leucyl-tRNA Synthetase Activity Research Articles

Assignment of a human genetic locus to chromosome 5 which corrects the heat sensitive lesion associated with reduced leucyl-tRNA synthetase activity in ts025Cl Chinese hamster cells.

A heat-sensitive (hs) leucyl-tRNA synthetase (leuRS) deficient CHO mutant, ts025Cl, was fused with human leukocytes and hybrids isolated in HAT medium at the nonpermissive temperature. Nineteen heat-resistant (hr) and 14 hs subclones were isolated from four independent primary hybrids and tested for the expression of 24 human isozymes which have been assigned to 17 human chromosomes. Four hr independent subclones and three hs independent subclones were analyzed for the presence of human chromosomes. A pattern of concordant segregation was noted for the hr phenotype, human hexosaminidase B (EC 3.2.1.30) and human chromosome 5. Based on these results, we have defined the human genetic locus which corrects the heat-sensitive lesion in ts025Cl as hr025Cl and have assigned this locus to human chromosome 5. Two hr hybrids exhibited leuRS activity 2.5 and 4 times the leuRS activity of ts025Cl but a wild-type level of activity was not restored. One hs hybrid had only 73% of the leuRS activity exhibited by ts025Cl while another hs hybrid had 1.8 times the leuRS activity of ts025Cl.

Cytokinin control of aminoacyl-tRNA synthetase s and ribulose-bisphosphate carboxylase in developing and and greening excised Cucurbita Cotyledons

The activities of ribulose-1,5-bisphosphate carboxylase (RuBPCase 2 )), plastid and cytoplasmic leucyl- and valyl-tRNA synthetases have been compared during etioplast and chloroplast development in excised Cucurbita pepo cotyledons under the influence of benzylaminopurine action. Our results demonstrate that the activities of the plastid-associated enzymes are stimulated by the hormone in etiolated and greening cotyydons. Cytoplasmic leucyl-tRNA synthetase activity is not :specifically increased in light or upon hormone addition. The rate of RuBPCase stimulation in light or after cytokinin treatment differs from that of plastid leucyl-tRNA synthetase, especially in later stages of cotyledon development. There is a preponderance of cytokinin control over light control of chloroplast enzyme formation under our experimental conditions. It is suggested that benzylaminopurine specifically stimulates protein biosynthesis in plastids, in addition to a less promoted synthesis of proteins of cytoplasmic origin. The variation in hormone- stimulated activities observed for particular plastid enzymes may be due to different rates ,of formation at their sites of synthesis.

Isolation and characterization of revertants of the mammalian temperature sensitive leucyl‐tRNA synthetase mutant tsHI

Nine spontaneous and seven ethyl methanesulfonate induced revertants of the Chinese hamster ovary cell line mutant (tsHl), which possesses a temperature sensitive leucyl-tRNA synthetase, were isolated and characterized with respect to growth rate, leucyl-tRNA synthetase activity and thermolability, intracellular leucine pool size, and rRNA content. Although most revertants had increased leucyl-tRNA synthetase activity, and of those tested, all but one had increased thermostability, each appears to be unique. One revertant may be an intergenic suppressor since it appears to contain an elevated level of tsHl-like synthetase. There was no evidence for any of the revertants having increased rRNA and tRNA contents, however, many showed leucine pools two to three times larger than wild type cells. Since similar increases have been observed in tsHl cells they are believed to result from regulation of leucine pool size by the leucyl-tRNA synthetase and are of a magnitude sufficient to affect significantly the growth of revertants at 38.5 degrees C.

The effect of amino acids on the temperature sensitive phenotype of the mammalian leucyl‐tRNA synthetase mutant ts Hl 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.

Untersuchungen über eine thermolabile Leucyl-tRNA-Synthetase in einer Mutante von Bacillus subtilis mit temperatursensitiven Wachstums- und Vermehrungseigenschaften

In a mutant of Bacillus subtilis SB 19, strain ts 33-6 with temperature-sensitive growth a temperature-labile leucyl-tRNA synthetase is described. Ts 33-6 grows at 46°C with remarkably reduced intensity without septation processes causing the formation of filaments. At 47°C growth processes are completely arrested. In comparison with this temperature-sensitive growth the activity of leucyl-tRNA synthetase decreases rapidly at 43°C to 47°C, whereas in the same temperature range leucyl-tRNA synthetase of wild type or valyl-tRNA synthetase of mutant are quite stable. These results may indicate that the reduced activity of leucyl-tRNA synthetase at higher temperatures causes temperature-sensitive growth. It could be possible that an indirect inhibition of cell division occurs as a consequence of the reduced growth rate. These assumptions, however, must be verified by the quantitative measurements of the leucyl-tRNA level in vivo under permissive (30–45°C) as well as restrictive conditions (46–47°C).

Control of downstream amplification in the [formula omitted] operon in isoleucyl-, valyl-, and leucyl-tRNA synthetase mutants of [formula omitted] K-12

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

A reexamination of the leucine tRNAs and the leucyl-tRNA synthetase in developing Tenebrio molitor

The translational control mechanism previously proposed for the synthesis of adult cuticular proteins in Tenebrio molitor is dependent upon the appearance of a major, novel leucine tRNA and a change in leucyl-tRNA synthetase activity just prior to adult emergence. The properties of the leucyl-tRNA synthetase extracted from pupae were reexamined. Under optimal aminoacylation conditions, no new leucine isoaccepting tRNAs were observed during development. However, under suboptimal conditions, a differential charging of the leucine tRNA species was noted. The chromatographic profiles of leucyl-tRNAs aminoacylated in vivo in both early and late pupae were found to be the same and were identical to the profiles obtained by charging tRNAs in vitro. Previous evidence for a translational control system operating in Tenebrio is discussed in relation to these results.

Inhibition of leucyl-tRNA synthetase in Escherichia coli by the cytostatic 5,8-dioxo-6-amino-7-chloroquinoline

At concentrations of 1–1.6 μg/ml, 5,8-dioxo-6-amino-7-chloroquinoline causes auxotrophy for leucine in Escherichia coli MRE 600. With increasing concentrations of this quinone additional amino acids are required for growth. The amount of leucine in the pool of free amino acids is not decreased after treatment of E. coli with the quinone. Transfer RNA Leu, however, is charged with leucine less than 10% in quinone-treated cells of E. coli, whereas in control cells the degree of aminoacylation is about 85%. From these data we conclude that the quinone causes auxotrophy for leucine by interacting with the charging process of tRNA Leu. Quinone was found to inhibit leucyl-tRNA synthetase activity in purified extracts of E. coli with E. coli tRNA as substrate.

Similarities in properties and a functional difference in purified leucyl-tRNA synthetase isolated from two developmental stages of Tenebrio molitor

In Tenebrio molitor, as well as in other biological systems, there are indications that differences in leucyl-tRNA synthetase activity may play a role in translational control. However, it has not been clear whether the difference in activity is due to the appearance of a multiplicity of enzymes during development or to the alteration of a single enzyme. The purification of leucyl-tRNA synthetase from day 1 and day 7 after the larval pupal molt of Tenebrio molitor is described. The enzyme from both developmental stages was purified over a 1000-fold. The two enzyme preparations are identical in molecular weight (99,000). They show the same characteristics after aging. The pH optimum, heat inactivation behavior, and dependency on divalent cations are the same for both enzymes. They also show identical kinetics with similar values of Km for leucine, ATP, Mg 2+, and tRNA day 1. However, leucyl-tRNA synthetase purified from day 7 exhibits an additional function in recognizing a new species of isoaccepting tRNA in day 7 tRNA. We have tentatively concluded that the two enzymes are probably different forms of the same enzyme and the additional activity is due to alteration of the enzyme at the macromolecular level during development.

Leucyl-transfer ribonucleic acid synthetase in senescing tobacco leaves.

1. The activity of leucyl-tRNA synthetase obtained from tobacco leaves declined by 50% over a period of 4 days senescence induced by detachment. In addition tRNA(Leu) from senescing leaves was charged to a lesser extent than tRNA(Leu) extracted from mature leaves immediately after detachment. 2. tRNA(Leu) was charged with a synthetase preparation from either mature or senescent leaves and chromatographed on an RPC 3 column. The elution profile showed that the marked decline in specific activity of leucyl-tRNA synthetase in senescent leaves was not associated with a loss of acylation of any isoacceptor of tRNA(Leu).

Inactivation of aminoacyl-tRNA Synthetases by amino acid chloromethylketones

Several amino acid chloromethylketones having the general structure ▪ were synthesized with the intent of labeling the active sites of the aminoacyl-tRNA synthetases. Of the compounds prepared, only the valyl chloromethylketone was sufficiently stable in pH 7.3 buffer for extensive inactivation studies. The l-valyl chloromethylketone caused little inactivation of the lysyl- and phenylalanyl-tRNA synthetases, but rapidly inactivated the valyl- and leucyl-tRNA synthetases. The amino acid acceptor activity of leucyl-tRNA synthetase was inhibited approximately ten times as rapidly as that of the valyl-tRNA synthetase. The inhibition appears to be irreversible since neither dialysis against buffer nor mercaptoethanol, which was found to destroy the inhibitor, caused reactivation of the enzymes. Both d-valyl chloromethylketone and chloroacetone were found to inhibit the aminoacyl-tRNA synthetases, although the rate of inactivation by chloroacetone was significantly lower than for the valyl analogs. Dissociation constants of complexes between valyl-tRNA synthetase and l- and d-valyl chloromethylketone were 20 and 100 mM, respectively. Chloroacetone did not exhibit saturation kinetics with this enzyme. With the leucyl-tRNA synthetases, l- and d-valyl chloromethylketone and chloroacetone were found to have dissociation constants of 7.5, 4.5, and 103 mM, respectively. The valyl- and leucyl-tRNA synthetases were protected from inactivation by their natural substrates (cognate amino acid, ATP + Mg 2+ , or both). These data suggest that l-valyl chloromethylketone inhibits by reaction at the active sites of the valyl- and leucyl-tRNA synthetases.

A mammalian cell mutant with a temperature-sensitive leucyl-transfer RNA synthetase.

A cell mutant of the Chinese hamster ovary line, which is temperature sensitive for protein synthesis, is specifically defective in vivo in its ability to charge tRNA with leucine. Cytoplasmic extracts exhibited temperature-sensitive leucyl-tRNA synthetase activity. It is, therefore, highly likely that the mutant has a structural alteration in leucyl-tRNA synthetase. The low leakiness and low reversion rate of this mutant, combined with the specificity of the defect in its protein-synthesizing machinery, make it an appealing tool for investigating regulatory mechanisms in animal cells.

Hydrocortisone induction of rat-liver leucyl-transfer RNA and its synthetases.

Within 3 hr after the intraperitoneal administration of hydrocortisone to female rats, a new leucine-accepting tRNA and a new leucyl-tRNA synthetase activity appear in the liver cytosol. The new isoaccepting tRNA can be acylated only with the synthetase derived from livers of hormone-treated animals. Both components are transient; by 12 hr after hydrocortisone administration, the isoaccepting tRNA and its synthetase disappear from livers of treated animals.

Leucyl-transfer ribonucleic acid synthetase from a wild-type and temperature-sensitive mutant of Salmonella typhimurium.

Leucyl-transfer ribonucleic acid (tRNA) synthetase was purified 100-fold from extracts of Salmonella typhimurium. The partially purified enzyme had the following K(m) values: leucine, 1.1 x 10(-5)m; adenosine triphosphate, 6.5 x 10(-4)m; tRNA(I) (Leu), 4.1 x 10(-8)m; tRNA(II) (Leu), 4.3 x 10(-8)m; tRNA(III) (Leu), 5.3 x 10(-8)m; and tRNA(IV) (Leu), 2.9 x 10(-8)m. The tRNA(Leu) fractions were isolated from Salmonella bulk tRNA by chromatography on reversed-phase columns and benzoylated diethylaminoethyl cellulose. The enzyme had a pH optimum of 8.5 and an activation energy of 10,400 cal per mole, and was inactivated exponentially at 49.5 C with a first-order rate constant of 0.064 min(-1). Strain CV356 (leuS3 leuABCD702 ara-9 gal-205) was isolated as a mutant resistant to dl-4-azaleucine and able to grow at 27 C but not at 37 C. Extracts of strain CV356 had no leucyl-tRNA synthetase activity (charging assay) when assayed at 27 or 37 C. Temperature sensitivity and enzyme deficiency were caused by mutation in the structural gene locus specifying leucyl-tRNA synthetase. A prototrophic derivative of strain CV356 (CV357) excreted branched-chain amino acids and had high pathway-specific enzyme levels when grown at temperatures where its doubling time was near normal. At growth-restricting temperatures, both amino acid excretion and enzyme levels were further elevated. The properties of strain CV357 indicate that there is only a single leucyl-tRNA synthetase in S. typhimurium.

Leucyl-tRNA synthetase activity in old cotyledons: evidence on repressor accumulation

Previous studies from this laboratory have shown that the complement of tRNA Leu isoaccepting species changes during cotyledon senescence. Concomitantly there is a reduction in the capacity of leucyl-tRNA synthetase from 21-day-old cotyledons to acylate tRNA 1–4 Leu fully. Leucyl-tRNA synthetase can be fractionated into 3 peaks of activity on hydroxylapatite. All 3 peaks of activity are present in both young (5-day) and old (21-day) cotyledons. This finding eliminates the possibility that 21-day-old cotyledons are specifically deficient in a charging enzyme for tRNA 1–4 Leu. Other experiments effectively rule out the possibility that tRNA 1–4 Leu consists of co-eluting subspecies of tRNA, as was earlier suggested. Inorganic pyrophosphate is inhibitory in the range 10 −5–10 −3 M and inhibition is overcome by the addition of pyrophosphate, but only to the normal level of charging for the old synthetase preparation. Since no loss in a specific synthetase activity can be demonstrated to account for the deficiency in charging of tRNA 1–4 Leu in the older cotyledons, these results are interpreted on the basis of a model of repressor accumulation and its subsequent interaction with aminoacyl-tRNA molecules.

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.

Isolation of an organ-specific leucyl-tRNA synthetase from soybean seedling.

The leucyl-tRNA synthetase activity from cotyledons of 4-day-old soybean seedlings has been fractionated into three components. One of these exclusively acylates two of the six tRNA(Leu) species present in this tissue. The remaining two enzyme fractions charge the other four tRNA(Leu) species equally well. Soybean hypocotyls appear to contain only the last two enzyme fractions.

The stimulatory effect of ammonium or potassium ions on the activity of leucyl-tRNA synthetase from Escherichia coli

NH 4 + at an optimal concentration of 0.005 M stimulated the activity of leucyl-tRNA synthetase ( l-leucine: tRNA ligase (AMP), EC 6.1.1.4) in Escherichia coli 6-fold. The activities of isoleucyl- and phenylalanyl-tRNA synthetases were stimulated to lesser extents, by factors of 2.9 and 1.5, respectively, whereas those of valyl-, glycyl-, lysyl-, alanyl-, and seryl-tRNA synthetases were not affected. Stimulation of the enzymatic activity by NH 4 + was shown in the formation of leucyl-tRNA, but not in the formation of leucyl hydroxamate, suggesting that the NH 4 + is involved in the attachment of the activated leucine to the tRNA molecules. K + can partially substitute for NH 4 + in the stimulatory effect of the latter on leucyl-tRNA synthetase. At an optimal concentration of 0.08 M, K + stimulated the enzymatic activity 3-fold. Na + or Li + was not effective at the same concentration.