally, our study shows that many components of parasitic cells, which are identified by automated annotation of pathogenic genomes, may lack part of their biological functions due to continuous genome decay.

Intracellular organisms, such as obligate parasites and endosymbionts, typically possess small genomes due to continuous genome decay caused by an environment with alleviated natural selection. Previously, a few species with highly reduced genomes, including the intracellular pathogens Mycoplasma and Microsporidia, have been shown to carry degenerated editing domains in aminoacyl-tRNA synthetases. These defects in the protein synthesis machinery cause inaccurate translation of the genetic code, resulting in significant statistical errors in protein sequences that are thought to help parasites to escape immune response of a host. In this study we analyzed 10,423 complete bacterial genomes to assess conservation of the editing domains in tRNA synthetases, including LeuRS, IleRS, ValRS, ThrRS, AlaRS, and PheRS. We found that, while the editing domains remain intact in free-living species, they are degenerated in the overwhelming majority of host-restricted bacteria. Our work illustrates that massive genome erosion triggered by an intracellular lifestyle eradicates one of the most fundamental components of a living cell: the system responsible for proofreading of amino acid selection for protein synthesis. This finding suggests that inaccurate translation of the genetic code might be a general phenomenon among intercellular organisms with reduced genomes.

Microsporidia are fungi-like parasites that have the smallest known eukaryotic genome, and for that reason they are used as a model to study the phenomenon of genome decay in parasitic forms of life. Similar to other intracellular parasites that reproduce asexually in an environment with alleviated natural selection, Microsporidia experience continuous genome decay that is driven by Muller's ratchet-an evolutionary process of irreversible accumulation of deleterious mutations that lead to gene loss and the miniaturization of cellular components. Particularly, Microsporidia have remarkably small ribosomes in which the rRNA is reduced to the minimal enzymatic core. In this study, we analyzed microsporidian ribosomes to study an apparent impact of Muller's ratchet on structure of RNA and protein molecules in parasitic forms of life. Through mass spectrometry of microsporidian proteome and analysis of microsporidian genomes, we found that massive rRNA reduction in microsporidian ribosomes appears to annihilate the binding sites for ribosomal proteins eL8, eL27, and eS31, suggesting that these proteins are no longer bound to the ribosome in microsporidian species. We then provided an evidence that protein eS31 is retained in Microsporidia due to its non-ribosomal function in ubiquitin biogenesis. Our study illustrates that, while Microsporidia carry the same set of ribosomal proteins as non-parasitic eukaryotes, some ribosomal proteins are no longer participating in protein synthesis in Microsporidia and they are preserved from genome decay by having extra-ribosomal functions. More generally, our study shows that many components of parasitic cells, which are identified by automated annotation of pathogenic genomes, may lack part of their biological functions due to continuous genome decay.

Microsporidia are parasitic fungi-like organisms that invade the interior of living cells and cause chronic disorders in a broad range of animals, including humans. These pathogens have the tiniest known genomes among eukaryotic species, for which they serve as a model for exploring the phenomenon of genome reduction in obligate intracellular parasites. Here we report a case study to show an apparent effect of overall genome reduction on the primary structure and activity of aminoacyl-tRNA synthetases, indispensable cellular proteins required for protein synthesis. We find that most microsporidian synthetases lack regulatory and eukaryote-specific appended domains and have a high degree of sequence variability in tRNA-binding and catalytic domains. In one synthetase, LeuRS, an apparent sequence degeneration annihilates the editing domain, a catalytic center responsible for the accurate selection of leucine for protein synthesis. Unlike accurate LeuRS synthetases from other eukaryotic species, microsporidian LeuRS is error-prone: apart from leucine, it occasionally uses its near-cognate substrates, such as norvaline, isoleucine, valine, and methionine. Mass spectrometry analysis of the microsporidium Vavraia culicis proteome reveals that nearly 6% of leucine residues are erroneously replaced by other amino acids. This remarkably high frequency of mistranslation is not limited to leucine codons and appears to be a general property of protein synthesis in microsporidian parasites. Taken together, our findings reveal that the microsporidian protein synthesis machinery is editing-deficient, and that the proteome of microsporidian parasites is more diverse than would be anticipated based on their genome sequences.

Inhibition of tRNA aminoacylation has proven to be an effective antimicrobial strategy, impeding an essential step of protein synthesis. Mupirocin, the well-known selective inhibitor of bacterial isoleucyl-tRNA synthetase, is one of three aminoacylation inhibitors now approved for human or animal use. However, design of novel aminoacylation inhibitors is complicated by the steadfast requirement to avoid off-target inhibition of protein synthesis in human cells. Here we review available data regarding known aminoacylation inhibitors as well as key amino-acid residues in aminoacyl-tRNA synthetases (aaRSs) and nucleotides in tRNA that determine the specificity and strength of the aaRS-tRNA interaction. Unlike most ligand-protein interactions, the aaRS-tRNA recognition interaction represents coevolution of both the tRNA and aaRS structures to conserve the specificity of aminoacylation. This property means that many determinants of tRNA recognition in pathogens have diverged from those of humans—a phenomenon that provides a valuable source of data for antimicrobial drug development.

Genetically encoded non-canonical amino acids are powerful tools of protein research and engineering; in particular they allow substitution of individual chemical groups or atoms in a protein of interest. One such amino acid is the tryptophan (Trp) analog 3-benzothienyl-l-alanine (Bta) with an imino-to-sulfur substitution in the five-membered ring. Unlike Trp, Bta is not capable of forming a hydrogen bond, but preserves other properties of a Trp residue. Here we present a pyrrolysyl-tRNA synthetase-derived, engineered enzyme BtaRS that enables efficient and site-specific Bta incorporation into proteins of interest in vivo. Furthermore, we report a 2.1 A-resolution crystal structure of a BtaRS•Bta complex to show how BtaRS discriminates Bta from canonical amino acids, including Trp. To show utility in protein mutagenesis, we used BtaRS to introduce Bta to replace the Trp28 residue in the active site of Staphylococcus aureus thioredoxin. This experiment showed that not the hydrogen bond between residues Trp28 and Asp58, but the bulky aromatic side chain of Trp28 is important for active site maintenance. Collectively, our study provides a new and robust tool for checking the function of Trp in proteins.

We have changed the amino acid set of the genetic code of Escherichia coli by evolving cultures capable of growing on the synthetic non-canonical amino acid L-β-(thieno[3,2-b]pyrrolyl)-alanine ([3,2]Tpa) as a sole surrogate for the canonical amino acid L-tryptophan (Trp). A long-term cultivation experiment in defined synthetic media resulted in the evolution of cells capable of surviving Trp → [3,2] Tpa substitutions in their proteomes in response to the 20,899 TGG codons of the E. coli W3110 genome. These evolved bacteria with new-to-nature amino acid composition are capable of robust growth in the complete absence of Trp. Our experimental results illustrate an approach for the evolution of synthetic cells with alternative biochemical building blocks.

Objective: We aimed to decipher the molecular genetic basis of disease in a cohort of children with a uniform clinical presentation of neonatal irritability, spastic or dystonic quadriplegia, virtually absent psychomotor development, axonal neuropathy, and elevated blood/CSF lactate. Methods: We performed whole-exome sequencing of blood DNA from the index patients. Detected compound heterozygous mutations were confirmed by Sanger sequencing. Structural predictions and a bacterial activity assay were performed to evaluate the functional consequences of the mutations. Mass spectrometry, Western blotting, and protein oxidation detection were used to analyze the effects of selenoprotein deficiency. Results: Neuropathology indicated laminar necrosis and severe loss of myelin, with neuron loss and astrogliosis. In 3 families, we identified a missense (p.Thr325Ser) and a nonsense (p.Tyr429*) mutation in SEPSECS , encoding the O -phosphoseryl-tRNA:selenocysteinyl-tRNA synthase, which was previously associated with progressive cerebellocerebral atrophy. We show that the mutations do not completely abolish the activity of SEPSECS, but lead to decreased selenoprotein levels, with demonstrated increase in oxidative protein damage in the patient brain. Conclusions: These results extend the phenotypes caused by defective selenocysteine biosynthesis, and suggest SEPSECS as a candidate gene for progressive encephalopathies with lactate elevation.

The diversity of the genetic code systems used by microbes on earth is yet to be elucidated. It is known that certain methanogenic archaea employ an alternative system for cysteine (Cys) biosynthesis and encoding; tRNACys is first acylated with phosphoserine (Sep) by O-phosphoseryl-tRNA synthetase (SepRS) and then converted to Cys-tRNACys by Sep-tRNA:Cys-tRNA synthase (SepCysS). In this study, we searched all genomic and metagenomic protein sequence data in the Integrated Microbial Genomes (IMG) system and at the NCBI to reveal new clades of SepRS and SepCysS proteins belonging to diverse archaea in the four major groups (DPANN, Euryarchaeota, TACK, and Asgard) and two groups of bacteria (“Candidatus Parcubacteria” and Chloroflexi). Bacterial SepRS and SepCysS charged bacterial tRNACys species with cysteine in vitro. Homologs of SepCysE, a scaffold protein facilitating SepRS⋅SepCysS complex assembly in Euryarchaeota class I methanogens, are found in a few groups of TACK and Asgard archaea, whereas the C-terminally truncated homologs exist fused or genetically coupled with diverse SepCysS species. Investigation of the selenocysteine (Sec)- and pyrrolysine (Pyl)-utilizing traits in SepRS-utilizing archaea and bacteria revealed that the archaea carrying full-length SepCysE employ Sec and that SepRS is often found in Pyl-utilizing archaea and Chloroflexi bacteria. We discuss possible contributions of the SepRS-SepCysS system for sulfur assimilation, methanogenesis, and other metabolic processes requiring large amounts of iron-sulfur enzymes or Pyl-containing enzymes., IMPORTANCE Comprehensive analyses of all genomic and metagenomic protein sequence data in public databases revealed the distribution and evolution of an alternative cysteine-encoding system in diverse archaea and bacteria. The finding that the SepRS-SepCysS-SepCysE- and the selenocysteine-encoding systems are shared by the Euryarchaeota class I methanogens, the Crenarchaeota AK8/W8A-19 group, and an Asgard archaeon suggests that ancient archaea may have used both systems. In contrast, bacteria may have obtained the SepRS-SepCysS system from archaea. The SepRS-SepCysS system sometimes coexists with a pyrrolysine-encoding system in both archaea and bacteria. Our results provide additional bioinformatic evidence for the contribution of the SepRS-SepCysS system for sulfur assimilation and diverse metabolisms which require vast amounts of iron-sulfur enzymes and proteins. Among these biological activities, methanogenesis, methylamine metabolism, and organohalide respiration may have local and global effects on earth. Taken together, uncultured bacteria and archaea provide an expanded record of the evolution of the genetic code.

Progress in genetic code expansion requires accurate, selective, and high-throughput detection of non-standard amino acid (NSAA) incorporation into proteins. Here, we discover how the N-end rule pathway of protein degradation applies to commonly used NSAAs. We show that several NSAAs are N-end stabilizing and demonstrate that other NSAAs can be made stabilizing by rationally engineering the N-end rule adaptor protein ClpS. We use these insights to engineer a synthetic quality control method, termed “Post-Translational Proofreading” (PTP). By implementing PTP, false positive proteins resulting from misincorporation of structurally similar standard amino acids or undesired NSAAs rapidly degrade, enabling high-accuracy discrimination of desired NSAA incorporation. We illustrate the utility of PTP during evolution of the biphenylalanine orthogonal translation system used for synthetic biocontainment. Our new OTS is more selective and confers lower escape frequencies and greater fitness in all tested biocontained strains. Our approach presents a new paradigm for molecular recognition of amino acids in target proteins.

Directed evolution of orthogonal aminoacyl-tRNA synthetases (AARSs) enables site-specific installation of non-canonical amino acids (ncAAs) into proteins. Traditional evolution techniques typically produce AARSs with greatly reduced activity and selectivity compared to their wild-type counterparts. We designed phage-assisted continuous evolution (PACE) selections to rapidly produce highly active and selective orthogonal AARSs through hundreds of generations of evolution. PACE of a chimeric Methanosarcina spp. pyrrolysyl-tRNA synthetase (PylRS) improved its enzymatic efficiency (kcat/KMtRNA) 45-fold compared to the parent enzyme. Transplantation of the evolved mutations into other PylRS-derived synthetases improved yields of proteins containing non-canonical residues up to 9.7-fold. Simultaneous positive and negative selection PACE over 48 h greatly improved the selectivity of a promiscuous Methanocaldococcus jannaschii tyrosyl-tRNA synthetase variant for site-specific incorporation of p-iodo-L-phenylalanine. These findings offer new AARSs that increase the utility of orthogonal translation systems and establish the capability of PACE to efficiently evolve orthogonal AARSs with high activity and amino acid specificity.

The tRNA identity elements for some amino acids are distinct between the bacterial and archaeal domains. Searching in recent genomic and metagenomic sequence data, we found some candidate phyla radiation (CPR) bacteria with archaeal tRNA identity for Tyr-tRNA and Trp-tRNA synthesis. These bacteria possess genes for tyrosyl-tRNA synthetase (TyrRS) and tryptophanyl-tRNA synthetase (TrpRS) predicted to be derived from DPANN superphylum archaea, while the cognate tRNATyr and tRNATrp genes reveal bacterial or archaeal origins. We identified a trace of domain fusion and swapping in the archaeal-type TyrRS gene of a bacterial lineage, suggesting that CPR bacteria may have used this mechanism to create diverse proteins. Archaeal-type TrpRS of bacteria and a few TrpRS species of DPANN archaea represent a new phylogenetic clade (named TrpRS-A). The TrpRS-A open reading frames (ORFs) are always associated with another ORF (named ORF1) encoding an unknown protein without global sequence identity to any known protein. However, our protein structure prediction identified a putative HIGH-motif and KMSKS-motif as well as many α-helices that are characteristic of class I aminoacyl-tRNA synthetase (aaRS) homologs. These results provide another example of the diversity of molecular components that implement the genetic code and provide a clue to the early evolution of life and the genetic code.

Many prokaryotes lack a tRNA synthetase to attach asparagine to its cognate tRNA(Asn), and instead synthesize asparagine from tRNA(Asn)-bound aspartate. This conversion involves two enzymes: a nondiscriminating aspartyl-tRNA synthetase (ND-AspRS) that forms Asp-tRNA(Asn), and a heterotrimeric amidotransferase GatCAB that amidates Asp-tRNA(Asn) to form Asn-tRNA(Asn) for use in protein synthesis. ND-AspRS, GatCAB, and tRNA(Asn) may assemble in an ∼400-kDa complex, known as the Asn-transamidosome, which couples the two steps of asparagine biosynthesis in space and time to yield Asn-tRNA(Asn). We report the 3.7-Å resolution crystal structure of the Pseudomonas aeruginosa Asn-transamidosome, which represents the most common machinery for asparagine biosynthesis in bacteria. We show that, in contrast to a previously described archaeal-type transamidosome, a bacteria-specific GAD domain of ND-AspRS provokes a principally new architecture of the complex. Both tRNA(Asn) molecules in the transamidosome simultaneously serve as substrates and scaffolds for the complex assembly. This architecture rationalizes an elevated dynamic and a greater turnover of ND-AspRS within bacterial-type transamidosomes, and possibly may explain a different evolutionary pathway of GatCAB in organisms with bacterial-type vs. archaeal-type Asn-transamidosomes. Importantly, because the two-step pathway for Asn-tRNA(Asn) formation evolutionarily preceded the direct attachment of Asn to tRNA(Asn), our structure also may reflect the mechanism by which asparagine was initially added to the genetic code.

Pyrrolysyl-tRNA synthetase (PylRS) and its cognate tRNA Pyl have emerged as ideal translation components for genetic code innovation. Variants of the enzyme facilitate the incorporation >100 noncanonical amino acids (ncAAs) into proteins. PylRS variants were previously selected to acylate N e -acetyl-Lys (AcK) onto tRNA Pyl . Here, we examine an N e -acetyl-lysyl-tRNA synthetase (AcKRS), which is polyspecific (i.e., active with a broad range of ncAAs) and 30-fold more efficient with Phe derivatives than it is with AcK. Structural and biochemical data reveal the molecular basis of polyspecificity in AcKRS and in a PylRS variant [iodo-phenylalanyl-tRNA synthetase (IFRS)] that displays both enhanced activity and substrate promiscuity over a chemical library of 313 ncAAs. IFRS, a product of directed evolution, has distinct binding modes for different ncAAs. These data indicate that in vivo selections do not produce optimally specific tRNA synthetases and suggest that translation fidelity will become an increasingly dominant factor in expanding the genetic code far beyond 20 amino acids.

Methanogenic archaea lack cysteinyl-tRNA synthetase; they synthesize Cys-tRNA and cysteine in a tRNA-dependent manner. Two enzymes are required: Phosphoseryl-tRNA synthetase (SepRS) forms phosphoseryl-tRNA(Cys) (Sep-tRNA(Cys)), which is converted to Cys-tRNA(Cys) by Sep-tRNA:Cys-tRNA synthase (SepCysS). This represents the ancestral pathway of Cys biosynthesis and coding in archaea. Here we report a translation factor, SepCysE, essential for methanococcal Cys biosynthesis; its deletion in Methanococcus maripaludis causes Cys auxotrophy. SepCysE acts as a scaffold for SepRS and SepCysS to form a stable high-affinity complex for tRNA(Cys) causing a 14-fold increase in the initial rate of Cys-tRNA(Cys) formation. Based on our crystal structure (2.8-Å resolution) of a SepCysS⋅SepCysE complex, a SepRS⋅SepCysE⋅SepCysS structure model suggests that this ternary complex enables substrate channeling of Sep-tRNA(Cys). A phylogenetic analysis suggests coevolution of SepCysE with SepRS and SepCysS in the last universal common ancestral state. Our findings suggest that the tRNA-dependent Cys biosynthesis proceeds in a multienzyme complex without release of the intermediate and this mechanism may have facilitated the addition of Cys to the genetic code.

Selenocysteine (Sec) is naturally co-translationally incorporated into proteins by recoding the UGA opal codon with a specialized elongation factor (SelB in bacteria) and an RNA structural signal (SECIS element). We have recently developed a SECIS-free selenoprotein synthesis system that site-specifically—using the UAG amber codon—inserts Sec depending on the elongation factor Tu (EF-Tu). Here, we describe the engineering of EF-Tu for improved selenoprotein synthesis. A Sec-specific selection system was established by expression of human protein O6-alkylguanine-DNA alkyltransferase (hAGT), in which the active site cysteine codon has been replaced by the UAG amber codon. The formed hAGT selenoprotein repairs the DNA damage caused by the methylating agent N-methyl-N′-nitro-N-nitrosoguanidine, and thereby enables Escherichia coli to grow in the presence of this mutagen. An EF-Tu library was created in which codons specifying the amino acid binding pocket were randomized. Selection was carried out for enhanced Sec incorporation into hAGT; the resulting EF-Tu variants contained highly conserved amino acid changes within members of the library. The improved UTu-system with EF-Sel1 raises the efficiency of UAG-specific Sec incorporation to >90%, and also doubles the yield of selenoprotein production.

Thiolation of carbon-2 of uridine located in the first position of the anticodons of tRNA UUG Gln , tRNA UUC Glu , and tRNA UUU Lys is a conserved RNA modification event requiring the 2-thiouridine synthetase Ncs6/Ctu1 in archaea and eukaryotes. Ncs6/Ctu1 activates uridine by adenylation, but its role in sulfur transfer is unclear. Here we show that Mmp1356, the Ncs6/Ctu1 homolog in the archaeon Methanococcus maripaludis, forms a persulfide enzyme adduct with an active site cysteine; this suggests that Mmp1356 directly participates in sulfur transfer as a persulfide carrier. Transposon mutagenesis shows that Mmp1356 is likely to be an essential protein.

I was first introduced to RNA in 1962. As a newly minted PhD in synthetic organic chemistry I had just joined in Madison, Wisconsin a team of four postdocs charged with the challenging task of chemically synthesizing 64 ribotrinucleotides by the most modern methods (manual synthesis, of course, requiring gallons of dry pyridine every day). My advisor, Har Gobind Khorana, a man of boundless energy, vision and courage, was convinced that these RNA triplets would be essential for the elucidation of the genetic code. It was an exciting time in molecular biology as the first details of the mechanism of protein synthesis and the genetic code emerged. Then, after three years of intense work with great team spirit the code was cracked! At the end of my time in Wisconsin I had become a molecular biologist, spellbound by the genetic code and by tRNA. I would never lose interest in this molecule, and it guided my research for the next five decades.

Here we report the crystal structures of the Thermus thermophilus ribosome at 2.3-2.5Å-resolution, which have enabled a comprehensive modeling of rRNA modifications. The structures reveal contacts of modified nucleotides with mRNA and tRNAs or protein pY, and contacts within the ribosome interior stabilizing the functional fold of rRNA. Our work provides a resource to explore the roles of rRNA modifications and yields the most complete atomic model of bacterial ribosome.

Most archaea and bacteria use a modified C in the anticodon wobble position of isoleucine tRNA to base pair with A but not with G of the mRNA. This allows the tRNA to read the isoleucine codon AUA without also reading the methionine codon AUG. To understand why a modified C, and not U or modified U, is used to base pair with A, we mutated the C34 in the anticodon of Haloarcula marismortui isoleucine tRNA (tRNA2Ile) to U, expressed the mutant tRNA in Haloferax volcanii, and purified and analyzed the tRNA. Ribosome binding experiments show that although the wild-type tRNA2Ile binds exclusively to the isoleucine codon AUA, the mutant tRNA binds not only to AUA but also to AUU, another isoleucine codon, and to AUG, a methionine codon. The G34 to U mutant in the anticodon of another H. marismortui isoleucine tRNA species showed similar codon binding properties. Binding of the mutant tRNA to AUG could lead to misreading of the AUG codon and insertion of isoleucine in place of methionine. This result would explain why most archaea and bacteria do not normally use U or a modified U in the anticodon wobble position of isoleucine tRNA for reading the codon AUA. Biochemical and mass spectrometric analyses of the mutant tRNAs have led to the discovery of a new modified nucleoside, 5-cyanomethyl U in the anticodon wobble position of the mutant tRNAs. 5-Cyanomethyl U is present in total tRNAs from euryarchaea but not in crenarchaea, eubacteria, or eukaryotes., National Institutes of Health (U.S.) (GM17151), National Institutes of Health (U.S.) (GM22854), National Institutes of Health (U.S.) (ES017010), Singapore-MIT Alliance for Research and Technology, Singapore. National Research Foundation, United States. Dept. of Energy (DE-FG36-08GO88055)

Genetic code expansion for synthesis of proteins containing noncanonical amino acids is a rapidly growing field in synthetic biology. Creating optimal orthogonal translation systems will require re-engineering central components of the protein synthesis machinery on the basis of a solid mechanistic biochemical understanding of the synthetic process.

Sense codon recoding is the basis for genetic code expansion with more than two different noncanonical amino acids. It requires an unused (or rarely used) codon, and an orthogonal tRNA synthetase:tRNA pair with the complementary anticodon. The Mycoplasma capricolum genome contains just six CGG arginine codons, without a dedicated tRNA(Arg). We wanted to reassign this codon to pyrrolysine by providing M. capricolum with pyrrolysyl-tRNA synthetase, a synthetic tRNA with a CCG anticodon (tRNA(Pyl)(CCG)), and the genes for pyrrolysine biosynthesis. Here we show that tRNA(Pyl)(CCG) is efficiently recognized by the endogenous arginyl-tRNA synthetase, presumably at the anticodon. Mass spectrometry revealed that in the presence of tRNA(Pyl)(CCG), CGG codons are translated as arginine. This result is not unexpected as most tRNA synthetases use the anticodon as a recognition element. The data suggest that tRNA misidentification by endogenous aminoacyl-tRNA synthetases needs to be overcome for sense codon recoding.

The discovery of diverse codon reassignment events has demonstrated that the canonical genetic code is not universal. Studying coding reassignment at the molecular level is critical for understanding genetic code evolution, and provides clues to genetic code manipulation in synthetic biology. Here we report a novel reassignment event in the mitochondria of Ashbya (Eremothecium) gossypii, a filamentous-growing plant pathogen related to yeast (Saccharomycetaceae). Bioinformatics studies of conserved positions in mitochondrial DNA-encoded proteins suggest that CUU and CUA codons correspond to alanine in A. gossypii, instead of leucine in the standard code or threonine in yeast mitochondria. Reassignment of CUA to Ala was confirmed at the protein level by mass spectrometry. We further demonstrate that a predicted tRNA(Ala)UAG is transcribed and accurately processed in vivo, and is responsible for Ala reassignment. Enzymatic studies reveal that tRNA(Ala)UAG is efficiently recognized by A. gossypii mitochondrial alanyl-tRNA synthetase (AgAlaRS). AlaRS typically recognizes the G3:U70 base pair of tRNA(Ala); a G3A change in Ashbya tRNA(Ala)UAG abolishes its recognition by AgAlaRS. Conversely, an A3G mutation in Saccharomyces cerevisiae tRNA(Thr)UAG confers tRNA recognition by AgAlaRS. Our work highlights the dynamic feature of natural genetic codes in mitochondria, and the relative simplicity by which tRNA identity may be switched.

Pyrrolysyl-tRNA synthetase (PylRS) is a class IIc aminoacyl-tRNA synthetase that is related to phenylalanyl-tRNA synthetase (PheRS). Genetic selection provided PylRS variants with a broad range of specificity for diverse non-canonical amino acids (ncAAs). One variant is a specific phenylalanine-incorporating enzyme. Structural models of the PylRSamino acid complex show that the small pocket size and π-interaction play an important role in specific recognition of Phe and the engineered PylRS active site resembles that of Escherichia coli PheRS.

Histones are the main protein components of chromatin. They undergo numerous posttranslational modifications, including phosphorylation, acetylation, and methylation, which often affect each other[1, 2]. Such cross-regulation of histone modification is known to play a central role in many physiological processes. Phosphorylation of histone H3 at serine 10 (H3S10) and acetylation of N-terminal lysine residues in histone H3 are representative markers of transcriptional activation in eukaryotic cells. Many in vivo studies have suggested a possible link between these modifications[3-5], but this association is not yet established. Early in vitro studies using small synthetic H3 peptides provided mixed and conflicting results regarding the association between phosphorylation and acetylation[3, 4, 6], and recent peptide experiments have failed to find any correlation[7]. However, since synthetic peptides differ from the physiological substrates that undergo modifications, they can hardly represent the real physiological interactions between chromatin and modifying enzymes. Such peptides often show more than a thousand-fold lower binding abilities for modifying proteins compared to nucleosomal substrates[6]. Inconsistent results and a lack of direct evidence have led to the development of two opposing models (synergistic and independent) for these modifications[6, 8]. Thus, despite extensive studies, the correlation between phosphorylation and acetylation in histone H3 remains unclear.

The degeneracy of the genetic code allows nucleic acids to encode amino acid identity as well as non-coding 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 ofEscherichia coliwith all 123 instances of AGR codons removed from all essential genes. We readily replaced 110 AGR codons with the synonymous CGU, but the remaining thirteen “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 multi-dimensional “safe replacement zone” (SRZ) within which alternative codons are more likely to be viable. To further evaluate synonymous and non-synonymous alternatives to essential AGRs, we implemented a CRISPR/Cas9-based method to deplete a diversified population of a wild type allele, allowing us to exhaustively evaluate the fitness impact of all 64 codon alternatives. Using this method, we confirmed 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.Significance StatementThis work presents the genome-wide replacement of all rare AGR arginine codons in the essential genes ofEscherichia coliwith synonymous CGN alternatives. Synonymous codon substitutions can lethally impact non-coding function by disrupting mRNA secondary structure and ribosomal binding site-like motifs. Here we quantitatively define the range of tolerable deviation in these metrics and use this relationship to provide critical insight into codon choice in recoded genomes. This work demonstrates that genome-wide removal of AGR is likely to be possible, and provides a framework for designing genomes with radically altered genetic codes.

© The Author(s) 2016. We report the identification of novel tRNA species with 12-base pair amino-acid acceptor branches composed of longer acceptor stem and shorter Tstem. 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 inEscherichia 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.

Radicals push proteins beyond genes Chemically modifying proteins after their translation can expand their structural and functional roles (see the Perspective by Hofmann and Bode). Two related methods describe how to exploit free radical chemistry to form carbon-carbon bonds between amino acid residues and a selected functional group. Wright et al. added a wide range of functional groups to proteins containing dehydroalanine precursors, with borohydride mediating the radical chemistry. Yang et al. employed a similar approach, using zinc in combination with copper ions. Together, these results will be useful for introducing functionalities and labels to a wide range of proteins. Science , this issue pp. 597 and 623 ; see also p. 553