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Aminoacyl-tRNA Synthetases

´ Louis Pasteur, Strasbourg, France (Present address: Albert Einstein College John G Arnez, Universite of Medicine, Bronx, New York, USA) ´ Louis Pasteur, Strasbourg, France Dino Moras, Universite

Introductory article
Article Contents
. Introduction . Class I and Class II of Aminoacyl-tRNA Synthetases . Catalytic and Special RNA Recognition Domains . tRNA-binding Modes of the Two Classes

Aminoacyl-tRNA synthetases catalyse a key reaction in protein biosynthesis. They match the 20 amino acids to the genetic code by specifically attaching them to their adaptors, transfer ribonucleic acid (tRNA) molecules.

Introduction
Aminoacyl-tRNA synthetases catalyse the highly specific attachment of amino acids to their corresponding adaptor molecules, the transfer ribonucleic acids (tRNA). The attachment reaction is called aminoacylation and proceeds in two steps (reactions [I] and [II]), in the presence of divalent magnesium (Mg 2+) ions. amino acid 1 ATP 0 aminoacyl-adenylate 1 PPi [I] aminoacyl-adenylate 1 tRNA 0 aminoacyltRNA 1 AMP ATP is adenosine triphosphate, AMP is adenosine monophosphate and PPi is inorganic pyrophosphate (diphosphate). In step [I], the amino acid is activated through the formation of aminoacyl-adenylate, which is a mixed anhydride of the a-carboxylate group of the amino acid and the a-phosphate group of ATP. The pyrophosphate is released, while the aminoacyl-adenylate remains bound to the active site of the enzyme. In step [II], the amino acid portion is transferred to the 2’ or 3’ sugar hydroxyl group of the 3’-terminal adenosine nucleotide of the tRNA. This is the rate-limiting step. Aminoacylation consumes energy, which is supplied by the hydrolysis of ATP. Both steps are bimolecular nucleophilic substitutions, which means that two molecules react, of which one is the attacking nucleophile that substitutes another group already present; the latter becomes the leaving group. In order for the aminoacyl-tRNA synthetases to catalyse the reaction, they must position the substrates in correct mutual orientations and release the reaction products in a timely fashion. They must also stabilize the reactants and transition states of both steps. First, the amino acid and ATP are bound. Both of these substrates possess negatively charged groups that are to be brought close together and react: the a-phosphate of ATP and the a-carboxylate group of the amino acid. The charge of the phosphate group has to be neutralized, since the amino acid carboxylate is the attacking nucleophile. The [II]

charge neutralization is accomplished by positively charged protein side-chains and divalent metal cations; these withdraw electrons from the phosphate group such that it is receptive to the pair of electrons of the nucleophile. The resulting transition state is pentacoordinate at the aphosphorus, which means that there are five chemical groupings transiently attached to the phosphorus atom: the amino acid, adenosine, pyrophosphate and two oxygens. It also has many negative charges that are similarly stabilized by the same groups of the enzyme: two on the a-phosphate, one on the b-phosphate and two on the g-phosphate. It is resolved through the dissociation of the pyrophosphate. The aminoacyl-adenylate remains in the active site of the enzyme and needs to be protected from reacting with water. This is an SN2 reaction (substitution, nucleophilic, bimolecular) and results in an inversion of configuration at the a-phosphorus. Its mechanism corresponds to an in-line associative nucleophilic substitution; it is associative as opposed to dissociative because the leaving group is displaced after the nucleophilic attack and transition state formation rather than before, and in-line because the nucleophile attacks on the side directly opposite the leaving group. Second, the tRNA is bound such that its terminal ribose (sugar) hydroxyls are close to the carbonyl carbon of the aminoacyl-adenylate. Some of the tRNA phosphate charges need to be balanced in order for it to bind. The negatively charged phosphate of the aminoacyl-adenylate is neutralized as well. However, this step does not involve phosphates but a hydroxyl and a carbonyl group. This too is a nucleophilic substitution and takes place at the acarbonyl carbon of the amino acid moiety of the mixed anhydride. In this case, the hydroxyl is the nucleophile; the carbonyl carbon has a slight positive formal charge and is thus a good electrophile. The transition state consists of four groups covalently bound to the a-carbonyl carbon: the amino acid, the tRNA, the AMP and the hydroxyl derived from the carbonyl group. The AMP is the leaving group in this substitution. The resolution of the transition state results in an ester linkage between the ribose hydroxyl
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ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Nature Publishing Group / www.els.net

which adds an additional aspect to the specificity of recognition and thus further strengthens it. the exact nature of the nucleotide bases. Second was the determination of the three-dimensional structures of a few key enzymes of this family. This structure constitutes a platform for binding ATP. Often an amino acid is attached to several tRNA species. some of them enable its cognate enzyme to accept it (positive determinants) while others cause all other aminoacyltRNA synthetases to reject it (negative determinants). Most identity nucleotides or elements are found in the acceptor arm or the anticodon arm of the tRNA. which they share with many other nucleotide-binding proteins. Some applied to one half and some to the other half. Finally.net . ‘MSK’ LeuRS IleRS ValRS CysRS MetRS GluRS GlnRS ArgRS TyrRS TrpRs Class II Active site: antiparallel b sheet Motifs: 1. whose definition is based on two different sets of limited amino acid sequence similarities and two active site architectures (Table 1). the aminoacylated tRNA is released. All these tRNA species form a family of isoacceptors that share the same identity and are recognized by the same aminoacyl-tRNA synthetase. Since there are 20 amino acids specified by the genetic code. The common features. ATP. a parallel b sheet surrounded by a helices. since it is specified by several three-letter keywords called codons in the genetic code. 3 HisRS ProRs SerRs ThrRS GlyRS AspRS AsnRs LysRS AlaRS PheRS Ribose a a a a a2 a a a a2 a2 2’ 2’ 2’ 2’ or 3’ 2’ 2’ 2’ 2’ 2’ or 3’ 2’ a2 a2 a2 a2 a2 a2 a2 a2 a4 (ab)2 3’ 3’ 3’ 3’ 3’ 3’ 3’ 3’ 3’ 2’ Class I and Class II of Aminoacyl-tRNA Synthetases As individual members of these enzymes were discovered and examined. occasionally they can also be found in the structural core region formed by the D and T arms. these three components constitute a cognate aminoacylation system. Class I Class I aminoacyl-tRNA synthetases are primarily monomeric proteins and share conserved amino acid sequence motifs histidine-isoleucine-glycine-histidine (abbreviated His-Ile-Gly-His or HIGH) and lysine-methionine-serinelysine-serine (Lys-Met-Ser-Lys-Ser or KMSKS). The two classes represent two solutions to the same problem of aminoacylation (Figure 1). The active-site architectures are based on the classical nucleotide-binding fold. The two ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Nature Publishing Group / www. Amino acids have the same core chemical groups and tRNAs are folded in the same L-shaped three-dimensional structures. Identity elements are also distributed differently in different isoacceptor tRNA families. All of these enzymes catalyse the same basic reaction and share a common substrate. arising from two sources of information. did not extend over the entire 2 family. there are generally 20 aminoacyl-tRNA synthetases in a given cell. Each enzyme is highly specific for its amino acid and matching set of tRNAs called isoacceptor tRNAs. also known as the Rossmann fold.els. Some enzymes are more specific for a particular structural feature of a given tRNA than the nucleotide sequence.Aminoacyl-tRNA Synthetases and amino acid carboxyl groups. however. amino acids have different side-chains. ranging from small monomers (single protein subunits) to large tetramers (four protein subunits).e. i. Each tRNA harbours within the common structural framework a variety of features that form its separate identity. each of which is recognized by its proper (cognate) aminoacyltRNA synthetase and rejected by the remaining 19. they were noted for their diversity in amino acid sequence. size and quaternary structural organization. However. First was the expanding database of amino acid sequences for all aminoacyl-tRNA synthetases obtained from several species. 2. Some common features were nevertheless discovered. Table 1 Classification of aminoacyl-tRNA synthetase Quaternary structure OH aminoacylated Class I Active site: nucleotide-binding fold Motifs: ‘HIGH’. partitioning the family into two classes. This seemed paradoxical in view of the same basic reaction that they catalyse and similar substrates they employ. These common features enable these compounds to engage as substrates in other reactions of protein biosynthesis.

two between the b. The aminoacyladenylate has a bent conformation. The triphosphate coordinates at least one divalent metal cation. they bind ATP in a unique bent conformation.net . These groups also stabilize the transition state of the first reaction (step [I]). Motif 2 consists of two b strands connected by a long loop. labelled motif 1. These enzymes bind their cognate amino acids in open and relaxed binding pockets. The two class I characteristic motifs HIGH and KMSKS are highlighted in red and blue. glutamate.Adapted from Arnez JG and Moras D (1997) Trends in Biochemical Sciences 22: 211 – 216. Motif 1 consists of an a helix followed by a b strand and contains a conserved proline. 2 and 3. tryptophan. motif 2 in green and motif 3 in blue.Aminoacyl-tRNA Synthetases Figure 1 Active-site domains of aminoacyl-tRNA synthetases complexed with the acceptor arms of their cognate tRNAs. The active-site architectures are based on a newly discovered fold observed to date only in these enzymes and a few related proteins. it forms a major part of the dimer interface. The insertion is shown in light blue. conserved motifs are part of the fold and interact with the nucleotide (Figure 1a). the first motif 2 arginine with the aphosphate. The three-dimensional structures of seven class I aminoacyl-tRNA synthetases are known.els. The nucleotide-binding fold (the Rossmann fold) is shown in yellow and the acceptor binding insertion is light blue. These residues and the cation withdraw electrons from the a-phosphate so that the amino acid a-carboxylate can attack nucleophilically at the phosphorus. They bind ATP in an extended conformation. The fold is a seven-stranded antiparallel b sheet surrounded by a helices and represents a new platform for binding ATP. arginine and isoleucine. (b) Class II aspartyl-tRNA synthetase. these are the enzymes for tyrosine.and gphosphates and one between the a. Shown also are ATP and key interactions with tRNA. glutamine. In the second reaction (step [II]) the amino acid is transferred to the 2’ hydroxyl of the terminal ribose of tRNA. which can be explained by the amino acid approaching from the side in order to react with the a-phosphate of ATP. The adenine of ATP lies against the glycine of the HIGH motif. (a) Class I glutaminyl-tRNA synthetase. and the motif 2 loop and motif 3 arginines with the g-phosphate. The adenine of ATP lies against the conserved phenylalanine of motif 2. The conserved arginines of motif 2 and 3 interact with the triphosphate of ATP. the two motifs contain a number of conserved amino acid residues involved in amino acid and ATP positioning and catalysis (Figure 1b). Class II Class II aminoacyl-tRNA synthetases are primarily dimeric proteins and share three amino acid sequence motifs. and motif 3 comprises a b strand followed by an a helix. The first motif 2 arginine and the metal ion coordinated to the a-phosphate withdraw electrons from the a-phosphate and thus make it electrophilic (seeking electrons). The enzyme–ATP complex generally coordinates three metal ions. for this hydroxyl is the closest to the aminoacyl-adenylate. The histidines of the HIGH motif and the lysines of the KMSKS motif interact with the triphosphate of ATP. which is similar to other proteins that have the Rossmann fold.and b-phosphates. methionine. respectively. The three class II characteristic motifs are highlighted: motif 1 in red. The nucleo3 ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Nature Publishing Group / www. Class II enzymes bind their cognate amino acids in specific rigid binding pockets. Unlike class I enzymes.

els. for this hydroxyl is the closest to the aminoacyladenylate. respectively. Among these are domains that interact with the anticodon loop of tRNA. The enzyme modules are rendered as follows: the core active-site domains in yellow. aspartate. Furthermore. many of these interact with various parts of tRNA molecules. The motif 2 and 3 arginines and the metal ions also stabilize the transition state of the first reaction (step [I]). The aminoacyl-adenylate has an extended conformation. glycine. lysine. These extra modules diversify the two basic architectures and ensure specific tRNA recognition. proline and asparagine. these are the enzymes for serine. insertions that form additional contacts with the acceptor arm of tRNA. left) and aspartyl-tRNA synthetase:tRNAAsp complex (class II. phenylalanine. the active-site insertions in light blue.net . histidine. Adapted from Arnez JG and Moras D (1997) Trends in Biochemical Sciences 22: 211 – 216. The core domain or module contains the active site and defines the class according to the architecture of the site. the linkers in green and the anticodon-binding domains in orange. Catalytic and Special RNA Recognition Domains Aminoacyl-tRNA synthetases are modular enzymes (Figure 2). In the second reaction (step [II]) the amino acid is transferred to the 3’ hydroxyl of the terminal ribose of tRNA. right) were superposed on tRNAPhe (the first tRNA known in three-dimensional structural detail and commonly used as reference. and modules that recognize certain specific features of a given tRNA. the variable loop and CCA end are highlighted in blue and green. The three-dimensional structures of eight class II aminoacyl-tRNA synthetases are known. some enzymes possess modules that carry out editing functions Figure 2 Modular organization of aminoacyl-tRNA synthetases and mirror symmetrical binding modes characteristic of the two classes.and g-phosphates are bent away. The tRNAs of glutaminyl-tRNA synthetase:tRNAGln complex (class I. middle). Additional modules are attached to the core. 4 ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Nature Publishing Group / www. since the b. which can be explained by the amino acid approaching head-on in order to react with the aphosphate of ATP.Aminoacyl-tRNA Synthetases philic carboxylate of the amino acid can thus attack at the a-phosphorus. The two enzymes were then translated away from tRNAPhe for this picture.

leucine. valine. The CCA end is bent into the active site. With the possible exception of tyrosyl-tRNA and tryptophanyl-tRNA synthetases. which consists of the central nucleotide-binding fold into which is inserted a special accessory subdomain. nucleotides whose bases form direct hydrogen bonds with the enzyme are shown in red. The anticodon-binding domain of glutaminyl-tRNA synthetase is a double b barrel (Figure 2. methionyl-tRNA and isoleucyl-tRNA synthetases are ahelical bundles. its anticodon undergoes a substantial conformational change. (a) tRNAGln. The two amino acids are very similar and are both activated in the Figure 3 Areas of tRNAs that interact with cognate aminoacyl-tRNA synthetases. although their three-dimensional structures are not presently known.net 5 . whereas those of glutamyl-tRNA. The nucleotide bases spread such that they maximize contacts with the enzyme and thus enable the enzyme to recognize the specific nature of the bases (Figure 3a). Isoleucyl-tRNA synthetase needs to specifically recognize isoleucine and discriminate against valine. Enzymes of both second and third subgroups have insertions in the nucleotide-binding domain for specific contacts with the acceptor arm of tRNA and dedicated anticodon-binding modules. Only in this way can the 2’ hydroxyl group of the terminal adenosine come within range to accept the amino acid from the aminoacyladenylate. These contacts include specific hydrogen bonds and van der Waals interactions. Specific elements of this module interact with the minor groove of the end of the acceptor arm double helix and the cytidine-cytidine-adenosine (CCA) of the 3’ terminus of the tRNA (Figures 1a and 3a). Analogous conformational changes are likely to occur in other enzyme–tRNA complexes. nucleotides that enable the tRNA to attain the conformation needed for productive interaction with the enzyme are drawn in green. (b) tRNAAsp and (c) tRNASer. cysteine and methionine). ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Nature Publishing Group / www.els. left). The acceptor arm of tRNA is recognized by the catalytic module. The two enzymes are dimeric and cannot function as monomers. The latter two subgroups are nevertheless not very well defined. The enzymes of the third subgroup would then comprise the enzymes that are specific for mostly aliphatic or sulfur-containing amino acids (isoleucine. The second monomer of the dimer functions as the anticodon-binding domain while the first monomer performs the catalysis. The second subgroup could conceivably contain the three enzymes specific for charged amino acids arginine and glutamate. which are attached at the Cterminal end of the core module. When glutamine-specific tRNA (tRNAGln) binds to glutaminyl-tRNA synthetase. These subgroups are also loosely associated with the type of amino acid that is attached to the tRNA. and polar amino acid glutamine.Aminoacyl-tRNA Synthetases or have roles not directly relevant to aminoacylation. and the portions of the phosphodiester backbone in contact with the enzyme are highlighted as purple spheres. essentially forming a hairpin that is stabilized by an intramolecular hydrogen bond. they can activate the amino acids into their respective adenylates only if they have bound tRNA first. Class I One subgroup of class I aminoacyl-tRNA synthetases comprises the two enzymes for aromatic amino acids tyrosine and tryptophan. which means their membership may change as more structural information becomes available. only one of the two active sites is functional at any one time. In each case. Additional similarities in the core domains that extend beyond the definition of classes and the nature of the added modules further partition each class into three subgroups. These enzymes have insertions in the core nucleotide-binding fold but no additional tRNA-binding module. class I enzymes possess anticodon-binding domains.

glycyl-tRNA. The smaller a subunit has essentially one module. for example. Histidyl-tRNA. These contacts include specific hydrogen bonds and van der Waals interactions.Aminoacyl-tRNA Synthetases first reaction into their corresponding aminoacyl-adenylates. it is the catalytic subunit. The third subgroup comprises multidimeric enzymes. Aspartyl-tRNA. When aspartic acid-specific tRNA (tRNAAsp) binds to aspartyl-tRNA synthetase. its anticodon undergoes a dramatic conformational change so as to maximize contacts with the anticodon-binding domain and thus enable the domain to recognize the specific nature of the nucleotide bases. The second subgroup charges small and polar amino acids (serine. Most of these enzymes have additional insertion domains that may interact with the tRNA. with the exception of seryl-tRNA synthetase. has a binding site that is specific for the noncognate valyl-adenylate. Subgrouping in this class is better defined than that in class I and is clearly related to additional architectural features of these enzymes. obviates the need for the CCA end to bend. a homodimer of two a – b dimers. Its family of isoacceptor tRNAs possesses a variety of anticodon sequences. the amino acid recognition portion of the site is too small to accommodate isoleucine. only valine is attached to valine-specific tRNA (tRNAVal). In addition. The 3’ hydroxyl of the terminal adenosine comes within the necessary range for accepting the amino acid from the aminoacyl-adenylate. The CCA end inserts straight into the active site and is held in place by structural elements surrounding the active site. threonine has a hydroxyl where valine has a methyl group. to varying degrees. In addition. the enzyme recognizes the acceptor arm and part of the T and D arms. however. Instead. a domain that is similar to some eukaryotic signal transduction proteins. major cold shock protein. It has to distinguish between valine and threonine. these enzymes have similar anticodon-binding modules attached to the Ntermini of their active site domains. ribosomal protein S17. Two well-defined anticodon-binding domains have been observed among this class of enzymes. with an insertion. where enzymes from prokaryotic organisms have large insertions and those from eukaryotic and archaeal organisms have small insertions. The disposition of the aminoacyl-adenylate. although the enzyme makes only one nucleotide base-specific contact with the helical portion of the arm. as has been observed in the case of aspartyl-tRNA synthetases. it also contains a class II type core module. which is roughly parallel to that of the CCA end. the catalytic core. which is larger than valine by a methyl group. and an RNA-binding domain that resembles the N-terminal domain of the U1A protein (a component of the eukaryotic small nuclear ribonucleoprotein particles that participate in premessenger RNA splicing). the b subunit. asparaginyl-tRNA and lysyl-tRNA synthetases share a five-stranded b barrel anticodon-binding domain that is attached to the N-terminal end of the core module (Figure 2. Glycyl-tRNA synthetase has an insertion between motifs 1 and 2. Seryl-tRNA synthetase does not interact with the anticodon of its cognate tRNAs (Figure 3c). ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Nature Publishing Group / www. while aspartyl-tRNA. some prokaryotic glycyl-tRNA synthetases are also multidimeric and may belong to this subgroup. The structures of the latter three are a helical. have similar anticodon-binding domains attached to the C-termini of their core modules. and polar amino acid asparagine.e. These domains may also vary from one division of the living world to another. proline. Phenylalanyl-tRNA synthetase is an (ab)2 tetramer. a domain that is similar to the anticodon-binding N-terminal domain of aspartyl-tRNA synthetase. these enzymes. The motif 2 loop interacts with the major groove of the end of the acceptor arm double helix (Figures 1b and 3b). A second domain. staphylococcal nuclease and some enterotoxins. although it is truncated and therefore inactive. which are specific for alanine and phenylalanine.net . namely the large extra arm in the place of the variable loop. it has a special coiled-coil module attached at its N-terminal end that recognizes a specific feature of serine-specific tRNA (tRNASer). The fold of this domain is similar to that of some other nucleic acid-binding and even oligosaccharidebinding proteins. As a result. right). This arm is an important structural element. histidine. lysyl-tRNA and histidyl-tRNA synthetases have insertions between motifs 2 and 3. which prevents valine from getting attached to isoleucine-specific tRNA (tRNAIle). i. which is inserted into the core catalytic domain. The domain is attached to the C-terminal end of the catalytic module. The acceptor-binding function of class II enzymes is part of the class II core domain architecture. Valyladenylate is then hydrolysed. Related enzyme valyl-tRNA synthetase also possesses an editing function. prolyl-tRNA 6 and threonyl-tRNA synthetases have a mixed a helix– b strand structure with a central five-stranded mixed b sheet surrounded by three a helices.els. The editing site must therefore specifically bind this hydroxyl and thus exclude valine. is a multidomain subunit. glycine and threonine). The other monomer. These amino acids have approximately the same size. Class II One subgroup of class II aminoacyl-tRNA synthetases is specific for the charged amino acids aspartate and lysine. Analogous conformational changes are likely to occur in other enzyme–tRNA complexes. this subunit possesses several additional domains: two similar domains that contain helix-turn-helix motifs characteristic of some DNA-binding proteins.

Annual Review of Biochemistry 48: 601–648. Arnez JG and Moras D (1997) Structural and functional considerations of the aminoacylation reaction. Moras D (1992) Structural and functional relationships between aminoacyl-tRNA synthetases. Mechulam Y and Blanquet S (1995) Aminoacyl-tRNA synthetase: occurrence. In: So ¨ ll D and RajBhandary UL (eds) tRNA: Structure. mechanism. and evolutionary relationships in aminoacyl-tRNA synthetases.Aminoacyl-tRNA Synthetases tRNA-binding Modes of the Two Classes Further Reading The enzymes of the two classes bind the acceptor arm of tRNA in two ways that constitute mirror images of each other (Figure 2). Quarterly Review of Biophysics 30: 195–240. Class I enzymes bind the tRNA on the side opposite of the variable loop and interact with the minor groove of the acceptor arm double helix. ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Nature Publishing Group / www.net 7 . 251–291. Class II enzymes bind the tRNA on the variable loop side and interact with the major groove of the acceptor arm double helix. Trends in Biochemical Sciences 17: 159–164. Trends in Biochemical Sciences 22: 211–216. The mirror symmetrical feature already becomes apparent in the first step of the reaction (step [I]). Carter CW. This leads to the distinct regiospecificity of the aminoacylation reaction. Schimmel P and So ¨ ll D (1979) Aminoacyl-tRNA synthetases: general features and recognition of transfer RNAs. class I enzymes form bent aminoacyl-adenylates. Biosynthesis and Function. Arnez JG and Cavarelli J (1997) Structures of RNA-binding proteins.els. and function. Annual Review of Biochemistry 62: 715–748. pp. Starting with an extended ATP conformation. Jr (1993) Cognition. Meinnel T. Class II enzymes form extended aminoacyl-adenylates from ATP in a bent conformation. Washington DC: American Society for Microbiology. structure.