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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.

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.

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...

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.

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.

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.

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.

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

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.

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.

The tRNA-like structure (TLS) of turnip yellow mosaic virus (TYMV) RNA was previously shown to be efficiently charged by yeast valyl-tRNA synthetase (ValRS). This RNA has a noncanonical structure at its 3'-terminus but mimics a tRNA L-shaped fold, including an anticodon loop containing the major identity nucleotides for valylation, and a pseudoknotted amino acid accepting domain. Here we describe an in vitro selection experiment aimed (i) to verify the completeness of the valine identity set, (ii) to elucidate the impact of the pseudoknot on valylation, and (iii) to investigate whether functional communication exists between the two distal anticodon and amino acid accepting domains. Valylatable variants were selected from a pool of 2 x 10(13) RNA molecules derived from the TYMV TLS randomized in the anticodon loop nucleotides and in the length (1-6 nucleotides) and sequence of the pseudoknot loop L1. After nine rounds of selection by aminoacylation, 42 have been isolated. Among them, 17 RNAs could be efficiently charged by yeast ValRS. Their sequence revealed strong conservation of the second and the third anticodon triplet positions (A(56), C(55)) and the very 3'-end loop nucleotide C(53). A large variability of the other nucleotides of the loop was observed and no wild-type sequence was recovered. The selected molecules presented pseudoknot domains with loop L1 varying in size from 3-6 nucleotides and some sequence conservation, but did neither reveal the wild-type combination. All selected variants are 5-50 times more efficiently valylated than the wild-type TLS, suggesting that the natural viral sequence has emerged from a combination of evolutionary pressures among which aminoacylation was not predominant. This is in line with the role of the TLS in viral replication.

The specific aminoacylation of tRNA by yeast tyrosyl-tRNA synthetase does not rely on the presence of modified residues in tRNA(Tyr), although such residues stabilize its structure. Thus, the major tyrosine identity determinants were searched by the in vitro approach using unmodified transcripts produced by T7 RNA polymerase. On the basis of the tyrosylation efficiency of tRNA variants, the strongest determinants are base pair C1-G72 and discriminator residue A73 (the 5'-phosphoryl group on C1, however, is unimportant for tyrosylation). The three anticodon bases G34, U35, and A36 contribute also to the tyrosine identity, but to a lesser extent, with G34 having the most pronounced effect. Mutation of the GUA tyrosine anticodon into a CAU methionine anticodon, however, leads to a loss of tyrosylation efficiency similar to that obtained after mutation of the C1-G72 or A73 determinants. Transplantation of the six determinants into four different tRNA frameworks and activity assays on heterologous Escherichia coli and Methanococcus jannaschii tRNA(Tyr) confirmed the completeness of the tyrosine set and the eukaryotic character of the C1-G72 base pair. On the other hand, it was found that tyrosine identity in yeast does not rely on fine architectural features of the tRNA, in particular the size and sequence of the D-loop. Noticeable, yeast TyrRS efficiently charges a variant of E. coli tRNA(Tyr) with a large extra-region provided its G1-C72 base pair is changed to a C1-G72 base pair. Finally, tyrosylation activity is compatible with a +1 shift of the anticodon in the 3'-direction but is strongly inhibited if this shift occurs in the opposite 5'-direction.

As part of a study of the effects of microgravity on protein crystallization, the growth of tetragonal crystals of thaumatin was monitored by CCD-time-lapse video in environments where convection is negligible. In Space Shuttle missions entitled United States Microgravity Laboratory-2 and Life and Microgravity Sciences, free interface diffusion and dialysis techniques were utilized to grow crystals in the advanced protein crystallization facility (APCF). Ng et al. (Acta Crystallogr. D 53 (1997) 724) have shown that the crystals recovered in these experiments are of superior crystallographic quality (at the level of their diffraction intensity, resolution, and mosaicity) with regard to earth controls. Here, the number of crystals, their size, growth rate, and protein solubility in microgravity were compared with data of dialysis experiments performed in parallel on earth. Image analysis shows that in microgravity about one-quarter to half of the crystals have nucleated and grown in the bulk of the solution, the remaining being attached to the walls of crystallization vessels. The growth of free-floating crystals was 2.5 times faster, has resulted in 15-fold larger crystals, and consumed more protein than that of attached crystals in earth controls. Distances between immobile free-floating crystals in microgravity were related to the size of the latter. Experimental results are in favor of a correlation between more favorable growth parameters in microgravity and better diffraction properties. The displacements of free-floating crystals at various velocities and in various directions on unrelated trajectories are indicative of drift and stirring motion. On the basis of an overview of reactors monitored on four APCF missions some forces causing motion are proposed. Advantages of crystallization in microgravity are discussed and recommendations for future experiments given.

Overproducing Escherichia coli tRNAGlu in its homologous host results in the presence of several distinctly modified forms of this molecule that we name modivariants. The predominant tRNAGlu modivariant in wild-type E. coli contains five modified nucleosides: Psi13, mnm5s2U34, m2A37, T54 and Psi55. Four other overproduced modivariants differ from it by, respectively, either the presence of an additional Psi, or the presence of s2U34, or the lack of A37 methylation combined with either s2U34 or U34. Chemical probing reveals that the anticodon loop of the predominant modivariant is less reactive to the probes than that of the four others. Furthermore, the modivariant with neither mnm5s2U34 nor m2A37 has additional perturbations in the D- and T-arms and in the variable region. The lack of a 2-thio group in nucleoside 34, which is mnm5s2U in the predominant tRNAGlu modivariant, decreases by 520-fold the specificity of E. coli glutamyl-tRNA synthetase for tRNAGlu in the aminoacylation reaction, showing that this thio group is the identity element in the modified wobble nucleotide of E. coli tRNAGlu. The modified nucleosides content also influences the recognition of ATP and glutamate by this enzyme, and in this case also, the predominant modivariant is the one that allows the best specificity for these two substrates. These structural and kinetic properties of tRNAGlu modivariants indicate that the modification system of tRNAGlu optimizes the stability of tRNAGlu and its action as cofactor of the glutamyl-tRNA synthetase for the recognition of glutamate and ATP.

Assuming that the L-shaped three-dimensional structure of tRNA is an architectural framework allowing the proper presentation of identity nucleotides to aminoacyl-tRNA synthetases implies that altered and/or simplified RNA architectures can fulfill this role and be functional substrates of these enzymes, provided they contain correctly located identity elements. In this work, this paradigm was submitted to new experimental verification. Yeast aspartyl-tRNA synthetase was the model synthetase, and the extent to which the canonical structural framework of cognate tRNA Asp can be altered without losing its ability to be aminoacylated was investigated. Three novel architectures recognized by the synthetase were found. The first resembles that of metazoan mitochondrial tRNA Ser lacking the D-arm. The second lacks both the D- and T-arms, and the 5'-strand of the amino acid acceptor arm. The third structure is a construct in which the acceptor and anticodon helices are joined by two connectors. Aspartylation specificity of these RNAs is verified by the loss of aminoacylation activity upon mutation of the putative identity residues. Kinetic data indicate that the first two architectures are mimics of the whole tRNA Asp molecule, while the third one behaves as an aspartate minihelix mimic. Results confirm the primordial role of the discriminator nucleotide G73 in aspartylation and demonstrate that neither a helical structure in the acceptor domain nor the presence of a D- or T-arm is mandatory for specific aspartylation, but that activity relies on the presence of the cognate aspartate GUC sequence in the anticodon loop.

Quasi-planar wave reflection profile and X-ray topography studies have been done to characterize the mosaicity of solution- and gel-grown crystals of three proteins, turkey egg-white (TEW) lysozyme, thaumatin, and a bacterial aspartyl-tRNA synthetase (AspRS) as well as of one virus, tomato bushy stunt virus (TBSV). These materials are representative of a large range of molecular weight, overall particle shapes, crystals habits, packings, and solvent contents. Measurements of the full-width at half-maximum (FWHM) of reflections show that these different crystals have all a weak mosaicity. Topographs display the same features as those of the well-studied hen egg-white (HEW) lysozyme crystals: misorientation generated at the seed level for TEW lysozyme or thaumatin crystals and/or strains at growth sector boundaries for AspRS crystals. No growth defects are evidenced for TBSV crystals. For the study of crystals diffracting at lower resolution (AspRS and virus), a less absorbant sample holder, which facilitates crystal positioning in the X-ray beam, has been developed. The results obtained for solution- and gel-grown crystals do not show important differences. However, for TEW lysozyme and thaumatin crystals, one notices a larger dispersion of results in the solution case and an overall tendency for improved reproducibility of quality for gel-grown crystals.

The wild-type transcript of Escherichia coli tRNASec, characterized by a peculiar core architecture and a large variable region, was shown to be aspartylatable by yeast AspRS. Similar activities were found for tRNASec mutants with methionine, leucine, and tryptophan anticodons. The charging efficiency of these molecules was found comparable to that of a minihelix derived from tRNAAsp and is accounted for by the presence of the discriminator residue G73, which is a major aspartate identity determinant. Introducing the aspartate identity elements from the anticodon loop (G34, U35, C36, C38) into tRNASec transforms this molecule into an aspartate acceptor with kinetic properties identical to tRNAAsp. Expression of the aspartate identity set in tRNASec is independent of the size of its variable region. The functional study was completed by footprinting experiments with four different nucleases as structural probes. Protection patterns by AspRS of transplanted tRNASec and tRNAAsp were found similar. They are modified, particularly in the anticodon loop, upon changing the aspartate anticodon into that of methionine. Altogether, it appears that recognition of a tRNA by AspRS is more governed by the presence of the aspartate identity set than by the structural framework that carries this set.

Numerous molecules have been described in literature as additives that were indispensable either for nucleation or growth of macromolecular crystals. In some cases, such additives were shown to improve the quality of the X-ray diffraction and to extend diffraction limits. We have investigated the effects of more than fifty compounds, belonging to several chemical families, on the crystallization of four model proteins (hen and turkey egg-white lysozymes, thaumatin, and aspartyl-tRNA synthetase from Thermus thermophilus). In addition, we have studied the crystallization of a ribonucleic acid from yeast, the transfer RNA specific for phenylalanine in the presence of synthetic polyamines. Crystals grown in the presence of the additives were optically evaluated and X-ray diffraction analyses were performed on selective crystals to compare their space group, cell parameters, and diffraction limit with those of controls. Whereas no changes in space group nor cell parameters were observed for the model proteins, significant improvements in diffraction limit were found when the transfer RNA was crystallized with certain synthetic polyamines.

tRNA post-transcriptional modification enzymes of Xenopus laevis were proposed previously to belong to two major groups according to their sensitivity to structural perturbations in their substrates. To further investigate the structural variations tolerated by these enzymes, the tRNA-like domain of turnip yellow mosaic virus RNA (88 nucleotides in length) has been microinjected into the oocytes of Xenopus laevis. This RNA possesses 12 potential target nucleotides for modification within a structure including a pseudoknotted folding, an extended anticodon stem, and unusual D-loop/T-loop interactions. Results indicate that only cytosine-42, a position equivalent to C-49 in canonical tRNAs, was quantitatively modified into m5C in the microinjected RNA. Modification was detected to high levels, indicating that at least one enzyme tolerates non-canonical structural features.

The last 82 nucleotides of the 6.3 kb genomic RNA of plant turnip yellow mosaic virus (TYMV), the so-called 'tRNA-like' domain, presents functional, structural and primary sequence homologies with canonical tRNAs. In particular, one of the stem-loops resembles the TPsi(pseudouridine)-branch of tRNA, except for the presence of a guanosine at position 37 (numbering is from the 3'-end) instead of the classical uridine-55 in tRNA (numbering is from the 5'-end). Both the wild-type TYMV-RNA fragment and a variant, TYMV-mut G37U in which G-37 has been replaced by U-37, have been tested as potential substrates for the yeast tRNA modification enzymes. Results indicate that two modified nucleotides were formed upon incubation of the wild-type TYMV-fragment in a yeast extract: one Psi which formed quantitatively at position 65, and one ribothymidine (T) which formed at low level at position U-38. In the TYMV-mutant G37U, besides the quantitative formation of both Psi-65 and T-38, an additional Psi was detected at position 37. Modified nucleotides Psi-65, T-38 and Psi-37 in TYMV RNA are equivalent to Psi-27, T-54 and Psi-55 in tRNA, respectively. Purified yeast recombinant tRNA:Psisynthases (Pus1 and Pus4), which catalyze respectively the formation of Psi-27 and Psi-55 in yeast tRNAs, are shown to catalyze the quantitative formation of Psi-65 and Psi-37, respectively, in the tRNA-like 3'-domain of mutant TYMV RNA in vitro . These results are discussed in relation to structural elements that are needed by the corresponding enzymes in order to catalyze these post-transcriptional modification reactions.

Arginylation of tRNA transcripts by yeast arginyl-tRNA synthetase can be triggered by two alternate recognition sets in anticodon loops: C35 and U36 or G36 in tRNA(Arg) and C36 and G37 in tRNA(Asp) (Sissler M, Giegé R, Florentz C, 1996, EMBO J 15:5069-5076). Kinetic studies on tRNA variants were done to explore the mechanisms by which these sets are expressed. Although the synthetase interacts in a similar manner with tRNA(Arg) and tRNA(Asp), the details of the interaction patterns are idiosyncratic, especially in anticodon loops (Sissler M, Eriani G, Martin F, Giegé R, Florentz C, 1997, Nucleic Acids Res 25:4899-4906). Exchange of individual recognition elements between arginine and aspartate tRNA frameworks strongly blocks arginylation of the mutated tRNAs, whereas full exchange of the recognition sets leads to efficient arginine acceptance of the transplanted tRNAs. Unpredictably, the similar catalytic efficiencies of native and transplanted tRNAs originate from different k(cat) and Km combinations. A closer analysis reveals that efficient arginylation results from strong anticooperative effects between individual recognition elements. Nonrecognition nucleotides as well as the tRNA architecture are additional factors that tune efficiency. Altogether, arginyl-tRNA synthetase is able to utilize different context-dependent mechanistic routes to be activated. This confers biological advantages to the arginine aminoacylation system and sheds light on its evolutionary relationship with the aspartate system.

Phenylalanine identity of yeast tRNAPhe is governed by five nucleotides including residues A73, G20, and the three anticodon nucleotides (Sampson et al., 1989, Science 243, 1363–1366). Analysis of in vitro transcripts derived from yeast tRNAPhe and Escherichia colitRNAAla bearing these recognition elements shows that phenylalanyl-tRNA synthetase is sensitive to additional nucleotides within the acceptor stem. Insertion of G2-C71 has dramatic negative effects in both tRNA frameworks. These effects become compensated by a second-site mutation, the insertion of the wobble G3-U70 pair, which by itself has no effect on phenylalanylation. From a mechanistic point of view, the G2-C71/G3-U70 combination is not a “classical” recognition element since its antideterminant effect is compensated for by a second-site mutation. This enlarges our understanding of tRNA identity that appears not only to be the outcome of a combination of positive and negative signals forming the so-called recognition/identity set but that is also based on the presence of nonrandom combinations of sequences elsewhere in tRNA. These sequences, we name “permissive elements,” are retained by evolution so that they do not hinder aminoacylation. Likely, no nucleotide within a tRNA is of random nature but has been selected so that a tRNA can fulfill all its functions efficiently.

Direct sequencing of human mitochondrial tRNALysshows the absence of editing and the occurrence of six modified nucleotides (m1A9, m2G10, Psi27, Psi28 and hypermodified nucleotides at positions U34 and A37). This tRNA folds into the expected cloverleaf, as confirmed by structural probing with nucleases. The solution structure of the corresponding in vitro transcript unexpectedly does not fold into a cloverleaf but into an extended bulged hairpin. This non-canonical fold, established according to the reactivity to a large set of chemical and enzymatic probes, includes a 10 bp aminoacyl acceptor stem (the canonical 7 bp and 3 new pairs between residues 8-10 and 65-63), a 13 nt large loop and an anticodon-like domain. It is concluded that modified nucleotides have a predominant role in canonical folding of human mitochondrial tRNALys. Phylogenetic comparisons as well as structural probing of selected in vitro transcribed variants argue in favor of a major contribution of m1A9 in this process.

Crystals were obtained by different methods under conditions where nucleation and growth occur from precipitated macromolecular material. The phenomenon was observed with compounds of different size and nature, such as thaumatin, concanavalin A, an α-amylase, a thermostable aspartyl-tRNA synthetase, the nucleo-protein complex between a tRNA Asp transcript and its cognate yeast aspartyl-tRNA synthetase, and tomato bushy stunt virus. In each system, after a rather rapid precipitation step at high supersaturation lasting one to several days, a few microcrystals appear after prolonged equilibration at constant temperature. With α-amylase, the virus and the thermostable synthetase, crystallization is accompanied by appearance of depletion zones around the growing crystals and growth of the largest crystals at the expense of the smaller ones. These features are evidences for crystal growth by Ostwald ripening. In the case of thaumatin, concanavalin A and the nucleo-protein complex, crystallization occurs by a phase transition mechanism since it is never accompanied by the disappearance of the smallest crystals. A careful analysis with thermostable aspartyl-tRNA synthetase indicates that its crystallization at 4°C under high supersaturation starts by a phase transition mechanism with the formation of small crystals within an amorphous protein precipitate. Ostwald ripening follows over a period of up to three/four months with a growth rate of about 0.8 A/s that is 13 times slower than that of crystals growing at 20°C in the absence of precipitate without ripening. At the end of the ripening process at 4°C, only one unique synthetase crystal remains per microassay with dimensions as large as 1 mm.

A micromethod was developed for the batch crystallization of proteins under conditions were the solution has no contact with the container walls. Drops of crystallization solutions (5 to 100 μl) are placed at the interface between two layers of inert and non-miscible silicone fluids contained in square glass or plastic cuvettes. The densities of the fluids are either lower or higher than those of the major precipitating agents of macromolecules, including aqueous solutions containing salts, polyethylene glycols or alcohols. Several proteins and a spherical plant virus were crystallized in the temperature range 4°C–20°C using this set-up. A thermostated device was built for the dynamic control of the temperature of crystallization drops and the monitoring of crystal growth by video-microscopy. In all cases, the habit of the crystals grown in floating drops are identical to those of controls grown in sealed glass tubes without silicone fluid. The comparison of the number of crystals in drops kept under one layer of fluid and in floating drops of the same volume indicates that heterogeneous nucleation is minimized when protein crystallization is performed in floating drops. The advantages and limitations of this novel containerless crystallization method are discussed.