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.     

Properties of purified chloroplastic and cytoplasmicvalyl- and leucyl-tRNA synthetases from Euglena gracilis

The catalytic properties of purified chloroplastic and cytoplasmic valyl- and leucyl-tRNA synthetasesfrom Euglena gracilis were studied and compare     

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.     

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.     

Derepression of Synthesis of the Aminoacyl-Transfer Ribonucleic Acid Synthetases for the Branched-Chain Amino Acids of Escherichia coli

The kinetics of derepression of valyl-, isoleucyl-, and leucyl-transfer ribonucleic acid (tRNA) synthetase formation was examined during valine-, isoleucine-, and leucine-limited growth. When valine was limiting growth, valyl-tRNA synthetase formation was maximally derepressed within 5 min, whereas the rates of synthesis of isoleucyl-, and leucyl-tRNA synthetases were unchanged. Isoleucine-restricted growth caused a maximal derepression of isoleucyl-tRNA synthetase formation in 5 min and derepression of valyl-tRNA synthetase formation in 15 min with no effect on leucyl-tRNA synthetase formation. When leucine was limiting growth, leucyl-tRNA synthetase formation was immediately derepressed, whereas valyl- and isoleucyl-tRNA synthetase formation was unaffected by manipulation of the leucine supply to the cells. These results support our previous findings that valyl-tRNA synthetase formation is subject to multivalent repression control by both isoleucine and valine. In contrast, repression control of iso-leucyl- and leucyl-tRNA synthetase formation is specifically mediated by the supply of the cognate amino acid.     

Valyl-tRNA synthetase gene of Escherichia coli K12. Primary structure and homology within a family of aminoacyl-TRNA synthetases

The DNA nucleotide sequence of the valS gene encoding valyl-tRNA synthetase of Escherichia coli has been determined. The deduced primary structure of valyl-tRNA synthetase was compared to the primary sequences of the known aminoacyl-tRNA synthetases of yeast and bacteria. Significant homology was detected between valyl-tRNA synthetase of E. coli and other known branched-chain aminoacyl-tRNA synthetases. In pairwise comparisons the highest level of homology was detected between the homologous valyl-tRNA synthetases of yeast and E. coli, with an observed 41% direct identity overall. Comparisons between the valyl- and isoleucyl-tRNA synthetases of E. coli yielded the highest level of homology detected between heterologous enzymes (19.2% direct identity overall). An alignment is presented between the three branched-chain aminoacyl-tRNA synthetases (valyl- and isoleucyl-tRNA synthetases of E. coli and yeast mitochondrial leucyl-tRNA synthetase) illustrating the close relatedness of these enzymes. These results give credence to the supposition that the branched-chain aminoacyl-tRNA synthetases along with methionyl-tRNA synthetase form a family of genes within the aminoacyl-tRNA synthetases that evolved from a common ancestral progenitor gene.     

Spontaneous inactivation of enkephalin.

The role of isoleucyl-, valyl-, and leucyl-tRNA synthetases in attenuation of the $\text{ilvEDA}$ operon was examined. The results indicate that the activities of isoleucyl- and valyl-tRNA synthetases are necessary to maintain attenuation of the $\text{ilvEDA}$ operon. Leucyl-tRNA synthetase activity is nonessential for attenuation. These studies imply that uncharged tRNA^Ile and tRNA^Val each may cause deattenuation.     

Biochemical comparison of the Neurospora crassa wild type and the temperature-sensitive and leucine-auxotroph mutant leu-5. Purification of the cytoplasmic and mitochondrial leucyl-tRNA synthetases and comparison of the enzymatic activities and the degradation patterns

The cytoplasmic leucyl-tRNA synthetases of Neurospora crassa wild type (grown at 37 degrees C) and mutant (grown at 28 degrees C) were purified approximately 1770-fold and 1440-fold respectively. Additional enzyme preparations were carried out with mutant cells grown for 24 h at 28 degrees C and transferred then to 37 degrees C for 10-70 h of growth. The mitochondrial leucyl-tRNA synthetase of the wild type was purified approximately 722-fold. The mitochondrial mutant enzyme was found only in traces. The cytoplasmic leucyl-tRNA synthetase from the mutant (grown at 37 degrees C) in vivo is subject of a proteolytic degradation. This leads to an increased pyrophosphate exchange, without altering aminoacylation. Proteolysis in vitro by trypsin or subtilisin of isolated cytoplasmic wild-type and mutant leucyl-tRNA synthetases, however, did not establish and difference in the degradation products and in their catalytic properties. Comparing the cytoplasmic wild-type and mutant enzymes (grown at 28 degrees C) via steady-state kinetics did not show significant differences between these synthetases either. The rate-determining step appears to be after the transfer of the aminoacyl group to the tRNA, e.g. a conformational change or the release of the product. Besides leucine only isoleucine is activated by the enzymes with a discrimination of approximately 1:600; however, no Ile-tRNALeu is released. Similarly these enzymes, when tested with eight ATP analogs, cannot be distinguished. For both enzymes six ATP analogs are neither substrates nor inhibitors. Two analogs are substrates with identical kinetic parameters. The mitochondrial wild-type leucyl-tRNA synthetase is different from the cytoplasmic enzyme, as particularly exhibited by aminoacylating Escherichia coli tRNALeu but not N. crassa cytoplasmic tRNALeu. The presence of traces of the analogous mitochondrial mutant enzyme could be demonstrated. Therefore, the difference between wild-type and mutant leu-5 does not rest in the catalytic properties of the cytoplasmic leucyl-tRNA synthetases. Differences in other properties of these enzymes are not excluded. In contrast the activity of the mitochondrial leucyl-tRNA synthetase of the mutant is approximately 1% of that of the wild-type enzyme.     

Regulation of Synthesis of the Aminoacyl-Transfer Ribonucleic Acid Synthetases for the Branched-Chain Amino Acids of Escherichia coli

The regulation of synthesis of valyl-, leucyl-, and isoleucyl-transfer ribonucleic acid (tRNA) synthetases was examined in strains of Escherichia coli and Salmonella typhimurium. When valine and isoleucine were limiting growth, the rate of formation of valyl-tRNA synthetase was derepressed about sixfold; addition of these amino acids caused repression of synthesis of this enzyme. The rate of synthesis of the isoleucyl- and leucyl-tRNA synthetases was derepressed only during growth restriction by the cognate amino acid. Restoration of the respective amino acid to these derepressed cultures caused repression of synthesis of the aminoacyl-tRNA synthetase, despite the resumption of the wild-type growth rate.     

Translational competition by mRNA species encoding albumin, ferritin, haemopexin and globin

The photoreactive gamma-(p-azidoanilidate) analog of ATP, AzAnATP, was used to affinity-label the chloroplastic and cytoplasmic leucyl-tRNA synthetases of Euglena gracilis. The analog is able to replace the substrate ATP in the tRNA leucylation reaction catalyzed by both enzymes. In the presence of ATP, it is a competitive inhibitor against ATP as well as leucine for the two isoenzymes, as is also shown for the photoinactive gamma-anilidate analog of ATP, AnATP, which does not serve as substrate in the enzyme reaction. During ultraviolet irradiation, the enzymes are irreversibly inactivated by AzAnATP in a concentration-dependent and time-dependent manner indicative of photoaffinity labeling. Both ATP and leucine, but not tRNA, protect the enzymes against ultraviolet-induced inactivation by AzAnATP. Comparative kinetic characterization of the inactivation process reveals differences in the active centers of the two intracellular isoenzymes.     

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.     

I. A study of the stages in the quantitative isolation of aminoacyl-tRNA synthetase activities from mouse liver.

The elution profiles of 17 aminoacyl-tRNA synthetases from chromatography of 149 000 x g supernatant on Sephadex G-200 were determined as well as the influence of different methods of homogenization and of chromatography on DEAE-cellulose on the elution profiles. With gentle homogenization all synthetases were eluted in the void volume in four different peaks, containing (a) leucyl- and phenylalanyl-, (b) lysyl-, prolyl-, isoleucyl-, methionyl-, glycl-, and valyl-, (c) arginyl-, alanyl-, and asparaginyl- and (d) aspartyl-, histidyl-, seryl-, threonyl-, glutaminyl-, and tyrosyl- tRNA synthetases. With less gentle homogenization, peaks of lower molecular weight appeared. More than two peaks for each aminoacyl-tRNA synthetases were never found. Of the aminoacyl-tRNA synthetases examined, alanyl-,arginyl-, aspartyl-, leucyl- and lysyl-tRNA synthetases were not inactivated by chromatography on DEAE-cellulose, whereas phenylalanyl- and seryl-tRNA synthetases lost 60% of their activity.     

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.     

Purification and properties of leucyl-tRNA synthetase from the cow mammary gland

Three forms (E1, E2 and E3) of leucyl-tRNA synthetase (LeuRS) were separated by DEAE-cellulose chromatography of total aminoacyl-tRNA synthetases from cow lactating mammary gland. The method of purification of all three components is described. E1 is a dimeric molecule (alpha 2) of molecular weight 182 000. Two other forms of molecular weight 67 000 and 64,000 consist of a single polypeptide chain as determined by polyacrylamide gel electrophoresis. Optimum conditions and kinetic parameters of leucyl-tRNA formation were studied for every enzyme form. The low values of Vmax and thermostability are characteristic of E3. All forms of LeuRS interact with 6 isoaccepting tRNA(Leu) from lactating mammary gland and can activate leucine in the absence of tRNA. E2 and E3 are supposed to derive from the native enzyme by endogenous proteolysis. The physico-chemical properties of native LeuRS from lactating mammary gland are compared with those of LeuRS's from other sources.     

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.     

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 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 absence of structural relationship between mitochondrial and mitochondrial and cytoplasmic leucyl-tRNA synthetases from Tetrahymena pyriformis.

Two leucyl-tRNA synthetases (EC 6.1.1.4) have been purified to near homogeneity, the one from mitochondria and the other from cytoplasm of Tetrahymena pyriformis. Both enzymes were found to be structurally unrelated, single polypeptides with molecular weights of approximately 100,000 as determined by gel permeation, sucrose gradient centrifugation, and sodium dodecyl sulfate-polyacrylamide-gel electrophoresis. These enzymes behaved differently in elution profiles through hydroxyapatite- and diethylaminoethyl cellulose-column chromatography and isoelectric focusing. The two enzymes also showed some differences in responses to various salts for charging and in pH optima and temperature sensitivity, but no significant difference was found in their affinities (K_m) for ATP and leucine. These enzymes recognized different leucyl-tRNA isoaccepting species as revealed by reversed-phase column chromatography. The mitochondrial enzyme can charge six isoaccepting leucyl-tRNA species, while the cytoplasmic enzyme can recognize only four species.     

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.     

Reversion of the effects of a threonine deaminase regulatory mutant by a mutation in ilvH in Escherichia coli K-12

In a strain carrying an ilvA538 mutation, the ilvGEDA operon expression is decreased (hyperattenuated) and the activity and/or expression of isoleucyl- and valyl- tRNA synthetases is decreased. We have isolated two revertants of ilvA538 owing to mutations in the ilvH gene, whose product is acetohydroxy acid synthase III. The regulatory properties of these revertants are consistent with a dual role for threonine deaminase as an effector of the ilvGEDA operon and the isoleucyl- and valyl- tRNA synthetase structural genes.