Aminoacyl-tRNA Synthetases: General Features and Relationships

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Chapter 58

SUSAN A. MARTINIS and PAUL SCHIMMEL

INTRODUCTION

Overview

The aminoacyl-tRNA synthetases are an ancient family of essential proteins (38a, 129, 192). They act as interpreters of the genetic code by relating amino acids to specific trinucleotides through the aminoacylation reaction. All 21 tRNA synthetases (one for each amino acid except lysine, which has two) have been cloned from Escherichia coli and sequenced (145). The sequences show clearly that the enzymes can be divided into two classes based on characteristic motifs found in the catalytic domains of members of the same class (36, 69). A second domain of these enzymes is not conserved, even for tRNA synthetases of the same class. The relationship of these tRNA synthetase domains to the two domains of the L-shaped tRNA molecule suggests a model for the historical development of tRNA synthetases and the genetic code (32). In addition, comparisons of the E. coli enzymes with their counterparts in higher eukaryotes (including humans) suggest that some enzymes evolved along distinctly different paths (see below).

Abbreviations

Individual tRNA synthetases are identified by the three-letter amino acid code. For example, alanyl-tRNA synthetase is represented by Ala-tRNA synthetase. Specific mutations are identified by single-letter amino acid code for the wild-type residue followed by its position and the substituted amino acid. For example, alanine substituted by glycine at position 50 is indicated by A50G. The "Ψ" symbol represents pseudouridine.

Protein Synthesis

The tRNA synthetases catalyze the initial step of protein synthesis by covalently linking an amino acid to its cognate tRNA (196). The reaction is carried out in a two-step mechanism which is energetically driven by consumption of ATP:

amino acid + ATP ↔ AMP-amino acid + PP_i

AMP-amino acid + tRNA ↔ tRNA-amino acid + AMP

In the first step, the high-energy bond of ATP is hydrolyzed to form an activated aminoacyl adenylate complex and PP_i. The amino acid is then transferred to the 3' terminus of the tRNA, forming "charged" tRNA and releasing AMP.

The charged tRNA acts as a shuttle by delivering the amino acid to the ribosome, an RNA scaffold where specific macromolecules converge for protein translation, including mRNA which serves as a template for polypeptide synthesis (157). The process is initiated by a unique initiator tRNA (tRNA^fMet) charged with N-formylmethionine (89, 125). The fMet-tRNA^fMet is bound to the so-called peptidyl site (P-site) of the ribosome. Subsequently, charged tRNAs bind to the aminoacyl site (A-site). Peptide bonds are formed by the transfer of the peptidyl moiety from the tRNA in the P-site to the amino group of the charged tRNA in the A-site. The resultant peptidyl-tRNA is then translocated from the A-site to the P-site so that another charged tRNA can bind to the A-site for the process to be repeated and the polypeptide extended (161). Thus, the sequence of the nascent polypeptide is dependent on which amino acid is associated with which tRNA. This association is determined by amino acid and tRNA specificities of the aminoacyl-tRNA synthetases.

Genetic Code

The typical tRNA is composed of 76 nucleotides which terminate at the 3' end in an NCCA-76 single-stranded tetranucleotide (213). The N-73 nucleotide is referred to as the discriminator base and can be any of the four bases (55). The RNA sequence can be arranged into a cloverleaf secondary structure because of self-complementary base pairs. As shown in Fig. 1A, characteristic features of the secondary structure include the TΨC loop, D (dihydrouridine) loop, and anticodon loop (123, 179, 180). These loops are attached respectively to short helical sections designated the TΨC stem, D stem, and anticodon stem. The cloverleaf secondary structure folds into an L-shaped tertiary structure which divides the molecule into two domains (122, 183). One domain is composed of a minihelix which contains the 12-bp amino acid acceptor/TΨC stem helix which is capped by the TΨC loop. The second domain fuses the D stem and loop with the anticodon stem and loop and contains the anticodon trinucleotide. The "corner" of the molecule brings together the D and TΨC loops and is stabilized in part by highly specific, novel pairing interactions between bases.

Fig. 1tRNA. (A) The secondary structure of tRNA is illustrated on the left in the cloverleaf form. The right structure depicts the tertiary folding of the tRNA. (B) RNA hairpin minihelix substrate for aminoacylation.

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On the ribosome, the genetic code is "decoded" by RNA-RNA base-pairing interactions between the tRNA anticodon nucleotide triplet and the complementary mRNA codon. Typically, the first two bases of the mRNA triplet codon dictate the amino acid identity, while the third base can be varied to any of the four nucleotides. For serine, leucine, and arginine, there are six distinct codons. Only methionine and tryptophan are represented by a single triplet codon. The number of tRNAs which read the 61 sense codon triplets is less than 61. Because of the wobble interaction at the third position of the codon-anticodon interaction, the number of tRNA isoacceptors is reduced (54). With the wobble interaction, a G in the first position of the anticodon pairs with a C or U in the third position of the codon triplet, and a first-position anticodon U can pair with G or A.

A single tRNA synthetase aminoacylates all of the tRNA isoacceptors specific for the cognate amino acid. Perhaps because of anticodon sequence variations in the tRNA isoacceptor, and because anticodon recognition was a later development in the evolution of aminoacylation systems, some tRNA synthetases do not utilize the anticodon for tRNA discrimination (193). The five isoacceptors which read the six serine codons collectively permute nucleotides at all positions of the anticodon triplet, so that the anticodon per se offers no basis for discrimination. The structure of the cocrystal of Thermus thermophilus Ser-tRNA synthetase with tRNA^Ser shows no protein-RNA contacts at the anticodon (17, 56). Likewise, the anticodon appears dispensable for recognition by Leu-tRNA synthetase (7). On the other hand, there are four alanine codons (GCN) which are read by two tRNA^Ala isoacceptors. The GC anticodon dinucleotide is unique to tRNA^Ala, and therefore Ala-tRNA synthetase could in principle distinguish tRNA^Ala from all others based on this dinucleotide. However, the anticodon is not required for aminoacylation with alanine (81), and RNA footprinting shows that the enzyme makes no contact with the anticodon (163).

Thus, for alanine, leucine, and serine, the genetic code triplet itself has no direct role in specifying the amino acid. For other tRNAs, the anticodon triplet plays a role in determining aminoacylation specificity and efficiency, although in many of these instances specific aminoacylation can be achieved with partial tRNAs that lack the anticodon. In these cases, RNA minihelix or microhelix substrates, composed of just the acceptor-TΨC stem-loop or acceptor stem, respectively, are substrates for aminoacylation (Fig. 1B) (81, 83, 140, 153, 173a). Aminoacylation specificity is determined by two to four nucleotides in these small RNA substrates. These observations have led to the concept of an operational RNA code for amino acids whereby sequences or structures in tRNA acceptor stems are related to specific amino acids (60a, 194, 195a). This operational RNA code may have originated in an RNA world or during the early part of the transition from the RNA world to the theater of proteins (see below).

SEQUENCES, STRUCTURES, AND CLASSIFICATION OF tRNA SYNTHETASES

Gene Isolation

The elucidation of the primary sequences of each of the 20 tRNA synthetases from different organisms has been crucial for establishing evolutionary relationships among the entire family of enzymes. The first complete sequence was determined for Bacillus stearothermophilus Trp-tRNA synthetase by classical methods of peptide mapping which involved proteolytic cleavage, followed by peptide isolation and sequencing (228). Rapid progress was not made in obtaining more sequences until genes were cloned by recombinant DNA methods and the Maxam-Gilbert chemical (141) and Sanger enzymatic (191) methods of DNA sequencing were developed. These DNA sequencing methods in early work were combined with partial sequencing of internal portions of the proteins themselves to establish a "reading-frame check" on the translated DNA sequence. For example, mass spectrometry was used to establish randomly located oligopeptide sequences. Alternatively, the masses of oligopeptides were determined, and these could be placed on the translated DNA sequence at scattered, unique locations (84, 170, 224). With improvements in the reliability of DNA sequencing, most tRNA synthetase amino acid sequences were eventually obtained solely by translation of the gene sequences.

Many of the tRNA synthetase genes were obtained by complementation using strains that were deficient in a tRNA synthetase activity. Table 1 lists the missense and deletion alleles obtained in E. coli. These mutant alleles may be divided into four categories: auxotrophic, temperature-sensitive, analog-resistant, and large or complete deletions (173, 196). Auxotrophic, temperature-sensitive, and analog-resistant mutant enzymes have been isolated by conventional methods. The mutant strains can be used as hosts to isolate genes whose protein products can complement or functionally substitute for the host’s deficient tRNA synthetase. Specifically, libraries of genomic DNA cloned into plasmid or phage vectors are used to transform the mutant host strain. If the mutant host strain is temperature sensitive, for example, growth at the restrictive temperature would indicate expression of a functional tRNA synthetase from the introduced plasmid or phage. The complemented strain can then be isolated and the plasmid DNA recovered to obtain the clone for further manipulation and investigation.

Table 1E. coli aminoacyl-tRNA synthetase mutant and null strains

Deletion or null alleles were created by site-specific recombination between a linear DNA fragment and a homologous region on the chromosome (110, 217). Specifically, an antibiotic resistance marker was sandwiched between DNA sequences homologous to the flanking regions on the chromosome of the tRNA synthetase gene of interest. Double reciprocal recombination replaced the target tRNA synthetase gene with the linear fragment. The null alleles are sustained by an extrachromosomal maintenance plasmid which encodes the missing essential gene. Most typically, this maintenance plasmid has a temperature-sensitive replicon, so that the resulting null strain is temperature sensitive. These null strains have been particularly useful for testing the activity of a mutant tRNA synthetase on a second, compatible plasmid. In particular, complications arising from an introduced mutant enzyme interacting in trans with a chromosomally encoded temperature-sensitive or auxotrophy-conferring mutant enzyme can be avoided (109).

As multiple sequences of the same tRNA synthetase across different species were determined, relationships were established which enabled the design of degenerate primers to isolate tRNA synthetases from new organisms by PCR (203, 206, 207). This approach has proven to be a powerful technique for cloning tRNA synthetase genes from a range of eukaryotic and prokaryotic organisms but is dependent on knowing a set of related primary sequences.

Primary Structures

The primary sequence for each of the tRNA synthetases from E. coli has been determined (Table 2) (145). With one exception, all of the tRNA synthetases are represented by a single copy of the gene. In the E. coli genome, separate constitutive (S) and thermoinducible (U) genes for two distinct Lys-tRNA synthetases are encoded (48, 116, 134, 190a).

Table 2Aminoacyl-tRNA synthetases from E. colia

The family of tRNA synthetases has been divided into two classes based on mechanism and on sequence and structural homology (36, 69). With one exception, class I tRNA synthetases initially charge the 2' hydroxyl, while the 3' position is aminoacylated by enzymes from the second class (94, 151, 210). (Phe-tRNA synthetase is an exception in that it was reported to charge the 2' position.) Between and among the two classes, Table 2 emphasizes the diversity of sizes and quaternary structures for the members of this family of enzymes. Polypeptide chain lengths range from 334 amino acids for Trp-tRNA synthetase (90) to 951 amino acids for Val-tRNA synthetase (91, 95). Class I contains monomeric and homodimeric enzymes. Class II has mostly homodimers. In addition, the class II Gly-tRNA (224) and Phe-tRNA (75, 144) synthetases are distinguished by their α ₂ β ₂ quaternary structure, and Ala-tRNA synthetase is an α ₄ tetramer (108).

Table 2 also defines a series of subclasses within each class of the tRNA synthetases (127a, 151). These groupings are based on related primary sequence, organization of class-defining structural features, similarities in the anticodon binding domains, and mechanistic distinctions. Three enzymes of class II cannot be easily categorized. The most notable exception is Phe-tRNA synthetase, which contains some class-defining sequence but is reported to aminoacylate the 2' rather than the 3' hydroxyl and has an α ₂ β ₂ quaternary structure (75, 94, 144, 151, 210). Although E. coli Gly-tRNA synthetase has a similar quaternary structure and also lacks at least one of the distinct class II sequence motifs, it is unusual because in other organisms, the glycine enzymes are homodimers (145), contain all three sequence motifs (see below), and most closely align with subclass A.

Critical Sequence Motifs and Classifications

Class I.

The diversity and sizes within the family of tRNA synthetases imposed difficulties for establishing sequence alignments between the enzymes. The primary sequences not only were diversified but had tRNA synthetase-specific deletions and insertions. The first connection between the enzymes was derived from the tertiary crystal structures of the native B. stearothermophilus Tyr-tRNA synthetase and the 547-amino-acid N-terminal fragment of E. coli Met-tRNA synthetase, which revealed similar nucleotide binding folds in each catalytic domain (see below) (181, 235). This structural overlap was used to locate a short homologous peptide with four conserved residues (HIGH) between Met- and Tyr-tRNA synthetases (12, 13, 20). The tetrapeptide is located between a β strand and α helix in the first half of the nucleotide binding fold. However, this peptide was too short to be useful for finding statistically significant sequence relationships between other tRNA synthetases.

When the 939-amino-acid E. coli Ile-tRNA synthetase was sequenced, an 11-amino-acid match that ended in HIGH was found between Ile- and Met-tRNA synthetases (212, 225). Whereas the 4-amino-acid HIGH sequence was too short for meaningful database searches, the overall length of the 11-amino-acid signature sequence provided a threshold for statistical reliability. Significant homology was also apparent with E. coli Gln-tRNA synthetase. The sequence of Saccharomyces cerevisiae Gln-tRNA synthetase was subsequently shown to have an exact 15-amino-acid match with the E. coli enzyme in a segment that encompasses the entire 11-amino-acid signature sequence (138). The two proteins otherwise have an overall sequence identity of about 40%. The a priori probability of an exact match of 15 amino acids is vanishingly small. Thus, the primary structure of the eukaryotic yeast glutamine enzyme provided further evidence for disproportionate selective pressure on the signature sequence region and strengthened the rationale for using this element as a basis for identifying proteins as tRNA synthetases.

A second conserved sequence between E. coli Tyr- and Met-tRNA synthetases was found from cross-linking studies between these enzymes and 3' oxidized derivatives of the cognate tRNAs (103, 104). Both of these tRNA synthetases were covalently labeled at a specific lysine residue which is implied to be proximal to the acceptor stem binding site for the tRNA. Comparison to the available crystal structures for B. stearothermophilus Tyr-tRNA synthetase and E. coli Met-tRNA synthetase showed that each lysine residue was in the second half of the nucleotide binding fold. A primary sequence alignment of this region in Tyr-, Met-, Ile-, and Trp-tRNA synthetases determined that the lysine as well as the sequence around it was homologous (104). With the exception of Tyr-tRNA synthetase, a pentapeptide, KMSKS, was completely conserved. In subsequent investigations, the covalently labeled lysine residue, Lys-335 in Met-tRNA synthetase, was substituted by site-directed mutagenesis and resulted in decreased activity (143). Kinetic studies indicated that the side chain amino group stabilized the transition state in the formation of methionyl adenylate.

By 1987, the two class-defining polypeptide sequences were well defined (192). Although the KMSKS region is not as highly conserved as the HIGH region of the signature sequence, where present, the KMSKS region is always accompanied by the signature sequence which ends in HIGH. All of the tRNA synthetases from E. coli have now been sequenced, and half of them contain these two critical sequences (127a, 145, 151). Although the signature sequence and KMSKS pentapeptide are in opposite halves of the nucleotide binding fold, both are located in the active site.

Class II.

The other 10 tRNA synthetases lack the signature sequence and KMSKS pentapeptide. Sequences of some of these unrelated tRNA synthetases, including Ala-tRNA (170), Gly-tRNA (224), His-tRNA (84), and yeast Asp-tRNA (70) synthetases, were known before all of those in class I had been obtained. But it was not until the gene for E. coli Pro-tRNA synthetase was sequenced (69) that clear relationships emerged, which established a distinct second class of tRNA synthetases.

Sequence comparisons determined that Pro-tRNA synthetase was most related to Thr- and Ser-tRNA synthetases, albeit with conserved sequences which were highly degenerate (69). However, a systematic pairwise analysis was carried out between these three proteins as well as the other tRNA synthetases that lacked the class I-defining sequences. Between several pairs of enzymes, three regions of homology were delineated. These motifs are designated in the order they occur in the sequence as motif 1, motif 2, and motif 3. Although the motifs have a conserved core, they vary in length and are marked by as little as a single invariant residue. The motif sequences are defined as follows: motif 1, gφxxφxxPφφ; motif 2, (F/Y/H)Rx(E/D)(4–12x)(R/H)xxxFxxx(D/E); and motif 3, λxφgφgφeRφφφφφ. (The abbreviations are as follows: x, variant; φ, hydrophobic; and λ, small amino acids. Lowercase letters indicate that the amino acid is partially conserved. None of these motifs are found in the class I family.) With the exception of E. coli Gly- and Phe-tRNA synthetases, which only contain a discernible motif 3, all enzymes incorporate all three motifs (178).

Ser- and Asp-tRNA synthetases are both class II enzymes. Analysis of their crystal structures showed a design completely distinct from that of class I enzymes (15, 17, 56, 57, 60, 151, 189). In particular, the active-site region consists of an eight-stranded β structure with three α helices. The conserved sequence motifs form a helix-loop-strand (motif 1), strand-loop-strand (motif 2), and strand-helix (motif 3). Residues in motifs 2 and 3 are directly involved in substrate interactions in the catalytic site.

Structures and Class-Defining Catalytic Cores

Crystal structures have been reported for 10 different aminoacyl-tRNA synthetases, with five from each class (Table 3). These include cocrystals having one or more of the three substrates (ATP, amino acid, and tRNA) of the aminoacylation reaction. Five of the structures include E. coli proteins, and the others are proteins from T. thermophilus, B. stearothermophilus, and the yeast S. cerevisiae. The structures of the T. thermophilus and S. cerevisiae Asp-tRNA synthetases are close homologs of the E. coli counterpart. Likewise, the T. thermophilus Ser-tRNA synthetase is closely similar in structure to that of the E. coli protein. The similarities in structures for a given tRNA synthetase across species often include regions which diverge significantly in sequence, thus demonstrating the diversity of sequences which can accommodate a given structural design.

Table 3Crystallization and structures of tRNA synthetases

Modular Arrangement of Domains in Both Classes of Synthetases

Although the structures of the class-defining catalytic domains are completely distinct for enzymes in the two classes (see below), enzymes in both classes have the same general overall organization. This organization consists of two major domains (Fig. 2). The two major domains are defined by the structures themselves and are seen as discrete structural units which may incorporate subdomains within a global overall structure. The class-defining catalytic domain occurs in either the N-terminal or C-terminal half of the protein. Insertions into that domain contain residues which enable the tRNA acceptor stem to dock near the activated amino acid.

Fig. 2Domains of a tRNA synthetase. Each tRNA synthetase consists of two distinct domains. One contains the catalytic core and sequence insertions which interact with the acceptor stem of the cognate tRNA. The second domain is highly diverse and idiosyncratic for each enzyme and provides for interactions outside the acceptor stem.

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The second domain is joined to the class-defining catalytic domain, and this domain is idiosyncratic to the enzyme; that is, even members of the same class have completely different second domains. This domain provides for interactions with parts of the tRNA outside of the acceptor-TΨC minihelix, including interactions with the anticodon or the variable loop. In the discussion below, some of the structural details of the catalytic domains of enzymes of both classes are discussed along with some of the structural features of the idiosyncratic domains of particular tRNA synthetases.

Class I.

The first crytallographic and X-ray diffraction investigation for an aminoacyl-tRNA synthetase was reported for Tyr-tRNA synthetase from B. stearothermophilus (106, 149, 175). An active trypsin-cleaved product of E. coli Met-tRNA synthetase was also crystallized at this time (222), but preliminary X-ray diffraction analysis was not completed until the next decade (181). These early structures provided a trace of the polypeptide chain including information about the topology of the protein tertiary structure, subunit arrangements, and even sites of substrate binding. However, resolution was limited by lack of a primary sequence.

The genes for B. stearothermophilus Tyr-tRNA synthetase (227) and E. coli Met-tRNA synthetase (12, 58) were subsequently cloned and sequenced. The X-ray crystal structures were reinvestigated, leading to more detailed information about the active sites and potential relationships that exist between the tRNA synthetases. In particular, a structural motif consisting of a six-stranded parallel β sheet with four intervening α helices (Fig. 3A) was found in both the Tyr-tRNA (16) and Met-tRNA (20) synthetase X-ray crystal structures. This motif resembled a nucleotide binding domain that is also called a Rossmann fold (34). This fold was first discovered in the dehydrogenases and later in the kinases and other proteins that bind nucleotides such as ATP, GTP, and NADH (184). Cocrystallization of Tyr-tRNA synthetase with tyrosyl adenylate determined that the aminoacylation reaction intermediate was bound in the suspected nucleotide binding pocket similar to the nicotinamide orientation in the dehydrogenases (27).

Fig. 3Catalytic domain structures of the tRNA synthetases. The arrows and cylinders represent ? strands and ? helices, respectively. Dotted lines indicate sequence or structural features which were omitted for clarity because they are extraneous to the actual active-site pocket. (A) Class I tRNA synthetase based on the methionine enzyme (29). The class I enzymes contain a parallel ? sheet with intervening ? helices similar to a Rossmann nucleotide binding fold. The long dashed lines represent CP1 or CP2 insertions. In this depiction of the structure, all elements of secondary structure are shown except for those in CP1 and CP2, a short ? strand (shown dotted) in the region of the KMSKS sequence, and two ? helices (shown dotted) located between the end of CP1 and the immediately following ? strand. The signature sequence (HIGH) and the conserved KMSKS region are marked. (B) Class II enzyme based on the Asp-tRNA synthetase catalytic domain (186). The class II active site is centered around a ? sheet (that is primarily antiparallel) and three ? helices. Motifs 1, 2, and 3 are as indicated.

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The two-domain structure of E. coli Met-tRNA synthetase was evident in the high-resolution structure (30). The N-terminal 360 amino acids contain the class-defining sequence motifs and active site. This domain is joined to a C-terminal structural unit which has a high proportion of α helices. The class-defining active-site domain of Tyr-tRNA synthetase is also in the N-terminal part of the protein and extends approximately to residue 220 (27). The second domain was not sufficiently ordered in the crystal to determine its structure.

More recently, the X-ray cocrystal structure for E. coli Gln-tRNA synthetase with ATP and tRNA^Gln was solved to a resolution of 2.8 Å (1 Å = 0.1 nm) (185). A nucleotide binding fold similar to that in the Tyr- and Met-tRNA synthetase structures was found in the N-terminal half of the protein. The nucleotide binding domain is folded into two halves. The first half primarily interacts with the adenine moiety of ATP through hydrogen bond contacts with the peptide backbone of three β strands and two α helices. The remaining half of the Rossmann fold-like structure also consists of three β strands and two α helices. This half, as well as a sequence called CP1 (connective polypeptide 1; see below), which is inserted between the two halves of the Rossmann fold, binds the acceptor stem of tRNA^Gln through sequence-specific contacts. The second domain follows a helical subdomain and consists of a multistranded β barrel.

Seven of the ten class I E. coli tRNA synthetases are monomers. The monomeric structure is consistent with all tRNA binding determinants seen in the Gln-tRNA synthetase-tRNA^Gln complex being contained within one polypeptide (164, 185). The exceptions to the monomer rule are Met-, Trp-, and Tyr-tRNA synthetases, each of which is an α ₂ dimer. In the case of Met-tRNA synthetase, a C-terminal deletion removes the oligomerization domain and yields a monomeric protein which is active in vivo and in vitro (42, 58). Thus, this enzyme can be viewed as operationally equivalent to other class I monomeric enzymes. However, the dimeric structure of Tyr-tRNA synthetase is needed for activity, because the bound tRNA bridges across the subunit interface (14, 27, 41). The cognate tRNA for Trp-tRNA synthetase is also expected to span the dimeric interface, utilizing separate subunits for anticodon and acceptor stem binding (64). Although the cocrystal structure has not been resolved, the crystal structure for the dimerized Trp-tRNA synthetase shows that each monomer is only about 40 Å long. A monomer would not simultaneously accommodate the tRNA 3' terminus and anticodon because they are separated by approximately 75 Å.

Class II.

The second class of tRNA synthetases was firmly defined when the crystal structure of the E. coli Ser-tRNA synthetase active site was shown to have no relationship to the Rossmann fold of class I enzymes (57). X-ray diffraction investigations with an ATP-bound Ser-tRNA synthetase cocrystal from T. thermophilus displayed the details of a novel ATP binding site (Fig. 3B) (15, 17, 56). The pocket is composed of an eight-stranded antiparallel β sheet that is flanked by α helices. An arginine at the N-terminal side of the loop of motif 2, and which is almost universally conserved among the class II tRNA synthetases, forms a salt bridge with the α-phosphate of ATP, while the adenine ring is stabilized by a stacking interaction with a phenylalanine in the second β strand of motif 2. A well-conserved carboxyl side chain of glutamic acid in the loop of motif 2 interacts with the adenine ring through a hydrogen bond with N-6 and also via a water molecule with N-7.

Motif 3 is comprised of a β strand followed by an α helix and is characterized by a GLER sequence. This motif is the only one that has been universally detected in all of the class II enzymes. The crystal structures of Ser-tRNA (15, 17, 56) and Asp-tRNA (189) synthetases implicate its role in amino acid and ATP binding. Mutations in this region resulted in a reduction in binding and/or a high K_m for amino acid or ATP (5, 70, 113, 115, 128).

Yeast Asp-tRNA synthetase was the first class II enzyme to be cocrystallized with its cognate tRNA (189), and a nucleotide binding structure similar to that found in Ser-tRNA synthetase was identified. The combination of these two class II crystal structures provides a model for the active sites of all of the class II tRNA synthetases.

Models of tRNA Synthetases of Unknown Structure

Class I.

The known crystal structures from each class of tRNA synthetases, combined with an understanding of the evolutionary relationships between the enzymes, allowed models for tRNA synthetases of unknown structure to be constructed (37). As discussed above, the class I Tyr- and Met-tRNA synthetases contain a structurally homologous ATP binding pocket (20). In contrast to Tyr-tRNA synthetase, the conserved primary sequence between E. coli Ile- and Met-tRNA synthetases extends beyond the 11-amino-acid HIGH signature sequence, thus suggesting that the Ile-tRNA synthetase also contains a homologous nucleotide binding fold (212, 225).

In the absence of a crystal structure, the conserved regions of the 939-amino-acid Ile-tRNA synthetase were placed on the known active-site tertiary structure of the truncated 547-amino-acid Met-tRNA synthetase (212). The positioning of the Ile-tRNA synthetase polypeptide on the structure of Met-tRNA synthetase was complicated by the much longer length of the Ile-tRNA synthetase chain. Although the isoleucine enzyme has an extension of 45 amino acids at the N terminus which is not present in Met-tRNA synthetase, the majority of the extra length is found in an insertion which splits the nucleotide binding fold into two halves (37, 212). This insertion, designated CP1, is approximately 300 and 126 amino acids for Ile- and Met-tRNA synthetases, respectively. A second, smaller insertion (designated CP2) is located between the first β strand and α helix of the second half of the nucleotide binding fold. As a consequence of the N-terminal extension and the longer lengths of the connective polypeptides, the nucleotide binding fold of Ile-tRNA synthetase encompasses about 630 amino acids, compared with 360 amino acids for the active site of the methionine enzyme.

Cys-tRNA synthetase is the smallest monomeric enzyme (461 amino acids [72, 102]) of class I and was also modeled on the basis of the structure of Met-tRNA synthetase (102). The primary sequence and structural organization of the polypeptide helped to characterize a subclass among the class I enzymes which includes Met-, Ile-, Val-, Leu-, and Cys-tRNA synthetases (Table 2) (36, 72, 102, 151). The CP1 insertion in each enzyme is distinct in length and dramatically influences the overall size of each protein. For example, Cys-tRNA synthetase (461 amino acids) contains a 69-amino-acid CP1 domain, while Ile-tRNA synthetase is composed of 939 residues and incorporates the largest CP1 insertion (300 amino acids). The CP2 insertion is smaller than the CP1 insertion but ranges in length from about 40 to 70 amino acids. Although the exact roles of the CP1 and CP2 insertion sequences have not been defined for this subclass, in the crystal structure of class I Gln-tRNA synthetase, parts of the CP1 domain interact with the tRNA acceptor helix (185).

In the case of Ile-tRNA synthetase, the N-terminal 50 amino acids of CP1 appear essential for activity (212). Proteins which collectively have deletions which encompass the remainder of CP1 are active. Thus, CP1 appears to be a region needed for activity but also one that can be expanded and contracted in size. Consistent with this interpretation, Starzyk et al. showed that new sequences could be inserted into CP1 of Met-tRNA synthetase (211). As expected, the recovery of activity in proteins having insertions depended strongly on the locations within CP1 at which the insertions were placed.

Comparison of the modeled Ile-tRNA synthetase with the crystal structure of Tyr-tRNA synthetases identified residues that may be important for amino acid binding (49). Asp-78 forms a hydrogen bond to the amino group of tyrosine. Random substitutions were introduced into the corresponding region in Ile-tRNA synthetase by cassette mutagenesis. An Ile-tRNA synthetase with a G94R mutation was recovered with a greater than 6,000-fold-higher K_m for isoleucine. The second domain of Met-tRNA synthetase is rich in α helices and contains the determinants for interaction with the anticodon of tRNA^Met (29, 88, 121, 148, 199, 219). For the related Cys-, Ile-, Leu-, and Val-tRNA synthetases, the C-terminal second domain is also predicted to be primarily made up of α helices (102). Amino acid sequence alignments between Met- and Ile-tRNA synthetases suggested a region which may be functionally homologous in anticodon binding (7a, 201). Alignment-guided mutagenesis generated a protein with a K732T mutation that had a high K_m for tRNA^Ile, but amino acid and ATP binding were not affected. Lys-732 of Ile-tRNA synthetase aligns near Trp-461 of Met-tRNA synthetase, a residue implicated in anticodon binding (88, 147, 148). Further experiments then showed that anticodon recognition of tRNA^Met and tRNA^Ile could be switched by a single amino acid swap between the corresponding anticodon-binding regions of the two enzymes (7a).

The X-ray crystal structures for the active-site domain of E. coli Gln-tRNA (185) and Met-tRNA (30, 181, 235) synthetases have been superimposed on the basis of their similar Rossmann folds. This superimposition included a unit that is adjacent to the ATP binding pocket of Met-tRNA synthetase (165). The cocrystal structure for the tRNA^Gln-bound Gln-tRNA synthetase shows that this region contacts the extreme inside turn of the L-shaped tRNA. In the absence of a tRNA-bound cocrystal structure, this interaction was used to model the tRNA binding site for Met-tRNA synthetase by docking it to tRNA^Gln. Acceptor stem recognition of tRNA^Gln is mediated by water molecules and main chain interactions. Likewise, the docked tRNA^Met acceptor stem appeared to form related interactions with polar side chains of Met-tRNA synthetase in the N-terminal half of the protein. Cross-linking and site-directed mutagenesis studies also supported the anticodon’s proximal location to Trp-461 (88, 146) and Lys-465 (133) in the C-terminal second domain.

Class II.

As described above, the class II tRNA synthetases are characterized by three conserved motifs. In the absence of a crystal structure for Ala-tRNA synthetase, the predicted conserved motifs and secondary structure and biochemical cross-linking studies were used to model an active site after the structurally conserved catalytic cores of the known crystal structures for Ser- and Asp-tRNA synthetases (178). These three class II enzymes contain little or no primary sequence homology other than in the highly degenerate motifs 1, 2, and 3.

Because motif 1 is at the dimer interface in the crystal structures of yeast Asp-tRNA synthetase (189) and E. coli (56, 57, 168) and T. thermophilus (15, 17, 56) Ser-tRNA synthetases, it was thought to be important for dimerization. This motif was identified in the N-terminal region of Ala-tRNA synthetase (178), but a series of deletion mutations had previously demonstrated that the sequence needed for oligomerization was at the C terminus of the protein (108, 109). Thus, motif 1 is not sufficient for oligomerization of this enzyme. An analysis of motif 1 in yeast Asp-tRNA synthetase also questions the role of motif 1 in oligomerization (68).

An idiographic representation of the predicted eight-stranded β structure with three α helices of Ala-tRNA synthetase has been constructed (178, 202). Collectively, over 40 mutations in motif 2 and the region between motifs 2 and 3 were individually constructed and tested (59, 202). These mutations were mostly at residues conserved among Ala-tRNA synthetases which had chemical functional groups. Although motif 2 is of a different size and has only two identities with its counterpart in yeast Asp-tRNA and T. thermophilus Ser-tRNA synthetases, the mutation analysis of this motif is explainable in terms of those structures and showed the importance of predicted motif 2 for adenylate synthesis (178). Randomized mutagenesis of this region also demonstrated its importance for adenylate transfer (137). The mutagenesis of specific residues in motif 2 of Ala-tRNA synthetase and mutagenesis of their predicted counterparts in known motif 2 of yeast Asp-tRNA synthetase yielded similar results (43, 59). Evidence was obtained for sequence context determining how the energy of adenylate binding is partitioned between ground and transition states in the two enzymes. Given the great dissimilarity of the sequence, the context effect is not surprising. In addition, a conserved aspartate among Ala-tRNA synthetases at the beginning of motif 3 was shown to be important for adenylate synthesis and particularly for the adenylate transfer reaction (59). The functional significance of motif 3 for adenylate synthesis has been demonstrated by mutagenesis in the yeast Asp-tRNA synthetase system (43).

RELATIONSHIP OF E. COLI ENZYMES TO THEIR HUMAN COUNTERPARTS

General Observations

While the class-defining domains of several recently obtained human tRNA synthetase sequences appear to be derived from the same ancestral sequence as their E. coli counterparts, sequences outside of the catalytic core, which make up the second major domain including the anticodon binding site, diverge significantly (207). For example, the class I-defining catalytic domain of E. coli and four other isoleucyl-tRNA synthetases (including human) have an identity of about 20% (across the five sequences). The close similarity encompasses the CP1 and CP2 insertions as well. On the other hand, the second domain in these enzymes is far less conserved and, in its second half, shows almost no identities across the five sequences. Thus, far greater selective pressure is exerted on the domain for adenylate synthesis and acceptor helix interactions than on that needed for interactions outside of the acceptor helix.

The homotetrameric structure of the E. coli alanine enzyme is unique among class II tRNA synthetases, most of which are dimeric. The sequence of the N-terminal 461-amino-acid active fragment of the 875-amino-acid E. coli Ala-tRNA synthetase has 41% identity to the N-terminal 498 amino acids of the 968-residue human cytoplasmic enzyme (203). Although the C-terminal halves of the respective proteins are less similar, they are nonetheless derived from the same ancestral sequence except for one region. This section comprises residues 699 to 808 of the E. coli protein, which is completely distinct from the human sequence. The replaced sequence cassette consists of the domain needed for oligomerization of the E. coli enzyme to form the α ₄ tetramer (108). In contrast, the human enzyme is a monomer (203). The same cassette is replaced in the monomeric enzyme from Bombyx mori (46). The reason for the tetrameric structure of the E. coli protein is not known, but the E. coli enzyme binds to a sequence palindrome in its promoter and autoregulates its gene transcription (172). Possibly the tetrameric structure is required for the promoter interaction.

The monomeric structure of the human (203) (and rat [61] and B. mori [46]) alanine enzyme is unique for a class II enzyme. Because the three conserved motifs of the class II enzymes are near the interface of the α ₂ dimer, and because motif 1 is needed for contact of one subunit with a tRNA bound to the other subunit in the yeast Asp-tRNA synthetase-tRNA^Asp cocrystal (189), a rationale for the dimeric structure of class II enzymes was apparent. Possibly the monomeric eukaryotic alanine enzyme uses sequences unique to it to form an internal structure which allows it to reconstruct an interface that resembles the dimeric enzymes.

E. coli Gly-tRNA synthetase has an unusual α ₂ β ₂ tetramer structure (162). The coding regions for the α and β subunits occur in tandem and are transcribed from a single promoter (118, 224). A UAA stop codon and nine nucleotides separate the two coding regions. Thus, the E. coli enzyme could be viewed as a single polypeptide which has been interrupted by a stop signal and site for translation reinitiation. When expressed in cells harboring an ochre suppressor, or when the UAA codon is altered to a sense codon by site-directed mutagenesis, the two chains are fused together to form a single polypeptide (117, 217). Thus, the α ₂ β ₂ tetramer may be considered to be made up of a dimer of one chain and, therefore, to resemble other class II enzymes.

The B. mori and yeast cytoplasmic glycine enzymes are α ₂ dimers(62, 119). However, the sequences of these two eukaryote Gly-tRNA synthetases and of the human enzyme provide no evidence that the E. coli protein is encoded by a gene homologous to those encoding the three eukaryotic enzymes (174, 206). Even in the regions of motifs 2 and 3 of the E. coli protein, there are no similarities to the eukaryote counterparts (206). The human and B. mori glycine enzyme sequences are more related to E. coli Thr- and Pro-tRNA synthetases in the motif 2 region than to that of putative motif 2 of E. coli Gly-tRNA synthetase (154, 206).

Comparison of tRNA synthetase sequences between humans and E. coli shows that the Ala-, Asp-, Cys-, Gln-, Glu-, His-, Ile-, Thr-, and Val-tRNA synthetases have class-defining catalytic domains encoded by the same ancestral gene (206). The sequences for Gly-, Pro-, and Trp-tRNA synthetases from E. coli diverge widely from their human counterparts, although limited similarities in a class-defining motif can be found in the cases of the Pro- and Trp-tRNA synthetases. Thus, Gly-tRNA synthetase is the most different and appears to be an extreme example of an enzyme which followed a different path of evolutionary development (206). These differences in the enzymes could arise from a longer evolutionary time frame for some than for others and, additionally or alternatively, a different origin for the prokaryote (eubacterial) enzyme compared with its eukaryote cytoplasmic counterpart in some instances.

Human Glu-tRNA Synthetase Is More Similar to E. coli Glutamine than to Glutamate Enzyme

The evolutionary relationship betwen Gln- and Glu-tRNA synthetase is of particular interest because gram-positive eubacteria, cyanobacteria, halobacteria, and the organelles of eukaryotes lack a separate Gln-tRNA synthetase. Instead, a single Glu-tRNA synthetase aminoacylates both tRNA^Glu and tRNA^Gln (129, 198, 220). A separate transamidation reaction converts Glu-tRNA^Gln to Gln-tRNA^Gln. In contrast, E. coli has separate Glu- and Gln-tRNA synthetases (26, 101, 231).

The sequence of human Glu-tRNA synthetase was originally identified as a glutamine enzyme, because the human enzyme’s sequence matched better with that of E. coli Gln- than Glu-tRNA synthetase (76). Similarly, the sequence of Drosophila melanogaster Glu-tRNA synthetase is closer to that of E. coli Gln- than Glu-tRNA synthetase (45). Lamour et al. (127) obtained the sequence of human Gln-tRNA synthetase and showed its close relationship to the S. cerevisiae (138) and D. melanogaster (45) glutamine enzyme sequences. Thus, the sequences of the eukaryote Gln-tRNA synthetases are clearly related to each other and to that of the E. coli enzyme. Pairwise alignments of sequences of 14 Gln- and Glu-tRNA synthetases established two major sequence families. The two human enzymes and the D. melanogaster glutamate enzyme were grouped with E. coli Gln-tRNA synthetase. (No sequence is yet available for D. melanogaster Gln-tRNA synthetase.) This grouping with the close similarity of the two eukaryote cytoplasmic Glu-tRNA synthetases to E. coli Gln-tRNA synthetase suggests that E. coli and presumably other eubacteria have acquired Gln-tRNA synthetase by horizontal gene transfer (127).

RNA WORLD AND ASSEMBLY OF tRNA SYNTHETASES

Self-replicating "living" systems are now thought to have begun in an RNA world (139, 157, 195, 195a, 226). The minihelix motif has been suggested as an ancient tag of RNA genomes (139, 226), the contemporary examples of which are seen in the minihelix-like structures needed for replication of RNA genomes by Qβ phage replicase (22, 150), plant virus RNA replicase (77), Tetrahymena telomerase (18, 19) and the Mauriceville reverse transcriptase of Neurospora crassa mitochondria (1, 126, 223). Aminoacylation of minihelices by ribozymes could have provided aminoacyl-RNA esters which promoted formation of peptide bonds through the assembly of aminoacyl-RNA arrays in a system of noncoded peptide synthesis (105a, 195). This system in turn could have led to coded peptide synthesis with minihelix-like molecules. Eventually, the minihelix was combined with a second domain—the anticodon-containing template reading head—to establish the full tRNA structure.

Thus, the earliest tRNA synthetases could have been ribozymes (60a, 105a), and most likely, as peptides were formed, they became RNA-protein complexes and eventually proteins devoid of an RNA cofactor. The enzymes and their precursors developed the operational RNA code to relate sequences and structures in the minihelices to specific amino acids (194, 195a). They were small proteins which formed aminoacyl adenylates and could not interact with nucleotides more than a few base pairs away from the amino acid attachment site. The proteins eventually were enlarged through the acquisition of a second domain, perhaps in concert with the addition of the anticodon-containing domain to the minihelix (32, 194).

Figure 4 shows the domain-domain interactions between the tRNA and tRNA synthetase. At least 11 examples representing both classes of enzymes have been found to specifically aminoacylate small minihelix substrates which represent the acceptor stem domain of the tRNA (90a, 140, 173a). In addition, Ala-tRNA synthetase was truncated to generate a monomeric N-terminal domain which has full adenylate synthesis activity (33). This domain charges tRNA^Ala and the alanine RNA minihelix with similar efficiencies, thus demonstrating the tightly associated domain-domain interactions between the conserved catalytic domain of the protein and the tRNA acceptor stem.

Fig. 4Domain-domain interactions between a tRNA synthetase and tRNA. (A) As described in the text, the tRNA and tRNA synthetase can be dissected into two major domains (32, 194, 195a). The conserved active-site domain binds the acceptor stem domain of the tRNA, while the idiosyncratic nonconserved domain interacts with other parts of the tRNA, including the anticodon domain. The dashed line indicates that some tRNA synthetases do not interact with the tRNA anticodon. (B) Several tRNA synthetases specifically aminoacylate small RNA acceptor minihelices (140), and for Ala-tRNA synthetase, the catalytic domain has been isolated and shown to charge the acceptor helix (33). (C) An anticodon hairpin helix binds specifically to the nonconserved tRNA synthetase domain (140) and has also been shown to interact with an isolated anticodon binding domain of Met-tRNA synthetase (86).

Source:

X-ray cocrystal structures show that the tRNA anticodon domain interacts with a second domain distinct from the active site of Gln-tRNA (186) and Asp-tRNA (189) synthetases. Small RNA anticodon stem-loops competitively inhibit tRNA binding for Met-tRNA (146) and Gln-tRNA (229) synthetases and also stimulate minihelix aminoacylation by Val-tRNA (85) and Ile-tRNA (158) synthetases (albeit by a small amount). Recently, Met-tRNA synthetase was genetically truncated, yielding the protein domain which specifically interacted with a small RNA hairpin helix that mimicked the tRNA anticodon domain (86).

The linkage of the anticodon-containing domain to the minihelix joined the nucleotides of the genetic code to the operational RNA code embedded in the minihelix (60a, 194, 195a). With this scheme for the evolution of the tRNA molecule, the association between a particular amino acid and a trinucleotide could be stochastic. While many tRNA synthetases added a domain which could interact with the anticodon, the catalytic domain along with its interactions with the RNA minihelix is believed to be the earliest historical component which retains an essential role in contemporary tRNA synthetases.

ACKNOWLEDGMENTS

We thank J. Kranz for reviewing the manuscript prior to submission. This work was supported by grants GM15539 and GM23562 from the National Institutes of Health.

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