The tRNA identity landscape for aminoacylation and beyond.

tRNAs are key partners in ribosome-dependent protein synthesis. This process is highly dependent on the fidelity of tRNA aminoacylation by aminoacyl-tRNA synthetases and relies primarily on sets of identities within tRNA molecules composed of determinants and antideterminants preventing mischarging by non-cognate synthetases. Such identity sets were discovered in the tRNAs of a few model organisms, and their properties were generalized as universal identity rules. Since then, the panel of identity elements governing the accuracy of tRNA aminoacylation has expanded considerably, but the increasing number of reported functional idiosyncrasies has led to some confusion. In parallel, the description of other processes involving tRNAs, often well beyond aminoacylation, has progressed considerably, greatly expanding their interactome and uncovering multiple novel identities on the same tRNA molecule. This review highlights key findings on the mechanistics and evolution of tRNA and tRNA-like identities. In addition, new methods and their results for searching sets of multiple identities on a single tRNA are discussed. Taken together, this knowledge shows that a comprehensive understanding of the functional role of individual and collective nucleotide identity sets in tRNA molecules is needed for medical, biotechnological and other applications.

History of tRNA research in strasbourg.

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.

Structure of Escherichia coli Arginyl-tRNA Synthetase in Complex with tRNAArg: Pivotal Role of the D-loop.

Aminoacyl-tRNA synthetases are essential components in protein biosynthesis. Arginyl-tRNA synthetase (ArgRS) belongs to the small group of aminoacyl-tRNA synthetases requiring cognate tRNA for amino acid activation. The crystal structure of Escherichia coli (Eco) ArgRS has been solved in complex with tRNAArg at 3.0-Å resolution. With this first bacterial tRNA complex, we are attempting to bridge the gap existing in structure-function understanding in prokaryotic tRNAArg recognition. The structure shows a tight binding of tRNA on the synthetase through the identity determinant A20 from the D-loop, a tRNA recognition snapshot never elucidated structurally. This interaction of A20 involves 5 amino acids from the synthetase. Additional contacts via U20a and U16 from the D-loop reinforce the interaction. The importance of D-loop recognition in EcoArgRS functioning is supported by a mutagenesis analysis of critical amino acids that anchor tRNAArg on the synthetase; in particular, mutations at amino acids interacting with A20 affect binding affinity to the tRNA and specificity of arginylation. Altogether the structural and functional data indicate that the unprecedented ArgRS crystal structure represents a snapshot during functioning and suggest that the recognition of the D-loop by ArgRS is an important trigger that anchors tRNAArg on the synthetase. In this process, A20 plays a major role, together with prominent conformational changes in several ArgRS domains that may eventually lead to the mature ArgRS:tRNA complex and the arginine activation. Functional implications that could be idiosyncratic to the arginine identity of bacterial ArgRSs are discussed.

What macromolecular crystallogenesis tells us - what is needed in the future.

Crystallogenesis is a longstanding topic that has transformed into a discipline that is mainly focused on the preparation of crystals for practising crystallo-graphers. Although the idiosyncratic features of proteins have to be taken into account, the crystallization of proteins is governed by the same physics as the crystallization of inorganic materials. At present, a diversified panel of crystallization methods adapted to proteins has been validated, and although only a few methods are in current practice, the success rate of crystallization has increased constantly, leading to the determination of ∼105 X-ray structures. These structures reveal a huge repertoire of protein folds, but they only cover a restricted part of macromolecular diversity across the tree of life. In the future, crystals representative of missing structures or that will better document the structural dynamics and functional steps underlying biological processes need to be grown. For the pertinent choice of biologically relevant targets, computer-guided analysis of structural databases is needed. From another perspective, crystallization is a self-assembly process that can occur in the bulk of crowded fluids, with crystals being supramolecular assemblies. Life also uses self-assembly and supramolecular processes leading to transient, or less often stable, complexes. An integrated view of supramolecularity implies that proteins crystallizing either in vitro or in vivo or participating in cellular processes share common attributes, notably determinants and antideterminants that favour or disfavour their correct or incorrect associations. As a result, under in vivo conditions proteins show a balance between features that favour or disfavour association. If this balance is broken, disorders/diseases occur. Understanding crystallization under in vivo conditions is a challenge for the future. In this quest, the analysis of packing contacts and contacts within oligomers will be crucial in order to decipher the rules governing protein self-assembly and will guide the engineering of novel biomaterials. In a wider perspective, understanding such contacts will open the route towards supramolecular biology and generalized crystallogenesis.

Aminoacyl-tRNA Synthetases in the Bacterial World.

Aminoacyl-tRNA synthetases (aaRSs) are modular enzymes globally conserved in the three kingdoms of life. All catalyze the same two-step reaction, i.e., the attachment of a proteinogenic amino acid on their cognate tRNAs, thereby mediating the correct expression of the genetic code. In addition, some aaRSs acquired other functions beyond this key role in translation. Genomics and X-ray crystallography have revealed great structural diversity in aaRSs (e.g., in oligomery and modularity, in ranking into two distinct groups each subdivided in 3 subgroups, by additional domains appended on the catalytic modules). AaRSs show huge structural plasticity related to function and limited idiosyncrasies that are kingdom or even species specific (e.g., the presence in many Bacteria of non discriminating aaRSs compensating for the absence of one or two specific aaRSs, notably AsnRS and/or GlnRS). Diversity, as well, occurs in the mechanisms of aaRS gene regulation that are not conserved in evolution, notably between distant groups such as Gram-positive and Gram-negative Bacteria. The review focuses on bacterial aaRSs (and their paralogs) and covers their structure, function, regulation, and evolution. Structure/function relationships are emphasized, notably the enzymology of tRNA aminoacylation and the editing mechanisms for correction of activation and charging errors. The huge amount of genomic and structural data that accumulated in last two decades is reviewed, showing how the field moved from essentially reductionist biology towards more global and integrated approaches. Likewise, the alternative functions of aaRSs and those of aaRS paralogs (e.g., during cell wall biogenesis and other metabolic processes in or outside protein synthesis) are reviewed. Since aaRS phylogenies present promiscuous bacterial, archaeal, and eukaryal features, similarities and differences in the properties of aaRSs from the three kingdoms of life are pinpointed throughout the review and distinctive characteristics of bacterium-like synthetases from organelles are outlined.

Interplay between Catalysts and Substrates for Activity of Class Ib Aminoacyl-tRNA Synthetases and Implications for Pharmacology.

Aminoacyl-tRNA synthetase:transfer RNA (aaRS:tRNA) systems became recently essential targets in molecular medicine, because perturbed recognition of cognate tRNAs by aaRSs and poor precision in tRNA aminoacylation do not guarantee accurate protein biosynthesis, thus leading to diseases. Sets of identity determinants situated at particular zones of tRNA are responsible for functional accuracy. Recent work in X-ray crystallography has revealed various snapshots of aaRS:ligand complexes which represent the stages required for aminoacylation. Here we focus on a small group of class I aaRSs conserved in evolution, the ArgRSs, GluRSs, GlnRSs, and atypical LysRSs found mostly in Archaea and in a few Bacteria, that catalyze amino acid activation only in the presence of their cognate tRNAs. Structural and functional features of these aaRSs, ranked in subclass Ib, together with their peculiar mode of tRNA recognition and identity expression are reviewed and compared. Strategies to inhibit class Ib aaRS:tRNA aminoacylation systems, their dysfunction leading to human diseases, and the implications for pharmacology are outlined.

tRNA biology in mitochondria.

Mitochondria are the powerhouses of eukaryotic cells. They are considered as semi-autonomous because they have retained genomes inherited from their prokaryotic ancestor and host fully functional gene expression machineries. These organelles have attracted considerable attention because they combine bacterial-like traits with novel features that evolved in the host cell. Among them, mitochondria use many specific pathways to obtain complete and functional sets of tRNAs as required for translation. In some instances, tRNA genes have been partially or entirely transferred to the nucleus and mitochondria require precise import systems to attain their pool of tRNAs. Still, tRNA genes have also often been maintained in mitochondria. Their genetic arrangement is more diverse than previously envisaged. The expression and maturation of mitochondrial tRNAs often use specific enzymes that evolved during eukaryote history. For instance many mitochondria use a eukaryote-specific RNase P enzyme devoid of RNA. The structure itself of mitochondrial encoded tRNAs is also very diverse, as e.g., in Metazoan, where tRNAs often show non canonical or truncated structures. As a result, the translational machinery in mitochondria evolved adapted strategies to accommodate the peculiarities of these tRNAs, in particular simplified identity rules for their aminoacylation. Here, we review the specific features of tRNA biology in mitochondria from model species representing the major eukaryotic groups, with an emphasis on recent research on tRNA import, maturation and aminoacylation.

A historical perspective on protein crystallization from 1840 to the present day.

Protein crystallization has been known since 1840 and can prove to be straightforward but, in most cases, it constitutes a real bottleneck. This stimulated the birth of the biocrystallogenesis field with both 'practical' and 'basic' science aims. In the early years of biochemistry, crystallization was a tool for the preparation of biological substances. Today, biocrystallogenesis aims to provide efficient methods for crystal fabrication and a means to optimize crystal quality for X-ray crystallography. The historical development of crystallization methods for structural biology occurred first in conjunction with that of biochemical and genetic methods for macromolecule production, then with the development of structure determination methodologies and, recently, with routine access to synchrotron X-ray sources. Previously, the identification of conditions that sustain crystal growth occurred mostly empirically but, in recent decades, this has moved progressively towards more rationality as a result of a deeper understanding of the physical chemistry of protein crystal growth and the use of idea-driven screening and high-throughput procedures. Protein and nucleic acid engineering procedures to facilitate crystallization, as well as crystallization methods in gelled-media or by counter-diffusion, represent recent important achievements, although the underlying concepts are old. The new nanotechnologies have brought a significant improvement in the practice of protein crystallization. Today, the increasing number of crystal structures deposited in the Protein Data Bank could mean that crystallization is no longer a bottleneck. This is not the case, however, because structural biology projects always become more challenging and thereby require adapted methods to enable the growth of the appropriate crystals, notably macromolecular assemblages.

Predicting protein crystallizability and nucleation.

The outcome of protein crystallization attempts is often uncertain due to inherent features of the protein or to the crystallization process that are not fully under control of the experimentalist. The aim of this contribution is to propose user-friendly tools that can increase the success rate of a protein crytallization project. Different bioinformatic approaches to predict the crystallization feasibility (before any crystallization attempts are undertaken) are discussed and a novel approach to assess the nucleation process of a given protein is proposed. Practical examples illustrate these two points.

Aminoacyl-tRNA Synthetases in the Bacterial World.

Aminoacyl-tRNAsynthetases (aaRSs) are modular enzymesglobally conserved in the three kingdoms of life. All catalyze the same two-step reaction, i.e., the attachment of a proteinogenic amino acid on their cognate tRNAs, thereby mediating the correct expression of the genetic code. In addition, some aaRSs acquired other functions beyond this key role in translation.Genomics and X-ray crystallography have revealed great structural diversity in aaRSs (e.g.,in oligomery and modularity, in ranking into two distinct groups each subdivided in 3 subgroups, by additional domains appended on the catalytic modules). AaRSs show hugestructural plasticity related to function andlimited idiosyncrasies that are kingdom or even speciesspecific (e.g.,the presence in many Bacteria of non discriminating aaRSs compensating for the absence of one or two specific aaRSs, notably AsnRS and/or GlnRS).Diversity, as well, occurs in the mechanisms of aaRS gene regulation that are not conserved in evolution, notably betweendistant groups such as Gram-positive and Gram-negative Bacteria.Thereview focuses on bacterial aaRSs (and their paralogs) and covers their structure, function, regulation,and evolution. Structure/function relationships are emphasized, notably the enzymology of tRNA aminoacylation and the editing mechanisms for correction of activation and charging errors. The huge amount of genomic and structural data that accumulatedin last two decades is reviewed,showing how thefield moved from essentially reductionist biologytowards more global and integrated approaches. Likewise, the alternative functions of aaRSs and those of aaRSparalogs (e.g., during cellwall biogenesis and other metabolic processes in or outside protein synthesis) are reviewed. Since aaRS phylogenies present promiscuous bacterial, archaeal, and eukaryal features, similarities and differences in the properties of aaRSs from the three kingdoms of life are pinpointedthroughout the reviewand distinctive characteristics of bacterium-like synthetases from organelles are outlined.

Structure of transfer RNAs: similarity and variability.

Transfer RNAs (tRNAs) are ancient molecules whose origin goes back to the beginning of life on Earth. Key partners in the ribosome-translation machinery, tRNAs read genetic information on messenger RNA and deliver codon specified amino acids attached to their distal 3'-extremity for peptide bond synthesis on the ribosome. In addition to this universal function, tRNAs participate in a wealth of other biological processes and undergo intricate maturation events. Our understanding of tRNA biology has been mainly phenomenological, but ongoing progress in structural biology is giving a robust physico-chemical basis that explains many facets of tRNA functions. Advanced sequence analysis of tRNA genes and their RNA transcripts have uncovered rules that underly tRNA 2D folding and 3D L-shaped architecture, as well as provided clues about their evolution. The increasing number of X-ray structures of free, protein- and ribosome-bound tRNA, reveal structural details accounting for the identity of the 22 tRNA families (one for each proteinogenic amino acid) and for the multifunctionality of a given family. Importantly, the structural role of post-transcriptional tRNA modifications is being deciphered. On the other hand, the plasticity of tRNA structure during function has been illustrated using a variety of technical approaches that allow dynamical insights. The large range of structural properties not only allows tRNAs to be the key actors of translation, but also sustain a diversity of unrelated functions from which only a few have already been pinpointed. Many surprises can still be expected.

Biocrystallography: past, present, future.

The evolution of biocrystallography from the pioneers' time to the present era of global biology is presented in relation to the development of methodological and instrumental advances for molecular sample preparation and structure elucidation over the last 6 decades. The interdisciplinarity of the field that generated cross-fertilization between physics- and biology-focused themes is emphasized. In particular, strategies to circumvent the main bottlenecks of biocrystallography are discussed. They concern (i) the way macromolecular targets are selected, designed, and characterized, (ii) crystallogenesis and how to deal with physical and biological parameters that impact crystallization for growing and optimizing crystals, and (iii) the methods for crystal analysis and 3D structure determination. Milestones that have marked the history of biocrystallography illustrate the discussion. Finally, the future of the field is envisaged. Wide gaps of the structural space need to be filed and membrane proteins as well as intrinsically unstructured proteins still constitute challenging targets. Solving supramolecular assemblies of increasing complexity, developing a "4D biology" for decrypting the kinematic changes in macromolecular structures in action, integrating these structural data in the whole cell organization, and deciphering biomedical implications will represent the new frontiers.

Mutation of the mitochondrial tyrosyl-tRNA synthetase gene, YARS2, causes myopathy, lactic acidosis, and sideroblastic anemia--MLASA syndrome.

Mitochondrial respiratory chain disorders are a heterogeneous group of disorders in which the underlying genetic defect is often unknown. We have identified a pathogenic mutation (c.156C>G [p.F52L]) in YARS2, located at chromosome 12p11.21, by using genome-wide SNP-based homozygosity analysis of a family with affected members displaying myopathy, lactic acidosis, and sideroblastic anemia (MLASA). We subsequently identified the same mutation in another unrelated MLASA patient. The YARS2 gene product, mitochondrial tyrosyl-tRNA synthetase (YARS2), was present at lower levels in skeletal muscle whereas fibroblasts were relatively normal. Complex I, III, and IV were dysfunctional as indicated by enzyme analysis, immunoblotting, and immunohistochemistry. A mitochondrial protein-synthesis assay showed reduced levels of respiratory chain subunits in myotubes generated from patient cell lines. A tRNA aminoacylation assay revealed that mutant YARS2 was still active; however, enzyme kinetics were abnormal compared to the wild-type protein. We propose that the reduced aminoacylation activity of mutant YARS2 enzyme leads to decreased mitochondrial protein synthesis, resulting in mitochondrial respiratory chain dysfunction. MLASA has previously been associated with PUS1 mutations; hence, the YARS2 mutation reported here is an alternative cause of MLASA.

Diversity and similarity in the tRNA world: overall view and case study on malaria-related tRNAs.

Transfer RNAs (tRNAs) are ancient macromolecules that have evolved under various environmental pressures as adaptors in translation in all forms of life but also towards alternative structures and functions. The present knowledge on both "canonical" and "deviating" signature motifs retrieved from vertical and horizontal sequence comparisons is briefly reviewed. Novel characteristics, proper to tRNAs from a given translation system, are revealed by a case study on the nuclear and organellar tRNA sets from malaria-related organisms. Unprecedented distinctive features for Plasmodium falciparum apicoplastic tRNAs appear, which provide novel routes to be explored towards anti-malarial drugs. The ongoing high-throughput sequencing programs are expected to allow for further horizontal comparisons and to reveal other signatures of either full or restricted sets of tRNAs.

Crystal growth of proteins, nucleic acids, and viruses in gels.

Medium-sized single crystals with perfect habits and no defect producing intense and well-resolved diffraction patterns are the dream of every protein crystallographer. Crystals of biological macromolecules possessing these characteristics can be prepared within a medium in which mass transport is restricted to diffusion. Chemical gels (like polysiloxane) and physical gels (such as agarose) provide such an environment and are therefore suitable for the crystallisation of biological macromolecules. Instructions for the preparation of each type of gel are given to urge crystal growers to apply diffusive media for enhancing crystallographic quality of their crystals. Examples of quality enhancement achieved with silica and agarose gels are given. Results obtained with other substances forming gel-like media (such as lipidic phases and cellulose derivatives) are presented. Finally, the use of gels in combination with capillary tubes for counter-diffusion experiments is discussed. Methods and techniques implemented with proteins can also be applied to nucleic acids and nucleoprotein assemblies such as viruses.

Microfluidic chips for the crystallization of biomacromolecules by counter-diffusion and on-chip crystal X-ray analysis.

Microfluidic devices were designed to perform on micromoles of biological macromolecules and viruses the search and the optimization of crystallization conditions by counter-diffusion, as well as the on-chip analysis of crystals by X-ray diffraction. Chips composed of microchannels were fabricated in poly-dimethylsiloxane (PDMS), poly-methyl-methacrylate (PMMA) and cyclo-olefin-copolymer (COC) by three distinct methods, namely replica casting, laser ablation and hot embossing. The geometry of the channels was chosen to ensure that crystallization occurs in a convection-free environment. The transparency of the materials is compatible with crystal growth monitoring by optical microscopy. The quality of the protein 3D structures derived from on-chip crystal analysis by X-ray diffraction using a synchrotron radiation was used to identify the most appropriate polymers. Altogether the results demonstrate that for a novel biomolecule, all steps from the initial search of crystallization conditions to X-ray diffraction data collection for 3D structure determination can be performed in a single chip.

Peculiar inhibition of human mitochondrial aspartyl-tRNA synthetase by adenylate analogs.

Human mitochondrial aminoacyl-tRNA synthetases (mt-aaRSs), the enzymes which esterify tRNAs with the cognate specific amino acid, form mainly a different set of proteins than those involved in the cytosolic translation machinery. Many of the mt-aaRSs are of bacterial-type in regard of sequence and modular structural organization. However, the few enzymes investigated so far do have peculiar biochemical and enzymological properties such as decreased solubility, decreased specific activity and enlarged spectra of substrate tRNAs (of same specificity but from various organisms and kingdoms), as compared to bacterial aaRSs. Here the sensitivity of human mitochondrial aspartyl-tRNA synthetase (AspRS) to small substrate analogs (non-hydrolysable adenylates) known as inhibitors of Escherichia coli and Pseudomonas aeruginosa AspRSs is evaluated and compared to the sensitivity of eukaryal cytosolic human and bovine AspRSs. L-aspartol-adenylate (aspartol-AMP) is a competitive inhibitor of aspartylation by mitochondrial as well as cytosolic mammalian AspRSs, with K(i) values in the micromolar range (4-27 microM for human mt- and mammalian cyt-AspRSs). 5'-O-[N-(L-aspartyl)sulfamoyl]adenosine (Asp-AMS) is a 500-fold stronger competitive inhibitor of the mitochondrial enzyme than aspartol-AMP (10nM) and a 35-fold lower competitor of human and bovine cyt-AspRSs (300 nM). The higher sensitivity of human mt-AspRS for both inhibitors as compared to either bacterial or mammalian cytosolic enzymes, is not correlated with clear-cut structural features in the catalytic site as deduced from docking experiments, but may result from dynamic events. In the scope of new antibacterial strategies directed against aaRSs, possible side effects of such drugs on the mitochondrial human aaRSs should thus be considered.

Toward a more complete view of tRNA biology.

Transfer RNAs are ancient molecules present in all domains of life. In addition to translating the genetic code into protein and defining the second genetic code together with aminoacyl-tRNA synthetases, tRNAs act in many other cellular functions. Robust phenomenological observations on the role of tRNAs in translation, together with massive sequence and crystallographic data, have led to a deeper physicochemical understanding of tRNA architecture, dynamics and identity. In vitro studies complemented by cell biology data already indicate how tRNA behaves in cellular environments, in particular in higher Eukarya. From an opposite approach, reverse evolution considerations suggest how tRNAs emerged as simplified structures from the RNA world. This perspective discusses what basic questions remain unanswered, how these answers can be obtained and how a more rational understanding of the function and dysfunction of tRNA can have applications in medicine and biotechnology.

Decreased aminoacylation in pathology-related mutants of mitochondrial tRNATyr is associated with structural perturbations in tRNA architecture.

A growing number of human pathologies are ascribed to mutations in mitochondrial tRNA genes. Here, we report biochemical investigations on three mt-tRNA(Tyr) molecules with point substitutions associated with diseases. The mutations occur in the atypical T- and D-loops at positions homologous to those involved in the tertiary interaction network of canonical tRNAs. They do not correspond to tyrosine identity positions and likely do not contact the mitochondrial tyrosyl-tRNA synthetase during the aminoacylation process. The impact of these substitutions on mt-tRNA(Tyr) tyrosylation and structure was investigated using the corresponding tRNA transcripts. In vitro tyrosylation efficiency is decreased 600-fold for mutant A22G (mitochondrial gene mutation T5874C), 40-fold for G15A (C5877T), and is without significant effect on U54C (A5843G). Comparative solution probings with lead and nucleases on mutant and wild-type tRNA(Tyr) molecules reveal a greater sensitivity to single-strand specific probes for mutants G15A and A22G. For both transcripts, the mutation triggers a structural destabilization in the D-loop that propagates toward the anticodon arm and thus hinders efficient tyrosylation. Further probing analysis combined with phylogenetic data support the participation of G15 and A22 in the tertiary network of human mt-tRNA(Tyr) via nonclassical Watson-Crick G15-C48 and G13-A22 pairings. In contrast, the pathogenic effect of the tyrosylable mutant U54C, where structure is only marginally affected, has to be sought at another level of the tRNA(Tyr) life cycle.