The structure of tRNA and its relationship with the biological necessity of specific tRNA aminoacylation reactions, in other words with identity, is reviewed. New structural data show the typical L-shaped tRNA architecture in great detail and highlight how adequate rigidity and plasticity of the molecule is essential for interaction with its biological partners, in particular with aminoacyl-tRNA synthetases. Identity is ensured by a small number of nucleosides predominantly located at the two distal extremities of the tRNA molecule. In several crystallographic complexes, these residues have been shown in contact with amino acids from the synthetases. In most cases, the interaction is accompanied by a conformational change of the tRNA. Assuming that the structural framework of tRNA displays identity elements to synthetases implies that altered and/or simplified RNA architectures can fulfill this role provided they contain correctly located identity elements. This paradigm holds true in nature where atypical and tRNA-like domains have been selected by evolution. Rationale-based engineering or selection by artificial evolution of novel RNA molecules recognized and aminoacylated by synthetases also verified it.

Introduction

Transfer RNAs (tRNAs) are ubiquitous molecules present in all forms of life. Unprecedented in biology, their necessity as the adapters translating the genetic code was predicted,¹ before their biochemical characterization.² Beside protein synthesis, specialized tRNAs and tRNA-like structures can participate in diverse metabolic pathways (reviewed in ref. 3). The universal occurrence of tRNA, its amino acid donor key role in protein synthesis, and its involvement in other cellular processes underline its ancient origin. Contemporary tRNAs participating in protein synthesis are structurally homogeneous but are versatile in function since during their functional cycle they interact with many enzymes (maturation and modification enzymes including RNase P, aminoacyl-tRNA synthetases), protein factors (for initiation, elongation, and termination) as well as with mRNA and the ribosome. Specialized tRNA functions such as initiation of protein synthesis,^4,5 incorporation of selenocysteine into proteins,⁶ are in general correlated with atypical structural features. In what follows, emphasis is given to the tRNAs directly involved in protein synthesis and to the problem of tRNA identity that underlies their capacity to be specifically aminoacylated by synthetases and consequently to be responsible for the correct translation of the genetic message from the DNA into the protein language.

Structure of tRNAs

Sequences and Modified Nucleosides

Since 1965, when yeast tRNA^Ala was sequenced,⁷ sequences of more than 4000 tRNAs from more than 300 organisms have been included in the tRNA database (http://www.uni-bayreuth.de/departments/biochemie/trna/),⁸ and these numbers increase steadily due to the many genome projects. A vast majority of tRNA sequences adopt the cloverleaf folding which highlights the structural and functional domains of the molecule (Fig. 1A).

Figure 1A

Structure of cytoplasmic tRNA participating in protein synthesis. (top) Cloverleaf folding emphasizing the different domains of the tRNA molecule as well as the position of conserved and semiconserved residues. Length of tRNA sequences ranges from 72 (more...)

Most information came from gene sequencing (3700 DNA versus 550 RNA sequences). The more difficult RNA sequencing revealed the presence of many modified nucleotides. To date, 81 such residues are in the tRNA modification database (http://medlib.med.utah.edu/RNAmods/).⁹ Some are common to almost all tRNA species, such as dihydrouridine (D) in D-loops and ribothymidine (T) in T-loops (for abbreviations of modified nucleosides, see ref. 8). Others are characteristic of individual tRNA species, of groups of organisms or of a given living kingdom (archaea, prokarya including the derived organelles or eukarya). For instance, wybutosine, a hypermodified G-residue formerly called the Y-base, and its derivatives, are exclusively found at position 37 of tRNA^Phe and queuosine derivatives, other G-analogs, are found at the first anticodon position of certain tRNA^Asn, tRNA^Asp, and tRNA^Tyr species. The triad Gm18, s²T54 and m¹A58 in the D- and T-loop characterizes tRNAs from thermophilic organisms, as found in tRNA^Asp from Thermus thermophilus,¹⁰ and likely contributes to their stability by increasing their melting temperature. ¹¹ The methylated residue m¹ at position 9 is typically mitochondrial. As demonstrated in the case of mitochondrial human tRNA^Lys, it prevents misfolding of the molecule into an elongated hairpin by preventing formation of an alternative pairing with U64 from the T-stem,¹² and therefore can be considered as an internal chaperone for correct folding. Likewise, archeosine, the G-like 7-deazaguanosine analog with ring substituted at position 7 by a formamidino group is exclusively archaeal and is found at position 15 in D-loops where it forms an atypical R15-Y48 Levitt tertiary pair.¹³

Sequence data soon revealed the presence of conserved (U8, A14, A21, U33, G53, T54, ψ55, C56, A58, C61, C74, C75 and A76) and semiconserved (Y11, R15, R24, Y32, R37, Y48, R57, and Y60) residues in tRNA. The steadily incoming sequences confirm their occurrence in almost all tRNAs, the main exceptions being organellar tRNAs (see e.g., website for mammalian mitochondrial tRNAs,¹⁴ [http://mamit-tRNA.u-strasbg.fr]) that often lack canonical D- and T-loop organizations. As demonstrated by crystallography, many of the conserved and semiconserved residues participate in the tertiary interactions that govern the three-dimensional structure of tRNA. In contrast, those in the anticodon loop play a role in mRNA decoding¹⁵ and sometimes in tRNA identity,¹⁶ the conserved 3'-terminal CCA triplet being crucial for tRNA recognition in the active site of synthetases. Helical regions of tRNAs are rich in G-U wobble base-pairs and to a lesser extent in other non-Watson-Crick pairs. The G-U pairs have unique structural characteristics that can be crucial to tRNA function.^17,18

Organellar tRNAs, like human mitochondrial tRNA^Ser or mitochondrial tRNAs from nematode worms, with part(s) of the canonical structure lacking, constitute remarkable exceptions to the conserved cloverleaf folding of cytoplasmic tRNAs.^19,20 However, sequence compensations allow these molecules to adopt L-shaped conformations.²¹ Noticeable, the sequence features in canonical tRNAs deviating from consensus, like the −1 residue at the 5'-terminus of histidine-specific tRNAs, ²² or the uncommon G15-G48 Levitt pair (instead of R15-Y48) in Escherichia coli tRNA^Cys were shown to be identity elements in these tRNAs.^23,24

Three-Dimensional Structures of Free tRNAs

As first proposed for yeast tRNA^Phe, the tRNA molecule has an L-shaped architecture that was established independently in three laboratories.^25-28 It is based on the cloverleaf folding and shows a symmetrical organization with two helical branches of similar size oriented perpendicularly (Fig. 1B). One branch is formed by the amino acid acceptor stem stacked over the T-arm and the dumbbell-like other branch by a stack of the anticodon- and D-arms. The same overall structural organization is found in yeast tRNA^Asp,^29,30 in Escherichia coli initiator tRNAf^Met,³¹ as well as in yeast initiator tRNA^Met,³² and in the recently solved structure of human tRNA^Lys3, the primer of HIV reverse-transcriptase.³³ An NMR investigation of the anticodon stem-loop of this human tRNA^Lys3 confirms the canonical U-turn structure of the anticodon loop,³⁴ as found in the crystal structure of the whole tRNA. This conclusion arising from solution data excludes that packing effects in crystal lattices trigger U-turn conformations. Interestingly, the NMR study shows further that the modified nucleosides (mnm⁵s²U34, t⁶A37, and ψ39) stabilize this structure.

Figure 1B

Example of two crystallographic conformers of tRNA emphasizing its structural plasticity: at the left, free tRNA^Asp mimicking a tRNA interacting with mRNA and at the right, the same tRNA complexed with cognate AspRS.

The above cited X-ray structures are of medium resolution (2.5Å at best) (Table 1) and except for tRNA^Lys3, were refined with nonoptimized methods. For long, these structures constituted the only structural RNA database. They revealed the conserved tRNA conformation and gave an architectural significance to the conserved and semiconserved residues of tRNA that interconnect the D- and T-loops. They showed also novel structural features in RNA, like noncanonical pairings and metal binding sites, and suggested existence of faint conformational differences between tRNAs due to differences in semiconserved residues and in localization of the G18G19 dinucleotide in the D-loop and length variations in this loop and the variable region.³⁵ Existence of such differences was clearly shown by structural mapping of tRNA by chemical probes.³⁶

Table 1

Summary of tRNA structures in their free state or in complexes with proteins.

The structure of yeast tRNA^Asp is of particular type (Fig. 1B). It deviates from that of tRNA^Phe by a significant opening of the angle between the two branches of the molecule and by changes in the D-/T-loop interaction with a disruption of the G19-C56 tertiary base-pair. These features result from crystal packing effects, but have biological meaning. Indeed, tRNA^Asp molecules interact via GUC/GUC anticodon/anticodon pairing in the crystal and this pairing mimics a tRNA/mRNA interaction. Thus, the crystal structure of tRNA^Asp would mimic the structure of a tRNA interacting with mRNA.³⁷ This interpretation was confirmed by solution data with tRNAs forming dimers, with in particular the demonstration of the opening of the G19-C56 pair by chemical probing.^37,38

Recently, the structure of yeast tRNA^Phe was revisited. New diffraction data were collected from monoclinic and orthorhombic crystals at synchrotron sources, and structural models were refined with advanced techniques. As a result, high-resolution electron density maps at ˜2Å resolution were obtained,^39,40 even from 15-year-old crystals that yielded at best 2.5Å data in the past.⁴⁰ The new versions of the tRNA^Phe structure overall confirm the previous structural information but reveal novel details. The triple and other tertiary base-pairs are now seen with high accuracy together with their hydration patterns and associated Mg²⁺ ions well defined (Fig. 2). Other striking improvements concern identification of new divalent cation binding sites and of many ordered water molecules.³⁹ Interestingly, in the case of the monoclinic crystals, four different cleavage sites, the major one in the D-loop, have been localized next to bound Mg²⁺ ions. The presence of such in situ Mg²⁺-induced cleavages was suggested in the orthorhombic tRNA^Phe crystals,⁴¹ and clearly demonstrated in tRNA^Asp crystals.³⁷ It is likely that this intrinsic chemical fragility explains many crystallization failures with free tRNA.

Figure 2

Details in the high-resolution structure of yeast tRNA^Phe as seen in the monoclinic crystal form. Typical examples of tertiary interactions are shown: the U8-A14-A21, A9-[A23-U12], and [C13-G22]-m⁷G46 triples and the G15-C48 Levitt pair. Notice the well-defined (more...)

Polyamines are obligatory additives for the crystallization of free tRNAs.⁴² Their role becomes clearly apparent in the high-resolution structure of tRNA^Phe which shows a spermine molecule in the major groove of the T-arm that connects a symmetry-related tRNA molecule in the crystal lattice.⁴⁰

Antibiotics from the aminoglycoside family are tRNA ligands, besides interacting with various other targets such as the ribosome (see Chapter 26 by D. Fourmy et al). The recently solved X-ray structure at 2.6Å resolution of a complex between yeast tRNA^Phe and neomycin B shows that the elongated antibiotic molecule is anchored in the tRNA core between residues G20, A44, and G45.⁴³ This binding site overlaps with known divalent metal ion binding sites and corresponds also to the location of major determinants for E. coli PheRS recognition. This suggests that binding of the antibiotic occurs by metal displacement and explains why neomycin and other structurally related aminoglycosides inhibit Pb²⁺ cleavage of tRNA^Phe by hindering binding of the metal cation. It explains also that neomycin B inhibits aminoacylation of E. coli tRNA^Phe.⁴³

The modular structure of tRNA in two domains connected by a network of tertiary interactions explains that the molecule can be dissected in individual parts with intrinsic structure and functional properties.⁴⁴ For instance, NMR⁴⁵ and crystallographic⁴⁶ investigations on a duplex RNA recapitulating the acceptor stem of E. coli tRNA^Ala informed about the structural environment of the G3-U70 base-pair that determines alanine identity. Other biochemical and biophysical studies confirmed the existence of anticodon- and T-loop structures and highlighted the importance of modified residues for their local conformation (see e.g., refs. 34,47–50).

The Structure of tRNA in Macromolecular Complexes

As examples, Figure 3A shows how E. coli tRNA^Gln and yeast tRNA^Asp bind to class I and class II synthetases.^51,52 In both tRNAs, binding to the synthetase triggers significant conformational changes in the anticodon loop. Bases become unstacked and point towards the recognition amino acids from the synthetases, and in the aspartate system the overall structure of complexed tRNA^Asp becomes more closed than in the free molecule (Fig. 1B). In tRNA^Gln, the terminal base-pair is disrupted to facilitate bending of the 3'-terminal CCA into the active site of GlnRS. Such bending is not needed in tRNA^Asp where the CCA-strand is in helical continuity with the acceptor stem to reach the catalytic site of AspRS. These structural differences in the acceptor stem of the two tRNAs rely to different binding modes in the catalytic site of class I and class II synthetases: in the minor groove in tRNA^Gln (class I) and the major groove in tRNA^Asp (class II). This differential binding has been verified by chemical probing of an unmodified tRNA^Asp transcript chargeable by both class I ArgRS and class II AspRS (Fig. 3B).⁵³

Figure 3

Comparison of tRNA binding to class I and class II synthetases. (A) Structure of tRNA^Asp (left) and tRNA^Gln (right) with residues in contact with AspRS and GlnRS shown in blue and yellow, respectively. (B) Mirror image recognition of a tRNA^Asp unmodified (more...)

In nature, tRNAs are modified and the obvious question is to know whether modifications affect binding to synthetases. A complex between unmodified tRNA^Gln and GlnRS shows no structural differences with a complex comprising modified tRNA, except the absence of bound water molecules crosslinking the N5 atom of ψ-residues to their 5'-phosphate. This finding suggests a possible role of pseudouridylation in tRNA stabilization through water-mediated binding of ψ-residues to the tRNA backbone.⁵⁴ Interestingly, one of the three ψs in tRNA^Gln is located in the anticodon loop at identity position 38,⁵⁵ and contacts GlnRS.⁵⁶ These data on tRNA^Gln are in line with the general assumption that modified tRNAs have more rigid structures than unmodified transcripts.^48,57

From a different viewpoint, two crystal structures of active tRNA^Gln mutants in complexes with GlnRS and an adenylate analog were solved.⁵⁸ These mutants with G15-G48 atypical Levitt pairs, and for one of them with an additional change in sequence and length of the variable region, show structural perturbations confined to the core of the molecule near to the Levitt pair and the variable region. They consist among others in a syn conformation of the guanine ring of G48 with respect to the ribose moiety. This conformation differs from what observed in the native structure, where C48 is present as an anti conformer. Surprisingly, this anti conformation was also found in the crystal structure of a tRNA with a native G15-G48 Levitt pair, namely that of E. coli tRNA^Cys complexed to EF-Tu.⁵⁹

Further information on tRNA structure came from crystallographic work on four class I,⁶⁰⁶³ and nine class II,⁶⁴⁷¹ tRNA/aaRS complexes, as well as from studies on complexes between tRNAs and other protein partners,^59,72,73 and recently even from X-ray studies of the complete ribosome,^74,75 (Table 1). As anticipated, the L-shaped structure of tRNA is confirmed, as well as the existence of conformational flexibility in the molecule (see below). Of particular interest are the structures of tRNA^Ser and tRNA^Cys, the two tRNAs with uncommon sequence characteristics. For tRNA^Ser, the structure of the complex with SerRS identified novel tertiary interactions in the core of the molecule. Notably, it showed the role of conserved residue G20b in the D-loop that determines the orientation of the long variable arm at ˜45° to the plane of the L-shaped tRNA molecule.⁶⁴ The case of E. coli tRNA^Cys is noteworthy, since, to solve its structure, it was crystallized on purpose as a complex with elongation factor. Overall, the structure is canonical, but with a large interstem angle of ˜100° and a geometry of its noncanonical G15-G48 Levitt pair that exposes the N2-N3 side of G15 and the O6-N7 side of G48 to the solvent.⁵⁹

Aminoacylation and Identity of tRNAs

The Aminoacylation Reaction and the Concept of Identity

Aminoacylation of tRNA occurs in two step reactions: first activation of the amino acids by ATP to form enzyme-bound aminoacyl-adenylates and second transfer of the activated amino acids to the 3'-terminal adenosine of tRNA. Amino acid attachment to tRNA occurs by ester bond formation with hydroxyl groups of the terminal ribose, either 2'-OH for the reactions catalyzed by class I synthetases or 3'-OH in the case of class II enzymes (reviewed in ref.85). The activation step is tRNA independent, except for ArgRS, GlnRS, and GluRS. Overall, aminoacylation reactions have to yield tRNAs correctly charged, otherwise wrong amino acids will be falsely incorporated into proteins.⁸⁶ This implies correct recognition of both amino acids and tRNAs by the synthetases. However, synthetases can misactivate amino acids, are able to recognize noncognate tRNAs and can catalyze mischarging reactions (reviewed in ref.35). A first answer to the dilemma was brought when it was realized that fidelity in tRNA aminoacylation, and consequently in protein synthesis, mainly relies to highest kinetic efficiency of the synthetases for their cognate substrates and is mostly governed by the k_cat of the tRNA charging reactions.⁸⁷ This answer was refined when proofreading (or editing) mechanisms were discovered (reviewed in ref. 85). Altogether, the phenomenological view of tRNA aminoacylation fidelity implies a strict correspondence between the charged amino acid and the codon read by the carrier tRNA according to the rules of the genetic code. This correspondence is mediated by identity determinants within tRNAs and is defined by the tRNA identity rules, which constitute a ‘second’ genetic code.

Major elements defining identity of all E. coli tRNAs have been deciphered and much is known about identity determinants of most yeast tRNAs and of a few tRNAs from other organisms.^16,8891 In short, identity of a tRNA is determined by a small number of nucleosides, and more precisely by chemical groups carried by these nucleosides that often have been seen interacting with amino acids on the synthetases. In each tRNA, these nucleosides constitute the so-called ‘identity set’ that can be completed by structural elements of the nucleic acid. Negative elements that prevent a tRNA to be mischarged by noncognate synthetases can participate in identity. Some of the positive identity determinants can be considered as ‘strong’ since their mutation strongly reduces the aminoacylation capacity of the mutant tRNA, others are ‘moderate’ or ‘weak’. As examples, Table 2 displays the strongest conserved identity determinants that have been characterized to date in prokaryotic tRNAs. At first glance, several features emerge: (i) Identity elements are mainly located at the two distal extremities of the tRNA. (ii) Except for glutamate and threonine identities, the discriminator base is a determinant, at least in E. coli tRNAs. (iii) Specific structural elements in tRNA often serve as identity determinants (i.e., the −1 residue in tRNA^His, the long extra-arm in tRNA^Ser, the G3-U70 wobble pair in tRNA^Ala). (iv) For tRNAs specific for amino acids coded by more than four codons (leucine, serine, arginine), anticodon residues either do not participate in identity (leucine, serine) or, only for the middle C35 and semiconserved U/G36 positions (arginine).

Table 2

Major identity determinants found in E. coli tRNAs and correlations with class-ranking of synthetases.

In most systems, modified nucleosides do not participate in identity and thus are not recognized by the cognate synthetases. But they can play a major role in negative discrimination by preventing tRNAs to be recognized by noncognate synthetases, as was shown in the isoleucine and aspartate systems.^92,93 At present, the universal nature of the identity rules is rather well established and only faint differences distinguish identity sets for a given amino acid specificity along evolution. However, much has to be learned about the structural and evolutionary relationships between identity sets, the precise molecular mechanisms by which they are expressed in different organisms, and the exact nature of identities of atypical tRNAs, in particular those of mitochondrial origin.

Structure and Identity of Atypical tRNAs and of tRNA-Like Domains

Figure 4 gives examples of the folding of natural RNAs with structural and/or functional characteristics different from those of canonical tRNAs. Structural deviations in these molecules likely reflect their specialized functions (protein synthesis in mitochondria, donor of selenocysteine in protein synthesis, tagging of abnormal proteins on the ribosome for proteolysis in the case of tmRNA, replication of viral RNA genomes for plant virus tRNA-like structures). All these RNAs are recognized by aminoacyl-tRNA synthetases and thus should contain identity determinants mimicking the determinants present in canonical tRNAs.

Figure 4

Schematized folding of atypical tRNAs emphasizing deviations from the canonical fold (dashed lines), domains mimicking tRNA features (in heavy lines), and location of identity determinants (full triangles) (adapted from ref. ). (A) Mitochondrial tRNAs, (more...)

Several bovine mitochondrial tRNAs have been studied by chemical probing, modeling and NMR methods. For tRNA^Ser(UCN), the structure is close to that of classical tRNA, but the connector between acceptor stem and D-stem has only one nucleotide and the anticodon stem contains six base-pairs.¹³⁷ In tRNA^Ser(AGY), the D-domain is missing and a novel pattern of tertiary interactions accounts for the three-dimensional structure of the molecule.¹³⁸ Interestingly, and in contrast with cytoplasmic tRNA^Ser species which have a large variable region, in the mitochondrial species, this region is always of small size. Further, because of the peculiar D- and T-loop sequences, interaction between these two loops is missing or strongly altered in most mitochondrial tRNAs. From the functional viewpoint, little is known about the identity determinants in these tRNAs, although it can be predicted from phylogenetic considerations and sequence comparisons that mitochondrial tRNAs contain identity elements found in E. coli tRNAs,¹⁴ but their importance awaits to be verified experimentally. This was already done for aspartate identity in a mitochondrial tRNA^Asp from a marsupial, which like in prokaryotic tRNA^Asp, relies on anticodon, notably on the central U35 residue.¹³⁹ Of different outcome, however, were studies on serine identity of mitochondrial bovine tRNA^Ser(AGY). Here, the importance of the T-loop and in particular of A58 is highlighted.^140-142 This differs from what found for serine identity of E. coli tRNA^Ser that is given by determinants in the acceptor stem and variable region.¹⁴³ For more details, see chapter 8 by C. Florentz and M. Sissler, on mitochondrial synthetases.¹⁴⁴

Selenocysteine inserting tRNAs differ from canonical tRNAs by an extended amino acid branch made of 13 base-pairs and novel tertiary interactions. They are recognized by SerRS, likely as are canonical tRNA^Ser species. The specific properties of these atypical molecules rely on their potential to interact with specialized elongation factors and more generally to participate in the pathway of selenocysteine synthesis and incorporation into proteins (reviewed in ref. 145; see also chapter 4 by S. Blanquet et al, and chapter 22 by I.P. Ivanov et al). Another family of atypical tRNAs is constituted by the tmRNAs that have both mRNA and tRNA properties (see e.g., ref. 146 for recent structural aspects about the tRNA-like domain within E. coli tmRNA, and chapter 21 by R.H. Buckingham and M. Ehrenberg). These molecules are aminoacylated by AlaRS,¹⁴⁷ and, as anticipated have a G3-U70 identity pair.

Viral tRNA-like structures were discovered at the 3'-end of genomic RNAs of several genera of plant viral RNAs (for early literature see e.g., refs. 148-151). Three groups of mimics have been characterized to date on the basis of their aminoacylation identity (valine, histidine, and tyrosine). Folding of these domains deviates markedly from the canonical tRNA cloverleaf. Closest sequence similarities with tRNA are found in the valine accepting structures in tymoviruses (e.g., TYMV). All the viral tRNA-like domains present a pseudoknotted acceptor stem. Recent advances in the field brought better understanding of the architectural features that actually mimic tRNA as well as of the rules that confer aminoacylation capacity to these molecules (reviewed in ref. 152). Because of the architectural properties of pseudoknotted stems,¹⁵³ all three families of tRNA-like domains (from TYMV, TMV, BMV) present in their acceptor arm a mimic of the −1 major histidine identity nucleotide and consequently were found histidylatable.²⁴ Interestingly, studies on tRNA showed that the phosphate group of residue −1 is in fact the actual histidine identity determinant.¹⁵⁴ Strength of this determinant is strongest in the TMV tRNA-like structure and is much weaker in the context of TYMV and BMV RNAs, especially when compared to the dominant identity in these tRNA-like molecules (valine and tyrosine, respectively). It seems therefore unlikely that this property has in vivo implications in contemporary systems,¹⁵⁵ and we believe it is more likely that histidine identity in TYMV and BMV RNAs is a functional remnant of the evolutionary history of these molecules. As to valine identity of TYMV RNA, mutagenesis experiments pointed to the importance of discriminator and anticodon residues,^156,157 like in canonical tRNAVal. A recent investigation based on in vitro selection of molecules derived from the TYMV tRNA-like structure randomized in the anticodon loop and in the pseudoknot confirmed the importance of the anticodon residues in valine identity.¹⁵⁸ It showed further that architectural rather than sequence features in the pseudoknot are important for efficient valylation. Finally, viruses belonging to the bromo-, cucumo- and hordeivirus genera possess tyrosine accepting tRNA-like structures at their 3'-extremity. All share a particularly intricate structure, as highlighted in the case of the BMV tRNA-like structure (Fig. 4F). In this structure, residues in the pseudoknotted acceptor arm,¹⁵⁹ functionally mimic the major tyrosine identity determinants found in yeast tRNA^Tyr, namely A73 and the first base-pair C1-G72 of the accepting stem.¹⁶⁰ Tyrosine identity in yeast, however, relies also on anticodon, but, strikingly, the BMV tRNA-like structure possesses neither a canonical anticodon loop nor a tyrosine anticodon triplet GUA.¹⁶⁰ In fact, recent experiments showed that yeast TyrRS interacts with a hairpin, oriented perpendicularly to the acceptor branch. This hairpin anchors the tRNA-like structure on the synthetase and contributes to the efficiency of the tyrosylation reaction, which otherwise relies only on identity elements in the acceptor branch.¹⁵⁹

Altogether, the conclusions arising from investigations on atypical tRNAs and tRNA-like domains favor the view of a strict conservation of the identity nucleotides for recognition of different types of RNA substrates by synthetases, whatever the structural scaffold in which these nucleotides are embedded.^16,161

Engineering Structure and Identity of tRNA

The concept of a structural scaffold carrying identity elements at its extremities, as visualized in Figure 5, provides a rational basis for engineering structure and identity of tRNA. It accounts for understanding the output of identity transplantation experiments. If presentation is sub-optimal, aminoacylation efficiency is poor, but can be improved by engineering the scaffold of the receiving tRNA. The concept is illustrated by identity switch experiments between tRNAs aminoacylated by class I GlnRS and class II AspRS,¹¹¹ and by the design of a tRNA derived from yeast tRNA^Asp having optimal phenylalanine identity,¹⁶² after remodeling the frameworks of the tRNAs. A further example is a E. coli tRNA^Gln variant that remained efficiently recognized by GlnRS upon transplantation of the large extra-arm of tRNA^Ser (characteristic of class II tRNAs) in its architectural core.¹⁶³ Generalizing such engineering, it was possible to construct four distinct designs of the core of class II tRNAs that present stable folding and are efficiently aminoacylated by GlnRS.¹⁶⁴

Figure 5

L-shaped RNA scaffold of tRNA and distribution of major identity residues at its distal extremities (adapted from ref. ). Size of spheres is proportional to the occurrence of identity elements - note the predominant occurrence at discriminator N73 position (more...)

Along the same lines, a molecule with multiple identity having lost its original identity (aspartate) and acquired three new identities (alanine, phenylalanine, and valine) was designed.¹⁶⁵ Two of these identities have well-separated determinants (phenylalanine and alanine) and are therefore expressed as efficiently as in their original framework, while the third one (valine) has determinants overlapping with those of phenylalanine and consequently is expressed less efficiently. Similarly, a full-length and bimolecular (obtained by annealing of two fragments) versions of a tRNA with dual phenylalanine and alanine identity were obtained upon introduction of the G3-U70 alanine identity determinant in the framework of yeast tRNA^Phe.¹⁰³ Going one step further, the framework of atypical tRNAs was shown to be recognized by AspRS when it was realized that mutations in the D- and T-loops of yeast tRNA^Asp, compatible with aminoacylation activity,¹⁶⁶ created a core sharing structural resemblance with that of tRNA^Sec. Direct identity swap experiments confirmed that the tRNA framework of tRNA^Sec is indeed fully recognized by AspRS.¹⁶⁷

In a more general perspective, the central core of the tRNA can be completely reorganized and again it can be anticipated that functional molecules can be obtained, provided that identity elements are properly located in the new scaffold. This engineering can be done either by combinatorial methods or by rationale based approaches (reviewed in ref. 168). Figure 6 shows examples of such engineered molecules that were derived from E. coli tRNA^Phe,¹⁶⁹ and yeast tRNA^Asp.^113,170 From a highly degenerate library of 63 nucleotides, several RNAs that tightly bind to E. coli PheRS were selected by a filter binding assay. Interestingly, all binders are missing canonical core features and contain an anticodon stem-loop of atypical sequence with a phenylalanine GAA anticodon.¹⁶⁹ But the presence of this triplet, part of the E. coli phenylalanine identity set (Table 2), is not sufficient to confer phenylalanylation capacity to these molecules. Presumably, their flexibility is restricted so that proper accommodation of the accepting 3'-end in the catalytic site of PheRS is prevented. This accommodation is possible for three types of tRNA^Asp-mimics having deep alteration in their central cores. A first architecture, well aspartylatable, resembles metazoan mitochondrial tRNA^Ser lacking the D-arm. The second one lacks both D- and T-arms and has its acceptor and anticodon helices joined by two connectors. This construct is a substrate of AspRS that behaves like a minihelix, since mutating the anticodon identity determinants does not affect aminoacylation. Removing the connector at the 5'-side provides more flexibility, and allows aminoacylation that is dependent on both G73 and anticodon identity elements.¹⁷⁰ Thus, neither a helical structure in the acceptor stem nor the presence of a D- or T-arm is mandatory for specific aspartylation. This conclusion is not restricted to the yeast aspartate system, since yeast PheRS is able to charge a tRNA^Phe fragment encompassing a single-stranded acceptor domain.¹⁷¹

Figure 6

Engineered tRNA structures that interact with a synthetase or are aminoacylatable. (A) PheRS-binders selected from a library based on the acceptor stem of E. coli tRNA^Phe with the other parts of the tRNA molecule degenerated (adapted from ref. ). (B) (more...)

A Few Remarks on Evolution

The structure of contemporary tRNA is the result of a long evolutionary history and it is most likely that the two domains of its modular L-shaped architecture have arisen independently with the acceptor branch appearing first. Likewise, modular structures, with well-defined catalytic cores, are found in synthetases. Here also the primitive versions of these enzymes would have been restricted to catalytic domains recognizing minimalist acceptor RNAs.^172,173 The implication is that the primordial signals defining aminoacylation of tRNA should be present in the minimal structural elements needed for function. As seen in Table 2, identity elements are indeed present in the acceptor arm of all tRNA species. On the other hand, the similar structure of all tRNAs and the structural similarities in the catalytic core of each of the two classes of synthetases, imply evolutionary relationships between the different tRNA aminoacylation systems. Again, this assumption finds support in the distribution and nature of tRNA identity elements (Table 2). Although a complete picture on determinants is presently only available for E. coli tRNAs and that information on tRNAs from other organisms except yeast,¹⁶ is scarcer, it appears clearly that many determinants are conserved in evolution. This is true in many systems for the discriminator base and adjacent base-pairs in the acceptor stem. Exceptions to this trend are the nonconservations of the discriminator base in tRNA^Gly and tRNA^His and even its noninvolvement in the case of tRNA^Thr identity. Interestingly, these idiosyncrasies seem to be compensated by conservation in evolution of determinants in the acceptor stem of these tRNAs recognized by class IIa synthetases.

In the context of evolution, the structural and functional relationships that were discovered in the glutamate/glutamine and aspartate/asparagine tRNA aminoacylation systems deserve special attention. In both pairs the synthetases are structurally related, belonging to subclass Ic for GluRS and GlnRS and subclass IIb for AspRS and AsnRS. Likewise the identity sets in the corresponding tRNAs, specific for either glutamate and glutamine or aspartate and asparagine, overlap and share strong homologies. Altogether this suggests common evolutionary origins of the glutamate/glutamine and aspartate/asparagine pairs. The fact that in some organisms GlnRS or AsnRS is absent and that aminoacylation of tRNA^Gln or tRNA^Asn occurs via a two-step process comprising tRNA mischarging by heterologous GluRS or AspRS followed by amidation of the mischarged amino acids by tRNA-dependent amidases (reviewed in ref. 174) supports this view. It suggests further that glutamine and asparagine identities originate from the glutamate and aspartate identities.

To conclude, we notice the idiosyncratic architectures of the anticodon-binding modules in synthetases that likely were appended to their catalytic cores late in the course of the primordial evolution of life. In most systems, these modules recognize identity determinants within the anticodon of the homologous tRNAs. Because of the universal nature of the genetic code, determinants in anticodons are obviously conserved in evolution. From that and other considerations, it can be suggested that the origin of tRNA aminoacylation systems is connected with the development of the genetic code (ref. 173 and references therein).

Acknowledgements

We thank C. Florentz for suggestions and critical reading of the manuscript. This work was supported by grants from the Centre National de la Recherche Scientifique (CNRS), Ministère de la Recherche (Programme PRFMMIP), Université Louis Pasteur, Strasbourg, and the European Community (BIO4-CT98-0189).

References

1.

Crick FHC. On protein synthesis. Symp Soc Exp Biol. 1958;12:128–163. [PubMed: 13580867]

2.

Hoagland MB, Stephenson ML, Scott JF. et al. A soluble ribonucleic acid intermediate in protein synthesis. J Biol Chem. 1958;231:241–257. [PubMed: 13538965]

3.

Söll D. Transfer RNA-an RNA for all seasons In: Gesteland R, Atkins J, eds.The RNA World Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press,1993157–184.

4.

Kiesewetter S, Ott G, Sprinzl M. The role of modified purine 64 in initiator/elongator discrimination of tRNA^Meti from yeast and wheat germ. Nucleic Acids Res. 1990;18:4677–4682. [PMC free article: PMC331916] [PubMed: 2395634]

5.

RajBhandary UL. Initiator transfer RNAs. J Bacteriol. 1994;176:547–552. [PMC free article: PMC205089] [PubMed: 7507918]

6.

H¨ttenhofer A, Böck A. RNA structures involved in selenoprotein synthesis In: Simons RW, Grunberg-Manago M, eds.RNA structure and Function Cold Spring Harbor: Cold Spring Harbor Laboratory Press,1998377–413.

7.

Holley RW, Apgar J, Everett GA. et al. Structure of a ribonucleic acid. Science. 1965;147:1462–1465. [PubMed: 14263761]

8.

Sprinzl M, Horn C, Brown M. et al. Compilation of tRNA sequences and sequences of tRNA genes. Nucleic Acids Res. 1998;26:148–153. [PMC free article: PMC147216] [PubMed: 9399820]

9.

Rozenski J, Crain PF, McCloskey JA. The RNA modification database: 1999 update. Nucleic Acids Res. 1999;27:196–197. [PMC free article: PMC148133] [PubMed: 9847178]

10.

Keith G, Yusupov M, Brion C. et al. Sequence of tRNA^Asp from Thermus thermophilus HB8. Nucleic Acids Res. 1993;21:4399. [PMC free article: PMC310085] [PubMed: 7692402]

11.

Horie N, Hara-Yokoyama M, Yokoyama S. et al. Two tRNA^Ile1 species from an extreme thermophile, Thermus thermophilus HB8: Effect of 2-thiolation of ribothymidine on the thermostability of tRNA. Biochemistry. 1985;24:5711–5715. [PubMed: 3853464]

12.

Helm M, Giegé R, Florentz C. A Watson-Crick base-pair disrupting methyl group (m¹A9) is sufficient for cloverleaf folding of human mitochondrial tRNA^Lys. Biochemistry. 1999;38:13338–13346. [PubMed: 10529209]

13.

Gregson JM, Crain PF, Edmonds CG. et al. Structure of the archaeal transfer RNA nucleoside G*-15 (2-amino-4,7-dihydro-4-oxo-7-beta-D-ribofuranosyl-1H-pyrrolo[2,3-d]pyrimidine-5-carboximidamide (archaeosine)). J Biol Chem. 1993;268:10076–10086. [PubMed: 7683667]

14.

Helm M, Brulé H, Friede D. et al. Search for characteristic structural features of mammalian mitochondrial tRNAs. RNA. 2000;6:1356–1379. [PMC free article: PMC1370008] [PubMed: 11073213]

15.

Yokoyama S, Nishimura S. Modified nucleosides and codon recognition In: Söll D, RajBhandary U, eds.tRNA: Structure, Biosynthesis, and Function Washington DC: Am Soc Microbiol Press,1995207–223.

16.

Giegé R, Sissler M, Florentz C. Universal rules and idiosyncratic features in tRNA identity. Nucleic Acids Res. 1998;26:5017–5035. [PMC free article: PMC147952] [PubMed: 9801296]

17.

Masquida B, Westhof E. On the wobble G-U and related pairs. RNA. 2000;6:9–15. [PMC free article: PMC1369889] [PubMed: 10668794]

18.

Varani G, MaClain WH. The G-U wobble base pair. A fundamental building block of RNA structure crucial to RNA function in diverse biological systems. EMBO Reports. 2000;1:18–23. [PMC free article: PMC1083677] [PubMed: 11256617]

19.

de Bruijn MH, Schreier PH, Eperon IC. et al. A mammalian mitochondrial serine transfer RNA lacking the “dihydrouridine” loop and stem. Nucleic Acids Res. 1980;8:5213–5222. [PMC free article: PMC324296] [PubMed: 6906662]

20.

Wolstenholme DR, Okimoto R, Mcfarlane JL. Nucleotide correlations that suggest tertiary interactions in the TV-replacement loop-containing mitochondrial tRNAs of the nematodes, Caenorhabditis elegans and Ascaris suum. Nucleic Acids Res. 1994;22:4300–4306. [PMC free article: PMC331950] [PubMed: 7937159]

21.

Steinberg S, Leclerc F, Cedergren R. Structural rules and conformational compensations in the tRNA L-form. J Mol Biol. 1997;266:269–282. [PubMed: 9047362]

22.

Cooley L, Appel B, Söll D. Post-transcriptional nucleotide addition is responsible for the formation of the 5'-terminus of histidine tRNA. Proc Natl Acad Sci USA. 1986;79:6475–6479. [PMC free article: PMC347149] [PubMed: 6292903]

23.

Hou Y-M, Westhof E, Giegé R. An unusual RNA tertiary interaction has a role for the specific aminoacylation of a transfer RNA. Proc Natl Acad Sci USA. 1993;90:6776–6780. [PMC free article: PMC47015] [PubMed: 8341698]

24.

Rudinger J, Felden B, Florentz C. et al. Strategy for RNA recognition by yeast histidyl-tRNA synthetase. Bioorg Med Chem. 1997;5:1001–1009. [PubMed: 9222493]

25.

Kim SH, Quigley GJ, Suddath FL. et al. Three dimensional structure of yeast phenylalanine transfer RNA. Folding of the polynucleotide chain. Science. 1973;179:285–288. [PubMed: 4566654]

26.

Kim SH, Suddath FL, Quigley GJ. et al. Three dimensional tertiary structure of yeast phenylalanine transfer RNA. Science. 1974;185:435–440. [PubMed: 4601792]

27.

Robertus JD, Ladner JE, Finch JT. et al. Structure of yeast phenylalanine tRNA at 3Å resolution. Nature. 1974;250:546–551. [PubMed: 4602655]

28.

Stout CD, Mizuno H, Rubin J. et al. Atomic coordinates and molecular conformation of yeast phenylalanyl tRNA. An independent investigation. Nucleic Acids Res. 1976;3:1111–1123. [PMC free article: PMC342970] [PubMed: 775444]

29.

Moras D, Comarmond MB, Fischer J. et al. Crystal structure of yeast tRNA^Asp. Nature. 1980;288:669–674. [PubMed: 7005687]

30.

Westhof E, Dumas P, Moras D. Crystallographic refinement of yeast aspartic acid transfer RNA. J Mol Biol. 1985;184:119–145. [PubMed: 3897553]

31.

Woo NH, Roe BA, Rich A. Three-dimensional structure of Escherichia coli initiator tRNA^fMet. Nature. 1980;286:346–351. [PubMed: 6157105]

32.

Basavappa R, Sigler PB. The 3Å crystal structure of yeast initiator tRNA: Functional implications in initiator/elongator discrimination. EMBO J. 1991;10:3105–3111. [PMC free article: PMC453028] [PubMed: 1915284]

33.

Bénas P, Bec G, Keith G. et al. The crystal structure of HIV reverse-transcription primer tRNA₃^Lys shows a canonical anticodon loop. RNA. 2000;6:1347–1355. [PMC free article: PMC1370007] [PubMed: 11073212]

34.

Sundaram M, Durant PC, Davis DR. Hypermodified nucleosides in the anticodon of tRNA^Lys stabilize a canonical U-turn structure. Biochemistry. 2000;39:12575–12584. [PubMed: 11027137]

35.

Giegé R, Puglisi JD, Florentz C. tRNA structure and aminoacylation efficiency. Prog Nucleic Acid Res Mol Biol. 1993;45:129–206. [PubMed: 8341800]

36.

Giegé R, Helm M, Florentz C. Chemical and enzymatic probing of RNA structure In: Söll D, Nishimura S, Moore P, eds.Prebiotic Chemistry, Molecular Fossils, Nucleosides, and RNA Oxford: Pergamon,199963–80.

37.

Moras D, Dock AC, Dumas P. et al. The structure of yeast tRNA^Asp. A model for tRNA interacting with mesenger RNA. J Biomol Struct Dyn. 1985;3:479–493. [PubMed: 3917033]

38.

Moras D, Dock A-C, Dumas P. et al. Anticodon-anticodon interaction induces conformational changes in tRNA: Yeast tRNA^Asp, a model for tRNA-mRNA recognition. Proc Natl Acad Sci USA. 1986;83:932–936. [PMC free article: PMC322984] [PubMed: 3513167]

39.

Shi HJ, Moore PB. The crystal structure of yeast phenylalanine tRNA at 1.93Å resolution: a classic structure revisited. RNA. 2000;6:1091–1105. [PMC free article: PMC1369984] [PubMed: 10943889]

40.

Jovine L, Djordjevic S, Rhodes D. The crystal structure of yeast phenylalanine tRNA at 2.0Å resolution: cleavage by Mg²⁺ in 15-year old crystals. J Mol Biol. 2000;301:401–414. [PubMed: 10926517]

41.

Sussman JL, Holbrook SR, Warrant RW. et al. Crystal structure of yeast phenylalanine transfer RNA. I. Crystallographic refinement. J Mol Biol. 1978;123:607–630. [PubMed: 357742]

42.

Dock-Bregeon A-C, Moras D, Giegé R. Nucleic acids and their complexes In: Ducruix A, Giegé R, eds.Crystallization of Nucleic Acids and Proteins. A Practical Approach (second edition)Oxford: IRL Press,1999209–243.

43.

Mikkelsen NE, Johansson K, Virtanen A. et al. Aminoglycoside binding displaces a divalent metal ion in tRNA-neomycin B complex. Nat Struct Biol. 2001;8:510–514. [PubMed: 11373618]

44.

Martinis SA, Schimmel P. Small RNA oligonucleotide substrates for specific aminoacylations In: Söll D, RajBhandary UL, eds.tRNA: Structure, Biosynthesis, and Function Washington, DC: Am Soc Microbiol Press,1995349–370.

45.

Ramos A, Varani G. Structure of the acceptor stem of Escherichia coli tRNA^Ala: role of the G3:U70 base pair in synthetase recognition. Nucleic Acids Res. 1997;25:2083–2090. [PMC free article: PMC146727] [PubMed: 9153306]

46.

Mueller U, Schubel H, Sprinzl M. et al. Crystal structure of acceptor stem of tRNA(Ala) from Escherichia coli shows unique G.U wobble base pair at 1.16 Å resolution. RNA. 1999;5:670–677. [PMC free article: PMC1369794] [PubMed: 10334337]

47.

Romby P, Carbon P, Westhof E. et al. Importance of conserved residues for the conformation of the T-loop in tRNAs. J Biomol Struct Dyn. 1987;5:669–687. [PubMed: 3078237]

48.

Agris PF. The importance of being modified: Roles of modified nucleosides and Mg²⁺ in RNA structure and function. Prog Nucleic Acid Res Mol Biol. 1996;53:79–129. [PubMed: 8650309]

49.

Koshlap KM, Guenther R, Sochacka E. et al. A distictive RNA fold: the solution structure of an analogue of the yeast tRNA^Phe TYC domain. Biochemistry. 1999;38:8647–8656. [PubMed: 10393540]

50.

Auffinger P, Westhof E. An extended structural signature for the tRNA anticodon loop. RNA. 2001;7:334–341. [PMC free article: PMC1370090] [PubMed: 11333014]

51.

Rould MA, Perona JJ, Söll D. et al. Structure of E. coli glutaminyl-tRNA synthetase complexed with tRNA^Gln and ATP at 2.8Å resolution. Science. 1989;246:1135–1142. [PubMed: 2479982]

52.

Ruff M, Krishnaswamy S, Boeglin M. et al. Class II aminoacyl transfer RNA synthetases: Crystal structure of yeast aspartyl-tRNA synthetase complexed with tRNA^Asp. Science. 1991;252:1682–1689. [PubMed: 2047877]

53.

Sissler M, Eriani G, Martin F. et al. Mirror image alternative interaction patterns of the same tRNA with either class I arginyl-tRNA synthetase or class II aspartyl-tRNA synthetase. Nucleic Acids Res. 1997;25:4899–4906. [PMC free article: PMC147145] [PubMed: 9396794]

54.

Arnez JG, Steitz TA. Crystal structure of unmodified tRNA^Gln complexed with glutaminyl-tRNA synthetase and ATP suggests a possible role for pseudo-uridines in stabilization of RNA structure. Biochemistry. 1994;33:7560–7567. [PubMed: 8011621]

55.

Hayase Y, Jahn M, Rogers MJ. et al. Recognition of bases in E. coli tRNA^Gln by glutaminyl-tRNA synthetase: A complete identity set. EMBO J. 1992;11:4159–4165. [PMC free article: PMC556926] [PubMed: 1396597]

56.

Rould MA, Perona JJ, Steitz TA. Structural basis of anticodon loop recognition by glutaminyl-tRNA synthetase. Nature. 1991;352:213–218. [PubMed: 1857417]

57.

Hall KB, Sampson JR, Uhlenbeck OC. et al. Structure of an unmodified tRNA molecule. Biochemistry. 1989;28:5794–5801. [PubMed: 2775736]

58.

Sherlin LD, Bullock TL, Newberry KJ. et al. Influence of transfer RNA tertiary structure on aminoacylation efficiency by glutaminyl and cysteinyl-tRNA synthetases. J Mol Biol. 2000;299:431–446. [PubMed: 10860750]

59.

Nissen P, Thirup S, Kjeldgaard M. et al. The crystal structure of Cys-tRNA^Cys-EF-Tu-GDPNP reveals general and specific features in the ternary complex and in tRNA. Structure. 1999;7:143–156. [PubMed: 10368282]

60.

Delagoutte B, Moras D, Cavarelli J. tRNA aminoacylation by arginyl-tRNA synthetase: induced conformations during substrates binding. EMBO J. 2000;19:5599–5610. [PMC free article: PMC305789] [PubMed: 11060012]

61.

Fukai S, Nureki O, Sekine S. et al. Structural basis for double-sieve discrimination of L-valine from L-isoleucine and L-threonine by the complex of tRNA(Val) and valyl-tRNA synthetase. Cell. 2001;103:793–803. [PubMed: 11114335]

62.

Silvian LF, Wang J, Steitz TA. Insights into editing from an ile-tRNA synthetase structure with tRNA^Ile and mupirocin. Science. 1999;285:1074–1077. [PubMed: 10446055]

63.

Sekine S, Nureki O, Shimada A. et al. Structural basis for anticodon recognition by discriminating glutamyl-tRNA synthetase. Nat Struct Biol. 2001;8:203–206. [PubMed: 11224561]

64.

Biou V, Yaremchuk A, Tukalo et al. The 2.9 Å crystal structure of T. thermophilus seryl-tRNA synthetase complexed with tRNA^Ser. Science. 1994;263:1404–1410. [PubMed: 8128220]

65.

Cusack S, Yaremchuck A, Tukalo M. The crystal structure of the ternary complex of T. thermophilus seryl-tRNA^Ser and a seryl-adenylate analogue reveals a conformational switch in the active site. EMBO J. 1996;15:2834–2842. [PMC free article: PMC450221] [PubMed: 8654381]

66.

Sankaranarayanan R, Dock-Bregeon A-C, Romby P. et al. The structure of threonyl-tRNA synthetase-tRNA^Thr complex enlightens its repressor activity and reveals an essential zinc ion in the active site. Cell. 1999;97:371–381. [PubMed: 10319817]

67.

Yaremchuk A, Cusack S, Tukalo M. Crystal structure of a eukaryote/archaeon-like prolyl-tRNA synthetase and its complex with tRNA(Pro)(CGG). EMBO J. 2000;19:4745–4758. [PMC free article: PMC302085] [PubMed: 10970866]

68.

Eiler S, Dock-Bregeon AC, Moulinier L. et al. Synthesis of aspartyl-tRNA^Asp in Escherichia coli-a snapshot of the second step. EMBO J. 1999;18:6532–6541. [PMC free article: PMC1171716] [PubMed: 10562565]

69.

Briand C, Poterszman A, Eiler S. et al. An intermediate step in the recognition of tRNA^Asp by aspartyl-tRNA synthetase. J Mol Biol. 2000;299:1051–1060. [PubMed: 10843857]

70.

Cusack S, Yaremchuk A, Tukalo M. The crystal structures of T. thermophilus lysyl-tRNA synthetase complexed with E. coli tRNA^Lys and a T. thermophilus tRNA^Lys transcript: anticodon recognition and conformational changes upon binding of a lysyl-adenylate analogue. EMBO J. 1996;15:6321–6334. [PMC free article: PMC452455] [PubMed: 8947055]

71.

Goldgur Y, Mosyak L, Reshetnikova L. et al. The crystal structure of phenylalanyl-tRNA synthetase from Thermus thermophilus complexed with cognate tRNA^Phe. Structure. 1997;5:59–68. [PubMed: 9016717]

72.

Nissen P, Kjeldgaard M, Thirup S. et al. Crystal structure of the ternary complex of Phe-tRNA^Phe, EF-Tu, and a GTP analog. Science. 1995;270:1464–1472. [PubMed: 7491491]

73.

Schmitt E, Panvert M, Blanquet S. et al. Crystal structure of methionyl-tRNA^fMet transformylase complexed with the initiator formyl-methionyl-tRNA^fMet. EMBO J. 1998;17:6819–6826. [PMC free article: PMC1171029] [PubMed: 9843487]

74.

Cate JH, Yusupov MM, Yusupova GZ. et al. X-ray crystal structures of 70S ribosome functional complexes. Science. 1999;285:2095–2104. [PubMed: 10497122]

75.

Yusupov MM, Yusupova GZ, Baucom A. et al. Crystal structure of the ribosome at 5.5Å resolution. Science. 2001;292:883–896. [PubMed: 11283358]

76.

Streeck RE, Zachau HG. Characterization of the native and denatured conformations of tRNA^Ser and tRNA^Phe from yeast. Eur J Biochem. 1972;30:382–391. [PubMed: 4351442]

77.

Madore E, Florentz C, Giegé R. et al. Magnesium-dependent alternate foldings of active and inactive Escherichia coli tRNA^Glu revealed by chemical probing. Nucleic Acids Res. 1999;27:3583–3588. [PMC free article: PMC148604] [PubMed: 10446250]

78.

Krauss G, Pingoud A, Boehme D. et al. Equivalent and non-equivalent binding sites for tRNA on aminoacyl-tRNA synthetases. Eur J Biochem. 1975;55:517–29. [PubMed: 1100384]

79.

Friederich MW, Vacano E, Hagerman PJ. Global flexibility of tertiary structure in RNA: yeast tRNA^Phe as a model system. Proc Natl Acad Sci USA. 1998;95:3572–3577. [PMC free article: PMC19877] [PubMed: 9520407]

80.

Limmer S, Hofmann HP, Ott G. et al. The 3'-terminal end (NCCA) of tRNA determines the structure and stability of the aminoacyl acceptor stem. Proc Natl Acad Sci USA. 1993;90:6199–6202. [PMC free article: PMC46895] [PubMed: 7687063]

81.

Puglisi EV, Puglisi JD, Williamson JR. et al. NMR analysis of tRNA acceptor stem microhelices: discriminator base change affects tRNA conformation at the 3'end. Proc Natl Acad Sci USA. 1994;91:11467–11471. [PMC free article: PMC45252] [PubMed: 7972085]

82.

Chang KY, Varani G, Bhattacharya S. et al. Correlation of deformability at a tRNA recognition site and aminoacylation specificity. Proc Natl Acad Sci USA. 1999;96:11764–11769. [PMC free article: PMC18360] [PubMed: 10518524]

83.

Horie N, Yamaizumi Z, Kuchino Y. et al. Modified nucleosides in the first positions of the anticodons of tRNA(Leu)₄ and tRNA(Leu)₅ from Escherichia coli. Biochemistry. 1999;38:207–217. [PubMed: 9890900]

84.

Liu M, Chu W-C, Liu JC-H. et al. Role of acceptor stem conformation in tRNA^Val recognition by its cognate synthetase. Nucleic Acids Res. 1997;25:4883–4890. [PMC free article: PMC147156] [PubMed: 9396792]

85.

First EA. Catalysis of tRNA aminoacylation by class I and class II aminoacyl-tRNA synthetases In: Sinnott M, ed.Comprehensive Biological Catalysis New-York: Academic Press,1998573–607.

86.

Chapeville F, Lipman F, Ehrenstein GV. et al. On the role of soluble ribonucleic acid in coding for amino acids. Proc Natl Acad Sci USA. 1962;48:1086–1092. [PMC free article: PMC220908] [PubMed: 13878159]

87.

Ebel J-P, Giegé R, Bonnet J. et al. Factors determining the specificity of the tRNA aminoacylation reaction. Biochimie. 1973;55:547–557. [PubMed: 4585176]

88.

Normanly J, Abelson J. tRNA identity. Annu Rev Biochem. 1989;58:1029–1049. [PubMed: 2673006]

89.

Schulman LH. Recognition of tRNAs by aminoacyl-tRNA synthetases. Prog Nucleic Acid Res Mol Biol. 1991;41:23–87. [PubMed: 1882076]

90.

McClain WH. Rules that govern tRNA identity in protein synthesis. J Mol Biol. 1993;234:257–280. [PubMed: 8230212]

91.

Saks ME, Sampson JR, Abelson JN. The transfer RNA identity problem: A search for rules. Science. 1994;263:191–197. [PubMed: 7506844]

92.

Muramatsu T, Nishikawa K, Nemoto F. et al. Codon and amino-acid specificities of a transfer RNA are both converted by a single post-transcriptional modification. Nature. 1988;336:179–181. [PubMed: 3054566]

93.

Perret V, Garcia A, Grosjean H. et al. Relaxation of transfer RNA specificity by removal of modified nucleotides. Nature. 1990;344:787–789. [PubMed: 2330033]

94.

Normanly J, Ogden RC, Horvath SJ. et al. Changing the identity of a transfer RNA. Nature. 1986;321:213–219. [PubMed: 3086742]

95.

Hou Y-M, Schimmel P. A simple structural feature is a major determinant of the identity of a transfer RNA. Nature. 1988;333:140–145. [PubMed: 3285220]

96.

McClain WH, Foss K. Changing the identity of a tRNA by introducing a G-U wobble pair near the 3' acceptor end. Science. 1988;240:793–796. [PubMed: 2452483]

97.

Sampson JR, DiRenzo AB, Behlen LS. et al. Nucleotides in yeast tRNA^Phe required for the specific recognition by its cognate synthetase. Science. 1989;243:1363–1366. [PubMed: 2646717]

98.

Pütz J, Puglisi JD, Florentz C. et al. Identity elements for specific aminoacylation of yeast tRNA^Asp by cognate aspartyl-tRNA synthetase. Science. 1991;252:1696–1699. [PubMed: 2047878]

99.

Giegé R, Florentz C, Kern D. et al. Aspartate identity of transfer RNAs. Biochimie. 1996;78:605–623. [PubMed: 8955904]

100.

Hou YM, Schimmel P. Evidence that a major determinant for the identity of a transfer RNA is conserved in evolution. Biochemistry. 1989;28:6800–6804. [PubMed: 2684266]

101.

Francklyn C, Schimmel P. Aminoacylation of RNA minihelices with alanine. Nature. 1989;337:478–481. [PubMed: 2915692]

102.

Beuning PJ, Yang F, Schimmel P. et al. Specific atomic groups and RNA helix geometry in acceptor stem recognition by a tRNA synthetase. Proc Natl Acad Sci USA. 1997;94:10150–10154. [PMC free article: PMC23330] [PubMed: 9294178]

103.

Pleiss JA, Wolfson AD, Uhlenbeck OC. Mapping contacts between Escherichia coli alanyl-tRNA synthetase and 2' hydroxyls using a complete tRNA molecule. Biochemistry. 2000;39:8250–8258. [PubMed: 10889033]

104.

Hou Y-M, Sterner T, Jansen M. Permutation of a pair of tertiary nucleotides in a transfer RNA. Biochemistry. 1995;34:2978–2984. [PubMed: 7534478]

105.

Lovato MA, Chihade JW, Schimmel P. Translocation within the acceptor helix of a major tRNA identity determinant. EMBO J. 2001;20:4846–4853. [PMC free article: PMC125604] [PubMed: 11532948]

106.

Sissler M, Giegé R, Florentz C. Arginine aminoacylation identity is context-dependent and ensured by alternate recognition sets in the anticodon loop of accepting tRNA transcripts. EMBO J. 1996;15:5069–5076. [PMC free article: PMC452246] [PubMed: 8890180]

107.

Sissler M, Giegé R, Florentz C. The tRNA sequence context defines the mechanistic routes by which yeast arginyl-tRNA synthetase charges tRNA. RNA. 1998;4:647–657. [PMC free article: PMC1369647] [PubMed: 9622124]

108.

Moor NA, Ankilova VN, Lavrik OI. Recognition of tRNA^Phe by phenyalanyl-tRNA synthetase of Thermus thermophilus. Eur J Biochem. 1995;234:897–902. [PubMed: 8575450]

109.

Sampson JR, Behlen LS, DiRenzo AB. et al. Recognition of yeast tRNA^Phe by its cognate yeast phenylalanyl-tRNA synthetase: an analysis of specificity. Biochemistry. 1992;31:4164–4167. [PubMed: 1567862]

110.

Frugier M, Helm M, Felden B. et al. Sequences outside recognition sets are not neutral for tRNA aminoacylation: evidence for non-permissive combinations of nucleotides in the acceptor stem of yeast tRNA^Phe. J Biol Chem. 1998;273:11605–11610. [PubMed: 9565578]

111.

Frugier M, Söll D, Giegé R. et al. Identity switches between tRNAs aminoacylated by class I glutaminyland class II aspartyl-tRNA synthetase. Biochemistry. 1994;33:9912–9921. [PubMed: 8060999]

112.

Pütz J, Puglisi JD, Florentz C. et al. Additive, cooperative and anti-cooperative effects between identity nucleotides of a tRNA. EMBO J. 1993;12:2949–2957. [PMC free article: PMC413550] [PubMed: 8335008]

113.

Frugier M, Florentz C, Giegé R. Efficient aminoacylation of resected RNA helices by class II aspartyl-tRNA synthetase dependent on a single nucleotide. EMBO J. 1994;13:2219–2226. [PMC free article: PMC395077] [PubMed: 8187774]

114.

Cavarelli J, Rees B, Ruff M. et al. Yeast tRNA^Asp recognition by its cognate class II aminoacyl-tRNA synthetase. Nature. 1993;362:181–184. [PubMed: 8450889]

115.

Rudinger J, Puglisi JD, Pütz J. et al. Determinant nucleotides of yeast tRNA^Asp interact directly with aspartyl-tRNA synthetase. Proc Natl Acad Sci USA. 1992;89:5882–5886. [PMC free article: PMC49401] [PubMed: 1631068]

116.

Pütz J, Florentz C, Benseler F. et al. A single methyl group prevents the mischarging of a tRNA. Nat Struct Biol. 1994;1:580–582. [PubMed: 7634096]

117.

Ibba M, Losey HC, Kawarabayasi Y. et al. Substrate recognition by class I lysyl-tRNA synthetase: A molecular basis for gene displacement. Proc Natl Acad Sci USA. 1999;96:418–423. [PMC free article: PMC15151] [PubMed: 9892648]

118.

Rudinger J, Hillenbrandt R, Sprinzl M. et al. Antideterminants present in minihelix^Sec hinder its recognition by prokaryotic elongation factor Tu. EMBO J. 1996;15:650–657. [PMC free article: PMC449983] [PubMed: 8599948]

119.

Mohan A, Whyte S, Wang X. et al. The 3' end CCA of mature tRNA is an antideterminant for eukaryotic 3'-tRNase. RNA. 1999;5:245–256. [PMC free article: PMC1369756] [PubMed: 10024176]

120.

Rogers MJ, Söll D. Discrimination between glutaminyl-tRNA synthetase and seryl-tRNA synthetase involves nucleotides in the acceptor helix of tRNA. Proc Natl Acad Sci USA. 1988;85:6627–6631. [PMC free article: PMC282030] [PubMed: 3045821]

121.

Jahn M, Rogers MJ, Söll D. Anticodon and acceptor stem nucleotides in tRNA^Gln are major recognition elements for E. coli glutaminyl-tRNA synthetase. Nature. 1991;352:258–260. [PubMed: 1857423]

122.

Rogers MJ, Adachi T, Inokuchi H. et al. Switching tRNA^Gln identity from glutamine to tryptophan. Proc Natl Acad Sci USA. 1992;89:3463–3467. [PMC free article: PMC48888] [PubMed: 1565639]

123.

Sherman JM, Thomann H-U, Söll D. Functionnal connectivity between tRNA binding domains in glutaminyl-tRNA synthetase. J Mol Biol. 1996;256:818–828. [PubMed: 8601833]

124.

Hong K-W, Ibba M, Weygand-Durasevic I. Transfer RNA-dependant cognate amino acid recognition by an aminoacyl-tRNA-synthetase. EMBO J. 1996;15:1983–1991. [PMC free article: PMC450117] [PubMed: 8617245]

125.

Liu J, Ibba M, Hong K-W. et al. The terminal adenosine of tRNA^Gln mediates tRNA-dependent amino acid recognition by glutaminyl-tRNA synthetase. Biochemistry. 1998;37:9836–9842. [PubMed: 9657697]

126.

Ibba M, Hong K-W, Sherman JM. et al. Interactions between tRNA identity nucleotides and their recognition sites in glutaminyl-tRNA synthetase determine the cognate amino acid affinitiy of the enzyme. Proc Natl Acad Sci USA. 1996;93:6953–6958. [PMC free article: PMC38915] [PubMed: 8692925]

127.

Mandal AK, Bhattacharyya A, Bhattacharyya S. et al. A cognate tRNA specific conformational change in glutaminyl-tRNA synthetase and its implication for specificity. Protein Science. 1998;7:1046–1051. [PMC free article: PMC2143984] [PubMed: 9568911]

128.

Sherman JM, Rogers MJ, Söll D. Competition of aminoacyl-tRNA synthetases for tRNA ensures the accuracy of aminoacylation. Nucleic Acids Res. 1992;20:2847–2852. [PMC free article: PMC336931] [PubMed: 1377381]

129.

Mirande M. Aminoacyl-tRNA synthetase family from prokaryotes and eukaryotes: Structural domains and their implications. Prog Nucleic Acid Res Mol Biol. 1991;40:95–142. [PubMed: 2031086]

130.

Whelihan EF, Schimmel P. Rescuing an essential enzyme-RNA complex with a non-essential appended domain. EMBO J. 1997;16:2968–2974. [PMC free article: PMC1169904] [PubMed: 9184240]

131.

Frugier M, Moulinier L, Giegé R. A domain in the N-terminal extension of class IIb eukaryotic aminoacyl-tRNA synthetases is important for tRNA binding. EMBO J. 2000;19:2371–2380. [PMC free article: PMC384352] [PubMed: 10811628]

132.

Chihade JW, Schimmel P. Assembly of a catalytic unit for RNA microhelix aminoacylation using nonspecific RNA binding domains. Proc Natl Acad Sci USA. 1999;96:12316–12321. [PMC free article: PMC22914] [PubMed: 10535919]

133.

Simos G, Sauer A, Fasiolo F. et al. A conserved domain within Arc1p delivers tRNA to aminoacyl-tRNA synthetases. Mol Cell. 1998;1:235–242. [PubMed: 9659920]

134.

Nomanbhoy T, Morales AJ, Abraham AT. et al. Simultaneous binding of two proteins to opposite sides of a single transfer RNA. Nat Struct Biol. 2001;8:344–348. [PubMed: 11276256]

135.

LaRiviere FJ, Wolfson AD, Uhlenbeck OC. Uniform binding of aminoacyl-tRNAs to elongation factor Tu by thermodynamic compensation. Science. 2001;294:165–168. [PubMed: 11588263]

136.

Khvorova AM, Motorin Y, Wolfson AD. Pyrophosphate mediates the effect of certain tRNA mutations on aminoacylation of yeast tRNA^Phe. Nucleic Acids Res. 1999;27:4451–4456. [PMC free article: PMC148729] [PubMed: 10536155]

137.

Hayashi I, Kawai G, Watanabe K. Higher-order structure and thermal instability of bovine mitochondrial tRNA^SerUGA investigated by proton NMR spectroscopy. J Mol Biol. 1998;284:57–69. [PubMed: 9811542]

138.

Hayashi I, Yokogawa T, Kawai G. et al. Assignment of imino proton signals of G-C base pairs and magnesium ion binding: an NMR study of bovine mitochondrial tRNA^SerGCU lacking the entire D arm. J Biochem. 1997;121:1115–1122. [PubMed: 9354385]

139.

Börner GV, Mörl M, Janke A. et al. RNA editing changes the identity of a mitochondrial tRNA in marsupials. EMBO J. 1996;15:5949–5957. [PMC free article: PMC452375] [PubMed: 8918472]

140.

Ueda T, Yotsumoto Y, Ikeda K. et al. The T-loop region of animal mitochondrial tRNA^Ser(AGY) is a main recognition site for homologous seryl-tRNA synthetase. Nucleic Acids Res. 1992;20:2217–2222. [PMC free article: PMC312334] [PubMed: 1375735]

141.

Yokogawa T, Shimada N, Takeuchi N. et al. Characterization and tRNA recognition of mammalian mitochondrial seryl-tRNA synthetase. J Biol Chem. 2000;275:19913–19920. [PubMed: 10764807]

142.

Shimada N, Suzuki T, Watanabe K. Dual mode of recognition of two isoacceptor tRNAs by mammalian mitochondrial seryl-tRNA synthetase. J Biol Chem. 2001;276:46770–46778. [PubMed: 11577083]

143.

Saks ME, Sampson JR. Variant minihelix RNAs reveal sequence-specific recognition of the helical tRNA^Ser acceptor stem by E. coli seryl-tRNA synthetase. EMBO J. 1996;15:2843–2849. [PMC free article: PMC450222] [PubMed: 8654382]

144.

Florentz C, Sissler M. chapter 8 of this book.

145.

Commans S, Böck A. Selenocysteine inserting tRNAs: an overview. FEMS Microbiology Reviews. 1999;23:335–351. [PubMed: 10371037]

146.

Zwieb C, Guven SA, Wower IK. et al. Three-dimensional folding of the tRNA-like domain of Escherichia coli tmRNA. Biochemistry. 2001;40:9587–9595. [PubMed: 11583158]

147.

Komine Y, Kitabatake M, Yokogawa T. et al. A tRNA-like structure is present in 10Sa RNA, a small stable RNA from Escherichia coli. Proc Natl Acad Sci USA. 1994;91:9223–9277. [PMC free article: PMC44784] [PubMed: 7524073]

148.

Pinck M, Yot P, Chapeville F. et al. Enzymatic binding of valine to the 3' end of TYMV RNA. Nature. 1970;226:954–956. [PubMed: 4315653]

149.

Dreher TW, Bujarski JJ, Hall TC. Mutant viral RNAs synthesized in vitro show altered aminoacylation and replicase template activities. Nature. 1984;311:171–175. [PubMed: 6472477]

150.

Mans MW, Pleij CWA, Bosch L. tRNA-like structures. Structure, function and evolutionary significance. Eur J Biochem. 1991;201:303–324. [PubMed: 1935928]

151.

Florentz C, Giegé R. tRNA-like structures in viral RNAs In: Söll D, RajBhandary UL, eds.tRNA: Structure, Biosynthesis, and Function Washington, DC: Am Soc Microbiol Press,1995141–163.

152.

Fechter P, Rudinger-Thirion J, Florentz C. et al. Novel features in the tRNA-like world of plant viral RNAs. CMLS, Cellular Mol Life Sciences. 2001;58:1547–1561. [PubMed: 11706983]

153.

Pleij CWA, Rietveld K, Bosch L. A new principle of folding based on pseudoknotting. Nucleic Acids Res. 1985;13:1717–1731. [PMC free article: PMC341107] [PubMed: 4000943]

154.

Fromant M, Plateau P, Blanquet S. Function of the extra 5'-phosphate carried by histidine tRNA. Biochemistry. 2000;39:4062–4067. [PubMed: 10747795]

155.

Dreher TW. Functions of 3'-untranslated regions of positive strand RNA viral genomes. Annu Rev Phytopathol. 1999;37:151–174. [PubMed: 11701820]

156.

Florentz C, Dreher TW, Rudinger J. et al. Specific valylation identity of turnip yellow mosaic virus RNA by yeast valyl-tRNA synthetase is directed by the anticodon in a kinetic rather than affinity-based discrimination. Eur J Biochem. 1991;195:229–234. [PubMed: 1991471]

157.

Dreher TW, Tsai C-H, Florentz C. et al. Specific valylation of turnip yellow mosaic virus RNA by wheat germ valyl-tRNA synthetase is determined by three anticodon loop nucleotides. Biochemistry. 1992;31:9183–9189. [PubMed: 1390705]

158.

Wientges J, P¨tz J, Giegé R. et al. Selection of viral RNA-derived tRNA-like structures with improved valylation activities. Biochemistry. 2000;39:6207–6218. [PubMed: 10821696]

159.

Fechter P, Giegé R, Rudinger-Thirion J. Specific tyrosylation of the bulky tRNA-like structure of Brome Mosaic Virus RNA relies solely on identity nucleotides present in its amino acid accepting stem. J Mol Biol. 2001;309:387–399. [PubMed: 11371160]

160.

Fechter P, Rudinger-Thirion J, Théobald-Dietrich A. et al. Identity of tRNA for yeast tyrosyl-tRNA synthetase: Tyrosylation is more sensitive to identity nucleotides than to structural features. Biochemistry. 2000;39:1725–1733. [PubMed: 10677221]

161.

Giegé R, Frugier M, Rudinger J. tRNA mimics. Curr Opin Struct Biol. 1998;8:286–293. [PubMed: 9666323]

162.

Perret V, Florentz C, Puglisi JD. et al. Effect of conformational features on the aminoacylation of tRNAs and consequences on the permutation of tRNA specificities. J Mol Biol. 1992;226:323–333. [PubMed: 1640453]

163.

Nissan TA, Oliphant B, Perona JJ. An engineered class I transfer RNA with a class II tertiary fold. RNA. 1999;5:434–445. [PMC free article: PMC1369771] [PubMed: 10094311]

165.

Nissan TA, Perona JJ. Alternative designs for construction of the class II transfer RNA tertiary core. RNA. 2000;6:1585–1596. [PMC free article: PMC1370028] [PubMed: 11105758]

165.

Frugier M, Florentz C, Schimmel P. et al. Triple aminoacylation specificity of a chimerized transfer RNA. Biochemistry. 1993;32:14053–14061. [PubMed: 8268184]

166.

Puglisi JD, Pütz J, Florentz C. et al. Influence of tRNA tertiary structure and stability on aminoacylation by yeast aspartyl-tRNA synthetase. Nucleic Acids Res. 1993;21:41–49. [PMC free article: PMC309063] [PubMed: 8441619]

167.

Rudinger-Thirion J, Giegé R. The peculiar architectural framework of tRNA^Sec is fully recognized by yeast AspRS. RNA. 1999;5:495–502. [PMC free article: PMC1369776] [PubMed: 10199566]

168.

Vörtler S, Pütz J, Giegé R. Manipulation of tRNA properties by structure-based and combinatorial in vitro approaches. Prog Nucleic Acid Res Mol Biol. 2001;70:291–334. [PubMed: 11642365]

169.

Tinkle Peterson E, Pan T, Coleman J. et al. In vitro selection of small RNAs that bind to Escherichia coli phenylalanyl-tRNA synthetase. J Mol Biol. 1994;242:186–192. [PubMed: 8089840]

170.

Wolfson AD, Khvorova AM, Sauter C. et al. Mimics of yeast tRNA^Asp and their recognition by aspartyl-tRNA synthetase. Biochemistry. 1999;38:11926–11932. [PubMed: 10508395]

171.

Renaud M, Ehrlich R, Bonnet J. et al. Lack of correlation between affinity of the tRNA for the aminoacyl-tRNA synthetase and aminoacylation capacity as studied with modified tRNA^Phe. Eur J Biochem. 1979;100:157–164. [PubMed: 385310]

172.

Schimmel P, Giegé R, Moras D. et al. An operational RNA code for amino acids and possible relationship to genetic code. Proc Natl Acad Sci USA. 1993;90:8763–8768. [PMC free article: PMC47440] [PubMed: 7692438]

173.

Ribas de Pouplana L, Schimmel P. Aminoacyl-tRNA synthetases: potential markers of genetic code development. Trends Biochem Sci. 2001;26:591–596. [PubMed: 11590011]

174.

Ibba M, Söll D. Aminoacyl-tRNA synthesis. Annu Rev Biochem. 2000;69:617–650. [PubMed: 10966471]