Cysteinyl-tRNA synthetase: determination of the last E. coli aminoacyl-tRNA synthetase primary structure.

The gene coding for E. coli cysteinyl-tRNA synthetase (cysS) was isolated by complementation of a strain deficient in cysteinyl-tRNA synthetase activity at high temperature (43 degrees C). Sequencing of a 2.1 kbp DNA fragment revealed an open reading frame of 1383 bp coding for a protein of 461 amino acid residues with a Mr of 52,280, a value in close agreement with that observed for the purified protein, which behaves as a monomer. The sequence of CysRS bears the canonical His-Ile- Gly -His (HIGH) and Lys-Met-Ser-Lys-Ser (KMSKS) motifs characteristic of the group of enzymes containing a Rossmann fold; furthermore, it shows striking homologies with MetRS (an homodimer of 677 residues) and to a lesser extent with Ile-, Leu-, and ValRS (monomers of 939, 860, and 951 residues respectively). With its monomeric state and smaller size, CysRS is probably more closely related to the primordial aminoacyl-tRNA synthetase from which all have diverged.     

A component of the multisynthetase complex is a multifunctional aminoacyl-tRNA synthetase.

In higher eukaryotes, nine aminoacyl-tRNA synthetases are associated within a multienzyme complex which is composed of 11 polypeptides with molecular masses ranging from 18 to 150 kDa. We have cloned and sequenced a cDNA from Drosophila encoding the largest polypeptide of this complex. We demonstrate here that the corresponding protein is a multifunctional aminoacyl-tRNA synthetase. It is composed of three major domains, two of them specifying distinct synthetase activities. The amino and carboxy-terminal domains were expressed separately in Escherichia coli, and were found to catalyse the aminoacylation of glutamic acid and proline tRNA species, respectively. The central domain is made of six 46 amino acid repeats. In prokaryotes, these two aminoacyl-tRNA synthetases are encoded by distinct genes. The emergence of a multifunctional synthetase by a gene fusion event seems to be a specific, but general attribute of all higher eukaryotic cells. This type of structural organization, in relation to the occurrence of multisynthetase complexes, could be a mechanism to integrate several catalytic domains within the same particle. The involvement of the internal repeats in mediating complex assembly is discussed.     

Crystallization of a tRNA . aminoacyl-tRNA synthetase complex. Characterization and first crystallographic data

A complex formed between the dimeric aspartyl-tRNA synthetase from yeast (Mr congruent to 125,000) and two molecules of its cognate yeast tRNAAsp (Mr = 24,160) was crystallized using ammonium sulfate as the precipitant. The crucial parameter which governs a successful crystallization is the enzyme tRNA stoichiometry. Crystals are only obtained when the starting solution precisely contains two tRNA molecules for one enzyme molecule. It was demonstrated by electrophoresis, biological activity assays, and crystallographic data that the crystals contain the two components in the same two to one stoichiometric ratio. The crystals, of cubic shape with edges up to 0.8 mm, belong to space group 1432. The cell parameter is 354 A and the asymmetric unit contains one particle of complex. The solvent content is about 78%, higher than the values commonly observed. Although particularly soft, the quality of the crystals is suitable for x-ray diffraction studies up to 7-A resolution.     

Cysteinyl-tRNA formation: the last puzzle of aminoacyl-tRNA synthesis

A 1H nuclear magnetic resonance study of the complex of cytochrome P450cam-putidaredoxin has been performed. Isocyanide is bound to cytochrome P450cam in order to increase the stability of the protein both in the reduced and the oxidized state. Diprotein complex formation was detected through variation of the heme methyl proton resonances which have been assigned in the two redox states. The electron transfer rate at equilibrium was determinated by magnetization transfer experiments. The observed rate of oxidation of reduced cytochrome P450 by the oxidized putidaredoxin is 27 (+/- 7) per s.     

Cysteinyl-tRNA synthetase from Bacillus stearothermophilus. A structural and functional monomer.

A procedure is described for the purification of cysteinyl-tRNA synthetase as a side product of a multi-enzyme isolation from Bacillus stearothermophilus. The native and denatured enzyme are both shown to have a molecular weight of 54000 by gel filtration and sodium dodecyl sulphate/polyacrylamide gel electrophoresis respectively. Fingerprinting and peptide counting indicate that the polypeptide chain has a nonrepeating primary structure. The enzyme has only one binding site for each of its substrates (cysteine, ATP and tRNACys) as judged by equilibrium dialysis, active-site titration and fluorescence quenching. No evidence for the dimerisation of the enzyme in the presence of these substrates could be found. We conclude that cysteinyl-tRNA synthetase, which is the smallest aminoacyl-tRNA synthetase yet described, is both structurally and functionally monomeric.     

Catalytic peptide of human glutaminyl-tRNA synthetase is essential for its assembly to the aminoacyl-tRNA synthetase complex

Human glutaminyl-tRNA synthetase (QRS) is one of several mammalian aminoacyl-tRNA synthetases (ARSs) that form a macromolecular protein complex. To understand the mechanism of QRS targeting to the multi-ARS complex, we analyzed both exogenous and endogenous QRSs by immunoprecipitation after overexpression of various Myc-tagged QRS mutants in human embryonic kidney 293 cells. Whereas a deletion mutant containing only the catalytic domain (QRS-C) was targeted to the multi-ARS complex, a mutant QRS containing only the N-terminal appended domain (QRS-N) was not. Deletion mapping showed that the ATP-binding Rossman fold was necessary for targeting of QRS to the multi-ARS complex. Furthermore, exogenous Myc-tagged QRS-C was co-immunoprecipitated with endogenous QRS. Since glutaminylation of tRNA was dramatically increased in cells transfected with the full-length QRS, but not with either QRS-C or QRS-N, both the QRS catalytic domain and the N-terminal appended domain were required for full aminoacylation activity. When QRS-C was overexpressed, arginyl-tRNA synthetase and p43 were released from the multi-ARS complex along with endogenous QRS, suggesting that the N-terminal appendix of QRS is required to keep arginyl-tRNA synthetase and p43 within the complex. Thus, the eukaryote-specific N-terminal appendix of QRS appears to stabilize the association of other components in the multi-ARS complex, whereas the C-terminal catalytic domain is necessary for QRS association with the multi-ARS complex.     

A bacterial amber suppressor in Saccharomyces cerevisiae is selectively recognized by a bacterial aminoacyl-tRNA synthetase.

Little is known about the conservation of determinants for the identities of tRNAs between organisms. We showed previously that Escherichia coli tyrosine tRNA synthetase can charge the Saccharomyces cerevisiae mitochondrial tyrosine tRNA in vivo, even though there are substantial sequence differences between the yeast mitochondrial and bacterial tRNAs. The S. cerevisiae cytoplasmic tyrosine tRNA differs in sequence from both its yeast mitochondrial and E. coli counterparts. To test whether the yeast cytoplasmic tyrosyl-tRNA synthetase recognizes the E. coli tRNA, we expressed various amounts of an E. coli tyrosine tRNA amber suppressor in S. cerevisiae. The bacterial tRNA did not suppress any of three yeast amber alleles, suggesting that the yeast enzymes retain high specificity in vivo for their homologous tRNAs. Moreover, the nucleotides in the sequence of the E. coli suppressor that are not shared with the yeast cytoplasmic tyrosine tRNA do not create determinants which are efficiently recognized by other yeast charging enzymes. Therefore, at least some of the determinants that influence in vivo recognition of the tyrosine tRNA are specific to the cell compartment and organism. In contrast, expression of the cognate bacterial tyrosyl-tRNA synthetase together with the bacterial suppressor tRNA led to suppression of all three amber alleles. The bacterial enzyme recognized its substrate in vivo, even when the amount of bacterial tRNA was less than about 0.05% of that of the total cytoplasmic tRNA.     

An aminoacyl-tRNA synthetase complex in Escherichia coli.

Aminoacyl-tRNA synthetases from several strains of Escherichia coli are shown to elute as a high-molecular-weight complex on 6% agarose columns (Bio-Gel A-5M). In contrast, very little synthetase activity was observed in such complexes on Sephadex G-200 columns, suggesting that these enzymes may interact with or are dissociated during chromatography on dextran. The size of the complex observed on Bio-Gel A-5M was influenced by the method of cell breakage and the salt concentrations present in buffers. The largest complexes (greater than 1,000,000 daltons) were seen with cells broken with a freeze press, whereas with sonicated preparations the average size of the complex was about 400,000 daltons. Extraction of synthetases at 0.15 M NaCl, to mimic physiological salt concentrations, also resulted in high-molecular-weight complexes, as demonstrated by both agarose gel filtration and ultracentrifugation analysis. Evidence is presented that dissociation of some synthetases does occur in the presence of higher salt levels (0.4 M NaCl). Partial purification of the synthetase complex on DEAE-Sephacel was accomplished with only minor dissociation of individual synthetases. These data suggest that a complex(es) of aminoacyl-tRNA synthetase does exist in bacterial cells, just as in eucaryotes, and that the complex may have escaped earlier detection due to its fragility during isolation.     

An aminoacyl-tRNA synthetase with a defunct editing site

Many aminoacyl-tRNA synthetases (aaRSs) contain two active sites, a synthetic site catalyzing aminoacyl-adenylate formation and tRNA aminoacylation and a second editing or proofreading site that hydrolyzes misactivated adenylates or mischarged tRNAs. The combined activities of these two sites lead to rigorous accuracy in tRNA aminoacylation, and both activities are essential to LeuRS and other aaRSs. Here, we describe studies of the human mitochondrial (hs mt) LeuRS indicating that the two active sites of this enzyme have undergone functional changes that impact how accurate aminoacylation is achieved. The sequence of the hs mt LeuRS closely resembles a bacterial LeuRS overall but displays significant variability in regions of the editing site. Studies comparing Escherichia coli and hs mt LeuRS reveal that the proofreading activity of the mt enzyme is disrupted by these sequence changes, as significant levels of Ile-tRNA(Leu) are formed in the presence of high concentrations of the noncognate amino acid. Experiments monitoring deacylation of Ile-tRNA(Leu) and misactivated adenylate turnover revealed that the editing active site is not operational. However, hs mt LeuRS has weaker binding affinities for both cognate and noncognate amino acids relative to the E. coli enzyme and an elevated discrimination ratio. Therefore, the enzyme achieves fidelity using a more specific synthetic active site that is not prone to errors under physiological conditions. This enhanced specificity must compensate for the presence of a defunct editing site and ensures translational accuracy.     

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.     

Metabolic regulation of aminoacyl-tRNA synthetase biosynthesis in bakers' yeast.

The specific activities of 15 aminoacyl-tRNA synthetases in Saccharomyces cerevisiae were measured after growth under a variety of conditions that produced a range of cell-doubling times. The specific activity of each synthetase increased as cell-doubling time decreased. Control experiments eliminate the possibility that these results are due to preferential recovery of synthetases, or to the presence of activators in the faster growing cultures or inhibitors in the slower growing ones. These observations run counter to the expectation that synthetases in bacteria and yeast are negatively regulated by free amino acids, or, more likely, by aminoacyl-tRNA. In fact, as the growth medium was enriched, generation times decreased, and synthetase and aminoacyl-tRNA levels increased. It is suggested that cytoplasmic aminoacyl-tRNA synthetases may be more or less coordinately controlled such that their response to growth follows the pattern observed for ribosome production and RNA synthesis. This suggests the possibility of coordinated response of genes for components of the protein synthetic apparatus.     

Structure-functional properties of eukaryotic aminoacyl-tRNA synthetase]

Data on structural and functional peculiarities of eukaryotic aminoacyl-tRNA synthetases (structure, supermolecular organization, and localization in eukaryotic cell) are reviewed. The functional significance of aminoacyl-tRNA synthetase association with high molecular weight complexes and other cellular components is discussed.     

Deinococcus glutaminyl-tRNA synthetase is a chimer between proteins from an ancient and the modern pathways of aminoacyl-tRNA formation

Glutaminyl-tRNA synthetase from Deinococcus radiodurans possesses a C-terminal extension of 215 residues appending the anticodon-binding domain. This domain constitutes a paralog of the Yqey protein present in various organisms and part of it is present in the C-terminal end of the GatB subunit of GatCAB, a partner of the indirect pathway of Gln-tRNA^Gln formation. To analyze the peculiarities of the structure–function relationship of this GlnRS related to the Yqey domain, a structure of the protein was solved from crystals diffracting at 2.3Å and a docking model of the synthetase complexed to tRNA^Gln constructed. The comparison of the modeled complex with the structure of the E. coli complex reveals that all residues of E. coli GlnRS contacting tRNA^Gln are conserved in D. radiodurans GlnRS, leaving the functional role of the Yqey domain puzzling. Kinetic investigations and tRNA-binding experiments of full length and Yqey-truncated GlnRSs reveal that the Yqey domain is involved in tRNA^Gln recognition. They demonstrate that Yqey plays the role of an affinity-enhancer of GlnRS for tRNA^Gln acting only in cis. However, the presence of Yqey in free state in organisms lacking GlnRS, suggests that this domain may exert additional cellular functions.     

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.     

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.     

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.     

A sulfhydryl presumed essential is not required for catalysis by an aminoacyl-tRNA synthetase

Several aminoacyl-tRNA synthetases are sensitive to reagents that modify sulfhydryl groups. We report here the significance of N-ethylmaleimide (NEM)-mediated inactivation of Escherichia coli glycyl-tRNA synthetase, and alpha 2 beta 2 enzyme. We confirmed earlier observations that NEM abolishes synthetase-catalyzed aminoacylation with pseudo-first order kinetics and provided a second method of proof that the site of inactivation is located in the beta-subunit. Using oligonucleotide-directed mutagenesis of the glyS gene, each beta-subunit cysteine codon (positions 98, 395, and 450) was replaced, individually, by an alanine codon. The three resulting mutant proteins are each active in vivo, and their in vitro aminoacylation activities are comparable to that of the native enzyme. A mutant incorporating all three amino acid substitutions is also active in vivo and in vitro. These results establish conclusively that a beta-subunit cysteine thiol is not required for the catalysis of aminoacylation. The Cys98----Ala and Cys450----Ala mutants are inactivated by NEM with the same kinetics as the wild-type protein. However, the Cys395----Ala mutant is refractory to NEM. This suggests that NEM inactivation of the native enzyme is due to alkylation of Cys395. Aware that inactivation may result from steric effects, we constructed a mutant with a bulkier amino acid residue at position 395 (Cys395----Gln). The aminoacylation activity of this protein is less than 10% of that of the wild-type enzyme. The glutamine substitution affects only the tRNA-dependent step of the reaction--the rate of glycyl adenylate synthesis is not lowered. In these features, the mutant resembles the NEM-inactivated protein. We propose that the NEM sensitivity of glycyl-tRNA synthetase, and possibly of other synthetases, arises from steric or conformational effects of the alkylated cysteine side chain.