Dominant Intermediate Charcot-Marie-Tooth disorder is not due to a catalytic defect in tyrosyl-tRNA synthetase.

Charcot-Marie-Tooth disorder (CMT) is the most common inherited peripheral neuropathy, afflicting 1 in every 2500 Americans. One form of this disease, Dominant Intermediate Charcot-Marie-Tooth disorder type C (DI-CMTC), is due to mutation of the gene encoding the cytoplasmic tyrosyl-tRNA synthetase (TyrRS). Three different TyrRS variants have been found to give rise to DI-CMTC: replacing glycine at position 41 by arginine (G41R), replacing glutamic acid at position 196 by lysine (E196K), and deleting amino acids 153-156 (Δ(153-156)). To test the hypothesis that DI-CMTC is due to a defect in the ability of tyrosyl-tRNA synthetase to catalyze the aminoacylation of tRNA(Tyr), we have expressed each of these variants as recombinant proteins and used single turnover kinetics to characterize their abilities to catalyze the activation of tyrosine and its subsequent transfer to the 3' end of tRNA(Tyr). Two of the variants, G41R and Δ(153-156), display a substantial decrease in their ability to bind tyrosine (>100-fold). In contrast, the E196K substitution does not significantly affect the kinetics for formation of the tyrosyl-adenylate intermediate and actually increases the rate at which the tyrosyl moiety is transferred to tRNA(Tyr). The observation that the E196K substitution does not decrease the rate of catalysis indicates that DI-CMTC is not due to a catalytic defect in tyrosyl-tRNA synthetase.

[A model of three-dimensional structure of Mycobacterium tuberculosis tyrosyl-tRNA synthetase].

The search of new effective antibacterial drugs against infectious agents also lately include inhibitors of some aminoacyl-tRNA synthetases. In this regard, tyrosyl-tRNA synthetase from M. tuberculosis (MtTyrRS) is one of especially attractive target due to its key role in cell metabolism and significant differences between spatial structures of eubacterial and human TyrRSs. In this article the theoretic homology modeling of spatial structure of catalytic module of MtTyrRS (region Met1-Ser321) has been carried out based on experimentally determined structures of homologous TyrRSs from other eubacteria, and the comparison of the structures of their active sites was performed. Most of MtTyrRS catalytic site residues, particularly those, which form special hydrogen bonds with low-molecular-weight ligands (Tyr36, Asp80, Tyrl71, Asp178, Gly194 and Asp196) as well catalytic residues Lys231 and Lys234 from KFGK motif of interdomain loop and Arg87, are conservative in evolution.

Toward the full set of human mitochondrial aminoacyl-tRNA synthetases: characterization of AspRS and TyrRS.

The human mitochondrion possesses a translational machinery devoted to the synthesis of 13 proteins. While the required tRNAs and rRNAs are produced by transcription of the mitochondrial genome, all other factors needed for protein synthesis are synthesized in the cytosol and imported. This is the case for aminoacyl-tRNA synthetases, the enzymes which esterify their cognate tRNA with the specific amino acid. The genes for the full set of cytosolic aaRSs are well defined, but only nine genes for mitochondrial synthetases are known. Here we describe the genes for human mitochondrial aspartyl- and tyrosyl-tRNA synthetases and the initial characterization of the enzymes. Both belong to the expected class of synthetases, have a dimeric organization, and aminoacylate Escherichia coli tRNAs as well as in vitro transcribed human mitochondrial tRNAs. Genes for the remaining missing synthetases were also found with the exception of glutaminyl-tRNA synthetase. Their sequence analysis confirms and further extends the view that, except for lysyl- and glycyl-tRNA synthetases, human mitochondrial and cytosolic enzymes are coded by two different sets of genes.

Expression, purification, and characterization of human tyrosyl-tRNA synthetase.

Human tyrosyl-tRNA synthetase is a homodimeric enzyme and each subunit is near 58 KD. It catalyzes the aminoacylation of tRNA(Tyr) by L-tyrosine. The His(6)-tagged human TyrS gene was obtained by RT-PCR from total RNA of human lung giant-cell cancer strain 95 D. It was confirmed by sequencing and cloned into the expression vector pET-24 a (+) to yield pET-24 a (+)-HTyrRS, which was transfected into Escherichia coli BL21-CodonPlus-RIL. The induced-expression level of His(6)-tagged human TyrRS was about 24% of total cell proteins under IPTG inducing. The recombinant protein was conveniently purified in a single step by metal (Ni(2+)) chelate affinity chromatography. About 22.3mg purified enzyme could be obtained from 1L cell culture. The k(cat) value of His(6)-tagged human TyrRS in the second step of tRNA(Tyr) aminoacylation was 1.49 s(-1). The K(m) values of tyrosine and tRNA(Tyr) were 0.3 and 0.9 microM. Six His residues at the C terminus of human TyrRS have little effect on the activities of the enzyme compared with other eukaryotic TyrRSs.

Stabilization of the transition state for the transfer of tyrosine to tRNA(Tyr) by tyrosyl-tRNA synthetase.

Aminoacylation of tRNA(Tyr) involves two steps: (1) tyrosine activation to form the tyrosyl-adenylate intermediate; and (2) transfer of tyrosine from the tyrosyl-adenylate intermediate to tRNA(Tyr). In Bacillus stearothermophilus tyrosyl-tRNA synthetase, Asp78, Tyr169, and Gln173 have been shown to form hydrogen bonds with the alpha-ammonium group of the tyrosine substrate during the first step of the aminoacylation reaction. Asp194 and Gln195 stabilize the transition state complex for the first step of the reaction by hydrogen bonding with the 2'-hydroxyl group of AMP and the carboxylate oxygen atom of tyrosine, respectively. Here, the roles that Asp78, Tyr169, Gln173, Asp194, and Gln195 play in catalysis of the second step of the reaction are investigated. Pre-steady-state kinetic analyses of alanine variants at each of these positions shows that while the replacement of Gln173 by alanine does not affect the initial binding of the tRNA(Tyr) substrate, it destabilizes the transition state complex for the second step of the reaction by 2.3 kcal/mol. None of the other alanine substitutions affects either the initial binding of the tRNA(Tyr) substrate or the stability of the transition state for the second step of the aminoacylation reaction. Taken together, the results presented here and the accompanying paper are consistent with a concerted reaction mechanism for the transfer of tyrosine to tRNA(Tyr), and suggest that catalysis of the second step of tRNA(Tyr) aminoacylation involves stabilization of a transition state in which the scissile acylphosphate bond of the tyrosyl-adenylate species is strained. Cleavage of the scissile bond on the breakdown of the transition state alleviates this strain.

Efficient synthesis of a 72-kDa mitochondrial polypeptide using the yeast Ty expression system.

Using the Ty system from yeast we report the efficient expression of a heterologous eukaryotic gene encoding a 72 kDa mitochondrial polypeptide. The pFM2IIBgIII expression vector was initially modified for this purpose by inserting the factor X(a) protease cleavage site. The TyA gene, which encodes the structural component of the yeast virus-like particles (VLPs), and the eukaryotic yst1 gene, encoding a 72 kDa mitochondrial tyrosyl-tRNA synthetase from the filamentous fungus Podospora anserina, were subsequently fused to the factor X(a) cleavage site. The resulting chimeric gene, in which the two polypeptide coding sequences are separated by the factor X(a) cleavage site, was expressed in yeast. High yield expression of this foreign protein, which was isolated from yeast transformants as hybrid TyVLPs, was verified after factor X(a) treatment by SDS polyacrylamide gel electrophoresis and antibody detection. The strategy presented here should be useful for expressing a wide variety of eukaryotic genes.

Involvement of Neurospora mitochondrial tyrosyl-tRNA synthetase in RNA splicing. A new method for purifying the protein and characterization of physical and enzymatic properties pertinent to splicing.

The Neurospora CYT-18 protein, the mitochondrial tyrosyl-tRNA synthetase, functions in the splicing of group I introns. Here, bacterially expressed CYT-18 protein, purified by a new procedure involving polyethyleneimine precipitation to remove tightly bound nucleic acids, was used to characterize properties pertinent to RNA splicing. Analytical ultracentrifugation and other methods showed that the CYT-18 protein is an asymmetric homodimer. The measured frictional ratio, f/fo = 1.55, corresponds to an axial ratio of 10 for a prolate ellipsoid or 12 for an oblate ellipsoid. Like bacterial TyrRSs, the CYT-18 protein exhibits half-sites reactivity, each homodimer having one active site for tyrosyl adenylation and RNA splicing. The splicing activity of CYT-18 was unaffected by aminoacylation substrates at concentrations used in aminoacylation reactions, whereas the TyrRS activity was inhibited by physiological concentrations of the splicing cofactor GTP, as well as CTP or UTP, or by low concentrations of a group I intron RNA. Kinetic measurements suggest that the binding of CYT-18 to a group I intron substrate is a two-step process, with an initial biomolecular step that is close to diffusion limited (3.24 +/- 0.03 x 10(7) M-1s-1) followed by a slower conformational change (0.54 +/- 0.07 s-1). After CYT-18 binding, splicing occurs at a rate of 0.0025 s-1, within 6-fold of the rate of self-splicing of the Tetrahymena large rRNA intron in vitro. The Kd for the complex between the CYT-18 protein and a group I intron substrate, calculated from koff/kon, was < 0.3 pM, substantially lower than determined by presumed equilibrium measurements [Guo, Q., & Lambowitz, A. M. (1992) Genes Dev. 6, 1357-1372]. As a result of this tight binding, the CYT-18 protein functions stoichiometrically in in vitro splicing reactions due to its extremely slow dissociation from the excised intron RNA. The very tight binding of the CYT-18 protein to the intron RNA raises the possibility that specific mechanisms exist for dissociating the protein from the excised intron in vivo.

Saccharomyces cerevisiae cytoplasmic tyrosyl-tRNA synthetase gene. Isolation by complementation of a mutant Escherichia coli suppressor tRNA defective in aminoacylation and sequence analysis.

Exploiting differences in tRNA recognition between prokaryotic and eukaryotic tyrosyl-tRNA synthetases (TyrRSs), we have isolated the gene for the cytoplasmic TyrRS of Saccharomyces cerevisiae by functional complementation in Escherichia coli of a mutant E. coli tRNA. The tRNA, derived from the E. coli initiator tRNA with changes to allow suppression of amber termination codons, is poorly aminoacylated in E. coli and hence, is only a weak amber suppressor. The same tRNA functions as a good suppressor in S. cerevisiae and is aminoacylated with tyrosine by yeast extracts. We expressed a yeast cDNA library in an E. coli strain carrying the mutant tRNA gene and several genes with amber mutations. cDNA clones were isolated which increased suppression and levels of aminoacylation of the mutant tRNA. Characterization of the gene identified a methionine-initiated open reading frame encoding a protein of 394 amino acids. Expression of this protein in E. coli demonstrated that tyrosine was incorporated during suppression and that yeast cytoplasmic TyrRS activity was produced. Yeast cytoplasmic TyrRS has sequences typical of class I aminoacyl-tRNA synthetases, but only weak overall sequence similarity to the corresponding eubacterial and mitochondrial TyrRSs. However, many of the residues known to line the tyrosyl-adenylate-binding pocket of the Bacillus stearothermophilus enzyme can be aligned in the yeast sequence. These include the aspartic acid and tyrosine residues thought to contact the tyrosine side chain to provide substrate specificity.

Purification of aminoacyl-tRNA synthetase kinase activities associated with threonyl- and tyrosyl-tRNA synthetases isolated from Bom:NMRI mouse liver.

Aminoacyl-tRNA synthetase kinase activities and aminoacyl-tRNA synthetase activities prepared from postribosomal supernatants of Bom:NMRI mouse liver comigrated through the following purification steps: 1 a) gel filtration on Sephadex G-200, 1 b) chromatofocusing, 2) affinity chromatography on immobilised total tRNA (mixture of tRNA's, specific for all aminoacyl-tRNA synthetases) and 3) affinity chromatography on immobilised tRNA, specific for each of threonyl- and tyrosyl-tRNA synthetases. The purification factors for threonyl- and tyrosyl-tRNA synthetases were about 17000x. The purified synthetases showed only one protein band following sodium dodecyl sulphate polyacrylamide gel electrophoresis. The purified threonyl- and tyrosyl-tRNA synthetase proteins were found also to possess threonyl- and tyrosyl-tRNA synthetase kinase activities, respectively. The purification of tRNA(Thr) and tRNA(Tyr) as well as a method for the renaturation and identification of threonyl- and tyrosyl-tRNA synthetase activities in protein bands obtained by SDS PAGE are described.

[Isolation and characteristics of functionally active proteolytically modified forms of tyrosyl-tRNA synthetase from bovine liver]. / Vydelenie i kharkteristkia funktsional'no aktivnoi proteoliticheski modifitsirovannoi formy tirozil-tRNK-sintetazy iz pecheni byka.

Functionally active proteolytic modified form of tyrosyl-tRNA-synthetase has been isolated in a homogeneous form from the bovine liver under incomplete blocking of endogenous proteolysis. The isolation scheme is described. From the data of gel electrophoresis under denaturing conditions the molecular weight of this form is 39 +/- 1.5 kDa and from the data of gel filtration under native conditions -84 kDa. Thus, this form as well as the native enzyme is a dimer of the alpha 2-type. As compared to the native enzyme (Mm 2 x 59 kDa) a proteolytically modified form has a fragment of the polypeptide chain about 20 kDa long split out (this fragment is not essential for catalytic activity). The values of catalytic characteristics of the modified form in tRNA(Tyr) aminoacylation reaction (Km = 1.19 microM and kcat = 2.99 min-1) are close to those obtained for the main form of the enzyme (0.69 microM and 2.97 min-1, respectively). Amino acid composition of the low-molecular form of tyrosyl-tRNA-synthetase has been determined. It was found that the fragment split out in limited proteolysis was characterized by very high content of positively charged lysine residues (46 residues). A proteolytically modified form of tyrosyl-tRNA-synthetase possesses, like the main form, the affinity to high-molecular rRNA but it is eluted from the column filled with rRNA-sepharose at lower salt concentration (50 mM KCl) as compared to the main form of the enzyme (100 mM KCl).

[Isolation of tyrosyl-tRNA-synthetase from Thermus thermophilus HB-27]. / Vydelenie tirozil-tRNK-sintetazy iz Thermus thermophilus HB-27.

A method for isolating tyrosyl-tRNA synthetase from Thermus thermophilus is described, including ammonium sulfate fractionation, chromatography on DEAE-sepharose, hydroxyapatite, heparin-sepharose and hydrophobic chromatography on Toyopearl HW-65. The yield of the purified enzyme was 1.6 mg per 1 kg of T. thermophilus cells. The enzyme is a dimer protein of the alpha 2 type with molecular weight of 100 kDa.

[Tyrosyl-tRNA synthetase from the bovine liver. Isolation and physico-chemical properties]. / Tirozil-tRNK-sintetaza iz pecheni byka. Vydelenie i fiziko-khimicheskie svoistva.

Tyrosyl-tRNA synthetase of beef liver has been isolated and its properties have been studied. Tyrosyl-tRNA synthetase is a structural dimer of alpha 2 type. Mr of the enzyme subunit is about 59 kDa. Km values for substrates have been determined and compared with kinetic properties of tyrosyl-tRNA synthetases from different sources. The polymorphism of tyrosyl-tRNA synthetase was studied. The enzyme was separated into two different forms by chromatography on phosphocellulose P 11. P1-form is active only in the amino acid activation reaction. This form is not due to the phosphorylation of the enzyme. The low molecular weight form (38 kDa) was also isolated. This form appeared due to the limited endogenic proteolysis of the main form and retained full activity in the aminoacylation reaction. Tyrosyl-tRNA synthetase from beef liver has non-specific affinity to rRNA-sepharose.

The amino acid sequence of the tyrosyl-tRNA synthetase from Bacillus stearothermophilus.

The primary structure of the tyrosyl-tRNA synthetase (TyrTS) of Bacillus stearothermophilus has been deduced from the nucleotide sequence of the cloned gene and from the amino acid sequence of peptides isolated from the purified enzyme. TyrTS (B. stearothermophilus) has a molecular weight of 47316 and the sequence is 56% homologous with that of TyrTS (Escherichia coli). The binding domain for the substrate intermediate tyrosyl adenylate is located in the N-terminal portion of the polypeptide and is highly conserved in both enzymes. Several lysine residues, which are shielded from acetylation in the TyrTS-tRNATyr complex, are also located in a stretch of highly conserved sequence.

Purification and some properties of rat liver tyrosyl-tRNA synthetase.

Rat liver cytoplasmic tyrosine:tRNA ligase (tyrosine:tRNA ligase, EC 6.1.1.1) was purified by ultracentrifugation, DEAE-cellulose chromatography and repeated phosphocellulose chromatography by more than 1500-fold. The molecular weight of the enzyme was approx. 150 000 as determined by Sephadex G-200 gel filtration. On the basis of sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the enzyme consisted of two subunits, each of 68 000 daltons. We found the following Km values for the enzyme: 13 micrometer for tyrosine and 1.7 mM for ATP in the ATP:PPi exchange reaction and 13 micrometer for tyrosine, 210 micrometer for ATP and 0.14 micrometer for tRNATyr in the aminoacylation reaction. The rate of tyrosyl-tRNA synthesis was 50-fold lower than that of ATP:PPi exchange. Addition of a saturating amount of tRNA did not affect the rate of ATP:PPi exchange.

Purification and properties of tyrosyl tRNA synthetase of rat liver.

Purification of rat liver tyrosine tRNA synthetase yields two protein fractions A and B and both fractions are required for charging of tyrosine to tRNAtyr. Fraction B catalyzes the activation of tyrosine. Fractions A and B have been purified to near homogeneity and they are composed of single polypeptide chains of 62,000 daltons each. Gel filtration studies suggest a molecular weight of 120,000 for the synthetase.

Tyroslyl-tRNA synthetase from baker's yeast. Rapid isolation by affinity elution, molecular weight of the enzyme, and determination of essential sulfhydryl groups.

Tyrosyl-tRNA synthetase (EC 6.1.1.1) has been isolated from baker's yeast with an overall purification factor of more than 5000. After opening the cells, pH 4.8 precipitation, ammonium sulfate fractionation, removal of the nucleic acids with DEAE-cellulose and chromatography on CM-Sephadex, the critical purification step is the elution of the cation-exchanger-bound tyrosyl-tRNA synthetase with tRNATyr. The homogeneous enzyme exhibits a molecular weight of 40 000 as estimated by sedimentation equilibrium centrifugation and dodecylsulfate-gel electrophoresis under reducing and non-reducing conditions. Gel filtration experiments show a molecular weight of about 100 000 indicating the existence of an active dimeric form. The possibility of proteolytic cleavage of the enzyme is excluded. The reaction of tyrosyl-tRNA synthetase with p-chloromercuribenzoate and N-ethylmaleimide reveals two repidly reacting sulfhydryl groups per subunit of molecular weight 40 000, as demonstrated by the inhibition of aminoacylation and the isolation of enzyme-inhibitor complexes. In addition an efficient purification method is described for isolating tRNATyr from soluble ribonucleic acid from baker's yeast in three chromatographic steps in a yield of 28%.

Mechanisms of suppression in Drosophila. IV. Specificity and properties of tyrosyl-tRNA synthetase.

Tyrosyl-tRNA synthetase from wide type Drosophila can distinguish tRNATyr of wild type from that of the suppressor mutant, su(s)2. The method of fractionation of the enzyme on DEAE-cellulose can produce three different forms of the enzyme. One of these forms is characterized in this study. We describe the apparent Km for K+, Mg2+, ATP, tyrosine and tRNA, as well as its heat stability and molecular weight. Spermine does not replace Mg2+ but is inhibitory, a Ki of 1 mM was obtained. Inhibition by p-chloromercuribenzoate results in a Ki of 1 muM. The properties of tyrosyl-tRNA synthetase of Drosophila are compared to the properties of this enzyme from E. coli, B. subtilis and yeast.

Gram-scale purification of methionyl-tRNA and tyrosyl-tRNA synthetases from Escherichia coli.

A procedure is presented for the purification of 3 g of homogeneous methionyl-tRNA synthetase and 1 g of homogeneous tyrosyl-tRNA synthetase from 50 kg batches of Escherichia coli. The procedure permits the isolation of many enzymes simultaneously and the elution positions of seven other aminoacyl-tRNA synthetases, catalase, rhodanese, phosphofructokinase, elongation factor Tu and cytochrome b-562 are indicated. The problems of extraction work on this scale are discussed.