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A stable conformer of Escherichia coli tRNA(Glu), obtained in the absence of Mg(2+), is inactive in the aminoacylation reaction. Probing it with diethylpyrocarbonate, dimethyl sulfate and ribonuclease V1 revealed that it has a hairpin structure with two internal loops; the helical segments at both extremities have the same structure as the acceptor stem and the anticodon arm of the native conformer of tRNA(Glu)and the middle helix is formed of nucleotides from the D-loop (G15-C20:2) and parts of the T-loop and stem (G51-C56), with G19 bulging out. This model is consistent with other known properties of this inactive conformer, including its capacity to dimerize. Therefore, this tRNA requires magnesium to acquire a conformation that can be aminoacylated, as others require a post-transcriptional modification to reach this active conformation.

In vitro transcription with T7 RNA polymerase is one of the most powerful tools in RNA research. This is mainly linked to the easy preparation of the enzyme, the quite unlimited range of sizes and sequences of the RNA that can be synthesized, as well as the efficiency and accuracy of synthesis. So far only two major limitations are common knowledge, namely poor transcription efficiency of non-G-rich initial sequences (Dunn & Studier, 1983) and 3′-end heterogeneities of the transcripts by 1 or 2 nt (Milligan et al., 1987; Draper et al., 1988; Kholod et al., 1998). These drawbacks have been overcome in most cases by improving the initiating sequence and/or purifying the transcription products to single-nucleotide resolution when necessary. The Uhlenbeck laboratory has published the first evidence for shifty errors by the T7 RNA polymerase at the very 5′ end of the synthesized RNA, an as yet unsuspected event (Pleiss et al., 1998). They demonstrated that in the particular case of tRNA sequences starting with G-rich stretches, the enzyme incorporates additional nontemplated G residues in up to 30% of the transcripts. The authors point to the fact that these errors, in combination with the 3′-end heterogeneity common to T7 transcripts, may be crucial, for instance, for functional studies of tRNAs, because some in vitro-transcribed tRNA molecules of rigorously correct final size may be erroneously considered as functional.

Eubacterial tmRNAs (10Sa RNAs) are unique because they function, at least in Escherichia coli , both as tRNA and mRNA (for a review, see Muto et al., 1998). These ∼360 ± 40-nt-long RNAs are charged with alanine at their 3′ ends by alanyl-tRNA synthetases or AlaRS (Komine et al., 1994; Ushida et al., 1994). Alanylation occurs thanks to the presence of the equivalent of the G 3 -U 70 pair, the major identity element for the alanylation of canonical tRNAs (Hou & Schimmel, 1988; McClain & Foss, 1988). Bacterial tmRNAs also have a short reading frame coding for 9 to 27 amino acids, depending on the species. E. coli tmRNA mediates recycling of ribosomes stalled at the end of terminatorless mRNAs, via a trans -translation process (Tu et al., 1995; Keiler et al., 1996; Himeno et al., 1997; Withey & Friedman, 1999). In E. coli , this amino acid tag is cotranslationally added to polypeptides synthesized from mRNAs lacking a termination codon, and the added 11-amino-acid C-terminal tag makes the protein a target for specific proteolysis (Keiler et al., 1996).

Transfer ribonucleic acids (tRNAs) are the key elements in protein biosynthesis. Indeed, these RNAs, charged with a specific amino acid, bind to messenger RNA and transfer their amino acid to the growing peptide chain. In order to fulfill this role, the tRNA has to interact with numerous macromolecular partners such as aminoacyl-tRNA synthetases (enzymes which catalyze binding of the specific amino acid), elongation factors, messenger RNA and different sites on the ribosome. The molecular mechanisms governing these various interactions, largely studied for cytosolic tRNAs, are based on the recognition of the three-dimensional structure of tRNA and on the recognition of signals on the surface of this structure (Soil and RajBhandary 1995). This has been best studied in the aminoacylation reaction, the key reaction of the whole process of protein synthesis (e.g. Saks et al. 1994; McClain 1995; Giege et al. 1998). In particular, it is well established that any perturbation affecting either the structure an/or the recognition signals has the potential to lead to a functional deficiency in these molecules.

The molecular recognition of specific transfer RNAs by the appropriate aminoacyl-tRNA synthetase is an important step in determining the accuracy of translation of the genetic message from nucleic acids into proteins. Recent studies using variant tRNAs with specific sequence modifications have indicated particular regions that determine their identity. Here we consider whether the base modifications commonly found in tRNAs contribute to their identity. Although unmodified tRNA(Asp) is charged with aspartate as efficiently as the modified native tRNA, it is mischarged with arginine with considerably increased efficiency. Our results indicate that post-transcriptional modification of tRNAs introduces structural 'anti-determinants', restricting the efficiency with which the tRNAs are charged with inappropriate amino acids.

Duplexes constituted by closed or open RNA circles paired to single-stranded oligonucleotides terminating with 3′-CCA OH form resected pseudoknots that are substrates of yeast histidyl-tRNA synthetase. Design of this RNA fold is linked to the mimicry of the pseudoknotted amino acid accepting branch of the tRNA-like domain from brome mosaic virus, known to be charged by tyrosyl-tRNA synthetases, with RNA minihelices recapitulating accepting branches of canonical tRNAs. Prediction of the histidylation function of the new family of minimalist tRNA-like structures relates to the geometry of resected pseudoknots that allows proper presentation to histidyl-tRNA synthetase of analogues of the histidine identity determinants N-1 and N73 present in tRNAs. This geometry is such that the analogue of the major N-1 histidine determinant in the RNA circles faces the analogue of the discriminator N73 nucleotide in the accepting oligonucleotides. The combination of identity elements found in tRNA His species from archaea, eubacteria, and organelles (G-1/C73) is the most efficient for determining histidylation of the duplexes. The inverse combination (C-1/G73) leads to the worst histidine acceptors with charging efficiencies reduced by 2–3 orders of magnitude. Altogether, these findings open new perspectives for understanding evolution of tRNA identity and serendipitous RNA functions.

International audience; Aspartyl-tRNA synthetase (AspRS) extracted from yeast is heterogeneous owing to proteolysis of its positively charged N-terminus; its crystals are of poor quality. To overcome this drawback, a rational strategy was developed to grow crystals of sufficient quality for structure determination. The strategy is based on improvement of the protein homogeneity and optimization of crystallization, taking advantage of predictions from crystal-growth theories. An active mutant lacking the first 70 residues was produced and initial crystallization conditions searched. The shape and habit of initial crystals were improved by establishing a phase diagram of protein versus crystallizing-agent concentrations. Growth of large well faceted crystals takes place at low supersaturations near the isochronic supersolubility curve. Further refinement led to reproducible growth of two crystalline forms of bipyramidal (I) or prismatic (II) habit. Both diffract X-­rays better than crystals previously obtained with native AspRS. Complete data sets were collected at 3 Å resolution for form I (space group P41212) and form II (space group P3221) and molecular-replacement solutions were found in both space groups.

A growing number of mutated mitochondrial tRNA genes have been found associated with severe human diseases. To investigate the potential interference of such mutations with the primordial function of tRNAs, i.e. their aminoacylation by cognate aminoacyl-tRNA synthetases, a human mitochondrial in vitro aminoacylation system specific for isoleucine has been established. Both native tRNA IIe and isoleucyl-tRNA synthetase activity have been recovered from human placental mitochondria and the kinetic parameters of tRNA aminoacylation determined. The effect of pathological point mutations present in the mitochondrial gene encoding tRNA IIe has been tackled by investigating the isoleucylation properties of wild-type and mutated in vitro transcripts. Data show that: (i) modified nucleotides contribute to efficient Isoleucylation; (ii) point mutation A4269G in the gene (A→G at nt 7 in the tRNA), associated with a cardiomyopathy, does not affect aminoacylation significantly; (iii) point mutation A4317G (A

The tRNA glycylation system is amongst the most complex aminoacylation systems since neither the oligomeric structure of the enzymes nor the discriminator base in tRNAs are conserved in the phylae. To understand better this structural diversity and its functional consequences, the prokaryotic glycylation system from Thermus thermophilus, an extreme thermophile, was investigated and its structural and functional inter-relations with those of other origins analyzed. Alignments of the protein sequence of the dimeric thermophilic glycyl-tRNA synthetase (Gly-tRNA synthetase) derived from its gene with sequences of other dimeric Gly-tRNA synthetases revealed an atypical character of motif 1 in all these class 2 synthetases. Interestingly, the sequence of the prokaryotic thermophilic enzyme resembles eukaryotic and archaebacterial Gly-tRNA synthetases, which are all dimeric, and diverges drastically from the tetrameric enzymes from other prokaryotes. Cross aminoacylations with tRNAs and synthetases of different origins provided information about functional interrelations between the glycylation systems. Efficient glycylations involving partners from T. thermophilus and Escherichia coli showed conservation of the recognition process in prokaryotes despite strong structural variations of the synthetases. However, Gly-tRNA synthetase from T. thermophilus acylates eukaryotic tRNA(Gly) while the charging ability of the E. coli enzyme is restricted to prokaryotic tRNA(Gly). A similar behaviour is found in eukaryotic systems where the restricted species specificity for tRNA glycylation of mammalian Gly-tRNA synthetase contrasts with the relaxed specificity of the yeast enzyme. The consensus sequence of the tRNAs charged by the various Gly-tRNA synthetases reveals conservation of only G1-C72 in the acceptor arm, C35 and C36 in the anticodon, and the (G10-Y25)-G45 triplet involved in tRNA folding. Conservation of these nucleotides indicates their key role in glycylation and suggests that they were part of the ancestral glycine identity set. These features are discussed in the context of the phylogenic connections between prokaryotes, eukaryotes, and archaebacteria, and of the particular place of T. thermophilus in this phylogeny.

The protein thaumatin was studied as a model macro-molecule for crystallization in microgravity-environment experiments conducted on two US Space Shuttle missions (USML-2 and LMS). In this investigation, we have evaluated and compared the quality of space- and earth-grown thaumatin crystals using X-ray diffraction analyses, and characterized them according to crystal size, diffraction resolution limit and mosaicity. Two different approaches for growing thaumatin crystals in the microgravity environment, dialysis and liquid-liquid diffusion, were employed as a joint experiment by our two investigative teams. Thaumatin crystals grown in a microgravity environment were generally larger in volume and the total number of crystals was less, relative to crystals grown on earth. They diffracted to significantly higher resolution and with improved diffraction properties, as judged by relative plots of I/sigma versus resolution. The mosaicity of space-grown crystals was significantly less than that of crystals grown on earth. Increased concentrations of protein in the crystallization chambers in microgravity led to larger crystals. The data presented here lend further support to the idea that protein crystals of improved quality can be obtained in a microgravity environment.

The Advanced Protein Crystallization Facility (APCF), a new protein crystallization device developed by ESA for the IML-2 Mission in 1994, was tested in its maiden flight on STS-57 Mission in SpaceHab-01 with a physico-chemical experiment on lysozyme crystallization. In pre-flight ground experiments, prior to the Shuttle Mission, the protocol for lysozyme crystallization with NaCl was based on its solubility diagram at 18°C and pH 4.5. Crystallization was conducted under microgravity in 25 APCF reactors using vapor diffusion, dialysis, and free liquid interface diffusion, with control on earth in 25 identical reactors. Identical supersaturation values were tested by the three crystallization techniques. Values of supersaturation derived from ground experiments allowed for conditions that yielded crystals in microgravity. The average number and size of crystals from the flight experiment and the earth control showed no significant difference; however many crystals were not free floating and grew on the walls of some of the protein chambers. The dialysis technique proved to be suitable, since no additional nucleation was generated by the membrane. Protein concentration measurements indicated that 13 days after activation of the experiment as much as 70–90% of the protein in supersaturated state had already crystallized. Data indicated differences in the crystallization behavior depending upon the crystallization set-up. Images of the protein chamber of 6 reactors, recorded during the flight, allowed us to evaluate the early stage of crystallization, to verify that recovered crystals had actually grown under microgravity conditions, and showed motions of crystals during the Mission. Using synchrotron radiation, resolution and rocking curve measurements of ground and space lysozyme crystals grown in APCF reactors showed no significant differences, although the values are much better than previously recorded diffraction limits and mosaicity data obtained with tetragonal lysozyme crystals grown in other set-ups and under different conditions. All controls foreseen throughout the microgravity experiment proved to be essential for the interpretation of the flight data, as concerning the effect of microgravity.

Two aspartyl-tRNA synthetases (AspRSs) were isolated from Thermus thermophilus HB8. Both are R2 dimers but differ in the length of their polypeptide chains (AspRS1, 68 kDa; and AspRS2, 51 kDa). Both chains start with Met and are deprived of common sequences to a significant extent. This rules out the possibility that AspRS2 is derived from AspRS1 by proteolysis, in agreement with specific recognition of each AspRS by the homologous antibodies. DNA probes derived from N-terminal amino acid sequences hybridize specifically to different genomic DNA fragments, revealing that the two AspRSs are encoded by distinct genes. Both enzymes are present in various strains from T. thermophilus and along the growth cycle of the bacteria, suggesting that they are constitutive. Kinetic investigations show that the two enzymes are specific for aspartic acid activation and tRNA Asp charging. tRNA aspartylation by the thermostable AspRSs is governed by thermodynamic parameters which values are similar to those measured for mesophilic aspartylation systems. Both thermophilic AspRSs are deprived of species specificity for tRNA aspartylation and exhibit N-terminal sequence signatures found in other AspRSs, suggesting that they are evolutionarily related to AspRSs from mesophilic prokaryotes and eukaryotes. Comparison of the efficiency of tRNA aspartylation by each enzyme under conditions approaching the physiological ones suggests that in ViVo tRNA Asp charging is essentially ensured by AspRS1, although AspRS2 is the major species. The physiological significance of the two different AspRSs in T. thermophilus is discussed. Accurate protein synthesis relies on specific aminoacyla- tion of tRNAs by aaRSs. 1 Most organisms possess an unique aaRS for each of the 20 amino acids involved in protein synthesis. In contrast, about 60 different tRNAs belonging to 20 families of isoaccepting species, specify incorporation of the amino acids in polypeptide chains. Each synthetase acylates the homologous isoaccepting tRNAs with the cognate amino acid. The functional uniqueness between synthetase, amino acid, and tRNAs provides aatRNAs with enough accuracy for high fidelity in translation. However

Histidine aminoacylation systems are of interest because of the structural diversity of the RNA substrates recognized by histidyl-tRNA synthetases. Among tRNAs participating in protein synthesis, those specific for histidine all share an additional residue at their 5′-extremities. On the other hand, tRNA-like domains at the 3′-termini of some plant viruses can also be charged by histidyl-tRNA synthetases, although they are not actors in protein synthesis. This is the case for the RNAs from tobacco mosaic virus and its satellite virus but also those of turnip yellow and brome mosaic viruses. All these RNAs have intricate foldings at their 3′-termini differing from that of canonical tRNAs and share a pseudoknotted domain which is the prerequisite for their folding into structures mimicking the overall L-shape of tRNAs. This paper gives an overview on tRNA identity and rationalizes the apparently contradictory structural and aminoacylation features of histidine-specific tRNAs and tRNA-like structures. The discussion mainly relies on histidylation data obtained with the yeast synthetase, but the conclusions are of a more universal nature. In canonical tRNA His , the major histidine identity element is the ‘minus’ 1 residue, since its removal impairs histidylation and conversely its addition to a non-cognate tRNA Asp confers histidine identity to the transplanted molecule. Optimal expression of histidine identity depends on the chemical nature of the — 1 residue and is further increased and/or modulated by the discriminator base N 73 and by residues in the anticodon. In the viral tRNA-like domains, the major identity determinant −1 is mimicked by a residue from the single-stranded L1 regions of the different pseudoknots. The consequences of this mimicry for the function of minimalist RNAs derived from tRNA-like domains are discussed. The characteristics of the histidine systems illustrate well the view that the core of the amino acid accepting RNAs is a scaffold that allows proper presentation of identity nucleotides to their amino acid identity counterparts in the synthetase and that different types of scaffoldings are possible.

Oligonucleotide derivatives conjugated to a chemical construction with two histamine residues imitating the catalytic center of ribonuclease A have been synthesized. In experiments with the conjugates complementary to the 3'-end and to the variable loop and the T loop of yeast tRNA(Phe), it was shown that the compounds can accomplish sequence-specific cleavage of the target RNA in physiologic conditions.

Glycyl-tRNA synthetase (Gly-tRNA synthetase) from Thermus thermophilus was purified to homogeneity and with high yield using a five-step purification procedure in amounts sufficient to solve its crystallographic structure [Logan, D.T., Mazauric, M.-H., Kern, D.Moras, D. (1995) EMBO J. 14, 4156-4167]. Molecular-mass determinations of the native and denatured protein indicate an oligomeric structure of the alpha 2 type consistent with that found for eukaryotic Gly-tRNA synthetases (yeast and Bombyx mori), but different from that of Gly-tRNA synthetases from mesophilic prokaryotes (Escherichia coli and Bacillus brevis) which are alpha 2 beta 2 tetramers. N-terminal sequencing of the polypeptide chain reveals significant identity, reaching 50% with those of the eukaryotic enzymes (B. mori, Homo sapiens, yeast and Caenorhabditis elegans) but no significant identity was found with both alpha and beta chains of the prokaryotic enzymes (E. coli, Haemophilus influenzae and Coxiella burnetii) albeit the enzyme is deprived of the N-terminal extension characterizing eukaryotic synthetases. Thus, the thermophilic Gly-tRNA synthetase combines strong structural homologies of eukaryotic Gly-tRNA synthetases with a feature of prokaryotic synthetases. Heat-stability measurements show that this synthetase keeps its ATP-PPi exchange and aminoacylation activities up to 70 degrees C. Glycyladenylate strongly protects the enzyme against thermal inactivation at higher temperatures. Unexpectedly, tRNA(Gly) does not induce protection. Cross-aminoacylations reveal that the thermophilic Gly-tRNA synthetase charges heterologous E. coli tRNA(gly(GCC)) and tRNA(Gly(GCC)) and yeast tRNA(Gly(GCC)) as efficiently as T. thermophilus tRNA(Gly). All these aminoacylation reactions are characterized by similar activation energies as deduced from Arrhenius plots. Therefore, contrary to the E. coli and H. sapiens Gly-tRNA synthetases, the prokaryotic thermophilic enzyme does not possess a strict species specificity. The results are discussed in the context of the three-dimensional structure of the synthetase and in the view of the particular evolution of the glycinylation systems.

The turnip yellow mosaic virus genomic RNA terminates at its 3' end in a tRNA-like structure that is capable of specific valylation. By directed mutation, the aminoacylation specificity has been switched from valine to methionine, a novel specificity for viral tRNA-like structures. The switch to methionine specificity, assayed in vitro under physiological buffer conditions with wheat germ methionyl-tRNA synthetase, required mutation of the anticodon loop and the acceptor stem pseudoknot. The resultant methionylatable genomes are infectious and stable in plants, but genomes that lack strong methionine acceptance (as previously shown with regard to valine acceptance) replicate poorly. The results indicate that amplification of turnip yellow mosaic virus RNA requires aminoacylation, but that neither the natural (valine) specificity nor interaction specifically with valyl-tRNA synthetase is crucial.

International audience; Yeast arginyl-tRNA synthetase recognizes the non-modified wild-type transcripts derived from both yeast tRNA(Arg) and tRNA(Asp) with equal efficiency. It discriminates its cognate natural substrate, tRNA(Arg), from non-cognate tRNA(Asp) by a negative discrimination mechanism whereby a single methyl group acts as an anti-determinant. Considering these facts, recognition elements responsible for specific arginylation in yeast have been searched by studying the in vitro arginylation properties of a series of transcripts derived from yeast tRNA(Asp), considered as an arginine isoacceptor tRNA. In parallel, experiments on similar tRNA(Arg) transcripts were performed. Unexpectedly, in the tRNA(Arg) context, arginylation is basically linked to the presence of residue C35, whereas in the tRNA(Asp) context, it is deeply related to that of C36 and G37 but is insensitive to the nucleotide at position 35. Each of these nucleotides present in one host, is absent in the other host tRNA. Thus, arginine identity is dependent on two different specific recognition sets according to the tRNA framework investigated.

Two crystallographically defined tRNAs, yeast tRNAAsp and tRNAPhe, were used as substrates for oxidative cleavage by Fe.bleomycin to facilitate definition at high resolution of the structural elements in RNAs conducive to bleomycin binding and cleavage. Yeast tRNAAsp underwent cleavage at G45 and U66; yeast tRNAPhe was cleaved at four sites, namely G19, A31, U52 and A66. Only two of these six sites involved oxidative cleavage of a 5'-G.Pyr-3' sequence, but three sites were at the junction between single- and double-stranded regions of the RNA, consistent with a binding model in which the bithiazole + C-terminal substituent of bleomycin bind to minor groove structures on the RNA. Also studied were four tRNA transcripts believed on the basis of biochemical and chemical mapping experiments to share structural elements in common with the mature tRNAs. Cleavage of these tRNAs by Fe.bleomycin gave patterns of cleavage very different from each other and than those of the mature tRNAs. This observation suggests strongly that Fe.bleomycin cannot be used for chemical mapping in the same fashion as more classical reagents, such as Pb2+ or dimethyl sulfate. However, the great sensitivity of Fe.bleomycin to changes in nucleic acid structure argues that those species which do show similar patterns of cleavage must be very close in structure.

The aspartate identity of tRNA for AspRS from Thermus thermophilus has been investigated by kinetic analysis of the aspartylation reaction of different tRNA molecules and their variants as well as of tRNAPhe variants with transplanted aspartate identity elements. It is shown that G10, G34, U35, C36, C38, and G73 determine recognition and aspartylation of yeast and T.thermophilus tRNA(Asp) by the thermophilic AspRS. This set of nucleotides specifies also tRNA aspartylation in the homologous yeast and Escherichia coli systems. Structural considerations indicate that the major aspartate identity elements interact with amino acids conserved in all AspRSs. It follows that the structural features of tRNA and synthetase specifying aspartylation are mainly conserved in various structural contexts and in organisms adapted to different life conditions. Mutations of tRNA identity elements provoke drastic losses of charging in the heterologous system involving yeast tRNA(Asp) and T. thermophilus AspRS. In the homologous systems, the mutational effects are less pronounced. However, effects in E. coli and T. thermophilus exceed those in yeast which are particularly moderate, indicating variations in the individual contributions of identity elements for aspartylation in prokaryotes and eukaryotes. Analysis of multiple tRNA mutants reveals cooperativity between the cluster of determinants of the anticodon loop and the additional determinants G10 and G73 for efficient aspartylation in the thermophilic system, suggesting that conformational changes trigger formation of the functional tRNA/synthetase complex.

Structures of large RNAs are not easily solved by X-ray crystallography or by NMR spectroscopy. This paper reviews the alternate methodology based on enzymatic and chemical mapping data collected on RNAs combined with graphical modeling for the construction of three-dimensional models. The different steps that lead to the establishment of the models are critically discussed. It is shown how the correctness of an RNA model can be strengthened by establishing correlations between the structure and the functionality of the molecule and its variants. Finally, the predictive potential of a model is discussed. The approach is illustrated by results obtained on plant viral tRNA-like structures, and particularly on that of brome mosaic virus (BMV) RNA.

During protein biosynthesis, all aminoacylated elongator tRNAs except selenocysteine-inserting tRNA Sec form ternary complexes with activated elongation factor. tRNA Sec is bound by its own translation factor, an elongation factor analogue, e.g. the SELB factor in prokaryotes. An apparent reason for this discrimination could be related to the unusual length of tRNA Sec amino acid-acceptor branch formed by 13 bp. However, it has been recently shown that an aspartylated minihelix of 13 bp derived from yeast tRNA Asp is an efficient substrate for Thermus thermophilus EF-Tu-GTP, suggesting that features other than the length of tRNA Sec prevent its recognition by EF-Tu-GTP. A stepwise mutational analysis of a minihelix derived from tRNA Sec in which sequence elements of tRNA Asp were introduced showed that the sequence of the amino acid- acceptor branch of Escherichia coli tRNA Sec contains a specific structural element that hinders its binding to T.thermophilus EF-Tu-GTP. This antideterminant is located in the 8th, 9th and 10th bp in the acceptor branch of tRNA Sec, corresponding to the last base pair in the amino acid acceptor stem and the two first pairs in the T-stem. The function of this C7.G66/G49.U65/C50.G64 box was tested by its transplantation into a minihelix derived from tRNA Asp, abolishing its recognition by EF-Tu-GTP. The specific role of this nucleotide combination is further supported by its absence in all known prokaryotic elongator tRNAs.

Information is still quite limited concerning the structural requirements in tRNA molecules for their post-transcriptional maturation by base and ribose modification enzymes. To address this question, we have chosen as the model system yeast tRNAAsp that has a known three-dimensional structure and the in vivo modifying machinery of the Xenopus laevis oocyte able to act on microinjected tRNA precursors. We have systematically compared the modification pattern of wild-type tRNAAsp with that of a series of structural mutants (21 altogether) altered at single or multiple positions in the D-, T-and the anticodon branch, as well as in the variable region. The experimental system allowed us to analyze the effects of structural perturbations in tRNA on the enzymatic formation of modified nucleosides at 12 locations scattered over the tRNA cloverleaf. We found that the formation of m1G37 and psi 40 in the anticodon loop and stem and psi 13 in the D-stem, were extremely sensitive to 3D perturbations. In contrast, the formation of T54, psi 55 and m1A58 in the T-loop, m5C49 in the T-stem and m2G6 in the amino acid accepting stem were essentially insensitive to change in the overall tRNA architecture; these modified nucleosides were also formed in appropriate minimalist (stems and loops) tRNA domains. The formation of m2G26 at the junction between the anticodon and the D-stem, of Q34 and manQ34 in the anticodon loop were sensitive only to drastic structural perturbation of the tRNA. Altogether, these results reflect the existence of different modes of tRNA recognition by the many different modifying enzymes. A classification of this family of maturation enzymes into two major groups, according to their sensitivities to structural perturbations in tRNA, is proposed.

Structure/function relationships accounting for specific tRNA charging by class II aspartyl-tRNA synthetases from Saccharomyces cerevisiae, Escherichia coli and Thermus thermophilus are reviewed. Effects directly linked to tRNA features are emphasized and aspects about synthetase contribution in expression of tRNA Asp identity are also covered. Major identity nucleotides conferring aspartate specificity to yeast, E coli and T thermophilus tRNAs comprise G34, U35, C36, C38 and G73, a set of nucleotides conserved in tRNA Asp molecules of other biological origin. Aspartate specificity can be enhanced by negative discrimination preventing, eg mischarging of native yeast tRNA Asp by yeast arginyl-tRNA synthetase. In the yeast system crystallography shows that identity nucleotides are in contact with identity amino acids located in the catalytic and anticodon binding domains of the synthetase. Specificity of RNA/protein interaction involves a conformational change of the tRNA that optimizes the H-bonding potential of the identity signals on both partners of the complex. Mutation of identity nucleotides leads to decreased aspartylation efficiencies accompanied by a loss of specific H-bonds and an altered adaptation of tRNA on the synthetase. Species-specific characteristics of aspartate systems are the number, location and nature of minor identity signals. These features and the structural variations in aspartate tRNAs and synthetases are correlated with mechanistic differences in the aminoacylation reactions catalyzed by the various aspartyl-tRNA synthetases. The reality of the aspartate identity set is verified by its functional expression in a variety of RNA frameworks. Inversely a number of identities can be expressed within a tRNA Asp framework. From this emerged the concept of the RNA structural frameworks underlying expression of identities which is illustrated with data obtained with engineered tRNAs. Efficient aspartylation of minihelices is explained by the primordial role of G73. From this and other considerations it is suggested that aspartate identity appeared early in the history of tRNA aminoacylation systems.

The phosphates of the tRNA(Phe) transcript from Thermus thermophilus interacting with the cognate synthetase were determined by footprinting. Backbone bond protection against cleavage by iodine of the phosphorothioate-containing transcripts was found in the anticodon stem-loop, the D stem-loop and the acceptor stem and weak protection was also seen in the variable loop. Most of the protected phosphates correspond to regions around known identity elements of tRNA(Phe). Enhancement of cleavage at certain positions indicates bending of tRNAPhe upon binding to the enzyme. When applied to the three-dimensional model of tRNA(Phe) from yeast the majority of the protections occur on the D loop side of the molecule, revealing that phenylalanyl-tRNA synthetase has a rather complex and novel pattern of interaction with tRNAPhe, differing from that of other known class II aminoacyl-tRNA synthetases.

Hydrolysis of RNA in imidazole buffer and by spermine-imidazole conjugates has been investigated. The RNA models were yeast tRNA(Asp) and a transcript derived from the 3'-terminal sequence of tobacco mosaic virus RNA representing a minihelix capable of being enzymatically aminoacylated with histidine. Imidazole buffer and spermine-imidazole conjugates in the presence of free imidazole cleave phosphodiester bonds in the folded RNAs in a specific fashion. Imidazole buffer induces cleavages preferentially in single-stranded regions because nucleotides in these regions have more conformational freedom and can assume more easily the geometry needed for formation of the hydrolysis intermediate state. Spermine-imidazole constructs supplemented with free imidazole cleave tRNA(Asp) within single-stranded regions after pyrimidine residues with a marked preference for pyrimidine-A sequences. Hydrolysis patterns suggest a cleavage mechanism involving an attack by the imidazole residue of the electrostatically bound spermine-imidazole and by free imidazole at the most accessible single-stranded regions of the RNA. Cleavages in a viral RNA fragment recapitulating a tRNA-like domain were found in agreement with the model of this molecule that accounts for its functional properties, thus illustrating the potential of the imidazole-derived reagents as structural probes for solution mapping of RNAs. The cleavage reactions are simple to perform, provide information reflecting the state of the ribose-phosphate backbone of RNA and can be used for mapping single- and double-stranded regions in RNAs.

Residue G-1 and discriminator base C73 are the major histidine identity elements in prokaryotes. Here we evaluate the importance of these two nucleotides in yeast histidine aminoacylation identity. Deletion of G-1 in yeast tRNA(His) transcript leads to a drastic loss of histidylation specificity (about 500-fold). Mutation of discriminator base A73, common to all yeast tRNA(His) species, into G73 has a more moderate but still significant effect with a 22-fold decrease in histidylation specificity. Changes at position 36 in the anticodon loop has negligible effect on histidylation. The role of residues -1 and 73 for specific aminoacylation by yeast HisRS was further investigated by studying the histidylation capacities of seven minihelices derived from the Turnip Yellow Mosaic Virus tRNA-like structure. Changes in the nature of nucleotides -1 and 73 modulate this activity but do not suppress it. The optimal mini-substrate for HisRS presents a G.A mismatch at the position equivalent to residues G-1.A73 in yeast tRNA(His), confirms the importance of this structural feature in yeast histidine identity. The fact that the minisubstrates contain a pseudoknot in which position -1 is mimicked by an internal nucleotide from the pseudoknot highlights further the necessity of a stacking interaction of this position over the amino acid accepting branch of the tRNA during the aminoacylation process. Individual transplantation of G-1 or A73 into yeast tRNA(Asp) transcript improves the histidylation efficiency of the engineered tRNA(Asp). However, a tRNA(Asp) transcript presenting simultaneously both residues G-1 and A73 becomes a less good substrate for HisRS, suggesting the importance of the structural context and/or the presence of antideterminants for an optimal expression of these two identity elements.

Arginyl-tRNA synthase will erroneously add Arg to tRNAAsp following a single nucleotide mutation of tRNAAsp.

A model of secondary structure is proposed for the 3'-terminal sequence of the satellite tobacco mosaic virus (STMV) RNA on the basis of phylogenetic comparisons with tobacco mosaic virus (TMV) genomic RNA. Sequence homologies and compensatory base changes found between the two related viral RNAs imply that the 3'-end of STMV RNA folds into a tRNA-like domain similar to that found in the TMV RNA. Accordingly, functional assays showed that STMV RNA can be aminoacylated in vitro with histidine by yeast histidyl-tRNA synthetase to plateaus reaching 30%. Histidylation properties of STMV RNA were compared to those of TMV RNA and of a canonical yeast tRNA(His) transcript which both are chargeable to nearly 100% plateau levels. Kinetic data indicate an excellent catalytic efficiency of STMV RNA charging expressed as Vmax/Km ratio, quasi-equivalent to that of TMV RNA, and only 17-fold reduced as compared to that of the yeast tRNAHis transcript. Biological implications of the structural mimicry between the tRNA-like regions of TMV and STMV RNAs are discussed in the light of the relationships of a satellite virus with its helper virus. This is the first report on a chargeable tRNA-like structure at the 3'-end of a satellite virus RNA.

The influence of nine synthetic polyamines on in vitro transcription with T7 RNA polymerase has been studied. The compounds used were linear or macrocyclic tetra- and hexaamine, varying in their size, shape and number of protonated groups. Their effect was tested on different types of templates, all presenting the T7 RNA promoter in a double-stranded form followed by sequences encoding short transcripts (25 to 35-mers) either on single- or double-stranded synthetic oligodeoxyribonucleotides. All polyamines used stimulate transcription of both types of templates at levels dependent on their size, shape, protonation degree, and concentration. For each compound, an optimal concentration could be defined; above this concentration, transcription inhibition occurred. Highest stimulation (up to 12-fold) was obtained by the largest cyclic compound called [38]N6C10.

This paper gives an overview of the science of crystals of biological macromolecules. The historical background of the field is outlined and the main achievements and open problems are discussed from both biological and physical–chemical viewpoints. Selected results, including data from the authors, illustrate this overview. The perspectives of crystallogenesis for structural biology, but also more general trends, are presented.

We show here that small RNA helices which recapitulate part or all of the acceptor stem of yeast aspartate tRNA are efficiently aminoacylated by cognate class II aspartyl-tRNA synthetase. Aminoacylation is strongly dependent on the presence of the single-stranded G73 'discriminator' identity nucleotide and is essentially insensitive to the sequence of the helical region. Substrates which contain as few as 3 bp fused to G73CCAOH are aspartylated. Their charging is insensitive to the sequence of the loop closing the short helical domains. Aminoacylation of the aspartate mini-helix is not stimulated by a hairpin helix mimicking the anticodon domain and containing the three major anticodon identity nucleotides. A thermodynamic analysis demonstrates that enzyme interactions with G73 in the resected RNA substrates and in the whole tRNA are the same. Thus, if the resected RNA molecules resemble in some way the earliest substrates for aminoacylation with aspartate, then the contemporary tRNA(Asp) has quantitatively retained the influence of the major signal for aminoacylation in these substrates.

Molecular recognition of Escherichia coli tRNA(Ile) by the cognate isoleucyl-tRNA synthetase (IleRS) was studied by analyses of chemical footprinting with N-nitroso-N-ethylurea and aminoacylation kinetics of variant tRNA(Ile) transcripts prepared with bacteriophage T7 RNA polymerase. IleRS binds to the acceptor, dihydrouridine (D), and anticodon stems as well as to the anticodon loop. The "complete set" of determinants for the tRNA(Ile) identity consists of most of the nucleotides in the anticodon loop (G34, A35, U36, t6A37 and A38), the discriminator nucleotide (A73), and the base-pairs in the middle of the anticodon, D and acceptor stems (C29.G41, U12.A23 and C4.G69, respectively). As for the tertiary base-pairs, two are indispensable for the isoleucylation activity, whereas the others are dispensable. Correspondingly, some of the phosphate groups of these dispensable tertiary base-pair residues were shown to be exposed to N-nitroso-N-ethylurea when tRNA(Ile) was bound with IleRS. Furthermore, deletion of the T psi C-arm only slightly impaired the tRNA(Ile) activity. Thus, it is proposed that the recognition by IleRS of all the widely distributed identity determinants is coupled with a global conformational change that involves the loosening of a particular set of tertiary base-pairs of tRNA(Ile).

RNA cleaving molecules were synthesized by conjugating imidazole residues imitating the essential imidazoles in the active center of pancreatic ribonuclease to an intercalating compound, derivative of phenazine capable of binding to the double stranded regions of polynucleotides. Action of the molecules on tRNA was investigated. It was found, that some of the compounds bearing two imidazole residues cleave tRNA under physiological conditions. The cleavage reaction shows a bell-shaped pH dependence with a maximum at pH 7.0 indicating participation of protonated and non-protonated imidazole residues in the process. Under the conditions stabilizing the tRNA structure, a tRNAAsp transcript was cleaved preferentially at the junctions of the stem and loop regions of the cloverleaf tRNA fold, at the five positions C56, C43, C20.1, U13, and U8, with a marked preference for C56. This cleavage pattern is consistent with a hydrolysis mechanism involving non-covalent binding of the compounds to the double-stranded regions of tRNA followed by an attack of the imidazole residues at the juxtaposed flexible single-stranded regions of the molecule. The compounds provide new probes for the investigation of RNA structure in solution and potential reactive groups for antisense oligonucleotide derivatives.

We report here the rational design and construction of a chimerized transfer RNA with tripartite aminoacylation specificity. A yeast aspartic acid specific tRNA was transformed into a highly efficient acceptor of alanine and phenylalanine and a moderate acceptor of valine. The transformation was guided by available knowledge of the requirements for aminoacylation by each of the three amino acids and was achieved by iterative changes in the local sequence context and the structural framework of the variable loop and the two variable regions of the dihydrouridine loop. The changes introduced to confer efficient acceptance of the three amino acids eliminate aminoacylation with aspartate. The interplay of determinants and antideterminants for different specific aminoacylations, and the constraints imposed by the structural framework, suggest that a tRNA with an appreciable capacity for more than three efficient aminoacylations may be inherently difficult to achieve.

RNA helical oligonucleotides that recapitulate the acceptor stems of transfer RNAs, and that are devoid of the anticodon trinucleotides of the genetic code, are aminoacylated by aminoacyl tRNA synthetases. The specificity of aminoacylation is sequence dependent, and both specificity and efficiency are generally determined by only a few nucleotides proximal to the amino acid attachment site. This sequence/structure-dependent aminoacylation of RNA oligonucleotides constitutes an operational RNA code for amino acids. To a rough approximation, members of the two different classes of tRNA synthetases are, like tRNAs, organized into two major domains. The class-defining conserved domain containing the active site incorporates determinants for recognition of RNA mini-helix substrates. This domain may reflect the primordial synthetase, which was needed for expression of the operational RNA code. The second synthetase domain, which generally is less or not conserved, provides for interactions with the second domain of tRNA, which incorporates the anticodon. The emergence of the genetic from the operational RNA code could occur when the second domain of synthetases was added with the anticodon-containing domain of tRNAs.

The nucleotides in a tRNA that specifically interact with the cognate aminoacyl-tRNA synthetase have been found largely located in the helical stems, the anticodon, or the discriminator base, where they vary from one tRNA to another. The conserved and semiconserved nucleotides that are responsible for the tRNA tertiary structure have been shown to have little role in synthetase recognition. Here we report that aminoacylation of Escherichia coli tRNA(Cys) depends on the anticodon, the discriminator base, and a tertiary interaction between the semiconserved nucleotides at positions 15 and 48. While all other tRNAs contain a purine at position 15 and a complementary pyrimidine at position 48 that establish the tertiary interaction known as the Levitt pair, E. coli tRNA(Cys) has guanosine -15 and -48. Replacement of guanosine -15 or -48 with cytidine virtually eliminates aminoacylation. Structural analyses with chemical probes suggest that guanosine -15 and -48 interact through hydrogen bonds between the exocyclic N-2 and ring N-3 to stabilize the joining of the two long helical stems of the tRNA. This tertiary interaction is different from the traditional base pairing scheme in the Levitt pair, where hydrogen bonds would form between N-1 and O-6. Our results provide evidence for a role of RNA tertiary structure in synthetase recognition.

We have investigated the functional relationship between nucleotides in yeast tRNAAsp that are important for aspartylation by yeast aspartyl-tRNA synthetase. Transcripts of tRNAAsp with two or more mutations at identity positions G73, G34, U35, C36 and base pair G10-U25 have been prepared and the steady-state kinetics of their aspartylation were measured. Multiple mutations affect the catalytic activities of the synthetase mainly at the level of the catalytic constant, kcat. Kinetic data were expressed as free energy variation at transition state of these multiple mutants and comparison of experimental values with those calculated from results on single mutants defined three types of relationships between the identity nucleotides of this tRNA. Nucleotides located far apart in the three-dimensional structure of the tRNA act cooperatively whereas nucleotides of the anticodon triplet act either additively or anti-cooperatively. These results are related to the specific interactions of functional groups on identity nucleotides with amino acids in the protein as revealed by the crystal structure of the tRNAAsp/aspartyl-tRNA synthetase complex. These relationships between identity nucleotides may play an important role in the biological function of tRNAs.

Mutations have been designed that disrupt the tertiary structure of yeast tRNA(Asp). The effects of these mutations on both tRNA structure and specific aspartylation by yeast aspartyl-tRNA synthetase were assayed. Mutations that disrupt tertiary interactions involving the D-stem or D-loop result in destabilization of the base-pairing in the D-stem, as monitored by nuclease digestion and chemical modification studies. These mutations also decrease the specificity constant (kcat/Km) for aspartylation by aspartyl-tRNA synthetase up to 10(3)-10(4) fold. The size of the T-loop also influences tRNA(Asp) structure and function; change of its T-loop to a tetraloop (-UUCG-) sequence results in a denatured D-stem and an almost 10(4) fold decrease of kcat/Km for aspartylation. The negative effects of these mutations on aspartylation activity are significantly alleviated by additional mutations that stabilize the D-stem. These results indicate that a critical role of tertiary structure in tRNA(Asp) for aspartylation is the maintenance of a base-paired D-stem.

A 3-D model of the tyrosylable tRNA-like domain of the genomic brome mosaic virus RNAs was built by computer modelling based on solution probing of the molecule with different chemical and enzymatic reagents. This model encompasses four major structural domains, including two peculiar substructures oriented perpendicularly and mimicking a tRNA structure, and a fifth domain which makes the connection with the rest of the viral RNA. After recalling the different steps that led to the present structural knowledge of the BMV tRNA-like domain, we review its novel structural features revealed by the modelling and that did not appear in older versions of 3-D models of this structure. These features comprise additional base-pairs, hairpin loops, new tertiary long-range interactions, and a second pseudoknot. The main goal of this paper is to strengthen the validity of the model by establishing correlations between the putative 3-D conformation and the functional properties of the domain. For that, we show how the present structural model rationalises mutagenic and footprinting data that have established the importance of specific regions of the RNA for its recognition and aminoacylation by yeast tyrosyl-tRNA synthetase. We discuss further how the model corroborates mutational analyses performed to understand recognition of this RNA domain by the (ATP,CTP):tRNA nucleotidyl-transferase and by the viral replicase. The published mutants of the BMV tRNA-like domain fall into two classes. In one class, the mutants leave unchanged the overall architecture of the molecule, thereby affecting functions directly. In the second class, the overall architecture of the mutants is perturbed, and thus functions are affected indirectly.

Publisher Summary This chapter discusses the role of tRNA structure in the recognition process with synthetases and on the implications for aminoacylation efficiency. Many examples are taken from our own research on several specific aminoacylation systems, for example aspartate, histidine, valine, but concepts are presented more globally with reference to the complete set of aminoacylation systems. It emphasizes on the importance of tRNA-like structures for understanding the interaction of canonical tRNAs with synthetase. Although tRNA-like molecules found in some plant viral RNAs do not participate in protein synthesis, they represent interesting natural mutants to be compared to canonical tRNAs. This is also the case of tRNAlike structures found in some messenger RNAs as well as of bizarre tRNAs from mitochondria . In addition, competition and kinetic effects may also contribute to the overall specificity of the various aminoacylation systems; the balance between the concentration of tRNAs and synthetases would be essential for ensuring optimal specificity. According to this view, individual aminoacylation systems do not work at their optimal chemical efficiency, but work instead to assure optimal discrimination among the different aminoacylation systems. Such a balance may be perturbed under certain physiological or pathological conditions. Finally, this chapter discusses a comparison of recent results with previous observations, and show how old concepts established phenomenologically can now be tested more explicitly.

For correct interpretation of the genetic code, the twenty aminoacyl-tRNA synthetases are required to strictly recognize their cognate tRNA and amino acid. The amino acid specificity of tRNA depends on a set of a relatively small number of nucleotide residues (determinants for the identity of tRNA) such as the anticodon residues, the discriminator base at position 73, base pairs 1:72, 2:71, and 3:70 in the acceptor stem, base pair 10:25 in the D stem, and residue 20 (Shimura et al., 1972; Normanly et al., 1986; Hou and Schimmel, 1988; McClain and Foss, 1988ab; Muramatsu et al., 1988b; Schulman and Pelka, 1988; Normanly and Abelson, 1989; Schimmel, 1989; Himeno et al., 1990; Jahn et al., 1991; Putz et al., 1991; Franklyn et al., 1992; Muramatsu et al., 1992; Schulman, 1991; Tamura et al., 1992). The three-dimensional structures of the complexes of E. coli tRNAG1n and yeast tRNAAsp with their cognate aminoacyl-tRNA synthetases, GlnRS and AspRS, respectively, have been determined by X-ray crystallography (Rould et al., 1989; Rould et al., 1991; Ruff et al., 1991). These synthetases recognize the identity determinants mainly in the two ends of the L-shaped structure of tRNA (the anticodon loop and the 5’/3’ terminal region) (Jahn et al., 1991; Putz et al., 1991) and concomitantly change the conformations of these regions (Rould et al., 1989; Rould et al., 1991; Ruff et al., 1991).

The valylation by wheat germ valyl-tRNA synthetase of anticodon loop mutants of turnip yellow mosaic virus RNA has been studied. RNA substrates 264 nucleotides long were made by T7 RNA polymerase from cDNA encompassing the 3' tRNA-like region of genomic RNA. Substitution singly, or in combination, of three nucleotides in the anticodon loop resulted in very poor valylation (Vmax/KM less than 10(-3) relative to wild type). These nucleotides thus represent the major valine identity determinants recognized by wheat germ valyl-tRNA synthetase; their relative contribution to valine identity, in descending order, was as follows: the middle nucleotide of the anticodon (A56 in TYMV RNA), the 3' anticodon nucleotide (C55), and the 3'-most anticodon loop nucleotide (C53). Substitutions in the wobble position (C57) had no significant effect on valylation kinetics, while substitutions of the discriminator base (A4) resulted in small decreases in Vmax/Km. Mutations in the major identity nucleotides resulted in large increases in KM, suggesting that wheat germ valyl-tRNA synthetase has a lowered affinity for variant substrates with low valine identity. Comparison with other studies using valyl-tRNA synthetases from Escherichia coli and yeast indicates that the anticodon has been phylogenetically conserved as the dominant valine identity region, while the identity contribution of the discriminator base has been less conserved. The mechanism by which anticodon mutations are discriminated also appears to vary, being affinity-based for the wheat germ enzyme, and kinetically-based for the yeast enzyme [Florentz et al. (1991) Eur. J. Biochem. 195, 229-234].

Chemical footprinting experiments on brewer's yeast tRNA(Asp) complexed to its cognate aspartyl-tRNA synthetase are reported: they demonstrate that bases of the anticodon loop, including the anticodon itself, are in close proximity with the synthetase. Contacts were determined using dimethylsulfate as the probe for testing reactivity of guanine and cytosine residues in free and complexed tRNA. Results correlate with the decrease in aspartylation activity of yeast tRNA(Asp) molecules mutated at these contact positions and will be compared with other structural data arising from solution and crystallographic studies on the aspartic acid complex.

The structure and function of in vitro transcribed tRNA(Asp) variants with inserted conformational features characteristic of yeast tRNA(Phe), such as the length of the variable region or the arrangement of the conserved residues in the D-loop, have been investigated. Although they exhibit significant conformational alterations as revealed by Pb2+ treatment, these variants are still efficiently aspartylated by yeast aspartyl-tRNA synthetase. Thus, this synthetase can accommodate a variety of tRNA conformers. In a second series of variants, the identity determinants of yeast tRNA(Phe) were transplanted into the previous structural variants of tRNA(Asp). The phenylalanine acceptance of these variants improves with increasing the number of structural characteristics of tRNA(Phe), suggesting that phenylalanyl-tRNA synthetase is sensitive to the conformational frame embedding the cognate identity nucleotides. These results contrast with the efficient transplantation of tRNA(Asp) identity elements into yeast tRNA(Phe). This indicates that synthetases respond differently to the detailed conformation of their tRNA substrates. Efficient aminoacylation is not only dependent on the presence of the set of identity nucleotides, but also on a precise conformation of the tRNA.

The interaction of wild-type and mutant yeast tRNA(Asp) transcripts with yeast aspartyl-tRNA synthetase (AspRS; EC 6.1.1.12) has been probed by using iodine cleavage of phosphorothioate-substituted transcripts. AspRS protects phosphates in the anticodon (G34, U35), D-stem (U25), and acceptor end (G73) that correspond to determinant nucleotides for aspartylation. This protection, as well as that in anticodon stem (C29, U40, G41) and D-stem (U11 to U13), is consistent with direct interaction of AspRS at these phosphates. Other protection, in the variable loop (G45), D-loop (G18, G19), and T-stem and loop (G53, U54, U55), as well as enhanced reactivity at G37, may result from conformational changes of the transcript upon binding to AspRS. Transcripts mutated at determinant positions showed a loss of phosphate protection in the region of the mutation while maintaining the global protection pattern. The ensemble of results suggests that aspartylation specificity arises from both protein-base and protein-phosphate contacts and that different regions of tRNA(Asp) interact independently with AspRS. A mutant transcript of yeast tRNA(Phe) that contains the set of identity nucleotides for specific aspartylation gave a phosphate protection pattern strikingly similar to that of wild-type tRNA(Asp). This confirms that a small number of nucleotides within a different tRNA sequence context can direct specific interaction with synthetase.

Minihelices mimicking the amino acid acceptor and anticodon branches of yeast tRNA(Val) have been synthesized by in vitro transcription of synthetic templates. It is shown that a minihelix corresponding to the amino acid acceptor branch and containing solely a valine-specific identity nucleotide can be aminoacylated by yeast valyl-tRNA synthetase. Its charging ability is lost after mutating this nucleotide. This ability is stimulated somewhat by the addition of a second hairpin helix that mimicks the anticodon arm, which suggests that information originating from the anticodon stem-loop can be transmitted to the active site of the enzyme by the core of the protein.

Mischarging of the valine specific tRNA-like structure of turnip yellow mosaic virus (TYMV) RNA has been tested in the presence of purified arginyl-, aspartyl-, histidinyl-, and phenylalanyl-tRNA synthetases from bakers' yeast. Important mischarging of a 264 nucleotide-long transcript was found with histidinyl-tRNA synthetase which can acylate this fragment up to a level of 25% with a loss of specificity (expressed as Vmax/KM ratios) of only 100 fold as compared to a yeast tRNA(His) transcript. Experiments on transcripts of various lengths indicate that the minimal valylatable fragment (n = 88) is the most efficient substrate for histidinyl-tRNA synthetase, with kinetic characteristics similar to those found for the control tRNA(His) transcript. Mutations in the anticodon or adjacent to the 3' CCA that severely affect the valylation capacity of the 264 nucleotide long TYMV fragment are without negative effect on its mischarging, and for some cases even improve its efficiency. A short fragment (n = 42) of the viral RNA containing the pseudoknot and corresponding to the amino acid accepting branch of the molecule is an efficient histidine acceptor.

The nucleotides crucial for the specific aminoacylation of yeast tRNA(Asp) by its cognate synthetase have been identified. Steady-state aminoacylation kinetics of unmodified tRNA transcripts indicate that G34, U35, C36, and G73 are important determinants of tRNA(Asp) identity. Mutations at these positions result in a large decrease (19- to 530-fold) of the kinetic specificity constant (ratio of the catalytic rate constant kcat and the Michaelis constant Km) for aspartylation relative to wild-type tRNA(Asp). Mutation to G10-C25 within the D-stem reduced kcat/Km eightfold. This fifth mutation probably indirectly affects the presentation of the highly conserved G10 nucleotide to the synthetase. A yeast tRNA(Phe) was converted into an efficient substrate for aspartyl-tRNA synthetase through introduction of the five identity elements. The identity nucleotides are located in regions of tight interaction between tRNA and synthetase as shown in the crystal structure of the complex and suggest sites of base-specific contacts.