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Bioenergetics, genetic coding, and catalysis are all difficult to imagine emerging without pre-existing historical context. That context is often posed as a "Chicken and Egg" problem; its resolution is concisely described by de Grasse Tyson: "The egg was laid by a bird that was not a chicken". The concision and generality of that answer furnish no details-only an appropriate framework from which to examine detailed paradigms that might illuminate paradoxes underlying these three life-defining biomolecular processes. We examine experimental aspects here of five examples that all conform to the same paradigm. In each example, a paradox is resolved by coupling "if, and only if" conditions for reciprocal transitions between levels, such that the consequent of the first test is the antecedent for the second. Each condition thus restricts fluxes through, or "gates" the other. Reciprocally-coupled gating, in which two gated processes constrain one another, is self-referential, hence maps onto the formal structure of "strange loops". That mapping uncovers two different kinds of forces that may help unite the axioms underlying three phenomena that distinguish biology from chemistry. As a physical analog for Gödel's logic, biomolecular strange-loops provide a natural metaphor around which to organize a large body of experimental data, linking biology to information, free energy, and the second law of thermodynamics.

We recently observed that errors in gene replication and translation could be seen qualitatively to behave analogously to the impedances in acoustical and electronic energy transducing systems. We develop here quantitative relationships necessary to confirm that analogy and to place it into the context of the minimization of dissipative losses of both chemical free energy and information. The formal developments include expressions for the information transferred from a template to a new polymer, I σ ; an impedance parameter, Z; and an effective alphabet size, n eff ; all of which have non-linear dependences on the fidelity parameter, q, and the alphabet size, n. Surfaces of these functions over the {n,q} plane reveal key new insights into the origin of coding. Our conclusion is that the emergence and evolutionary refinement of information transfer in biology follow principles previously identified to govern physical energy flows, strengthening analogies (i) between chemical self-organization and biological natural selection, and (ii) between the course of evolutionary trajectories and the most probable pathways for time-dependent transitions in physics. Matching the informational impedance of translation to the four-letter alphabet of genes uncovers a pivotal role for the redundancy of triplet codons in preserving as much intrinsic genetic information as possible, especially in early stages when the coding alphabet size was small.

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.

Genetic coding is generally thought to have required ribozymes whose functions were taken over by polypeptide aminoacyl-tRNA synthetases (aaRS). Two discoveries about aaRS and their interactions with tRNA substrates now furnish a unifying rationale for the opposite conclusion: that the key processes of the Central Dogma of molecular biology emerged simultaneously and naturally from simple origins in a peptide•RNA partnership, eliminating the epistemological utility of a prior RNA world. First, the two aaRS classes likely arose from opposite strands of the same ancestral gene, implying a simple genetic alphabet. The resulting inversion symmetries in aaRS structural biology would have stabilized the initial and subsequent differentiation of coding specificities, rapidly promoting diversity in the proteome. Second, amino acid physical chemistry maps onto tRNA identity elements, establishing reflexive, nanoenvironmental sensing in protein aaRS. Bootstrapping of increasingly detailed coding is thus intrinsic to polypeptide aaRS, but impossible in an RNA world. These notions underline the following concepts that contradict gradual replacement of ribozymal aaRS by polypeptide aaRS: 1) aaRS enzymes must be interdependent; 2) reflexivity intrinsic to polypeptide aaRS production dynamics promotes bootstrapping; 3) takeover of RNA-catalyzed aminoacylation by enzymes will necessarily degrade specificity; and 4) the Central Dogma's emergence is most probable when replication and translation error rates remain comparable. These characteristics are necessary and sufficient for the essentially de novo emergence of a coupled gene-replicase-translatase system of genetic coding that would have continuously preserved the functional meaning of genetically encoded protein genes whose phylogenetic relationships match those observed today., (© The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

The genetic code sectored via tRNA charging errors, and the code progressed toward closure and universality because of evolution of aminoacyl-tRNA synthetase (aaRS) fidelity and translational fidelity mechanisms. Class I and class II aaRS folds are identified as homologs. From sequence alignments, a structurally conserved Zn-binding domain common to class I and class II aaRS was identified. A model for the class I and class II aaRS alternate folding pathways is posited. Five mechanisms toward code closure are highlighted: 1) aaRS proofreading to remove mischarged amino acids from tRNA; 2) accurate aaRS active site specification of amino acid substrates; 3) aaRS-tRNA anticodon recognition; 4) conformational coupling proofreading of the anticodon-codon interaction; and 5) deamination of tRNA wobble adenine to inosine. In tRNA anticodons there is strong wobble sequence preference that results in a broader spectrum of contacts to synonymous mRNA codon wobble bases. Adenine is excluded from the anticodon wobble position of tRNA unless it is modified to inosine. Uracil is generally preferred to cytosine in the tRNA anticodon wobble position. Because of wobble ambiguity when tRNA reads mRNA, the maximal coding capacity of the three nucleotide code read by tRNA is 31 amino acids + stops.

The standard genetic code (SGC) is virtually universal among extant life forms. Although many deviations from the universal code exist, particularly in organelles and prokaryotes with small genomes, they are limited in scope and obviously secondary. The universality of the code likely results from the combination of a frozen accident, i.e., the deleterious effect of codon reassignment in the SGC, and the inhibitory effect of changes in the code on horizontal gene transfer. The structure of the SGC is nonrandom and ensures high robustness of the code to mutational and translational errors. However, this error minimization is most likely a by-product of the primordial code expansion driven by the diversification of the repertoire of protein amino acids, rather than a direct result of selection. Phylogenetic analysis of translation system components, in particular aminoacyl-tRNA synthetases, shows that, at a stage of evolution when the translation system had already attained high fidelity, the correspondence between amino acids and cognate codons was determined by recognition of amino acids by RNA molecules, i.e., proto-tRNAs. We propose an experimentally testable scenario for the evolution of the code that combines recognition of amino acids by unique sites on proto-tRNAs (distinct from the anticodons), expansion of the code via proto-tRNA duplication, and frozen accident.

Background: The development of orthogonal translation systems (OTSs) for genetic code expansion (GCE) has allowed for the incorporation of a diverse array of non-canonical amino acids (ncAA) into proteins. Transfer RNA, the central molecule in the translation of the genetic message into proteins, plays a significant role in the efficiency of ncAA incorporation., Scope of Review: Here we review the biochemical basis of OTSs for genetic code expansion. We focus on the role of tRNA and discuss strategies used to engineer tRNA for the improvement of ncAA incorporation into proteins., Major Conclusions: The engineering of orthogonal tRNAs for GCE has significantly improved the incorporation of ncAAs. However, there are numerous unintended consequences of orthogonal tRNA engineering that cannot be predicted ab initio., General Significance: Genetic code expansion has allowed for the incorporation of a great diversity of ncAAs and novel chemistries into proteins, making significant contributions to our understanding of biological molecules and interactions. 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., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The aminoacyl‐tRNA synthetases (aaRS) are a large group of enzymes that implement the genetic code in all known biological systems. They attach amino acids to their cognate tRNAs, moonlight in various translational and non‐translational activities beyond aminoacylation, and are linked to many genetic disorders. The aaRS have a subtle ontology characterized by structural and functional idiosyncrasies that vary from organism to organism, and protein to protein. Across the tree of life, the 22 coded amino acids are handled by 16 evolutionary families of Class I aaRS and 21 families of Class II aaRS. We introduce AARS Online, an interactive Wikipedia‐like tool curated by an international consortium of field experts. This platform systematizes existing knowledge about the aaRS by showcasing a taxonomically diverse selection of aaRS sequences and structures. Through its graphical user interface, AARS Online facilitates a seamless exploration between protein sequence and structure, providing a friendly introduction to the material for non‐experts and a useful resource for experts. Curated multiple sequence alignments can be extracted for downstream analyses. Accessible at www.aars.online, AARS Online is a free resource to delve into the world of the aaRS. [ABSTRACT FROM AUTHOR]

Aminoacyl-tRNA synthetases hold the key to the genetic code and assign nucleic acid-based codons to amino acids, the building blocks of proteins. In their ability to recognize identity elements on transfer RNAs (tRNAs), some as simple as a single base pair, they ensure that the same proteins are formed each time information embedded in DNA is transcribed into messenger RNA (mRNA) and translated into proteins (Figure 1A). Thus, aminoacyl-tRNA synthetase active sites are conserved; however, since their evolutionary origin, their functions have been co-opted, expanded on and played novel roles during evolution. Below, we provide an overview of the many functions of aminoacyl-tRNA synthetases — from their role in translation, one of the most fundamental processes of all life, to newly discovered, diverse functions. Rasangi Tennakoon and Haissi Cui introduce aminoacyl-tRNA synthetases, the class of enzymes that load amino acids onto tRNAs and thus interpret the genetic code. [ABSTRACT FROM AUTHOR]

Until now, research has not taken into consideration the physicochemical purine–pyrimidine symmetries of the genetic code in the transcription and translation processes of proteinogenesis. Our Supersymmetry Genetic Code table, developed in 2022, is common and unique for all RNA and DNA living species. Its basic structure is a purine–pyrimidine symmetry net with double mirror symmetry. Accordingly, the symmetry of the genetic code directly shows its organisation based on the principle of nucleotide Watson–Crick and codon–anticodon pairing. The maximal purine–pyrimidine symmetries of codons show that each codon has a strictly defined and unchangeable position within the genetic code. We discovered that the physicochemical symmetries of the genetic code play a fundamental role in recognising and differentiating codons from mRNA and the anticodon tRNA and aminoacyl-tRNA synthetases in the transcription and translation processes. These symmetries also support the wobble hypothesis with non-Watson–Crick pairing interactions between the translation process from mRNA to tRNA. The Supersymmetry Genetic Code table shows a specific arrangement of the second base of codons, according to which it is possible that an anticodon from tRNA recognises whether a codon from mRNA belongs to an amino acid with two or four codons, which is very important in the purposeful use of the wobble pairing process. Therefore, we show that canonical and wobble pairings essentially do not lead to misreading and errors during translation, and we point out the role of physicochemical purine–pyrimidine symmetries in decreasing disorder according to error minimisation and preserving the integrity of biological processes during proteinogenesis. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The editorial in "Frontiers in Microbiology" focuses on the role of transfer RNAs (tRNAs) and protein synthesis in microorganisms, highlighting their importance in various research fields. The document discusses aminoacyl-tRNA synthesis, tRNA modifications, and ribosomes in microorganisms, emphasizing their impact on gene expression, microbial stress responses, and drug design. The research presented in the editorial provides valuable insights into the molecular mechanisms underlying protein synthesis and microbial pathogenesis, offering potential avenues for future studies in the field. [Extracted from the article]

Aminoacyl-tRNA synthetases (aaRSs) play a central role in the translation of genetic code, serving as attractive drug targets. Within this family, the lysyl-tRNA synthetase (LysRS) constitutes a promising antimalarial target. ASP3026, an anaplastic lymphoma kinase (ALK) inhibitor was recently identified as a novel Plasmodium falciparum LysRS (PfLysRS) inhibitor. Here, based on cocrystal structures and biochemical experiments, we developed a series of ASP3026 analogues to improve the selectivity and potency of LysRS inhibition. The leading compound 36 showed a dissociation constant of 15.9 nM with PfLysRS. The inhibitory efficacy on PfLysRS and parasites has been enhanced. Covalent attachment of l-lysine to compound 36 resulted in compound 36K3, which exhibited further increased inhibitory activity against PfLysRS but significantly decreased activity against ALK. However, its inhibitory activity against parasites did not improve, suggesting potential future optimization directions. This study presents a new example of derivatization of kinase inhibitors repurposed to inhibit aaRS. Using a combination of biochemical and structural approaches, the authors demonstrate that derivatization of kinase inhibitors can yield new repurposed anti-malarial drugs that specifically target Plasmodium falciparum Lysine-tRNA synthetase. [ABSTRACT FROM AUTHOR]

Mitochondria play multiple critical roles in cellular activity. In particular, mitochondrial translation is pivotal in the regulation of mitochondrial and cellular homeostasis. In this forum article, we discuss human mitochondrial tRNA metabolism and highlight its tight connection with various mitochondrial diseases caused by mutations in aminoacyl-tRNA synthetases, tRNAs, and tRNA-modifying enzymes. [ABSTRACT FROM AUTHOR]

The advancement of genetic code expansion (GCE) technology is attributed to the establishment of specific aminoacyl-tRNA synthetase/tRNA pairs. While earlier improvements mainly focused on aminoacyl-tRNA synthetases, recent studies have highlighted the importance of optimizing tRNA sequences to enhance both unnatural amino acid incorporation efficiency and orthogonality. Given the crucial role of tRNAs in the translation process and their substantial impact on overall GCE efficiency, ongoing efforts are dedicated to the development of tRNA engineering techniques. This review explores diverse tRNA engineering approaches and provides illustrative examples in the context of GCE, offering insights into the user-friendly implementation of GCE technology. [ABSTRACT FROM AUTHOR]

How Nature discovered genetic coding is a largely ignored question, yet the answer is key to explaining the transition from biochemical building blocks to life. Other, related puzzles also fall inside the aegis enclosing the codes themselves. The peptide bond is unstable with respect to hydrolysis. So, it requires some form of chemical free energy to drive it. Amino acid activation and acyl transfer are also slow and must be catalyzed. All living things must thus also convert free energy and synchronize cellular chemistry. Most importantly, functional proteins occupy only small, isolated regions of sequence space. Nature evolved heritable symbolic data processing to seek out and use those sequences. That system has three parts: a memory of how amino acids behave in solution and inside proteins, a set of code keys to access that memory, and a scoring function. The code keys themselves are the genes for cognate pairs of tRNA and aminoacyl-tRNA synthetases, AARSs. The scoring function is the enzymatic specificity constant, kcat/kM, which measures both catalysis and specificity. The work described here deepens the evidence for and understanding of an unexpected consequence of ancestral bidirectional coding. Secondary structures occur in approximately the same places within antiparallel alignments of their gene products. However, the polar amino acids that define the molecular surface of one are reflected into core-defining non-polar side chains on the other. Proteins translated from base-paired coding strands fold up inside out. Bidirectional genes thus project an inverted structural duality into the proteome. I review how experimental data root the scoring functions responsible for the origins of coding and catalyzed activation of unfavorable chemical reactions in that duality. [ABSTRACT FROM AUTHOR]

Selenocysteine (Sec) was discovered as the 21st genetically encoded amino acid. In nature, site-directed incorporation of Sec into proteins requires specialized biosynthesis and recoding machinery that evolved distinctly in bacteria compared to archaea and eukaryotes. Many organisms, including higher plants and most fungi, lack the Sec-decoding trait. We review the discovery of Sec and its role in redox enzymes that are essential to human health and important targets in disease. We highlight recent genetic code expansion efforts to engineer site-directed incorporation of Sec in bacteria and yeast. We also review methods to produce selenoproteins with 21 or more amino acids and approaches to delivering recombinant selenoproteins to mammalian cells as new applications for selenoproteins in synthetic biology. [ABSTRACT FROM AUTHOR]

Aminoacyl-tRNA synthetases (aaRSs) originated from an ancestral bidirectional gene (mirror symmetry), and through the evolution of the genetic code, the twenty aaRSs exhibit a symmetrical distribution in a 6-dimensional hypercube of the Standard Genetic Code. In this work, we assume a primeval RNY code and the Extended Genetic RNA code type II, which includes codons of the types YNY, YNR, and RNR. Each of the four subsets of codons can be represented in a 4-dimensional hypercube. Altogether, these 4 subcodes constitute the 6-dimensional representation of the SGC. We identify the aaRSs symmetry groups in each of these hypercubes. We show that each of the four hypercubes contains the following sets of symmetries for the two known Classes of synthetases: RNY: dihedral group of order 4; YNY: binary group; YNR: amplified octahedral group; and RNR: binary group. We demonstrate that for each hypercube, the group of symmetries in Class 1 is the same as the group of symmetries in Class 2. The biological implications of these findings are discussed. [ABSTRACT FROM AUTHOR]

Antibiotics target key biological processes that include protein synthesis. Bacteria respond by developing resistance, which increases rapidly due to antibiotics overuse. Mupirocin, a clinically used natural antibiotic, inhibits isoleucyl-tRNA synthetase (IleRS), an enzyme that links isoleucine to its tRNAIle for protein synthesis. Two IleRSs, mupirocin-sensitive IleRS1 and resistant IleRS2, coexist in bacteria. The latter may also be found in resistant Staphylococcus aureus clinical isolates. Here, we describe the structural basis of mupirocin resistance and unravel a mechanism of hyper-resistance evolved by some IleRS2 proteins. We surprisingly find that an up to 103-fold increase in resistance originates from alteration of the HIGH motif, a signature motif of the class I aminoacyl-tRNA synthetases to which IleRSs belong. The structural analysis demonstrates how an altered HIGH motif could be adopted in IleRS2 but not IleRS1, providing insight into an elegant mechanism for coevolution of the key catalytic motif and associated antibiotic resistance. Aminoacyl-tRNA synthetases translate the genetic code. These enzymes harbor signature catalytic motifs dating from their ancient ancestors. A natural variation of one of the stated motifs was discovered and linked to antibiotic hyper-resistance. [ABSTRACT FROM AUTHOR]

In this work, we formulate the following question: How the distribution of aminoacyl-tRNA synthetases (aaRSs) went from an ancestral bidirectional gene (mirror symmetry) to the symmetrical distribution of aaRSs in a six-dimensional hypercube of the Standard Genetic Code (SGC)? We assume a primeval RNY code, two Extended Genetic RNA codes type 1 and 2, and the SGC. We outline the types of symmetries of the distribution of aaRSs in each code. The symmetry groups of aaRSs in each code are described, until the symmetries of the SGC display a mirror symmetry. Considering both Extended RNA codes the 20 aaRSs were already present before the Last Universal Ancestor. These findings reveal intricacies in the diversification of aaRSs accompanied by the evolution of the genetic code. [ABSTRACT FROM AUTHOR]

Specific amino acid (AA) binding by aminoacyl-tRNA synthetases (aaRSs) is necessary for correct translation of the genetic code. Sequence and structure analyses have revealed the main specificity determinants and allowed a partitioning of aaRSs into two classes and several subclasses. However, the information contributed by each determinant has not been precisely quantified, and other, minor determinants may still be unidentified. Growth of genomic data and development of machine learning classification methods allow us to revisit these questions. This work considered the subclass IIb, formed by the three enzymes aspartyl-, asparaginyl-, and lysyl-tRNA synthetase (LysRS). Over 35,000 sequences from the Pfam database were considered, and used to train a machine-learning model based on ensembles of decision trees. The model was trained to reproduce the existing classification of each sequence as AspRS, AsnRS, or LysRS, and to identify which sequence positions were most important for the classification. A few positions (5–8 depending on the AA substrate) sufficed for accurate classification. Most but not all of them were well-known specificity determinants. The machine learning models thus identified sets of mutations that distinguish the three subclass members, which might be targeted in engineering efforts to alter or swap the AA specificities for biotechnology applications. [Display omitted] • Over 35,000 class IIb synthetase sequences were collected and curated. • The sequences were used to train interpretable machine learning models. • The most potent variables included several previously neglected amino acid positions. [ABSTRACT FROM AUTHOR]

I analyzed the polyphyletic origin of glycyl-tRNA synthetase (GlyRS) and lysyl-tRNA synthetase (LysRS), making plausible the following implications. The fact that the genetic code needed to evolve aminoacyl-tRNA synthetases (ARSs) only very late would be in perfect agreement with a late origin, in the main phyletic lineages, of both GlyRS and LysRS. Indeed, as suggested by the coevolution theory, since the genetic code was structured by biosynthetic relationships between amino acids and as these occurred on tRNA-like molecules which were evidently already loaded with amino acids during its structuring, this made possible a late origin of ARSs. All this corroborates the coevolution theory of the origin of the genetic code to the detriment of theories which would instead predict an early intervention of the action of ARSs in organizing the genetic code. Furthermore, the assembly of the GlyRS and LysRS protein domains in main phyletic lineages is itself at least evidence of the possibility that ancestral genes were assembled using pieces of genetic material that coded these protein domains. This is in accordance with the exon theory of genes which postulates that ancestral exons coded for protein domains or modules that were assembled to form the first genes. This theory is exemplified precisely in the evolution of both GlyRS and LysRS which occurred through the assembly of protein domains in the main phyletic lineages, as analyzed here. Furthermore, this late assembly of protein domains of these proteins into the two main phyletic lineages, i.e. a polyphyletic origin of both GlyRS and LysRS, appears to corroborate the progenote evolutionary stage for both LUCA and at least the first part of the evolutionary stages of the ancestor of bacteria and that of archaea. Indeed, this polyphyletic origin would imply that the genetic code was still evolving because at least two ARSs, i.e. proteins that make the genetic code possible today, were still evolving. This would imply that the evolutionary stages involved were characterized not by cells but by protocells, that is, by progenotes because this is precisely the definition of a progenote. This conclusion would be strengthened by the observation that both GlyRS and LysRS originating in the phyletic lineages leading to bacteria and archaea, would demonstrate that, more generally, proteins were most likely still in rapid and progressive evolution. Namely, a polyphyletic origin of proteins which would qualify at least the initial phase of the evolutionary stage of the ancestor of bacteria and that of archaea as stages belonging to the progenote. • I analyzed the polyphyletic origin of glycyl-tRNA synthetase (GlyRS) and lysyl-tRNA synthetase (LysRS). • The fact that the genetic code needed to evolve aminoacyl-tRNA synthetases (ARSs) only very late would be in perfect agreement with a late origin, in the main phyletic lineages, of both GlyRS and LysRS. • As suggested by the coevolution theory, since the genetic code was structured by biosynthetic relationships between amino acids and as these occurred on tRNA-like molecules which were evidently already loaded with amino acids during its structuring, this made possible a late origin of ARSs. • All this corroborates the coevolution theory of the origin of the genetic code to the detriment of theories which would instead predict an early intervention of the action of ARSs in organizing the genetic code. • Furthermore, the assembly of the GlyRS and LysRS protein domains in main phyletic lineages is itself at least evidence of the possibility that ancestral genes were assembled using pieces of genetic material that coded these protein domains. • This is in accordance with the exon theory of genes which postulates that ancestral exons coded for protein domains or modules that were assembled to form the first genes. This theory is exemplified precisely in the evolution of both GlyRS and LysRS which occurred through the assembly of protein domains in the main phyletic lineages. • The late assembly of protein domains of these proteins into the two main phyletic lineages, i.e. a polyphyletic origin of both GlyRS and LysRS, appears to corroborate the progenote evolutionary stage for both LUCA and at least the first part of the evolutionary stages of the ancestor of bacteria and that of archaea. [ABSTRACT FROM AUTHOR]

This Special Issue is intended to highlight recent advances in genetic code expansion, particularly the site-specific incorporation of noncanonical amino acids (ncAAs) into proteins. Engineered variations in tRNA structure might help develop artificial aaRS/tRNA pairs, although difficulties in rearranging tRNA structure hamper this direction. Still, genetic code expansion has been focused on only two OTSs based on tyrosyl-tRNA synthetase (TyrRS) and pyrrolysyl-tRNA synthetase (PylRS), largely because these two synthetases recognize most parts of the available ncAA collection. 10.3390/ijms20010029 16 Schwark D.G., Schmitt M.A., Fisk J.D. Directed evolution of the Methanosarcina barkeri pyrrolysyl tRNA/aminoacyl tRNA synthetase pair for rapid evaluation of sense codon reassignment potential. [Extracted from the article]

Universally present aminoacyl-tRNA synthetases (aaRSs) stringently recognize their cognate tRNAs and acylate them with one of the proteinogenic amino acids. However, some organisms possess aaRSs that deviate from the accurate translation of the genetic code and exhibit relaxed specificity toward their tRNA and/or amino acid substrates. Typically, these aaRSs are part of an indirect pathway in which multiple enzymes participate in the formation of the correct aminoacyl-tRNA product. The indirect cysteine (Cys)-tRNA pathway, originally thought to be restricted to methanogenic archaea, uses the unique O -phosphoseryl-tRNA synthetase (SepRS), which acylates the non-proteinogenic amino acid O -phosphoserine (Sep) onto tRNACys. Together with Sep-tRNA:Cys-tRNA synthase (SepCysS) and the adapter protein SepCysE, SepRS forms a transsulfursome complex responsible for shuttling Sep-tRNACys to SepCysS for conversion of the tRNA-bound Sep to Cys. Here, we report a comprehensive bioinformatic analysis of the diversity of indirect Cys encoding systems. These systems are present in more diverse groups of bacteria and archaea than previously known. Given the occurrence and distribution of some genes consistently flanking SepRS, it is likely that this gene was part of an ancient operon that suffered a gradual loss of its original components. Newly identified bacterial SepRS sequences strengthen the suggestion that this lineage of enzymes may not rely on the m1G37 identity determinant in tRNA. Some bacterial SepRSs possess an N-terminal fusion resembling a threonyl-tRNA synthetase editing domain, which interestingly is frequently observed in the vicinity of archaeal SepCysS genes. We also found several highly degenerate SepRS genes that likely have altered amino acid specificity. Cross-analysis of selenocysteine (Sec)-utilizing traits confirmed the co-occurrence of SepCysE and the Sec-utilizing machinery in archaea, but also identified an unusual O -phosphoseryl-tRNASec kinase fusion with an archaeal Sec elongation factor in some lineages, where it may serve in place of SepCysE to prevent crosstalk between the two minor aminoacylation systems. These results shed new light on the variations in SepRS and SepCysS enzymes that may reflect adaptation to lifestyle and habitat, and provide new information on the evolution of the genetic code. [ABSTRACT FROM AUTHOR]

Universally present aminoacyl-tRNA synthetases (aaRSs) stringently recognize their cognate tRNAs and acylate them with one of the proteinogenic amino acids. However, some organisms possess aaRSs that deviate from the accurate translation of the genetic code and exhibit relaxed specificity toward their tRNA and/or amino acid substrates. Typically, these aaRSs are part of an indirect pathway in which multiple enzymes participate in the formation of the correct aminoacyl-tRNA product. The indirect cysteine (Cys)-tRNA pathway, originally thought to be restricted to methanogenic archaea, uses the unique O-phosphoseryl-tRNA synthetase (SepRS), which acylates the non-proteinogenic amino acid O-phosphoserine (Sep) onto tRNACys. Together with Sep-tRNA:Cys-tRNA synthase (SepCysS) and the adapter protein SepCysE, SepRS forms a transsulfursome complex responsible for shuttling Sep-tRNACys to SepCysS for conversion of the tRNA-bound Sep to Cys. Here, we report a comprehensive bioinformatic analysis of the diversity of indirect Cys encoding systems. These systems are present in more diverse groups of bacteria and archaea than previously known. Given the occurrence and distribution of some genes consistently flanking SepRS, it is likely that this gene was part of an ancient operon that suffered a gradual loss of its original components. Newly identified bacterial SepRS sequences strengthen the suggestion that this lineage of enzymes may not rely on the m1G37 identity determinant in tRNA. Some bacterial SepRSs possess an N-terminal fusion resembling a threonyl-tRNA synthetase editing domain, which interestingly is frequently observed in the vicinity of archaeal SepCysS genes. We also found several highly degenerate SepRS genes that likely have altered amino acid specificity. Cross-analysis of selenocysteine (Sec)-utilizing traits confirmed the co-occurrence of SepCysE and the Sec-utilizing machinery in archaea, but also identified an unusual O-phosphoseryl-tRNASec kinase fusion with an archaeal Sec elongation factor in some lineages, where it may serve in place of SepCysE to prevent crosstalk between the two minor aminoacylation systems. These results shed new light on the variations in SepRS and SepCysS enzymes that may reflect adaptation to lifestyle and habitat, and provide new information on the evolution of the genetic code.

The evolutionary history of Class I aminoacyl-tRNA synthetases (aaRS) through the reconstruction of ancestral sequences is presented. From structural molecular modeling, we sought to understand its relationship with the acceptor arms and the tRNA anticodon loop, how this relationship was established, and the possible implications in determining the genetic code and the translation system. The results of the molecular docking showed that in 7 out 9 aaRS, the acceptor arm and the anticodon loop bond practically in the same region. Domain accretion process in aaRS and repositioning of interactions between tRNAs and aaRS are illustrated. Based on these results, we propose that the operational code and the anticodon code coexisted, competing for the aaRS catalytic region, while consequently contributed to the stabilization of these proteins. [ABSTRACT FROM AUTHOR]

[Display omitted] • Codon–amino acid assignments and their redundancies are consistent with simultaneous use in all 6 reading frames. • In-frame bidirectional coding entails unique additional symmetries that distinguish it from frame-shifted overprinting. • Bidirectional coding likely differentiated amino acid types necessary for the earliest genetically coded peptides. • Frame-shifted overprinting, observed in extant viruses, segregates functionally important sequences enhancing fitness. • Long open reading frames opposite polymerase genes of some narnaviruses suggest an unknown role for bidirectional coding. Genetic coding generally uses only one of a gene's two strands; its complement serving as template for replication. Aminoacyl-tRNA synthetases, aaRS, apparently first emerged as pairs on bidirectional genes, in which anticodons in the template strand served as codons for an entirely different protein. Interpreting both strands in frame constrained such genes sufficiently that it was rapidly superseded, leaving only traces in the elevated pairing between codon middle bases in antiparallel alignments. Codon assignments actually promote using information from both strands in multiple reading frames. Related phenomena, known as overprinting, are widely associated with viruses. In-frame bidirectional coding and overprinting nevertheless imply different structural and functional relationships, and different roles in generating folded proteins throughout the evolution of the proteome. [ABSTRACT FROM AUTHOR]

Diverse models have been advanced for the evolution of the genetic code. Here, models for tRNA, aminoacyl-tRNA synthetase (aaRS) and genetic code evolution were combined with an understanding of EF-Tu suppression of tRNA 3rd anticodon position wobbling. The result is a highly detailed scheme that describes the placements of all amino acids in the standard genetic code. The model describes evolution of 6-, 4-, 3-, 2- and 1-codon sectors. Innovation in column 3 of the code is explained. Wobbling and code degeneracy are explained. Separate distribution of serine sectors between columns 2 and 4 of the code is described. We conclude that very little chaos contributed to evolution of the genetic code and that the pattern of evolution of aaRS enzymes describes a history of the evolution of the code. A model is proposed to describe the biological selection for the earliest evolution of the code and for protocell evolution. [ABSTRACT FROM AUTHOR]

The family of giant viruses is still expanding, and evidence of a translational machinery is emerging in the virosphere. The Klosneuvirinae group of giant viruses was first reconstructed from in silico studies, and then a unique member was isolated, Bodo saltans virus. Here we describe the isolation of a new member in this group using coculture with the free-living amoeba Vermamoeba vermiformis. This giant virus, called Yasminevirus, has a 2.1-Mb linear double-stranded DNA genome encoding 1,541 candidate proteins, with a GC content estimated at 40.2%. Yasminevirus possesses a nearly complete translational machinery, with a set of 70 tRNAs associated with 45 codons and recognizing 20 amino acids (aa), 20 aminoacyl-tRNA synthetases (aaRSs) recognizing 20 aa, as well as several translation factors and elongation factors. At the genome scale, evolutionary analyses placed this virus in the Klosneuvirinae group of giant viruses. Rhizome analysis demonstrated that the genome of Yasminevirus is mosaic, with ~34% of genes having their closest homologues in other viruses, followed by ~13.2% in Eukaryota, ~7.2% in Bacteria, and less than 1% in Archaea. Among giant virus sequences, Yasminevirus shared 87% of viral hits with Klosneuvirinae. This description of Yasminevirus sheds light on the Klosneuvirinae group in a captivating quest to understand the evolution and diversity of giant viruses. [ABSTRACT FROM AUTHOR]

The Standard Genetic Code (SGC) is robust to mutational errors such that frequently occurring mutations minimally alter the physio-chemistry of amino acids. The apparent correlation between the evolutionary distances among codons and the physio-chemical distances among their cognate amino acids suggests an early co-diversification between the codons and amino acids. Here we formulated the co-minimization of evolutionary distances between codons and physio-chemical distances between amino acids as a Traveling Salesman Problem (TSP) and solved it with a Hopfield neural network. In this unsupervised learning algorithm, macromolecules (e.g., tRNAs and aminoacyl-tRNA synthetases) associating codons with amino acids were considered biological analogs of Hopfield neurons associating "tour cities" with "tour positions". The Hopfield network efficiently yielded an abundance of genetic codes that were more error-minimizing than SGC and could thus be used to design artificial genetic codes. We further argue that as a self-optimization algorithm, the Hopfield neural network provides a model of origin of SGC and other adaptive molecular systems through evolutionary learning. [ABSTRACT FROM AUTHOR]

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

The tRNA molecules, in addition to translating the genetic code into protein and defining the second genetic code via their aminoacylation by aminoacyl‐tRNA synthetases, act in many other cellular functions and dysfunctions. This article, illustrated by personal souvenirs, covers the history of ~60 years tRNA research in Strasbourg. Typical examples point up how the work in Strasbourg was a two‐way street, influenced by and at the same time influencing investigators outside of France. All along, research in Strasbourg has nurtured the structural and functional diversity of tRNA. It produced massive sequence and crystallographic data on tRNA and its partners, thereby leading to a deeper physicochemical understanding of tRNA architecture, dynamics, and identity. Moreover, it emphasized the role of nucleoside modifications and in the last two decades, highlighted tRNA idiosyncrasies in plants and organelles, together with cellular and health‐focused aspects. The tRNA field benefited from a rich local academic heritage and a strong support by both university and CNRS. Its broad interlinks to the worldwide community of tRNA researchers opens to an exciting future. © 2019 IUBMB Life, 2019 © 2019 IUBMB Life, 71(8):1066–1087, 2019 [ABSTRACT FROM AUTHOR]

Aminoacyl-tRNA synthetases (aaRSs) catalyse the ATP-dependent coupling of an amino acid to its cognate tRNA. Being vital for protein translation aaRSs are considered a promising target for the development of novel antimicrobial agents. 5′- O -(N -aminoacyl)-sulfamoyl adenosine (aaSA) is a non-hydrolysable analogue of the aaRS reaction intermediate that has been shown to be a potent inhibitor of this enzyme family but is prone to chemical instability and enzymatic modification. In an attempt to improve the molecular properties of this scaffold we synthesized a series of base substituted aaSA analogues comprising cytosine, uracil and N 3 -methyluracil targeting leucyl-, tyrosyl- and isoleucyl-tRNA synthetases. In in vitro assays seven out of the nine inhibitors demonstrated K i app values in the low nanomolar range. To complement the biochemical studies, X-ray crystallographic structures of Neisseria gonorrhoeae leucyl-tRNA synthetase and Escherichia coli tyrosyl-tRNA synthetase in complex with the newly synthesized compounds were determined. These highlighted a subtle interplay between the base moiety and the target enzyme in defining relative inhibitory activity. Encouraged by this data we investigated if the pyrimidine congeners could escape a natural resistance mechanism, involving acetylation of the amine of the aminoacyl group by the bacterial N-acetyltransferases RimL and YhhY. With RimL the pyrimidine congeners were less susceptible to inactivation compared to the equivalent aaSA, whereas with YhhY the converse was true. Combined the various insights resulting from this study will pave the way for the further rational design of aaRS inhibitors. Image 1 • Nine aminoacyl-sulfamoylated pyrimidine nucleosides were synthesized. • Several compounds displayed nanomolar activity in in vitro assays. • LeuRS and TyrRS ligand bound X-ray crystallographic structures determined. • Activity determined in cell extracts in the presence of N-acetyl transferases. • N-acetyl transferase activity affected by both the base and the amino acid. [ABSTRACT FROM AUTHOR]

Aminoacyl-tRNA synthetases (ARSs) are universal enzymes that catalyze the attachment of amino acids to the 3' ends of their cognate tRNAs. The resulting aminoacylated tRNAs are escorted to the ribosome where they enter protein synthesis. By specifically matching amino acids to defined anticodon sequences in tRNAs, ARSs are essential to the physical interpretation of the genetic code. In addition to their canonical role in protein synthesis, ARSs are also involved in RNA splicing, transcriptional regulation, translation, and other aspects of cellular homeostasis. Likewise, aminoacylated tRNAs serve as amino acid donors for biosynthetic processes distinct from protein synthesis, including lipid modification and antibiotic biosynthesis. Thanks to the wealth of details on ARS structures and functions and the growing appreciation of their additional roles regulating cellular homeostasis, opportunities for the development of clinically useful ARS inhibitors are emerging to manage microbial and parasite infections. Exploitation of these opportunities has been stimulated by the discovery of new inhibitor frameworks, the use of semi-synthetic approaches combining chemistry and genome engineering, and more powerful techniques for identifying leads from the screening of large chemical libraries. Here, we review the inhibition of ARSs by small molecules, including the various families of natural products, as well as inhibitors developed by either rational design or highthroughput screening as antibiotics and anti-parasitic therapeutics. [ABSTRACT FROM AUTHOR]

Aminoacyl-tRNA synthetases (ARSs) are enzymes that ligate their cognate amino acids to tRNAs for protein synthesis. However, recent studies have shown that their functions are expanded beyond protein synthesis through the interactions with diverse cellular factors. In this review, we discuss how ARSs have evolved to expand and control their functions by forming protein assemblies. We particularly focus on a macromolecular ARS complex in eukaryotes, named multitRNA synthetase complex (MSC), which is proposed to provide a channel through which tRNAs reach bound ARSs to receive their cognate amino acid and transit further to the translation machinery. Approximately half of the ARSs assemble into the MSC through cis-acting noncatalytic domains attached to their catalytic domains and trans-acting factors. Evolution of the MSC included its functional expansion, during which the MSC interaction network was augmented by additional cellular pathways present in higher eukaryotes. We also discuss MSC components that could be functionally involved in the pathophysiology of tumorigenesis. For example, the activities of some trans-acting factors have tumor-suppressing effects or maintain DNA integrity and are functionally compromised in cancer. On the basis of Gene Ontology analyses, we propose that the regulatory activities of the MSC-associated ARSs mainly converge on five biological processes, including mammalian target of rapamycin (mTOR) and DNA repair pathways. Future studies are needed to investigate how the MSC-associated and free- ARSs interact with each other and other factors in the control of multiple cellular pathways, and how aberrant or disrupted interactions in the MSC can cause disease. [ABSTRACT FROM AUTHOR]

Abstract Aminoacyl-tRNA synthetases (aaRSs), the enzymes responsible for coupling tRNAs to their cognate amino acids, minimize translational errors by intrinsic hydrolytic editing. Here, we compared norvaline (Nva), a linear amino acid not coded for protein synthesis, to the proteinogenic, branched valine (Val) in their propensity to mistranslate isoleucine (Ile) in proteins. We show that in the synthetic site of isoleucyl-tRNA synthetase (IleRS), Nva and Val are activated and transferred to tRNA at similar rates. The efficiency of the synthetic site in pre-transfer editing of Nva and Val also appears to be similar. Post-transfer editing was, however, more rapid with Nva and consequently IleRS misaminoacylates Nva-tRNAIle at slower rate than Val-tRNAIle. Accordingly, an Escherichia coli strain lacking IleRS post-transfer editing misincorporated Nva and Val in the proteome to a similar extent and at the same Ile positions. However, Nva mistranslation inflicted higher toxicity than Val, in agreement with IleRS editing being optimized for hydrolysis of Nva-tRNAIle. Furthermore, we found that the evolutionary-related IleRS, leucyl- and valyl-tRNA synthetases (I/L/VRSs), all efficiently hydrolyze Nva-tRNAs even when editing of Nva seems redundant. We thus hypothesize that editing of Nva-tRNAs had already existed in the last common ancestor of I/L/VRSs, and that the editing domain of I/L/VRSs had primarily evolved to prevent infiltration of Nva into modern proteins. Graphical Abstract Unlabelled Image Highlights • Induced mistranslation of non-proteinogenic norvaline and valine was compared. • Norvaline and valine misincorporate at overlapping isoleucine positions. • The same frequency of norvaline mistranslation inflicts higher toxicity. • Norvaline is a preferred target of IleRS editing. • Norvaline could have participated in primitive translation. [ABSTRACT FROM AUTHOR]

Synthesis of proteins with non-canonical amino acids via genetic code expansion is at the forefront of synthetic biology. Progress in this field has enabled site-specific incorporation of over 200 chemically and structurally diverse amino acids into proteins in an increasing number of organisms. This has been facilitated by our ability to repurpose aminoacyl-tRNA synthetases to attach non-canonical amino acids to engineered tRNAs. Current efforts in the field focus on overcoming existing limitations to the simultaneous incorporation of multiple non-canonical amino acids or amino acids that differ from the l -α-amino acid structure (e.g. d -amino acid or β-amino acid). Here, we summarize the progress and challenges in developing more selective and efficient aminoacyl-tRNA synthetases for genetic code expansion. [ABSTRACT FROM AUTHOR]

Histidyl tRNA Synthetase (HARS) is a member of the aminoacyl tRNA synthetase (ARS) family of enzymes. This family of 20 enzymes is responsible for attaching specific amino acids to their cognate tRNA molecules, a critical step in protein synthesis. However, recent work highlighting a growing number of associations between ARS genes and diverse human diseases raises the possibility of new and unexpected functions in this ancient enzyme family. For example, mutations in HARS have been linked to two different neurological disorders, Usher Syndrome Type IIIB and Charcot Marie Tooth peripheral neuropathy. These connections raise the possibility of previously undiscovered roles for HARS in metazoan development, with alterations in these functions leading to complex diseases. In an attempt to establish Danio rerio as a model for studying HARS functions in human disease, we characterized the Danio rerio hars gene and compared it to that of human HARS. Using a combination of bioinformatics, molecular biology, and cellular approaches, we found that while the human genome encodes separate genes for cytoplasmic and mitochondrial HARS protein, the Danio rerio genome encodes a single hars gene which undergoes alternative splicing to produce the respective cytoplasmic and mitochondrial versions of Hars. Nevertheless, while the HARS genes of humans and Danio differ significantly at the genomic level, we found that they are still highly conserved at the amino acid level, underscoring the potential utility of Danio rerio as a model organism for investigating HARS function and its link to human diseases in vivo. [ABSTRACT FROM AUTHOR]

This year marks the 48th anniversary of Francis Crick's seminal work on the origin of the genetic code, in which he first proposed the "frozen accident" hypothesis to describe evolutionary selection against changes to the genetic code that cause devastating global proteome modification. However, numerous efforts have demonstrated the viability of both natural and artificial genetic code variations. Recent advances in genetic engineering allow the creation of synthetic organisms that incorporate noncanonical, or even unnatural, amino acids into the proteome. Currently, successful genetic code engineering is mainly achieved by creating orthogonal aminoacyl-tRNA/synthetase pairs to repurpose stop and rare codons or to induce quadruplet codons. In this review, we summarize the current progress in genetic code engineering and discuss the challenges, current understanding, and future perspectives regarding genetic code modification. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

In this work, we determine the biological and mathematical properties that are sufficient and necessary to uniquely determine both the primeval RNY (purine-any base-pyrimidine) code and the standard genetic code (SGC). These properties are: the evolution of the SGC from the RNY code; the degeneracy of both codes, and the non-degeneracy of the assignments of aminoacyl-tRNA synthetases (aaRSs) to amino acids; the wobbling property; the consideration that glycine was the first amino acid; the topological and symmetrical properties of both codes. [ABSTRACT FROM AUTHOR]

Phosphorylation of many aminoacyl tRNA synthetases (AARSs) has been recognized for decades, but the contribution of post-translational modification to their primary role in tRNA charging and decryption of genetic code remains unclear. In contrast, phosphorylation is essential for performance of diverse noncanonical functions of AARSs unrelated to protein synthesis. Phosphorylation of glutamyl-prolyl tRNA synthetase (EPRS) has been investigated extensively in our laboratory for more than a decade, and has served as an archetype for studies of other AARSs. EPRS is a constituent of the IFN-γ-activated inhibitor of translation (GAIT) complex that directs transcript-selective translational control in myeloid cells. Stimulus-dependent phosphorylation of EPRS is essential for its release from the parental multi-aminoacyl tRNA synthetase complex (MSC), for binding to other GAIT complex proteins, and for regulating the binding to target mRNAs. Importantly, phosphorylation is the common driving force for the context- and stimulus-dependent release, and non-canonical activity, of other AARSs residing in the MSC, for example, lysyl tRNA synthetase (KARS). Here, we describe the concepts and experimental methodologies we have used to investigate the influence of phosphorylation on the structure and function of EPRS. We suggest that application of these approaches will help to identify new functional phosphorylation event(s) in other AARSs and elucidate their possible roles in noncanonical activities. [ABSTRACT FROM AUTHOR]

The 11th IUBMB Focused Meeting on Aminoacyl-tRNA Synthetases was held in Clearwater Beach, Florida from 29 October–2 November 2017, with the aim of presenting the latest research on these enzymes and promoting interchange among aminoacyl-tRNA synthetase (ARS) researchers. Topics covered in the meeting included many areas of investigation, including ARS evolution, mechanism, editing functions, biology in prokaryotic and eukaryotic cells and their organelles, their roles in human diseases, and their application to problems in emerging areas of synthetic biology. In this report, we provide a summary of the major themes of the meeting, citing contributions from the oral presentations in the meeting.

Genetic code expansion (GCE) has become a central topic of synthetic biology. GCE relies on engineered aminoacyl-tRNA synthetases (aaRSs) and a cognate tRNA species to allow codon reassignment by co-translational insertion of non-canonical amino acids (ncAAs) into proteins. Introduction of such amino acids increases the chemical diversity of recombinant proteins endowing them with novel properties. Such proteins serve in sophisticated biochemical and biophysical studies both in vitro and in vivo, they may become unique biomaterials or therapeutic agents, and they afford metabolic dependence of genetically modified organisms for biocontainment purposes. In the Methanosarcinaceae the incorporation of the 22nd genetically encoded amino acid, pyrrolysine (Pyl), is facilitated by pyrrolysyl-tRNA synthetase (PylRS) and the cognate UAG-recognizing tRNAPyl. This unique aaRS-tRNA pair functions as an orthogonal translation system (OTS) in most model organisms. The facile directed evolution of the large PylRS active site to accommodate many ncAAs, and the enzyme's anticodon-blind specific recognition of the cognate tRNAPyl make this system highly amenable for GCE purposes. The remarkable polyspecificity of PylRS has been exploited to incorporate >100 different ncAAs into proteins. Here we review the Pyl-OT system and selected GCE applications to examine the properties of an effective OTS. [ABSTRACT FROM AUTHOR]

Aminoacyl-tRNA synthetases (aaRS) catalyze both chemical steps that translate the universal genetic code. Rodin and Ohno offered an explanation for the existence of two aaRS classes, observing that codons for the most highly conserved Class I active-site residues are anticodons for corresponding Class II active-site residues. They proposed that the two classes arose simultaneously, by translation of opposite strands from the same gene. We have characterized wild-type 46-residue peptides containing ATP-binding sites of Class I and II synthetases and those coded by a gene designed by Rosetta to encode the corresponding peptides on opposite strands. Catalysis by WT and designed peptides is saturable, and the designed peptides are sensitive to active-site residue mutation. All have comparable apparent second-order rate constants 2.9-7.0E-3 M-1 s-1 or ~750,000-1,300,000 times the uncatalyzed rate. The activities of the two complementary peptides demonstrate that the unique information in a gene can have two functional interpretations, one from each complementary strand. The peptides contain phylogenetic signatures of longer, more sophisticated catalysts we call Urzymes and are short enough to bridge the gap between them and simpler uncoded peptides. Thus, they directly substantiate the sense/antisense coding ancestry of Class I and II aaRS. Furthermore, designed 46-mers achieve similar catalytic proficiency to wild-type 46-mers by significant increases in both kcat and Km values, supporting suggestions that the earliest peptide catalysts activated ATP for biosynthetic purposes. [ABSTRACT FROM AUTHOR]