Amino acid discrimination by a class I aminoacyl-tRNA synthetase specified by negative determinants

The 2.5 A crystal structure of Escherichia coli glutaminyl-tRNA synthetase in a quaternary complex with tRNA(Gln), an ATP analog and glutamate reveals that the non-cognate amino acid adopts a distinct binding mode within the active site cleft. In contrast to the binding of cognate glutamine, one oxygen of the charged glutamate carboxylate group makes a direct ion-pair interaction with the strictly conserved Arg30 residue located in the first half of the dinucleotide fold domain. The nucleophilic alpha-carboxylate moiety of glutamate is mispositioned with respect to both the ATP alpha-phosphate and terminal tRNA ribose groups, suggesting that a component of amino acid discrimination resides at the catalytic step of the reaction. Further, the other side-chain carboxylate oxygen of glutamate is found in a position identical to that previously proposed to be occupied by the NH(2) group of the cognate glutamine substrate. At this position, the glutamate oxygen accepts hydrogen bonds from the hydroxyl moiety of Tyr211 and a water molecule. These findings demonstrate that amino acid specificity by GlnRS cannot arise from hydrogen bonds donated by the cognate glutamine amide to these same moieties, as previously suggested. Instead, Arg30 functions as a negative determinant to drive binding of non-cognate glutamate into a non-productive orientation. The poorly differentiated cognate amino acid-binding site in GlnRS may be a consequence of the late emergence of this enzyme from the eukaryotic lineage of glutamyl-tRNA synthetases.     

Amino acid sensing using aminoacyl-tRNA synthetase

The detection of amino acids using aminoacyl-tRNA synthetases (ARSs) as the molecular recognition element was proposed, and the binding activity and specificity of ARSs were evaluated. Using this rapid and easy method, from 15 to 50 μM tyrosine could be measured specifically. The method suggested in this article could be realized without an amino acid labeling process or a large volume of organic solvents, and the time for measurement was reasonable.     

Transfer RNA-dependent cognate amino acid recognition by an aminoacyl-tRNA synthetase.

An investigation of the role of tRNA in the catalysis of aminoacylation of Escherichia coli glutaminyl-tRNA synthetase (GlnRS) has revealed that the accuracy of specific interactions between GlnRS and tRNAGln determines amino acid affinity. Mutations in GlnRS at D235, which makes contacts with nucleotides in the acceptor stem of tRNAGln, and at R260 in the enzyme's active site were found to be independent during tRNA binding but interactive for aminoacylation. Characterization of mutants of GlnRS at position 235, showed amino acid recognition to be tRNA mediated. Aminoacylation of tRNA(CUA)Tyr [tyrT (UAG)] by GlnRS-D235H resulted in a 4-fold increase in the Km for the Gln, which was reduced to a 2-fold increase when A73 was replaced with G73. These and previous results suggest that specific interactions between GlnRS and tRNAGln ensure the accurate positioning of the 3' terminus. Disruption of these interactions can change the Km for Gln over a 30-fold range, indicating that the accuracy of aminoacylation is regulated by tRNA at the level of both substrate recognition and catalysis. The observed role of RNA as a cofactor in optimizing amino acid activation suggests that the tRNAGln-GlnRS complex may be partly analogous to ribonucleoprotein enzymes where protein-RNA interactions facilitate catalysis.     

tRNA discrimination at the binding step by a class II aminoacyl-tRNA synthetase.

Aminoacyl-tRNA synthetases preserve the fidelity of decoding genetic information by accurately joining amino acids to their cognate transfer RNAs. Here, tRNA discrimination at the level of binding by Escherichia coli histidyl-tRNA synthetase is addressed by filter binding, analytical ultracentrifugation, and iodine footprinting experiments. Competitive filter binding assays show that the presence of an adenylate analogue 5'-O-[N-(L-histidyl)sulfamoyl]adenosine, HSA, decreased the apparent dissociation constant (K(D)) for cognate tRNA(His) by more than 3-fold (from 3.87 to 1.17 microM), and doubled the apparent K(D) for noncognate tRNA(Phe) (from 7.3 to 14.5 microM). By contrast, no binding discrimination against mutant U73 tRNA(His) was observed, even in the presence of HSA. Additional filter binding studies showed tighter binding of both cognate and noncognate tRNAs by G405D mutant HisRS [Yan, W., Augustine, J., and Francklyn, C. (1996) Biochemistry 35, 6559], which possesses a single amino acid change in the C-terminal anticodon binding domain. Discrimination against noncognate tRNA was also observed in sedimentation velocity experiments, which showed that a stable complex was formed with the cognate tRNA(His) but not with noncognate tRNA(Phe). Footprinting experiments on wild-type versus G405D HisRS revealed characteristic alterations in the pattern of protection and enhancement of iodine cleavage at phosphates 5' to tRNA nucleotides in the anticodon and hinge regions. Together, these results suggest that the anticodon and core regions play major roles in the initial binding discrimination between cognate and noncognate tRNAs, whereas acceptor stem nucleotides, particularly at position 73, influence the reaction at steps after binding of tRNA.     

Amino acid-dependent transfer RNA affinity in a class I aminoacyl-tRNA synthetase

Steady-state and transient kinetic analyses of glutaminyl-tRNA synthetase (GlnRS) reveal that the enzyme discriminates against noncognate glutamate at multiple steps during the overall aminoacylation reaction. A major portion of the selectivity arises in the amino acid activation portion of the reaction, whereas the discrimination in the overall two-step reaction arises from very weak binding of noncognate glutamate. Further transient kinetics experiments showed that tRNA(Gln) binds to GlnRS approximately 60-fold weaker when noncognate glutamate is present and that glutamate reduces the association rate of tRNA with the enzyme by 100-fold. These findings demonstrate that amino acid and tRNA binding are interdependent and reveal an important additional source of specificity in the aminoacylation reaction. Crystal structures of the GlnRS x tRNA complex bound to either amino acid have previously shown that glutamine and glutamate bind in distinct positions in the active site, providing a structural basis for the amino acid-dependent modulation of tRNA affinity. Together with other crystallographic data showing that ligand binding is essential to assembly of the GlnRS active site, these findings suggest a model for specificity generation in which required induced-fit rearrangements are significantly modulated by the identities of the bound substrates.     

Outersphere and innersphere coordinated metal ions in an aminoacyl-tRNA synthetase ribozyme

Metal ions are essential cofactors for various ribozymes. Here we dissect the roles of metal ions in an aminoacyl-tRNA synthetase-like ribozyme (ARS ribozyme), which was evolved in vitro. This ribozyme can charge phenylalanine on tRNA in cis, where it is covalently attached to the 5′-end of tRNA (i.e. a form of precursor tRNA), as well as in trans, where it can act as a catalyst. The presence of magnesium ion is essential for this ribozyme to exhibit full catalytic activity. Metal-dependent kinetics, as well as structural mappings using Tb³⁺ in competition with Mg²⁺ or Co(NH₃)₆³⁺, identified two potential metal-binding sites which are embedded near the tRNA-binding site. The high affinity metal-binding site can be filled with either Mg²⁺ or Co(NH₃)₆³⁺ and thus the activity relies on a metal ion that is fully coordinated with water or ammonium ions. This site also overlaps with the amino acid-binding site, suggesting that the metal ion plays a role in constituting the catalytic core. The weak metal-binding site is occupied only by a metal ion(s) that can form innersphere contacts with ligands in the ribozyme and, hence, Mg²⁺ can enhance ribozyme activity, but Co(NH₃)₆³⁺ cannot. The experiments described in this work establish the roles of metal ions that have distinct coordination properties in the ARS ribozyme.     

tRNA-balanced expression of a eukaryal aminoacyl-tRNA synthetase by an mRNA-mediated pathway

Aminoacylation of transfer RNAs is a key step during translation. It is catalysed by the aminoacyl-tRNA synthetases (aaRSs) and requires the specific recognition of their cognate substrates, one or several tRNAs, ATP and the amino acid. Whereas the control of certain aaRS genes is well known in prokaryotes, little is known about the regulation of eukaryotic aaRS genes. Here, it is shown that expression of AspRS is regulated in yeast by a feedback mechanism that necessitates the binding of AspRS to its messenger RNA. This regulation leads to a synchronized expression of AspRS and tRNA(Asp). The correlation between AspRS expression and mRNA(AspRS) and tRNA(Asp) concentrations, as well as the presence of AspRS in the nucleus, suggests an original regulation mechanism. It is proposed that the surplus of AspRS, not sequestered by tRNA(Asp), is imported into the nucleus where it binds to mRNA(AspRS) and thus inhibits its accumulation.     

Kinetic discrimination of tRNA identity by the conserved motif 2 loop of a class II aminoacyl-tRNA synthetase

The selection of tRNAs by their cognate aminoacyl-tRNA synthetases is critical for ensuring the fidelity of protein synthesis. While nucleotides that comprise tRNA identity sets have been readily identified, their specific role in the elementary steps of aminoacylation is poorly understood. By use of a rapid kinetics analysis employing mutants in tRNA(His) and its cognate aminoacyl-tRNA synthetase, the role of tRNA identity in aminoacylation was investigated. While mutations in the tRNA anticodon preferentially affected the thermodynamics of initial complex formation, mutations in the acceptor stem or the conserved motif 2 loop of the tRNA synthetase imposed a specific kinetic block on aminoacyl transfer and decreased tRNA-mediated kinetic control of amino acid activation. The mechanistic basis of tRNA identity is analogous to fidelity control by DNA polymerases and the ribosome, whose reactions also demand high accuracy.     

Proofreading in trans by an aminoacyl-tRNA synthetase: a model for single site editing by isoleucyl-tRNA synthetase.

Editing of errors in amino acid selection by an aminoacyl-tRNA synthetase prevents attachment of incorrect amino acids to tRNA, thereby greatly enhancing accuracy of translation of the genetic code. Editing of the non-protein amino acid homocysteine, a frequent type of an error-correcting process, involves reaction of the side chain sulfhydryl group of homocysteine with its activated carboxyl group forming a cyclic thioester, homocysteine thiolactone. Here, it is shown that isoleucyl-tRNA synthetase (IleRS), which occasionally misactivates homocysteine in vitro and in vivo, catalyzes reactions of activated isoleucine with organic thiols (analogues of the side chain of homocysteine). That these enzymatic reactions occur between Ile-tRNAIle or Ile-AMP (bound in the synthetic sub-site) and a thiol (an analogue of the side chain of homocysteine, bound in the editing sub-site), indicates that the two sub-sites are physically close on the surface of IleRS, forming a single synthetic/editing active site of the enzyme. Although IleRS.Val-AMP undergoes thiolysis as efficiently as do IleRS.Ile-AMP and IleRS.Ile-tRNAIle, IleRS.Val-tRNAIle does not react with thiols. These and other data suggest that the mischarged valine residue in IleRS.Val-tRNAIle is, most likely, positioned off the enzyme.     

Active site of an aminoacyl-tRNA synthetase dissected by energy-transfer-dependent fluorescence.

Aminoacyl-tRNA synthetases establish the rules of the genetic code by catalyzing attachment of amino acids to specific transfer RNAs (tRNAs) that bear the anticodon triplets of the code. Each of the 20 amino acids has its own distinct aminoacyl-tRNA synthetase. Here we use energy-transfer-dependent fluorescence from the nucleotide probe N-methylanthraniloyl dATP (mdATP) to investigate the active site of a specific aminoacyl-tRNA synthetase. Interaction of the enzyme with the cognate amino acid and formation of the aminoacyl adenylate intermediate were detected. In addition to providing a convenient tool to characterize enzymatic parameters, the probe allowed investigation of the role of conserved residues within the active site. Specifically, a residue that is critical for binding could be distinguished from one that is important for the transition state of adenylate formation. Amino acid binding and adenylate synthesis by two other aminoacyl-tRNA synthetases was also investigated with mdATP. Thus, a key step in the synthesis of aminoacyl-tRNA can in general be dissected with this probe.     

Hydrolysis of non-cognate aminoacyl-adenylates by a class II aminoacyl-tRNA synthetase lacking an editing domain

Aminoacyl-tRNA synthetases, a group of enzymes catalyzing aminoacyl-tRNA formation, may possess inherent editing activity to clear mistakes arising through the selection of non-cognate amino acid. It is generally assumed that both editing substrates, non-cognate aminoacyl-adenylate and misacylated tRNA, are hydrolyzed at the same editing domain, distant from the active site. Here, we present the first example of an aminoacyl-tRNA synthetase (seryl-tRNA synthetase) that naturally lacks an editing domain, but possesses a hydrolytic activity toward non-cognate aminoacyl-adenylates. Our data reveal that tRNA-independent pre-transfer editing may proceed within the enzyme active site without shuttling the non-cognate aminoacyl-adenylate intermediate to the remote editing site.     

Zinc ion mediated amino acid discrimination by threonyl-tRNA synthetase

Accurate translation of the genetic code depends on the ability of aminoacyl-tRNA synthetases to distinguish between similar amino acids. In order to investigate the basis of amino acid recognition and to understand the role played by the zinc ion present in the active site of threonyl-tRNA synthetase, we have determined the crystal structures of complexes of an active truncated form of the enzyme with a threonyl adenylate analog or threonine. The zinc ion is directly involved in threonine recognition, forming a pentacoordinate intermediate with both the amino group and the side chain hydroxyl. Amino acid activation experiments reveal that the enzyme shows no activation of isosteric valine, and activates serine at a rate 1,000-fold less than that of cognate threonine. This study demonstrates that the zinc ion is neither strictly catalytic nor structural and suggests how the zinc ion ensures that only amino acids that possess a hydroxyl group attached to the beta-position are activated.     

tRNA-dependent amino acid discrimination by yeast seryl-tRNA synthetase.

The ability of aminoacyl-tRNA synthetases to distinguish between similar amino acids is crucial for accurate translation of the genetic code. Saccharomyces cerevisiae seryl-tRNA synthetase (SerRS) employs tRNA-dependent recognition of its cognate amino acid serine [Lenhard, B., Filipic, S., Landeka, I., Skrtic, I., S?ll, D. & Weygand-Durasevic, I. (1997) J. Biol. Chem.272, 1136-1141]. Here we show that dimeric SerRS enzyme complexed with one molecule of tRNASer is more specific and more efficient in catalyzing seryl-adenylate formation than the apoenzyme alone. Sequence-specific tRNA-protein interactions enhance discrimination of the amino acid substrate by yeast SerRS and diminish the misactivation of the structurally similar noncognate threonine. This may proceed via a tRNA-induced conformational change in the enzyme's active site. The 3'-terminal adenosine of tRNASer is not important in effecting the rearrangement of the serine binding site. Our results do not provide an indication for a readjustment of ATP binding in a tRNA-assisted manner. The stoichiometric analyses of the complexes between the enzyme and tRNASer revealed that two cognate tRNA molecules can be bound to dimeric SerRS, however, with very different affinities.     

tRNA-dependent aminoacyl-adenylate hydrolysis by a nonediting class I aminoacyl-tRNA synthetase

Glutaminyl-tRNA synthetase generates Gln-tRNA(Gln) 10(7)-fold more efficiently than Glu-tRNA(Gln) and requires tRNA to synthesize the activated aminoacyl adenylate in the first step of the reaction. To examine the role of tRNA in amino acid activation more closely, several assays employing a tRNA analog in which the 2'-OH group at the 3'-terminal A76 nucleotide is replaced with hydrogen (tRNA(2'HGln)) were developed. These experiments revealed a 10(4)-fold reduction in kcat/Km in the presence of the analog, suggesting a direct catalytic role for tRNA in the activation reaction. The catalytic importance of the A76 2'-OH group in aminoacylation mirrors a similar role for this moiety that has recently been demonstrated during peptidyl transfer on the ribosome. Unexpectedly, tracking of Gln-AMP formation utilizing an alpha-32P-labeled ATP substrate in the presence of tRNA(2'HGln) showed that AMP accumulates 5-fold more rapidly than Gln-AMP. A cold-trapping experiment revealed that the nonenzymatic rate of Gln-AMP hydrolysis is too slow to account for the rapid AMP formation; hence, the hydrolysis of Gln-AMP to form glutamine and AMP must be directly catalyzed by the GlnRS x tRNA(2'HGln) complex. This hydrolysis of glutaminyl adenylate represents a novel reaction that is directly analogous to the pre-transfer editing hydrolysis of noncognate aminoacyl adenylates by editing synthetases such as isoleucyl-tRNA synthetase. Because glutaminyl-tRNA synthetase does not possess a spatially separate editing domain, these data demonstrate that a pre-transfer editing-like reaction can occur within the synthetic site of a class I tRNA synthetase.     

Association of an aminoacyl-tRNA synthetase with a putative metabolic protein in archaea

Aminoacyl-tRNA synthetases are essential enzymes that catalyze attachment of amino acids to tRNAs for decoding of genetic information. In higher eukaryotes, several synthetases associate with non-synthetase proteins to form a high-molecular mass complex that may improve the efficiency of protein synthesis. This multi-synthetase complex is not found in bacteria. Here we describe the isolation of a non-synthetase protein from the archaeon Methanocaldococcus jannaschii that was copurified with prolyl-tRNA synthetase (ProRS). This protein, Mj1338, also interacts with several other tRNA synthetases and has an affinity for general tRNA, suggesting the possibility of forming a multi-synthetase complex. However, unlike the non-synthetase proteins in the eukaryotic complex, the protein Mj1338 is predicted to be a metabolic protein, related to members of the family of H(2)-forming N(5),N(10)-methylene tetrahydromethanopterin (5,10-CH(2)-H(4)MP) dehydrogenases that are involved in the one-carbon metabolism of the archaeon. The association of Mj1338 with ProRS, and with other components of the protein synthesis machinery, thus suggests the possibility of a closer link between metabolism and decoding in archaea than in eukarya or bacteria.     

Synthetic peptide model of an essential region of an aminoacyl-tRNA synthetase

A 40 amino acid sequence of the unsolved structure of Escherichia coli alanine-tRNA synthetase is essential for tRNA binding and encodes an immunological determinant that cross-reacts with antibodies raised against a eukaryote (insect Bombyx mori) alanine enzyme. The secondary structure of this sequence is predicted to be an amphiphilic alpha-helix that includes one aspartyl and eight glutamyl side chain carboxyl groups. The antibody reactivity and the conformation of a synthetic peptide model of this region (Glu346 to Ser385) were investigated. In addition, double Arg----Gln and Leu----Ala substitutions were separately placed in the enzyme on the hydrophilic and hydrophobic face, respectively, of the predicted helix. These mutations conserve the polar/nonpolar character of each face and retain the potential for helix formation. Circular dichroism spectra of the synthetic peptide model demonstrate the potential for amphiphilic helix formation for the segment from Glu346 to Ser385. The behavior of the mutations in the enzyme, together with earlier data and immunological assays presented here, suggests that one face of the putative helix is an antigenic region of the surface of the enzyme where it contributes to the interaction with alanine tRNA and that the specific sequence of the helix is an important determinant of enzyme stability.     

Kinetic quality control of anticodon recognition by a eukaryotic aminoacyl-tRNA synthetase

Aminoacyl-tRNA synthetases are an ancient class of enzymes responsible for the matching of amino acids with anticodon sequences of tRNAs. Eukaryotic tRNA synthetases are often larger than their bacterial counterparts, and several mammalian enzymes use the additional domains to facilitate assembly into a multi-synthetase complex. Human cysteinyl-tRNA synthetase (CysRS) does not associate with the multi-synthetase complex, yet contains a eukaryotic-specific C-terminal extension that follows the tRNA anticodon-binding domain. Here we show by mutational and kinetic analysis that the C-terminal extension of human CysRS is used to selectively improve recognition and binding of the anticodon sequence, such that the specificity of anticodon recognition by human CysRS is higher than that of its bacterial counterparts. However, the improved anticodon recognition is achieved at the expense of a significantly slower rate in the aminoacylation reaction, suggesting a previously unrecognized kinetic quality control mechanism. This kinetic quality control reflects an evolutionary adaptation of some tRNA synthetases to improve the anticodon specificity of tRNA aminoacylation from bacteria to humans, possibly to accommodate concomitant changes in codon usage.