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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.

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-A 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.

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 tRNA

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.

Transfer RNAs (tRNA) are small RNAs that provide the interface between DNA and ribosome-dependent protein synthesis, besides being involved in many other cellular processes. They interact with an impressive number of small molecule and macromolecular ligands and show structural and functional plasticity far to be deciphered. Here it is shown how tRNA biology was (and still is) idea and technology-driven and why crystallogenesis was at the heart of this science/technology interplay. Thus the quest to understand tRNA recognition/misrecognition by aminoacyl-tRNA synthetases (aaRS) stimulated biologists since the 1960s to crystallize tRNAs and their protein partners under their different functional states. This led to novel crystallization methods (vapor phase diffusion, microdialysis), characterization of system-specific additives (polyamines, metal ions, adenylate analogues), and discovery of ammonium sulfate as a crystallant for tRNA:aaRS complexes. Studies on the physical chemistry of tRNA and aaRS crysta...

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.

International audience; 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.

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.

Orthorhombic crystals of the enzyme aspartyl-tRNA synthetase (AspRS) were prepared in agarose gel, a chemical alternative to microgravity or nano-volume drops. Besides providing a convection-free medium, the network of the polysaccharide improved the stability of the crystalline lattice during soaking with L-aspartol adenylate, a synthetic and non-hydrolysable analog of the catalytic intermediate aspartyl adenylate. When crystals were embedded in the polysaccharide matrix the ligand reached their surfaces more uniformly. Gel-grown crystals exhibited well defined reflections even at high resolution and low mosaicity values, despite their fairly high solvent content and the relatively harsh flash cooling procedure. By contrast, soaked AspRS crystals prepared in solution broke apart within 10–30 s after the ligand was introduced into the mother liquor, and subsequently these fragments became an amorphous precipitate. A general objection to the use of gels in protein crystallization is that chemical interactions may occur between the polysaccharide matrix and proteins or ligands. The example of AspRS shows that this is not a major concern. This method may be generally applicable to crystal soaking with other small molecules or heavy atoms.

Aminoacyl-tRNA synthetases (aaRSs) catalyze the attachment of amino acids on transfer RNAs (tRNAs) and thus enable the correct expression of the genetic code during protein synthesis. After a brief historical review of early aaRS crystallizations we present results of a survey of the crystallization conditions of 220 aaRSs (either free or bound to small ligands) and of 59 aaRS:tRNA complexes whose structures are hosted by the RCSB Protein Data Bank. These structures at resolutions between 4.5 and 1.2 A are those of synthetases from the three kingdoms of life. Their phylogenic distribution is heterogeneous: most stem from bacteria and archaea and some from eukarya and organelles. Interesting correlations are found between biochemical, physical−chemical, and crystallographic parameters. They highlight common and more specific features of the crystallogenesis of free or liganded aaRSs. The effects of the chemical nature of the crystallant(s), of ligands and other additives, as well as of the presence of impu...

International audience; 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.

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.

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.

Discovery of aminoacyl-tRNA synthetases and importance of these enzymes for correct genetic code expression as well as early structural data and related enzymology will be reviewed. Despite structural diversity, all synthetases follow a two-step mechanism for tRNA aminoacylation. Specifi city, however, is not absolute since synthetases were shown to catalyze mischarging reactions. These reactions are characterized by low catalytic rates and can lead to incomplete charging levels. Incomplete charging can also occur for cognate tRNA aminoacylation and refl ects the equilibrium between the forward acylation and the reverse deacylation (enzymatic and chemical) reactions. Early strategies to characterize the structural features within a given tRNA that account for its preferential aminoacylation by the cognate synthetase will be reviewed. Among them were (i) sequence comparisons of tRNA (RNAs) recognized by a same synthetase, (ii) activity measurements of modifi ed tRNAs (by chemical or enzymatic means) and (iii) reconstitutions of active tRNAs from fragments. As a result, the importance of anticodon and tRNA amino acid accepting arms were highlighted as well as that of the overall tRNA architecture that can be mimicked by tRNA-like structures. Altogether early data gave the robust background to the modern concept of identity.

In metazoans, cell-cycle-dependent histones are produced from poly(A)-lacking mRNAs. The 3 0 end of histone mRNAs is formed by an endonucleolytic cleavage of longer precursors between a conserved stem–loop structure and a purine-rich histone downstream element (HDE). The cleavage requires at least two trans-acting factors: the stem–loop binding protein (SLBP), which binds to the stem– loop and the U7 snRNP, which anchors to histone pre-mRNAs by annealing to the HDE. Using RNA structure-probing techniques, we determined the secondary structure of the 3 0 -untranslated region (3 0 -UTR) of mouse histone pre-mRNAs H4–12, H1t and H2a–614. Surprisingly, the HDE is embedded in hairpin structures and is therefore not easily accessible for U7 snRNP anchoring. Probing of the 3 0 -UTR in complex with SLBP revealed structural rearrangements leading to an overall opening of the structure especially at the level of the HDE. Electrophoretic mobility shift assays demonstrated that the SLBP-induced opening of HDE actually facilitates U7 snRNA anchoring on the histone H4–12 premRNAs 3 0 end. These results suggest that initial binding of the SLBP functions in making the HDE more accessible for U7 snRNA anchoring.

The crystallographic quality of protein crystals that were grown in microgravity has been compared to that of crystals that were grown in parallel on earth gravity under otherwise identical conditions. A goal of this comparison was to assess if a more accurate 3D-structure can be derived from crystallographic analysis of the former crystals. Therefore, the properties of crystals prepared with the Advanced Protein Crystallisation Facility (APCF) on earth and in orbit during the last decade were evaluated. A statistical analysis reveals that about half of the crystals produced under microgravity had a superior X-ray diffraction limit with respect of terrestrial controls. Eleven protein structures could be determined at previously unachieved resolutions using crystals obtained in the APCF. Microgravity induced features of the most relevant structures are reported. A second goal of this study was to identify the cause of the crystal quality enhancement useful for structure determination. No correlations between the effect of microgravity and other system-dependent parameters, such as isoelectric point or crystal solvent content, were found except the reduced convection during the crystallisation process. Thus, crystal growth under diffusive regime appears to be the key parameter explaining the beneficial effect of microgravity on crystal quality. The mimicry of these effects on earth in gels or in capillary tubes is discussed and the practical consequences for structural biology highlighted.

Accurate translation of genetic information necessitates the tuned expression of a large group of genes. Amongst them, controlled expression of the enzymes catalyzing the aminoacylation of tRNAs, the aminoacyl-tRNA synthetases, is essential to insure translational fidelity. In the yeast Saccharomyces cerevisiae, expression of aspartyl-tRNA synthetase (AspRS) is regulated in a process necessitating recognition of the 5' extremity of AspRS messenger RNA (mRNA(AspRS)) by its translation product and adaptation to the cellular tRNA(Asp) concentration. Here, we have established the folding of the approximately 300 nucleotides long 5' end of mRNA(AspRS) and identified the structural signals involved in the regulation process. We show that the regulatory region in mRNA(AspRS) folds in two independent and symmetrically structured domains spaced by two single-stranded connectors. Domain I displays a tRNA(Asp) anticodon-like stem-loop structure with mimics of the aspartate identity determinants, that is restricted in domain II to a short double-stranded helix. The overall mRNA structure, based on enzymatic and chemical probing, supports a three-dimensional model where each monomer of yeast AspRS binds one individual domain and recognizes the mRNA structure as it recognizes its cognate tRNA(Asp). Sequence comparison of yeast genomes shows that the features within the mRNA recognized by AspRS are conserved in different Saccharomyces species. In the recognition process, the N-terminal extension of each AspRS subunit plays a crucial role in anchoring the tRNA-like motifs of the mRNA on the synthetase.

In the methanogenic archae Methanosarcina barkeri, insertion of pyrrolysine, the 22nd amino acid, results from the decoding of an amber UAG codon in the mRNA of monomethylamine methyltransferases (MtmB). Sequence comparisons combined with structural enzymatic and chemical probing on M. barkeri MtmB1 mRNA demonstrate the presence of a hairpin motif located immediately after the redefined UAG codon. This structure of 86 nucleotides differs slightly from a proposal given in the literature and comprises four successive stems separated by three internal loops and closed by a large apical loop. Sequence alignments of MtmB mRNAs of different Methanosarcinacae reveal a conservation of the motif in both sequence and folding levels. The functional role of this motif as a signal leading to pyrrolysine insertion is discussed.

Structural plasticity of transfer RNA (tRNA) molecules is essential for interactions with their biological partners in aminoacylation reactions and during ribosome-dependent protein synthesis. This holds true when tRNAs are recruited for other functions than translation. Here we review regulation pathways where tRNAs and tRNA mimics play a pivotal role. We further discuss the importance of the identity signals used in aminoacylation that are also required to specify regulatory mechanisms. Such mechanisms are diverse and intervene in transcription, splicing and translation. Altogether, the review highlights the many manners architectural features of tRNA were selected by evolution to control biological key processes.

Aminoacylation of transfer RNAs is a key step during translation. It is catalysed by the aminoacyl-tRNA synthetases (aaRSs) and requires the specific recognition of their cognate substrates, one or several tRNAs, ATP and the amino acid. Whereas the control of certain aaRS genes is well known in prokaryotes, little is known about the regulation of eukaryotic aaRS genes. Here, it is shown that expression of AspRS is regulated in yeast by a feedback mechanism that necessitates the binding of AspRS to its messenger RNA. This regulation leads to a synchronized expression of AspRS and tRNA(Asp). The correlation between AspRS expression and mRNA(AspRS) and tRNA(Asp) concentrations, as well as the presence of AspRS in the nucleus, suggests an original regulation mechanism. It is proposed that the surplus of AspRS, not sequestered by tRNA(Asp), is imported into the nucleus where it binds to mRNA(AspRS) and thus inhibits its accumulation.

Borrelia burgdorferi and other spirochetes contain a class I lysyl-tRNA synthetase (LysRS), in contrast to most eubacteria that have a canonical class II LysRS. We analyzed tRNA(Lys) recognition by B. burgdorferi LysRS, using two complementary approaches. First, the nucleotides of B. burgdorferi tRNA(Lys) in contact with B. burgdorferi LysRS were determined by enzymatic footprinting experiments. Second, the kinetic parameters for a series of variants of the B. burgdorferi tRNA(Lys) were then determined during aminoacylation by B. burgdorferi LysRS. The identity elements were found to be mostly located in the anticodon and in the acceptor stem. Transplantation of the identified identity elements into the Escherichia coli tRNA(Asp) scaffold endowed lysylation activity on the resulting chimera, indicating that a functional B. burgdorferi lysine tRNA identity set had been determined.

In vitro transcription has proven to be a successful tool for preparation of functional RNAs, especially in the tRNA field, in which, despite the absence of post-transcriptional modifications, transcripts are correctly folded and functionally active. Human mitochondrial (mt) tRNALys deviates from this principle and folds into various inactive conformations, due to the absence of the post-transcriptional modification m1A9 which hinders base-pairing with U64 in the native tRNA. Unavailability of a functional transcript is a serious drawback for structure/function investigations as well as in deciphering the molecular mechanisms by which point mutations in the mt tRNALys gene cause severe human disorders. Here, we show that an engineered in vitro transcribed “pseudo-WT“ tRNALys variant is efficiently recognized by lysyl-tRNA synthetase and can substitute for the WT tRNA as a valuable reference molecule. This has been exploited in a systematic analysis of the effects on aminoacylation of nine pathology-related mutations described so far. The sole mutation located in a loop of the tRNA secondary structure, A8344G, does not affect aminoacylation efficiency. Out of eight mutations located in helical domains converting canonical Watson–Crick pairs into G–U pairs or C•A mismatches, six have no effect on aminoacylation (A8296G, U8316C, G8342A, U8356C, U8362G, G8363A), and two lead to drastic decreases (5000- to 7000-fold) in lysylation efficiencies (G8313A and G8328A). This screening, allowing for analysis of the primary impact level of all mutations affecting one tRNA under comparable conditions, indicates distinct molecular origins for different disorders.

The product of the Escherichia coli yadB gene is homologous to the N-terminal part of bacterial glutamyl-tRNA synthetases (GluRSs), including the Rossmann fold with the acceptor-binding domain and the stem-contact fold. This GluRS-like protein, which lacks the anticodon-binding domain, does not use tRNA Glu as substrate in vitro nor in vivo , but aminoacylates tRNA Asp with glutamate. The yadB gene is expressed in wild-type E. coli as an operon with the dksA gene, which encodes a protein involved in the general stress response by means of its action at the translational level. The fate of the glutamylated tRNA Asp is not known, but its incapacity to bind elongation factor Tu suggests that it is not involved in ribosomal protein synthesis. Genes homologous to yadB are present only in bacteria, mostly in Proteobacteria. Sequence alignments and phylogenetic analyses show that the YadB proteins form a distinct monophyletic group related to the bacterial and organellar GluRSs (α-type GlxRSs superfamily) with ubiquitous function as suggested by the similar functional properties of the YadB homologue from Neisseria meningitidis .

The newly discovered tRNA(Pyl) is involved in specific incorporation of pyrrolysine in the active site of methylamine methyltransferases in the archaeon Methanosarcina barkeri. In solution probing experiments, a transcript derived from tRNA(Pyl) displays a secondary fold slightly different from the canonical cloverleaf and interestingly similar to that of bovine mitochondrial tRNA(Ser)(uga). Aminoacylation of tRNA(Pyl) transcript by a typical class II synthetase, LysRS from yeast, was possible when its amber anticodon CUA was mutated into a lysine UUU anticodon. Hydrolysis protection assays show that lysylated tRNA(Pyl) can be recognized by bacterial elongation factor. This indicates that no antideterminant sequence is present in the body of the tRNA(Pyl) transcript to prevent it from interacting with EF-Tu, in contrast with the otherwise functionally similar tRNA(Sec) that mediates selenocysteine incorporation.

The intensely sweet protein thaumatin has been crystallized in a hexagonal lattice after a temperature shift from 293 to 277 K. The structure of the protein in the new crystal was solved at 1.6 A resolution. The protein fold is identical to that found in three other crystal forms grown in the presence of crystallizing agents of differing chemical natures. The proportions of lattice interactions involving hydrogen bonds, hydrophobic or ionic groups differ greatly from one form to another. Moreover, the distribution of acidic and basic residues taking part in contacts also varies. The hexagonal packing is characterized by the presence of channels parallel to the c axis that are so wide that protein molecules can diffuse through them.

The effect of hydrostatic pressure and pH on the crystallization of tetragonal hen lysozyme crystals (HeL, M r 14,300) and hexagonal turkey lysozyme crystals (TeL, M r 14,200) in agarose gel was studied. Samples (adjusted to a pH in the range 4–6) were pressurized at 0.1–100 MPa for nine days at 293 K. The morphology and number of crystals as well as protein solubility were analyzed after depressurization. For both proteins and whatever the pH, a higher pressure resulted in greater numbers of crystals and greater solubility values. At any pressure, the solubility and number of crystals were lower at the highest pH. Thus the physical chemical parameters and the outcome of the crystallization process are more affected by pressure at pH 4.0 than at pH 6.0. In the case of HeL, either high pressure or high pH induces a transition from the initial tetragonal form to an orthorhombic one. The observed effects are related to minor differences in the macromolecular structures.

Hydrostatic pressure in the 0.1–75 MPa range has been used as a non-invasive tool to study the crystallization process of the tetragonal crystal form of the protein thaumatin (Mr 22 200). Crystals were prepared within agarose gel and at temperatures in the range from 283 to 303 K. The solubility, i.e. the concentration of soluble macromolecules remaining in equilibrium with the crystals, decreases when the pressure increases and when the temperature decreases. High pressure was used to probe the nucleation behaviour of thaumatin. The pressure dependence of the nucleation rate leads to an activation volume of −46.5 cm3 mol−1. It is shown that an increase in pressure decreases the enthalpy, the entropy and the free energy of crystallization of thaumatin. The data are discussed in the light of the results of crystallographic analyses and of the structure of the protein.

The combined effect of both pressure and pH on the crystallization in agarose gel of thaumatin (0.1–230 MPa and pH 5.7–8.0) and the lysozymes from turkey (0.1–100 MPa and pH 4.0–6.0) and hen egg-white (0.1–75 MPa and pH 4.0–6.0) was investigated. For each condition, crystal morphology was examined and four crystal growth parameters determined (crystal number as a measure of nucleation, solubility, supersaturation and pressure-induced volume changes of crystallization). Comparison of data reveals a different crystallization behavior of thaumatin and the two lysozymes. First, thaumatin is more pressure tolerant in agarose gel than the lysozymes, with crystals growing up to 230 MPa for the former and only up to 75/100 MPa for the latter. Second, while an increase in pressure and pH increases nucleation of thaumatin crystals and decreases their solubility, one observes an inverse effect with the two lysozymes. Here, an increase in pressure but a decrease in pH increases nucleation and solubility. Third, pH influences the pressure-induced volume changes of crystallization, the strongest pH-dependence being found for turkey lysozyme. However, the volume changes are negative for thaumatin and positive for both lysozymes. These volume changes are correlated with solubility changes and are explained by pH- and pressure-induced alterations of the conformation of the three proteins, including their solvation shells.

Dimeric class II aspartyl-tRNA synthetase (AspRS) from yeast has a modular architecture and includes an N-terminal appendix of 70 amino acid residues that protrudes from the anticodon-binding module. This extension, of predicted helical structure, is not essential for aminoacylation but contains an RNA-binding motif that promotes non-specific interactions with tRNAs. As shown here, this protein extension can also interact with the 5′ end of the AspRS mRNA. In vitro, optimal binding occurs on an mRNA domain comprising part of the 87 nucleotide long 5′UTR and the sequence encoding the N-terminal appendix. At the protein side, only the appendix and the anticodon-binding module participate in the interaction between AspRS and the mRNA domain. Binding is specific, since only tRNAAsp can dissociate the complex. In vivo, AspRS also binds specifically this mRNA domain and in doing so triggers a reduced translation of a fused GFP mRNA. From that, a mechanism for the regulation of this eukaryotic aminoacyl-tRNA synthetase is proposed. Implications for aspartylation accuracy in yeast are given.

The genetic code was established through aminoacylations of RNA substrates that emerged as tRNAs. The 20 aminoacyl-tRNA synthetases (one for each amino acid) are ancient proteins, the active-site domain of which catalyzes formation of an aminoacyl adenylate that subsequently reacts with the 3′ end of bound tRNA. Binding of tRNA depends on idiosyncratic (to the particular synthetase) domains and motifs that are fused to or inserted into the conserved active-site domain. Here we take the domain for synthesis of alanyl adenylate and fuse it to ``artificial'' peptide sequences (28 aa) that were shown previously to bind to the acceptor arm of tRNA Ala . Certain fusions confer aminoacylation activity on tRNA Ala and on hairpin microhelices modeled after its acceptor stem. Aminoacylation was sensitive to the presence of a specific G:U base pair known to be a major determinant of tRNA Ala identity. Aminoacylation efficiency and specificity also depended on the specific peptide sequence. The results demonstrate that barriers to RNA-specific aminoacylations are low and can be achieved by relatively simple peptide fusions. They also suggest a paradigm for rationally designed specific aminoacylations based on peptide fusions.

The human mitochondrial genome encodes 22 tRNAs interspersed among the two rRNAs and 11 mRNAs, often without spacers, suggesting that tRNAs must be efficiently excised. Numerous maternally transmitted diseases and syndromes arise from mutations in mitochondrial tRNAs, likely due to defect(s) in tRNA metabolism. We have systematically explored the effect of pathogenic mutations on tRNA(Ile) precursor 3' end maturation in vitro by 3'-tRNase. Strikingly, four pathogenic tRNA(Ile) mutations reduce 3'-tRNase processing efficiency (V(max) / K(M)) to approximately 10-fold below that of wild-type, principally due to lower V(max). The structural impact of mutations was sought by secondary structure probing and wild-type tRNA(Ile) precursor was found to fold into a canonical cloverleaf. Among the mutant tRNA(Ile) precursors with the greatest 3' end processing deficiencies, only G4309A displays a secondary structure substantially different from wild-type, with changes in the T domain proximal to the substitution. Reduced efficiency of tRNA(Ile) precursor 3' end processing, in one case associated with structural perturbations, could thus contribute to human mitochondrial diseases caused by mutant tRNAs.

Crystals of thaumatin and of turkey and hen lysozyme, prepared in a batch at 293 K under hydrostatic pressures in the 0.1–150 MPa range, have been analyzed to investigate how this parameter influences crystallization and if depressurization introduces defects in the crystalline lattice. The X-ray diffraction properties of depressurized crystals have been compared with those of unpressurized control crystals prepared under otherwise identical conditions at atmospheric pressure (0.1 MPa). Independently of pressure, the crystals of each protein belong to the same space group and have cell parameters identical to those of the controls. Their diffraction limit is more dependent upon crystal volume than upon pressure. Crystal mosaicity, expressed as the full-width at half-maximum w of the Bragg reflection profile, was used to quantify the defects in the lattice. While the quality of lysozyme crystals deteriorates as pressure increases, that of tetragonal thaumatin crystals becomes more homogeneous. The first result correlates with the apparition of cleavages oriented perpendicularily to the crystal's c -axis. The second one is interpreted by a gradual selection of well-formed nuclei. The 3D structure of thaumatin in a crystal that was grown under a pressure of 150 MPa and returned to atmospheric pressure was solved. The fold of the polypeptide chain and the distribution of water molecules was compared with those in a reference crystal grown at 0.1 MPa.The results suggest that the crystal lattice is elastic. A possible application of pressure to the preparation of high quality protein crystals is discussed in the light of these results.

Crystals of thaumatin and of lysozyme from turkey and hen egg white have been prepared in batch at 20°C under hydrostatic pressures in the 0.1–220 MPa range. The latter model protein served as a reference in this comparative study. Crystallization was performed in an agarose gel in contrast to former studies under pressure that were conducted in solution. After depressurization, the habit, number, length, shape and solubility of crystals were compared to those of control crystals that were prepared at atmospheric pressure (0.1 MPa). For the three proteins, the number of the crystals increases with pressure. For thaumatin and hen lysozyme, crystal length decreases but for turkey lysozyme it increases. While the solubility of the first protein decreases, that of both lysozymes increases. The relationship between solubility and pressure is linear in the three cases. The crystallization volumes Δ V are −11 cm 3 mol −1 for thaumatin, +15 cm 3 mol −1 for turkey lysozyme and +3 cm 3 mol −1 for hen lysozyme. For the lysozymes, they are explained by pressure-dependent conformational changes. The structure of thaumatin appears to be less deformable under pressure.

Aspartyl-tRNA synthetases were the model proteins in pilot crystallogenesis experiments. They are homodimeric enzymes of Mr~125 kDa that possess as substrates a transfer RNA, ATP and aspartate. They have been isolated from different sources and were crystallized either as free proteins or in association with their ligands. This review discusses their crystallisability with emphasis to crystal quality and structure determination. Crystallization in low diffusivity gelled media or in microgravity environments is highlighted. It has contributed to prepare high-resolution diffracting crystals with better internal order as reflected by their mosaicity. With AspRS from Thermus thermophilus, the better crystalline quality of the space-grown crystals within APCF is correlated with higher quality of the derived electron density maps. Usefulness for structural biology of targeted methods aimed to improve the intrinsic physical quality of protein crystals is highlighted.

One reason for introducing a gel in the crystallization medium of proteins is its ability to reduce convection in solution. This can lead to better nucleation and growth conditions, and to crystals having enhanced diffraction properties. We report here the X-ray characterization at room temperature of high-quality crystals of the intensely sweet thaumatin prepared in a sodium tartrate solution gelified with 0.15% (m/v) agarose. Using a synchrotron radiation, these crystals diffracted to a previously unachieved resolution. A diffraction dataset was collected from four crystals at a resolution of 1.2 A with a Rsym of 3.6% and a completeness of 99%. Refinement was carried out to a final crystallographic R-factor of 12.0%. The quality of the electron density map allowed for the observation of fine structural details in the protein and its solvation shell. Crystallization in gel might be used more generally to improve the quality of macromolecular crystals. Advantages provided by the gelified medium in the frame of structural studies are emphasized. Proteins 2002;48:146–150. © 2002 Wiley-Liss, Inc.

Aminoglycosides inhibit translation in bacteria by binding to the A site in the ribosome. Here, it is shown that, in yeast, aminoglycosides can also interfere with other processes of translation in vitro. Steady-state aminoacylation kinetics of unmodified yeast tRNA(Asp) transcript indicate that the complex between tRNA(Asp) and tobramycin is a competitive inhibitor of the aspartylation reaction with an inhibition constant (K(I)) of 36 nM. Addition of an excess of heterologous tRNAs did not reverse the charging of tRNA(Asp), indicating a specific inhibition of the aspartylation reaction. Although magnesium ions compete with the inhibitory effect, the formation of the aspartate adenylate in the ATP-PP(i) exchange reaction by aspartyl-tRNA synthetase in the absence of the tRNA is not inhibited. Ultraviolet absorbance melting experiments indicate that tobramycin interacts with and destabilizes the native L-shaped tertiary structure of tRNA(Asp). Fluorescence anisotropy using fluorescein-labelled tobramycin reveals a stoichiometry of one molecule bound to tRNA(Asp) with a K(D) of 267 nM. The results indicate that aminoglycosides are biologically effective when their binding induces a shift in a conformational equilibrium of the RNA.

Fidelity of transfer ribonucleic acid (tRNA) charging by amino acids ensures correct translation of the genetic code into proteins. Charging is catalysed by a set of enzymes known as aminoacyl-tRNA synthetases. Owing to the degeneracy of the genetic code, some of the different tRNAs have the same amino acid attached to them. Specificity of charging obeys universal rules and is ensured by positive elements, the identity determinants unique to each tRNA and responsible for its recognition by the cognate synthetase, and negative elements, the antideterminants that prevent false recognitions. To fulfil the aminoacylation specificity and prevent noncognate aminoacyl-tRNA delivery to the ribosome, some synthetases also mediate proofreading reactions that increase fidelity of the tRNA charging. In such reactions, misactivated amino acids or mischarged tRNAs are checked in specific sites and noncognate products are hydrolysed. However, mischarging is beneficial under certain stress circumstances or when catalysed by nondiscriminatory synthetases, and represents a driving force in evolution. Key Concepts: Translational expression of the genetic code refers to aminoacyl-tRNA- and ribosome-dependent decoding of genes into proteins, a process highly dependent on fidelity of tRNA aminoacylation by synthetases. The rules that account for the aminoacylation identity of tRNAs are referred to as the second genetic code. The RNA operational code is encoded in the acceptor stem of tRNA and is crucial for recognition by aminoacyl-tRNA synthetases and specific aminoacylation. Allostery in tRNA-synthetase systems concerns long-range transfer of chemical information (up to 75 A) to the synthetase catalytic site (through the body of tRNA and/or synthetase) triggered by contacts of tRNA identity determinants with the synthetase. Engineering the identity of tRNA-synthetase systems allows reprogramming the genetic code. Keywords: allostery; aminoacyl-tRNA synthetase; genetic code; identity antideterminant; identity determinants; protein synthesis; RNA recognition; translation; tRNA; tRNA post-transcriptional modification

This chapter summarizes the structural and functional mimicry of tRNA by plant viral tRNA-like structures, and emphasizes how studies on these structures were innovative in the domain of RNA folding and beneficial to the better understanding of canonical tRNA aminoacylation. It also discusses current ideas about the biological significance of these aminoacylatable structures as well as other viral RNA sequences mimicking tRNA, during the life cycles of their carrier viruses.

A quasi-planar X-ray study has been done to assess the quality of crystals of pure thaumatin and of hen egg-white lysozyme as well as of lysozyme which was intentionally contaminated by structurally unrelated (ovalbumin and conalbumin) or related (turkey egg-white lysozyme) macromolecules. To investigate the behavior of different growth sectors, we have chosen crystals exhibiting well defined habit, namely crystals grown either in agarose gel or in silica gel. The main defect evidenced is a misorientation originating at the level of the nucleus: the parts grown in the +c and −c directions seem to be individuals twisted always clockwise with respect to the growth direction. The measured lattice plane tilt increases up to 3.5 min of arc when the contaminant content increases. In addition to this defect, we could measure for lysozyme relative lattice parameters differences (Δd/d) of the order of 10−4 between prismatic and pyramidal growth sectors. All these defects do not seem to have a major influence on the resolution limit but they have consequences on optical properties.

Two active forms of homodimeric aspartyl-tRNA synthetase from Saccharomyces cerevisiae differing in length at their N-terminus crystallize in the same orthorhombic space group (P4 1 2 1 2) with identical cell parameters. Initial studies were hampered by the poor and anisotropic diffraction of the crystals of enzyme extracted from yeast cells. Isotropic diffraction at higher resolution was obtained when crystals were grown from an engineered protein deprived of its 70 N-terminal amino acids. The present work describes the packing contacts in crystals of the shortened protein whose structure was solved at 2.3 A resolution. Each subunit of the enzyme develops two lattice interactions covering a surface of 670 A 2 , about 7-fold smaller than that of the interface between monomers. The smallest lattice interaction, covering 150 A 2 , brings the anticodon binding domain adjacent to the N-terminus of one monomer in contact with a loop from the active-site domain of a neighboring monomer. Modeling of the extension in the solvent channels shows that the 150 A 2 intermolecular contact is perturbed in protein molecules possessing a floppy appendix while their second and larger 520 A 2 contact area is unaffected. Altogether the packing organization explains the poor diffraction properties of the native enzyme crystals and the enhanced diffraction of the crystals of shortened synthetase.

The thermostable aspartyl-tRNA synthetase (AspRS-1) from Thermus thermophilus is a 132 kDa homodimer with a subunit composed of 580 amino acids. It catalyses the aminoacylation of tRNAAsp with aspartic acid in the process of translating genetic information. Here we present data on crystals grown in the presence of two different crystallizing agents. A first crystal form (form A) grows in the presence of 0.8 M ammonium sulfate and exhibits the orthorhombic space group P212121. Monoclinic plates (form B) grow in an aqueous solution of 6% (m/v) PEG-8000. In this study, the monoclinic crystal structure (form B) was solved by molecular replacement using the orthorhombic crystal structure as a model and refined to a 2.65 A resolution limit. The contacts between molecules in both crystalline lattices are compared. Although the overall-accessible surface of the protein is more hydrophilic than average, the packing contacts in both lattices comprise mainly hydrophobic van der Waals interactions and only a few salt bridges and hydrogen bonds. Interaction areas are much larger in the orthorhombic than in the monoclinic lattice, and only 6 contact residues out of 134 are common.

In the frame of a study of the effects of microgravity on protein crystallization, tetragonal thaumatin crystals were, for the first time, prepared in agarose gel within the Advanced Protein Crystallization facility (APCF) on Space Shuttle mission STS-95 (October 1998). We have reported elsewhere that these crystals are of superior crystallographic quality, with regard to their diffraction intensity and limit as well as mosaic spread, when compared to control crystals grown on Earth [Lorber et al., Acta Crystallogr. (1999) D55, 1491]. The analysis of images taken during the growth of these crystals under microgravity indicates that their nucleation was more synchronous than that of control crystals which were prepared simultaneously on Earth under otherwise identical conditions. Nucleation occurred essentially in the bulk of the medium and crystal growth proceeded up to 2 times faster. Crystals prepared under microgravity had better optical properties than control crystals. Experimental results give evidence of the existence of zones depleted in protein located around growing crystals. Also, they are in favor of a correlation between the better diffraction properties of these crystals and the more favorable crystallization parameters in Space. The content of individual crystals was analyzed and the results of purity analyses are presented. The advantages of crystallization in a gel under microgravity and importance of image documentation for asserting growth cessation are discussed.

To elucidate the minimal substrate for the plant nuclear tRNA 3' processing enzyme, we synthesized a set of tRNA variants, which were subsequently incubated with the nuclear tRNA 3' processing enzyme. Our experiments show that the minimal substrate for the nuclear RNase Z consists of the acceptor stem and T arm. The broad substrate spectrum of the nuclear RNase Z raises the possibility that this enzyme might have additional functions in the nucleus besides tRNA 3' processing. Incubation of tRNA variants with the plant mitochondrial enzyme revealed that the organellar counterpart of the nuclear enzyme has a much narrower substrate spectrum. The mitochondrial RNase Z only tolerates deletion of anticodon and variable arms and only with a drastic reduction in cleavage efficiency, indicating that the mitochondrial activity can only cleave bona fide tRNA substrates efficiently. Both enzymes prefer precursors containing short 3' trailers over extended 3' additional sequences. Determination of cleavage sites showed that the cleavage site is not shifted in any of the tRNA variant precursors.

Transfer RNA (tRNA) is a small nucleic acid (typically 76 nucleotides) that forms binary complexes with proteins, such as aminoacyl tRNA synthetases (RS) and Trbp111. The latter is a widely distributed structure-specific tRNA-binding protein that is incorporated into cell signaling molecules. The structure of Trbp111 was modeled onto to the outer, convex side of the L-shaped tRNA. Here we present RNA footprints that are consistent with this model. This binding mode is in contrast to that of tRNA synthetases, which bind to the inside, or concave side, of tRNA. These opposite locations of binding for these two proteins suggest the possibility of a ternary complex. The formation of a tRNA synthetase–tRNA–Trbp111 ternary complex was detected by two independent methods. The results indicate that the tRNA is sandwiched between the two protein molecules. A thermodynamic and functional analysis is consistent with the tRNA retaining its native structure in the ternary complex. These results may have implications for how the translation apparatus is linked to other cellular machinery.

Growth kinetics and diffraction properties of monoclinic crystals of eubacterial Thermus thermophilus aspartyl-tRNA synthetase-1 (AspRS-1) prepared in the presence of polyethylene glycol and agarose are studied. Their solubility and two-dimensional phase diagram are compared with those of orthorhombic crystals which grow in the presence of sodium formate or ammonium sulfate. The growth mechanism of the novel crystals was monitored by atomic force microscopy. The gel stabilizes the crystal lattice under the cryogenic conditions used for structure determination at high resolution.

Imidazole and compounds containing imidazole residues have been shown to cleave RNA in an RNase A-mimicking manner. Di-imidazole lexitropsin is a compound which is derived from the polyamide drugs distamycin and netropsin essentially by the replacement of two pyrrole heterocycles with N-methyl-imidazole residues. This enables it to bind to the minor groove of B-DNA in a sequence-specific manner. We demonstrate here that this lexitropsin derivative has RNA cleavage activity, as tested on model RNAs. Optimal cleavage conditions and cleavage specificity resemble those known from other imidazole conjugates and are thus consistent with an RNase A type cleavage mechanism. The optimum concentration of the compound for cleavage is similar to previously investigated imidazole-based RNase mimics. As a whole new class of chemical compounds capable of interacting with nucleic acids through extensive hydrogen bonding, these imidazole containing compounds constitute promising scaffolds and ligands, for the construction of novel RNase mimics with high affinity.

Using in vitro tRNA transcripts and minihelices it was shown that the tyrosine identity for tRNA charging by tyrosyl-tRNA synthetase (TyrRS) from the archaeon Methanococcus jannaschii is determined by six nucleotides: the discriminator base A73 and the first base-pair C1-G72 in the acceptor stem together with the anticodon triplet. The anticodon residues however, participate only weakly in identity determination, especially residues 35 and 36. The completeness of the aforementioned identity set was verified by its tranfer into several tRNAs which then become as efficiently tyrosylatable as the wild-type transcript from M. jannaschii. Temperature dependence experiments on both the structure and the tyrosylation properties of M. jannaschii and yeast tRNA(Tyr) transcripts show that the archaeal transcript has greater structural stability and enhanced aminoacylation behaviour than the yeast transcript. Tyrosine identity in M. jannaschii is compared to that in yeast, and the conservation of the major determinant in both organisms, namely the C1-G72 pair, gives additional support to the existence of a functional connection between archaeal and eukaryotic aminoacylation systems.

A number of mitochondrial (mt) tRNAs have strong structural deviations from the classical tRNA cloverleaf secondary structure and from the conventional L-shaped tertiary structure. As a consequence, there is a general trend to consider all mitochondrial tRNAs as "bizarre" tRNAs. Here, a large sequence comparison of the 22 tRNA genes within 31 fully sequenced mammalian mt genomes has been performed to define the structural characteristics of this specific group of tRNAs. Vertical alignments define the degree of conservation/variability of primary sequences and secondary structures and search for potential tertiary interactions within each of the 22 families. Further horizontal alignments ascertain that, with the exception of serine-specific tRNAs, mammalian mt tRNAs do fold into cloverleaf structures with mostly classical features. However, deviations exist and concern large variations in size of the D- and T-loops. The predominant absence of the conserved nucleotides G18G19 and T54T55C56, respectively in these loops, suggests that classical tertiary interactions between both domains do not take place. Classification of the tRNA sequences according to their genomic origin (G-rich or G-poor DNA strand) highlight specific features such as richness/poorness in mismatches or G-T pairs in stems and extremely low G-content or C-content in the D- and T-loops. The resulting 22 "typical" mammalian mitochondrial sequences built up a phylogenetic basis for experimental structural and functional investigations. Moreover, they are expected to help in the evaluation of the possible impacts of those point mutations detected in human mitochondrial tRNA genes and correlated with pathologies.