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.     

Solvation change and ion release during aminoacylation by aminoacyl-tRNA synthetases

Discrimination between cognate and non-cognate tRNAs by aminoacyl-tRNA synthetases occurs at several steps of the aminoacylation pathway. We have measured changes of solvation and counter-ion distribution at various steps of the aminoacylation pathway of glutamyl- and glutaminyl-tRNA synthetases. The decrease in the association constant with increasing KCl concentration is relatively small for cognate tRNA binding when compared to known DNA–protein interactions. The electro-neutral nature of the tRNA binding domain may be largely responsible for this low ion release stoichiometry, suggesting that a relatively large electrostatic component of the DNA–protein interaction free energy may have evolved for other purposes, such as, target search. Little change in solvation upon tRNA binding is seen. Non-cognate tRNA binding actually increases with increasing KCl concentration indicating that charge repulsion may be a significant component of binding free energy. Thus, electrostatic interactions may have been used to discriminate between cognate and non-cognate tRNAs in the binding step. The catalytic constant of glutaminyl-tRNA synthetase increases with increasing osmotic pressure indicating a water release of 8.4 ± 1.4 mol/mol in the transition state, whereas little change is seen in the case of glutamyl-tRNA synthetase. We propose that the significant amount of water release in the transition state, in the case of glutaminyl-tRNA synthetase, is due to additional contact of the protein with the tRNA in the transition state.     

Aminoacylation of tRNAs as critical step of protein biosynthesis.

Isoleucyl-tRNA synthetases isolated from commercial baker's yeast and E coli were investigated for their sequences of substrate additions and product releases. The results show that aminoacylation of tRNA is catalyzed by these enzymes in different pathways, eg isoleucyl-tRNA synthetase from yeast can act with four different catalytic cycles. Amino acid specificities are gained by a four-step recognition process consisting of two initial binding and two proofreading steps. Isoleucyl-tRNA synthetase from yeast rejects noncognate amino acids with discrimination factors of D = 300-38000, isoleucyl-tRNA synthetase from E coli with factors of D = 600-68000. Differences in Gibbs free energies of binding between cognate and noncognate amino acids are related to different hydrophobic interaction energies and assumed conformational changes of the enzyme. A simple hypothetical model of the isoleucine binding site is postulated. Comparison of gene sequences of isoleucyl-tRNA synthetase from yeast and E coli exhibits only 27% homology. Both genes show the 'HIGH'- and 'KMSKS'-regions assigned to binding of ATP and tRNA. Deletion of 250 carboxyterminal amino acids from the yeast enzyme results in a fragment which is still active in the pyrophosphate exchange reaction but does not catalyze the aminoacylation reaction. The enzyme is unable to catalyze the latter reaction if more than 10 carboxyterminal residues are deleted.     

A fluorescence spectroscopic study of glutaminyl-tRNA synthetase from Escherichia coli and its implications for the enzyme mechanism

Interaction between Escherichia coli glutaminyl-tRNA synthetase (GlnRS) and its substrates have been studied by fluorescence quenching. In the absence of other substrates, glutamine, tRNA(Gln) and ATP bind with dissociation constants of 460, 0.22 and 180 microM, respectively. The presence of other substrates has either no effect or, at best a weak effect, on binding of ligands. Attempts to isolate enzyme-bound aminoacyl adenylate did not succeed. Binding of the phosphodiester, 5'-(methyl)adenosine monophosphate (MeAMP), to GlnRS was studied by fluorescence quenching and radioactive-ligand binding. tRNA also only has a weak effect on phosphodiester binding. Selectively pyrene-labeled GlnRS was used to obtain shape and size information for free GlnRS. A comparison with the GlnRS shape in the GlnRS/tRNA(Gln) crystal structure indicates that no major change in shape and size occurs upon tRNA(Gln) binding to GlnRS. 5,5'-Bis(8-anilino-1-naphthalene sulfonate) (bis-ANS), a non-covalent fluorescent probe, was also used to probe for conformational changes in GlnRS. This probe also indicated that no major conformational change occurs upon tRNA(Gln) binding. We conclude that lack of tRNA-independent pyrophosphate-exchange activity in this enzyme is not a result of either lack of glutamine or ATP binding in the absence of tRNA, or formation of aminoacyl adenylate and slow release of pyrophosphate. A conformational change is implied upon tRNA binding, which promotes pyrophosphate exchange. Fluorescence studies indicate that this conformational change must be limited and local in nature.     

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.     

Aminoacyl-tRNA synthetase-induced cleavage of tRNA.

Aminoacyl-tRNA synthetases interact with their cognate tRNAs in a highly specific fashion. We have examined the phenomenon that upon complex formation E. coli glutaminyl-tRNA synthetase destabilizes tRNA(Gln) causing chain scissions in the presence of Mg2+ ions. The phosphodiester bond cleavage produces 3'-phosphate and 5'-hydroxyl ends. This kind of experiment is useful for detecting conformational changes in tRNA. Our results show that the cleavage is synthetase-specific, that mutant and wild-type tRNA(Gln) species can assume a different conformation, and that modified nucleosides in tRNA enhance the structural stability of the molecule.     

In vitro conversion of a methionine to a glutamine-acceptor tRNA

A derivative of Escherichia coli tRNAfMet containing an altered anticodon sequence, CUA, has been enzymatically synthesized in vitro. The variant tRNA was prepared by excision of the normal anticodon, CAU, in a limited digestion of intact tRNAfMet with RNase A, followed by insertion of the CUA sequence into the anticodon loop with T4 RNA ligase and polynucleotide kinase. The altered methionine tRNA showed a large enhancement in the rate of aminoacylation by glutaminyl-tRNA synthetase and a large decrease in the rate of aminoacylation by methionyl-tRNA synthetase. Measurement of kinetic parameters for the charging reaction by the cognate and noncognate enzymes revealed that the modified tRNA is a better acceptor for glutamine than for methionine. The rate of mischarging is similar to that previously reported for a tryptophan amber suppressor tRNA containing the anticodon CUA, su+7 tRNATrp, which is aminoacylated with glutamine both in vivo and in vitro [Yaniv, M., Folk, W. R., Berg, P., & Soll, L. (1974) J. Mol. Biol. 86, 245-260; Yarus, M., Knowlton, R. E., & Soll, L. (1977) in Nucleic Acid-Protein Recognition (Vogel, H., Ed.) pp 391-408, Academic Press, New York]. The present results provide additional evidence that the specificity of aminoacylation by glutaminyl-tRNA synthetase is sensitive to small changes in the nucleotide sequence of noncognate tRNAs and that uridine in the middle position of the anticodon is involved in the recognition of tRNA substrates by this enzyme.     

tRNA aminoacylation by arginyl-tRNA synthetase: induced conformations during substrates binding

The 2.2 A crystal structure of a ternary complex formed by yeast arginyl-tRNA synthetase and its cognate tRNA(Arg) in the presence of the L-arginine substrate highlights new atomic features used for specific substrate recognition. This first example of an active complex formed by a class Ia aminoacyl-tRNA synthetase and its natural cognate tRNA illustrates additional strategies used for specific tRNA selection. The enzyme specifically recognizes the D-loop and the anticodon of the tRNA, and the mutually induced fit produces a conformation of the anticodon loop never seen before. Moreover, the anticodon binding triggers conformational changes in the catalytic center of the protein. The comparison with the 2.9 A structure of a binary complex formed by yeast arginyl-tRNA synthetase and tRNA(Arg) reveals that L-arginine binding controls the correct positioning of the CCA end of the tRNA(Arg). Important structural changes induced by substrate binding are observed in the enzyme. Several key residues of the active site play multiple roles in the catalytic pathway and thus highlight the structural dynamics of the aminoacylation reaction.     

G-1:C73 recognition by an arginine cluster in the active site of Escherichia coli histidyl-tRNA synthetase

Aminoacylation of a transfer RNA (tRNA) by its cognate aminoacyl-tRNA synthetase relies upon the recognition of specific nucleotides as well as conformational features within the tRNA by the synthetase. In Escherichia coli, the aminoacylation of tRNA(His) by histidyl-tRNA synthetase (HisRS) is highly dependent upon the recognition of the unique G-1:C73 base pair and the 5'-monophosphate. This work investigates the RNA-protein interactions between the HisRS active site and these critical recognition elements. A homology model of the tRNA(His)-HisRS complex was generated and used to design site-specific mutants of possible G-1:C73 contacts. Aminoacylation assays were performed with these HisRS mutants and N-1:C73 tRNA(His) and microhelix(His) variants. Complete suppression of the negative effect of 5'-phosphate deletion by R123A HisRS, as well as the increased discrimination of Q118E HisRS against a 5'-triphosphate, suggests a possible interaction between the 5'-phosphate and active-site residues Arg123 and Gln118 in which these residues create a sterically and electrostatically favorable pocket for the binding of the negatively charged phosphate group. Additionally, a network of interactions appears likely between G-1 and Arg116, Arg123, and Gln118 because mutation of these residues significantly reduced the sensitivity of HisRS to changes at G-1. Our studies also support an interaction previously proposed between Gln118 and C73. Defining the RNA-protein interactions critical for efficient aminoacylation by E. coli HisRS helps to further characterize the active site of this enzyme and improves our understanding of how the unique identity elements in the acceptor stem of tRNA(His) confer specificity.     

Achieving error-free translation; the mechanism of proofreading of threonyl-tRNA synthetase at atomic resolution

The fidelity of aminoacylation of tRNA(Thr) by the threonyl-tRNA synthetase (ThrRS) requires the discrimination of the cognate substrate threonine from the noncognate serine. Misacylation by serine is corrected in a proofreading or editing step. An editing site has been located 39 A away from the aminoacylation site. We report the crystal structures of this editing domain in its apo form and in complex with the serine product, and with two nonhydrolyzable analogs of potential substrates: the terminal tRNA adenosine charged with serine, and seryl adenylate. The structures show how serine is recognized, and threonine rejected, and provide the structural basis for the editing mechanism, a water-mediated hydrolysis of the mischarged tRNA. When the adenylate analog binds in the editing site, a phosphate oxygen takes the place of one of the catalytic water molecules, thereby blocking the reaction. This rules out a correction mechanism that would occur before the binding of the amino acid on the tRNA.     

Mutant enzymes and tRNAs as probes of the glutaminyl-tRNA synthetase: tRNA(Gln) interaction.

This paper focuses on several aspects of the specificity of mutants of Escherichia coli glutaminyl-tRNA synthetase (GlnRS) and tRNA(Gln). Temperature-sensitive mutants located in glnS, the gene for GlnRS, have been described previously. The mutations responsible for the temperature-sensitive phenotype were analyzed, and pseudorevertants of these mutants isolated and characterized. The nature of these mutations is discussed in terms of their location in the three-dimensional structure of the tRNA(Gln).GlnRS complex. In order to characterize the specificity of the aminoacylation reaction, mutant tRNA(Gln) species were synthesized with either a 2'-deoxy AMP or 3'-deoxy AMP as their 3'-terminal nucleotide. Subsequent assays for aminoacylation and ATP/PPi exchange activity established the esterification of glutamine to the 2'-hydroxyl of the terminal adenosine; there is no glutaminylation of the 3'-OH group. This correlates with the classification of GlnRS as a class I aminoacyl-tRNA synthetase. Mutations in tRNA(Gln) are discussed which affect the recognition of GlnRS and the current concept of glutamine identity in E coli is reviewed.     

The free yeast aspartyl-tRNA synthetase differs from the tRNA(Asp)-complexed enzyme by structural changes in the catalytic site, hinge region, and anticodon-binding domain

Aminoacyl-tRNA synthetases catalyze the specific charging of amino acid residues on tRNAs. Accurate recognition of a tRNA by its synthetase is achieved through sequence and structural signalling. It has been shown that tRNAs undergo large conformational changes upon binding to enzymes, but little is known about the conformational rearrangements in tRNA-bound synthetases. To address this issue the crystal structure of the dimeric class II aspartyl-tRNA synthetase (AspRS) from yeast was solved in its free form and compared to that of the protein associated to the cognate tRNA(Asp). The use of an enzyme truncated in N terminus improved the crystal quality and allowed us to solve and refine the structure of free AspRS at 2.3 A resolution. For the first time, snapshots are available for the different macromolecular states belonging to the same tRNA aminoacylation system, comprising the free forms for tRNA and enzyme, and their complex. Overall, the synthetase is less affected by the association than the tRNA, although significant local changes occur. They concern a rotation of the anticodon binding domain and a movement in the hinge region which connects the anticodon binding and active-site domains in the AspRS subunit. The most dramatic differences are observed in two evolutionary conserved loops. Both are in the neighborhood of the catalytic site and are of importance for ligand binding. The combination of this structural analysis with mutagenesis and enzymology data points to a tRNA binding process that starts by a recognition event between the tRNA anticodon loop and the synthetase anticodon binding module.     

Interaction of Escherichia coli glutaminyl-tRNA synthesis with noncognate tRNA''s.

Several noncognate tRNA's from Escherichia coli were mischarged with glutamine by E. coli glutaminyl-tRNA synthetase if dimethylsulfoxide was present in the reaction mixture. Kinetic analysis of the mischarging revealed that dimethyl sulfoxide stimulated the misacylation by affecting the maximum velocity. Several noncognate tRNA's were shown to interact with glutaminyl-tRNA synthetase as measured by their ability to protect the enzyme against thermal inactivation or to replace cognate tRNA in stimulating glutamine-dependent ATP-PPi exchange reaction. These tRNA's, however, did not coincide with those which were mischargeable with glutamine.     

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.     

Superspecificity of aminoacyl-tRNA-synthases

The correct aminoacylation of tRNA with the proper aminoacid by aminoacyl-tRNA synthetase is one of the key reactions which determines the overall high fidelity of protein biosynthesis. The initial selection of the amino acid is achieved in the active centre of the synthetase at the activation step due to differences in the side chains binding energies of specific substrate and the competing amino acids present in cell. If, nevertheless, the activation of amino acids structurally similar to the cognate one does proceed, additional mechanisms of correction which are based on the decomposition of unstable noncognate (intermediate or final) product of the tRNA aminoacylation reaction, by synthetase are switched on. In this review the literature on the specificity of aminoacyl-tRNA synthetases at amino acid activation step is analyzed along with the proofreading mechanisms which allow the elimination of the errors, leading to so called superspecifity of aminoacyl-tRNA synthetases.     

Yeast seryl tRNA synthetase: interactions between the ATP binding site and the sites for tRNASer and L-serine.

T1 ribonuclease digestion of yeast tRNASer in the presence of seryl tRNA synthetase was used for monitoring the relationship between the substrate binding sites on the synthetase. It was found that (a) ATP displaces the tRNA from the synthetase with an effector affinity constant corresponding to the Km for ATP of 10 micron; (b) AMP and a number of nucleoside triphosphates, while influencing the rate of aminoacylation, do not displace the tRNA from the enzyme; (c) ADP and PPi inhibit the aminoacylation and the binding of tRNASer; (d) adenylyl diphosphonate is bound to the synthetase and lowers the protection of the tRNA against the nuclease attack in a similar way as does ATP; (e) interactions between the sites of L-serine and tRNASer could only be shown when both sites for serine were saturated and, in addition, the ATP analog or ADP was present. It is concluded that in seryl tRNA synthetase binding sites for ATP interact with the ones for tRNA as well as with the ones for serine. These findings contribute to the understanding of the mechanism of aminoacylation.     

The homotetrameric phosphoseryl-tRNA synthetase from Methanosarcina mazei exhibits half-of-the-sites activity

Synthesis of cysteinyl-tRNA(Cys) in methanogenic archaea proceeds by a two-step pathway in which tRNA(Cys) is first aminoacylated with phosphoserine by phosphoseryl-tRNA synthetase (SepRS). Characterization of SepRS from the mesophile Methanosarcina mazei by gel filtration and nondenaturing mass spectrometry shows that the native enzyme exists as an alpha4 tetramer when expressed at high levels in Escherichia coli. However, active site titrations monitored by ATP/PP(i) burst kinetics, together with analysis of tRNA binding stoichiometry by fluorescence spectroscopy, show that the tetrameric enzyme binds two tRNAs and that only two of the four chemically equivalent subunits catalyze formation of phosphoseryl adenylate. Therefore, the phenomenon of half-of-the-sites activity, previously described for synthesis of 1 mol of tyrosyl adenylate by the dimeric class I tyrosyl-tRNA synthetase, operates as well in this homotetrameric class II tRNA synthetase. Analysis of cognate and noncognate reactions by ATP/PP(i) and aminoacylation kinetics strongly suggests that SepRS is able to discriminate against the noncognate amino acids glutamate, serine, and phosphothreonine without the need for a separate hydrolytic editing site. tRNA(Cys) binding to SepRS also enhances the capacity of the enzyme to discriminate among amino acids, indicating the existence of functional connectivity between the tRNA and amino acid binding sites of the enzyme.     

Unilateral aminoacylation specificity between bovine mitochondria and eubacteria

The present study shows unilateral aminoacylation specificity between bovine mitochondria and eubacteria (Escherichia coli and Thermus thermophilus) in five amino acid-specific aminoacylation systems. Mitochondrial synthetases were capable of charging eubacterial tRNA as well as mitochondrial tRNA, whereas eubacterial synthetases did not efficiently charge mitochondrial tRNA. Mitochondrial phenylalanyl-, threonyl-, arginyl-, and lysyl-tRNA synthetases were shown to charge and discriminate cognate E. coli tRNA species from noncognate ones strictly, as did the corresponding E. coli synthetases. By contrast, mitochondrial seryl-tRNA synthetase not only charged cognate E. coli serine tRNA species but also extensively misacylated noncognate E. coli tRNA species. These results suggest a certain conservation of tRNA recognition mechanisms between the mitochondrial and E. coli aminoacyl-tRNA synthetases in that anticodon sequences are most likely to be recognized by the former four synthetases, but not sufficiently by the seryl-tRNA synthetase. The unilaterality in aminoacylation may imply that tRNA recognition mechanisms of the mitochondrial synthetases have evolved to be, to some extent, simpler than their eubacterial counterparts in response to simplifications in the species-number and the structural elements of animal mitochondrial tRNAs.     

A succession of substrate induced conformational changes ensures the amino acid specificity of Thermus thermophilus prolyl-tRNA synthetase: comparison with histidyl-tRNA synthetase

We describe the recognition by Thermus thermophilus prolyl-tRNA synthetase (ProRSTT) of proline, ATP and prolyl-adenylate and the sequential conformational changes occurring when the substrates bind and the activated intermediate is formed. Proline and ATP binding cause respectively conformational changes in the proline binding loop and motif 2 loop. However formation of the activated intermediate is necessary for the final conformational ordering of a ten residue peptide ("ordering loop") close to the active site which would appear to be essential for functional tRNA 3' end binding. These induced fit conformational changes ensure that the enzyme is highly specific for proline activation and aminoacylation. We also present new structures of apo and AMP bound histidyl-tRNA synthetase (HisRS) from T. thermophilus which we compare to our previous structures of the histidine and histidyl-adenylate bound enzyme. Qualitatively, similar results to those observed with T. thermophilus prolyl-tRNA synthetase are found. However histidine binding is sufficient to induce the co-operative ordering of the topologically equivalent histidine binding loop and ordering loop. These two examples contrast with most other class II aminoacyl-tRNA synthetases whose pocket for the cognate amino acid side-chain is largely preformed. T. thermophilus prolyl-tRNA synthetase appears to be the second class II aminoacyl-tRNA synthetase, after HisRS, to use a positively charged amino acid instead of a divalent cation to catalyse the amino acid activation reaction.