Biochemical comparison of the Neurospora crassa wild-type and the temperature-sensitive leucine-auxotroph mutant leu-5. Detailed kinetic comparison of the leucyl-tRNA synthetases

The cytoplasmic leucyl-tRNA synthetases were purified from a wild-type Neurospora crassa and from a temperature-sensitive leucine-auxotroph (leu-5) mutant. A detailed steady-state kinetic study of the aminoacylation of the tRNALeu from N. crassa by the purified synthetases was carried out. These enzymes need preincubation with dithioerythritol and spermine before the assay in order to become fully active. The Kappm value for leucine was lowered by high ATP concentrations and correspondingly the Kappm,ATP was lowered by high leucine concentrations. The Kappm,Leu was lowered by high pH, a pK value of 6.7 (at 30 degrees C) was calculated for the ionizable group affecting the Km. At the concentrations of 2 mM ATP, 20 microM leucine, 0.3 microM tRNALeu, and pH 7 the apparent Km values were Kappm,ATP = 1.3 mM, Kappm,Leu = 49 microM and Kappm,tRNA = 0.15 microM. No essentially altered cytoplasmic leucyl-tRNA synthetase was produced by the temperature-sensitive mutant strain when kept at 37 degrees C. In none of these experiments could we find any difference between the wild-type enzyme and the enzyme from the mutant strain (whether grown at permissive temperature, 28 degrees C, or grown at permissive temperature for 24 h followed by growth at 37 degrees C). We therefore think that the small difference in the Km value for leucine of the wild-type and mutant enzyme, established in some earlier investigations, is not due to a difference in the kinetic properties of the enzyme molecules but to an external influence. The almost total lack of the mitochondrial leucyl-tRNA synthetase in the mutant strain besides the leucine autotrophy remains the only difference between the wild-type and mutant strains.     

Phaseolus vulgaris cytoplasmic leucyl-tRNA synthetase. Purification and comparison of its catalytic, structural, and immunological properties with those of the chloroplastic enzyme

The cytoplasmic leucyl-tRNA synthetase was purified from bean (Phaseolus vulgaris) leaves. After ammonium sulfate fractionation and chromatography on Sephadex G-50, DEAE-cellulose, hydroxylapatite, and phosphocellulose, complete purification was achieved by blue Sepharose CL-6B chromatography using specific elution with pure yeast tRNALeu1. The enzyme was purified 1050-fold and had a specific activity of 940 nmol of leucyl-tRNA formed/min/mg of protein. Polyacrylamide gel electrophoresis of the native enzyme showed one band, but the denatured enzyme showed two bands. These two protein bands are structurally related. The smallest protein appears to be a cleavage product from the largest one, suggesting the presence of a sensitive cleavage site in the cytoplasmic leucyl-tRNA synthetase. The cytoplasmic enzyme is a monomer (Mr = 130,000), larger than its chloroplastic counterpart (Mr = 120,000). The two enzymes differ in their substrate (tRNA) specificity, tryptic peptide map, and amino acid composition. Antibodies were raised against the cytoplasmic enzyme and against the chloroplastic enzyme and no cross-immunological reaction was detected, showing that the two enzymes do not share any antigenic determinant. Taken together, these results suggest that P. vulgaris cytoplasmic and chloroplastic leucyl-tRNA synthetases are coded for by different genes.     

Inhibition of leucyl-tRNA-synthetases by modified ATP analogs

The interaction between modifying ATP analogs containing alkylating or phosphorylating groups in the polyphosphate moiety of the ATP molecule and leucyl-tRNA synthetases from cytoplasm and chloroplasts of Euglena gracilis (strain Z) was studied. It was shown that most of the ATP analogs irreversibly inhibit the cytoplasmic enzyme, having no inhibiting effect on the chloroplast synthetase. The kinetic constants K1 and k2 for the interaction between the most effective irreversible inhibitors and the cytoplasmic enzyme were determined. The data on the protection of the enzyme activity by substrates against irreversible inhibition suggest, that the effect of the adenosine 5'-(beta-chloroethyl phosphate) is directed to the ATP-binding site of the cytoplasmic enzyme, whereas the mixed anhydride of AMP and mesithylene carbonic acid acts predominantly on the binding site of 3'-terminal adenosine of the tRNALeu molecule. ATP analogs may be effectively used for affinity labelling of the cytoplasmic leucyl-tRNA synthetase.     

Nuclear origin of specific yeast mitochondrial aminoacyl-tRNA synthetases.

Hydroxylapatite chromatographies of mitochondrial and total enzymes from a rho+ yeast, or from the related rho degrees mitochondrial DNA-less mutant, show the occurrence in the mitochondrial enzyme of one Phe-, one Met-, one Leu-tRNA synthetase peak which elutes distinctly from the cytoplasmic counterpart and charges well mitochondrial tRNA, whereas the cytoplasmic enzyme does not. The measurement of the mitochondrial synthetases activities in various enzymatic extracts shows that they are not repressed in rho+ cells grown on 10% glucose and that they are concentrated in the mitochondria (Phe- and Met- tRNA synthetases) but are also present outside the mitochondria. It is concluded that yeast mitochondrial protein biosynthesis involves the nuclear coded mitochondrial specific Phe-, Met- and Leu-tRNA synthetases and that the entrance of the synthetases into the mitochondria needs no factor depending on the mitochondrial DNA.     

Homology of yeast mitochondrial leucyl-tRNA synthetase and isoleucyl- and methionyl-tRNA synthetases of Escherichia coli

Respiratory deficient mutants of Saccharomyces cerevisiae previously assigned to complementation group G59 are pleiotropically deficient in respiratory chain components and in mitochondrial ATPase. This phenotype has been shown to be a consequence of mutations in a nuclear gene coding for mitochondrial leucyl-tRNA synthetase. The structural gene (MSL1) coding for the mitochondrial enzyme has been cloned by transformation of two different G59 mutants with genomic libraries of wild type yeast nuclear DNA. The cloned gene has been sequenced and shown to code for a protein of 894 residues with a molecular weight of 101,936. The amino-terminal sequence (30-40 residues) has a large percentage of basic and hydroxylated residues suggestive of a mitochondrial import signal. The cloned MSL1 gene was used to construct a strain in which 1 kb of the coding sequence was deleted and substituted with the yeast LEU2 gene. Mitochondrial extracts obtained from the mutant carrying the disrupted MSL1::LEU2 allele did not catalyze acylation of mitochondrial leucyl-tRNA even though other tRNAs were normally charged. These results confirmed the correct identification of MSL1 as the structural gene for mitochondrial leucyl-tRNA synthetase. Mutations in MSL1 affect the ability of yeast to grow on nonfermentable substrates but are not lethal indicating that the cytoplasmic leucyl-tRNA synthetase is encoded by a different gene. The primary sequence of yeast mitochondrial leucyl-tRNA synthetase has been compared to other bacterial and eukaryotic synthetases. Significant homology has been found between the yeast enzyme and the methionyl- and isoleucyl-tRNA synthetases of Escherichia coli. The most striking primary sequence homology occurs in the amino-terminal regions of the three proteins encompassing some 150 residues. Several smaller domains in the more internal regions of the polypeptide chains, however, also exhibit homology. These observations have been interpreted to indicate that the three synthetases may represent a related subset of enzymes originating from a common ancestral gene.     

Cysteinyl-tRNA synthetase is a direct descendant of the first aminoacyl-tRNA synthetase.

The gene encoding the cysteinyl-tRNA synthetase of E. coli was cloned from an E. coli genomic library made in lambda 2761, a lambda vector which can integrate and which carries a chloramphenicol resistance gene. A thermosensitive cysS mutant of E. coli was lysogenised and chloramphenicol-resistant colonies able to grow at 42 degrees C were selected to isolate phages containing the wild-type cysS gene. The sequence of the gene was determined. It codes for a 461 amino-acid protein and includes the sequences HIGH and KMSK known to be involved in the ATP and tRNA binding respectively of class I synthetases. The cysteinyl enzyme has segments in common with the cytoplasmic leucyl-tRNA synthetase of Neurospora crassa, the tryptophanyl-tRNA synthetase of Bacillus stearothermophilus, and the phenylalanyl-tRNA synthetase of Saccharomyces cerevisiae. Sequence comparisons show that the amino end of the cysteinyl-tRNA synthetase has similarities with prokaryotic elongation factors Tu; this region is close to the equivalent acceptor binding domain of the glutaminyl-tRNA synthetase of E. coli. There is a further similarity with the seryl enzyme (a class II enzyme) which has led us to propose that both classes had a common origin and that this was the ancestor of the cysteinyl-tRNA synthetase.     

人线粒体tRNALeu(UUR)基因A3243G点突变对其亮氨酰化活性的影响

化学法合成人线粒体野生型与A3243G点突变型tRNALeu(UUR)基因,体外转录生成相应的tRNALeu(UUR),表达并纯化人线粒体亮氨酰tRNA合成酶(mtLeuRS),用mtLeuRS催化野生型与突变型tRNALeu(UUR)与亮氨酸结合,分别检测两种类型tRNALeu(UUR)的氨酰化动力学常数。结果表明,野生型tRNALeu(UUR)的Km/Kcat仅为突变型tRNALeu(UUR)的63.9%,A3243G点突变使tRNALeu(UUR)接受亮氨酸的能力明显下降,提示此为A3243G点突变致病机制之一。 Abstract:The wild-type and mutant-type human mitochondrial tRNALeu(UUR) genes were synthesized and transcribed in vitro with T7 RNA polymerase.The kinetic parameters of human mitochondrial leucyl-tRNA synthetase(mtLeuRS) were determined with wild-type and mutant-type human mitochondrial tRNALeu(UUR) respectively.The results show that the value of Km/Kcat of mtLeuRS for the mutant-type tRNALeu(UUR) is 63.9% as compared with the wild-type.Human mitochondrial tRNALeu(UUR) gene A3243G point mutant can remarkably reduce it′s aminoacylation activity,suggesting it would be one of the mechanisms that the mutation could produce such clinical phenotypes.     

Mutation and “Reversion” at the leu-5 locus of Neurospora and its effect on the cytoplasmic and mitochondrial leucyl-tRNA synthetases

The Neurospora mitochondrial and cytoplasmic leucyl-tRNA synthetases differ from each other not only in location but also with respect to tRNA specificity, chromatographic mobility, leucine affinity, and sensitivity to phosphate inhibition. Strain 45208t, which bears a mutation in the leu-5 cistron, produces a cytoplasmic enzyme with reduced affinity for leucine and little if any mitochondrial enzyme activity. Reversion of the 45208t mutation was found to result not only in the reappearance of mitochondrial leucyl-tRNA synthetase activity but also in the production of a cytoplasmic synthetase with an affinity for leucine intermediate between mutant and wild type. The reversion studied, then, did not involve a return to the wild-type nucleotide sequence in the leu-5 cistron. The results obtained lend further support to the conclusion that the leu-5 cistron is involved in specifying, at least in part, the structure of both the cytoplasmic and mitochondrial leucyl-tRNA synthetases, despite the physical and functional differences between them.Research was supported in part by National Science Foundation Grant 27575.     

A complex from cultured Chinese hamster ovary cells containing nine aminoacyl-tRNA synthetases. Thermolabile leucyl-tRNA synthetase from the tsH1 mutant cell line is an integral component of this complex

The size distribution of the 20 aminoacyl-tRNA synthetases from wild-type Chinese hamster ovary (CHO) cells and from the mutant cell line tsH1, containing a temperature-sensitive leucyl-tRNA synthetase, was determined by gel filtration. Nine aminoacyl-tRNA synthetases, specific for arginine, aspartic acid, glutamic acid, glutamine, isoleucine, leucine, lysine, methionine and proline, which coeluted as high-Mr entities (Mr approximately 1.2 X 10(6)), were further co-purified to yield a multienzyme complex, the polypeptide composition of which was identical to that previously determined for the complex from rabbit liver. Immunoprecipitates obtained from crude extracts of wild-type and tsH1 mutant cells, using specific antibodies directed to the lysyl-tRNA or methionyl-tRNA synthetase components of the complex, displayed the same polypeptide compositions as that of the purified complex, thereby establishing the heterotypic nature of this complex. Although the activity of leucyl-tRNA synthetase from the mutant cells, grown at a permissive temperature, was low compared to that from the wild-type, the polypeptide of Mr 129 000, corresponding to this enzyme, was present in similar amounts and occurred exclusively as a component of the high-Mr complex. Finally, we report that attempts to demonstrate phosphorylation of the components of the complex from cultured CHO, HeLa and C3 cells were unsuccessful.     

Search for difference in aminoacylation of mitochondrial DNA-encoded wild-type and mutant human tRNALeu (UUR)

The pathogenetic mechanism of the most extensively investigated A3243G mutated tRNALeu(UUR) gene, which causes the MELAS encephalomyopathy, maternally inherited diabetes, or chronic progressive external ophlthalmoplegia, is still unresolved, despite the numerous investigations on the topic. Previous evidences presented in published work suggested that the mitochondrial DNA harboring A3243G mutation result decreases in the rates of mitochondrial protein synthesis. To search for differences in aminoacylation of mitochondrial DNA-encoded wild-type and mutant human tRNALeu(UUR), we have expressed and purified the two kinds of tRNAsLeu(UUR), and have expressed human mitochondrial leucyl-tRNA synthetase for in vitro assays of aminoacylation of wild-type and mutant human tRNALeu(UUR). The results indicate human mitochondrial tRNALeu(UUR) gene A3243G point mutant can remarkably reduce its aminoacylation, suggesting it could be one of the mechanisms that the mutation can produce in such clinical phenotypes.     

Mutants of Salmonella typhimurium with an Altered Leucyl-Transfer Ribonucleic Acid Synthetase

Two trifluoroleucine-resistant mutants of Salmonella typhimurium, strains CV69 and CV117, had an altered leucyl-transfer ribonucleic acid (tRNA) synthetase. The mutant enzymes had higher apparent K(m) values for leucine (ca. 10-fold) and lower specific activities (ca. twofold) than the parent enzyme when tested in crude extracts. Preparations of synthetase purified ca. 60-fold from the parent and strain CV117 differed sixfold in their leucine K(m) values. In addition, the mutant enzyme was inactivated faster than the parent enzyme at 50 C. The growth rates of strains CV69 and CV117 at 37 C were not significantly different from that of the parent, whereas at 42 C strain CV69 grew more slowly than the parent. Leucine-, valine-, and isoleucine-forming enzymes were partially derepressed when the mutants were grown in minimal medium; the addition of leucine repressed these enzymes to wild-type levels. During growth in minimal medium, the proportion of leucine tRNA that was charged in the mutants was about 75% of that in the parent. The properties of strain CV117 were shown to result from a single mutation located near gal at minute 18 on the genetic map. These studies suggest that leucyl-tRNA synthetase is involved in repression of the enzymes required for the synthesis of branched-chain amino acids.     

Altered leucyl-transfer RNA synthetase from a mammalian cell culture mutant.

Altered leucyl-tRNA synthetase from a mammalian cell culture temperature-sensitive mutant, tsHl, was compared with enzyme from normal wild type Chinese hamster ovary cells. The mutant enzyme had a Km for leucine four times larger than that of wild type and enzyme levels 3-10% that of wild type. The presence of tRNA was necessary during in vitro heating of the mutant enzyme to allow expression of thermolability while the presence of tRNA protected wild type enzyme against thermal inactivation. The tsHl enzyme was stable when heated alone or in the presence of tRNA, leucine, and ATP simultaneously. The mutant's enzymes aminoacylated tRNALeu, tRNAVal, and tRNAIle with fidelity in vitro as determined by cochromatography of the amino-acyl-tRNA isoacceptors on RPC-5 reversed phase chromatography. The mutant failed to show any defect other than the direct formation of leucyl tRNALeu by leucyl-tRNA synthetase.     

A viable amino acid editing activity in the leucyl-tRNA synthetase CP1-splicing domain is not required in the yeast mitochondria

Aminoacyl-tRNA synthetases are a family of enzymes that are responsible for translating the genetic code in the first step of protein synthesis. Some aminoacyl-tRNA synthetases have editing activities to clear their mistakes and enhance fidelity. Leucyl-tRNA synthetases have a hydrolytic active site that resides in a discrete amino acid editing domain called CP1. Mutational analysis within yeast mitochondrial leucyl-tRNA synthetase showed that the enzyme has maintained an editing active site that is competent for post-transfer editing of mischarged tRNA similar to other leucyl-tRNA synthetases. These mutations that altered or abolished leucyl-tRNA synthetase editing were introduced into complementation assays. Cell viability and mitochondrial function were largely unaffected in the presence of high levels of non-leucine amino acids. In contrast, these editing-defective mutations limited cell viability in Escherichia coli. It is possible that the yeast mitochondria have evolved to tolerate lower levels of fidelity in protein synthesis or have developed alternate mechanisms to enhance discrimination of leucine from non-cognate amino acids that can be misactivated by leucyl-tRNA synthetase.     

The effect of amino acids on the temperature sensitive phenotype of the mammalian leucyl-tRNA synthetase mutant tsHl and its revertants.

The temperature sensitive leucyl-tRNA synthetase mutant tsHl and two revertants have been compared to the parental Chinese hamster ovary cells with respect to the effects of amino acid concentrations in the medium on growth. Elevating the leucine concentration 30- or 100-fold allowed tsHl to grow exponentially at 38.5 degrees C, normally the nonpermissive temperature. Partial revertants that had recovered some enzyme activity required smaller supplements for growth. Measurements of the leucine pools indicated that they respond directly to the extracellular leucine concentration and may mediate the effect. Use of combinations of amino acids confirmed that isoleucine has a similar though weaker effect on tsHl and identified an even weaker protection by valine. The triple combination of leucine, isoleucine and valine was a much more efficient medium supplement and three times normal concentrations of these amino acids supported growth of tsHl at 38.5 degrees C. It is postulated that they are acting at their respective aminoacyl-tRNA synthetases to help stabilize a complex which also contains the mutant leucyl-tRNA synthetase. The pool size measurements also showed that the leucine pools of tsHl and a revertant increased 2-fold more in a response to increased temperature than those of WT. It is suggested that this is a regulatory response to low leucyl-tRNA synthetase activity and is important in determining growth phenotypes.     

Pyrophosphate-caused inhibition of the aminoacylation of tRNA by the leucyl-tRNA synthetase from Neurospora crassa

Inorganic pyrophosphate inhibits the aminoacylation of tRNALeu by the leucyl-tRNA synthetase from Neurospora crassa giving very low Kapp.i, PPi values of 3-20 microM. The inhibition by pyrophosphate, together with earlier kinetic data, suggest a reaction mechanism where leucine, ATP and tRNA are bound to the enzyme in almost random order, and pyrophosphate is dissociated before the rate-limiting step. A kinetic analysis of this mechanism shows that the measured Kapp.i values do not give the real dissociation constant but it is about 0.4 mM. Other dissociation constants are 90 microM for leucine, 2.2 mM for ATP and 1 microM for tRNALeu. At the approximate conditions of the living cell (2 mM ATP, 100 microM leucine and 150 microM PPi) the leucyl-tRNA synthetase is about 85% inhibited by pyrophosphate.     

In vitro synthesis of bean (Phaseolus vulgaris) chloroplastic and cytoplasmic leucyl-tRNA synthetases. Characterization and processing of a precursor polypeptide for the chloroplast enzyme

Bean (Phaseolus vulgaris) chloroplastic and cytoplasmic leucyl-tRNA synthetases differ in their structural and catalytic properties and do not share common antigenic determinants. Polyadenylated mRNAs, prepared from young bean leaves, have been translated in vitro in a rabbit reticulocyte lysate cell-free system. The newly synthesized polypeptides have been submitted to immunoadsorption on protein A-Sepharose in the presence of the antibodies raised against the chloroplastic or the cytoplasmic leucyl-tRNA synthetase. The specificity of the immunoadsorption has been checked by competition experiments involving the pure enzymes. Bean chloroplastic leucyl-tRNA synthetase is synthesized in vitro from a polyadenylated mRNA as a precursor polypeptide of 130 kDa, which is somewhat larger than the mature enzyme of 120 kDa. Bean cytoplasmic leucyl-tRNA synthetase is synthesized in vitro as a polypeptide which has the size of the mature monomer (130 kDa). Processing of the precursor polypeptide of the chloroplastic leucyl-tRNA synthetase, yielding the mature enzyme, has been obtained by performing the in vitro translation in the presence of canine pancreatic microsomal membranes. These results suggest that in vivo bean chloroplastic leucyl-tRNA synthetase could be synthesized in the cytoplasm as a precursor which would be transported into the chloroplasts.     

Chloroplastic and cytoplasmic valyl- and leucyl-tRNA synthetases from Euglena gracilis. Comparative study of their structural properties

Chloroplastic and cytoplasmic valyl- and leucyl-tRNA synthetases purified from Euglena gracilis show a monomeric structure. The molecular weights of the two valyl-tRNA synthetases are identical (126,000) while those of the leucyl-tRNA synthetases are different (100 000 for the chloroplastic and 116 000 for the cytoplasmic enzyme). The tryptic maps and the amino acid compositions reveal differences between the chloroplastic valyl- and leucyl-tRNA synthetases and their cytoplasmic homologues. These results suggest that a chloroplastic aminoacyl-tRNA synthetase and its cytoplasmic counterpart are coded for by distinct genes.     

Chloroplastic and cytoplasmic valyl- and leucyl- tRNA synthetases from Euglena gracilis: Comparative study of their structural properties

Chloroplastic and cytoplasmic valyl- and leucyl-tRNA synthetases purified from Euglena gracilis show a monomeric structure. The molecular weights of the two valyl-tRNA synthetases are identical (126 000) while those of the leucyl-tRNA synthetases are different (100 000 for the chloroplastic and 116 000 for the cytoplasmic enzyme). The tryptic maps and the amino acid compositions reveal differences between the chloroplastic valyl- and leucyl-tRNA synthetases and their cytoplasmic homologues. These results suggest that a chloroplastic aminoacyl-tRNA synthetase and its cytoplasmic counterpart are coded for by distinct genes.     

Chloroplast leucyl-tRNA synthetase from Euglena gracilis. Purification, kinetic analysis, and structural characterization

Euglena gracilis chloroplast leucyl-tRNA synthetase was purified to homogeneity by a series of steps including ammonium sulfate precipitation and chromatography on hydroxylapatite, DEAE-cellulose, Sepharose 6B, phosphocellulose, and Blue Dextran-Sepharose. The purified enzyme exhibits a specific activity of 1233 units/mg of protein, which is one of the highest specific activities obtained for an aminoacyl-tRNA synthetase prepared from plant cells. The enzyme has an apparent Km value of 8 x 10(-6) M for L-leucine, 1.3 x 10(-4) M for ATP, and 1.3 x 10(-6) M for tRNALeu. Chloroplast leucyl-tRNA synthetase appears to be a monomeric enzyme with a molecular weight of 100 000. The amino acid composition of chloroplast leucyl-tRNA synthetase has been determined. It is the first reported for a chloroplast aminoacyl-tRNA synthetase, and it reveals a relatively large proportion of apolar residues, as in the case of prokaryotic aminoacyl-tRNA synthetases.