aminoacyl-tRNA Synthetase Activity Research Articles

Characterization of a proteolipid complex of aminoacyl-tRNA synthetases and transfer RNA from rat liver

A high molecular weight complex containing aminoacyl-tRNA synthetases, peptidyl acetyltransferase, lipids and tRNA has been isolated from the 250 000 × g postmitochondrial supernatant from rat liver cells. Aminoacyl-tRNA synthetase activity directed towards arginine, aspartate, glutamine, glutamate, glycine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, and tyrosine is present. An endogenous pool of aminoacyladenylates is indicated by an ATP- 32PP i exchange catalyzed by the native complex, which shows a dramatic increase after addition of ATP. Lysine is the only amino acid which greatly increases the exchange rate catalyzed by the native complex in vitro, whereas components of the denatured complex activate all the 13 amino acids in the presence of ATP. Six of the eight lipid fractions were glycolipids; cholesterol and cholesterol esters were absent. The extracted RNA has many characteristics of tRNA. These findings provide evidence for the organization of aminoacyl-tRNA synthetases in a complex with peptidyl acetyltransferase that also contains lipids and tRNA and that can be readily isolated from the cytosol of rat liver cells.

Effect of starvation on tRNA synthesis, amino acid pool, tRNA charging levels and aminoacyl-tRNA synthetase activities in the posterior silk gland of Bombyx mori L

Changes in the translational machinery components of the Bombyx mori posterior silk gland were analysed during starvation and refeeding and compared to the regularly fed larvae. During starvation, tRNA and ribosomal RNA synthesis are stopped. The amounts of different RNA classes and of the different tRNA species slow down at the same rate. Thus various tRNA show similar half-lifes and the preexisting tRNA adaptation to fibroin mRNA translation persists during starvation. Similarly, the tRNA/rRNA ratio is constant during starvation and refeeding (12 tRNA molecules for one ribosome) as in silk glands of control animals. Aminoacyl-tRNA synthetases and tRNA charging levels are decreased during starvation. The maximal tRNA charging level obtained during maximal protein synthesis in control animals is regained after 24 h refeeding of starved larvae. Changes observed in the free amino acid pool are not similar from one amino acid to another and levels reached after starvation do not differ strongly from the controls. Our results suggest that the production of translation apparatus components is coordinated and adjusted to the protein synthesis activity. Whether this coordination occurs in the silk gland is discussed on the basis of the "metabolic regulation", primarily described in prokaryotes and Yeast. Transfer RNA charging levels seem to play a key role in the process of regulation and could be implicated in the mechanism of tRNA adaptation if this phenomenon results as expected from a transcriptional control.

The role of cyclic 3′,5′-AMP in the regulation of aminoacyl-tRNA synthetase activities in mouse uterus and liver following 17β-oestradiol treatment: Activation of a phosphoaminoacyl-tRNA synthetase phosphatase by phosphorylation with cyclic 3′,5′-AMP-dependent protein kinase

The changes in the activities of 17 aminoacyl-tRNA synthetases induced by phosphorylation [1] were reversed by the action of cyclic AMP in preparations from both uterus and liver. Cyclic AMP also inhibited the phosphorylation of aminoacyl-tRNA synthetase protein by endogenous non-cyclic AMP-dependent protein kinase and [γ- 32P]ATP. The effect was not due to a stimulation of phosphoaminoacyl-tRNA synthetase phosphatase or to an influence of cyclic AMP on aminoacyl-tRNA synthetases. The activity of phosphoaminoacyl-tRNA synthetase phosphatase was increased by treatment with endogenous cyclic AMP-dependent protein kinase, ATP and cyclic AMP. Affinity chromatography of the 32P-labeled phosphorylated phosphosynthetase phosphatase protein followed by gel electrophoresis showed that the activated phosphatase was phosphorylated. In the uterus, the changes in 17 aminoacyl-tRNA synthetase activities observed 5 min after dibutyryl cyclic AMP administration to ovariectomized mice were similar to those observed after 17β-oestradiol treatment, whereas in the liver the changes in these activities were the opposite to those found after treatment with 17β-oestradiol. A mechanism for the regulation of the 17 aminoacyl-tRNA synthetase activities is proposed, which suggests that the synthetase activities inhibited (group I) or stimulated (group II) by phosphorylation with a non-cyclic AMP-dependent aminoacyl-tRNA synthetase kinase are reactivated (group I) or inhibited (group II), respectively, by the action of a cyclic AMP-dependent phosphatase kinase through the increased activity of phosphorylated phosphoaminoacyl-tRNA synthetase phosphatase.

Evidence for structural gene alterations affecting aminoacyl-tRNA synthetases in CHO cell mutants and revertants.

Aminoacyl-tRNA synthetase (aaRS) activities in extracts of mutant strains of the Chinese hamster ovary line (CHO) were examined for alterations in thermal stability. Mutants having low activity for MetRS, AsnRS, or GlnRS contained aaRSs that were inactivated much more rapidly upon heating than those from wild-type cells. Revertant lines, isolated from cultures of these mutants (Asn-5, Met-2, and Gln-2) after treatment with nitrosoguanidine or ethyl methanesulfonate, had thermolabilities intermediate between mutant and wild-type, and consistently had higher activities than the mutants. With a modified in vivo aminoacylation procedure, two previously exceptional mutants. Arg-1 and His-1, showed pronounced reductions in the amount of arginyl-tRNA or histidyl-tRNA, respectively, under restrictive conditions, compared to wild type. Revertants of Arg-1 (like the mutant itself) had no measurable ArgRS in vitro activity (less than 0.4% of wild type) although in vivo aminoacylation in the one revertant tested was partially restored. These data provide evidence that the forward mutations have occurred in the structural genes of the aaRSs and that most of the reversions are probably the result of second-site point mutations in the aaRS genes.

Mutations in the structural genes of CHO cell histidyl-, valyl-, and leucyl-tRNA synthetases.

Forty-three temperature-sensitive mutants were isolated in the CHO cell line by selecting for noncycling cells using [3H]TdR and cytosine arabinoside. Cell division was extremely temperature sensitive in eight of the mutants, and these were studied in more detail. In seven of these eight mutants, the in vitro specific activity of a single aminoacyl-tRNA synthetase was greatly reduced; four had reduced levels of histidyl-tRNA synthetase, two of valyl-tRNA synthetase, and one of leucyl-tRNA synthetase. Cell hybridization studies showed that the mutants formed three complementation groups. In six of the seven mutants the aminoacyl-tRNA synthetase which had reduced activity was also more thermolabile than the wild-type enzyme. The spontaneous reversion frequency was low for these mutants, and in most cases could be increased by treatment with a chemical mutagen. The isolation of the valyl-tRNA synthetase mutant reported here brings to eight the number of different aminoacyl-tRNA synthetase mutants isolated in the CHO cell line.

Effect of extreme amino acid starvation on the protein synthetic machinery of CHO cells

When CHO cells are incubated under conditions of extreme amino acid starvation, effected by withdrawal of an amino acid from the medium together with genetic or chemical interference with the activity of the corresponding aminoacyl-tRNA synthetase, there is a rapid and profound decline in the functional capacity of the protein synthetic machinery. The effect was observed for all amino acids tested including leucine, asparagine, histidine, methionine and glutamine. This decline in protein synthetic potential appears to be due to a progressive permanent inactivation of the specific aminoacyl-tRNA synthetase concerned, as shown by a decline in the amount of cellular, specific aminoacyl-tRNA and a decline in the cell-free enzyme activity, measured after reversal of the starvation conditions. When cells are left for more than several hours under these starvation conditions, they shrink in size, lose viability and eventually disintegrate, with anomalous rapidity. We suggest that the progressive loss of protein synthetic capacity of the cells is the prime cause of these subsequent events. If the starvation conditions are reversed before cell death, regeneration of the protein synthetic potential occurs rapidly but requires protein synthesis itself, implying the existence of strong control mechanisms for cellular aminoacyl-tRNA synthetase activities.

Synthesis and activities of branched-chain aminoacyl-tRNA synthetases in threonine deaminase mutants of Escherichia coli

Valyl-, isoleucyl-, and leucyl-tRNA synthetase activities were examined in an Escherichia coli K-12 strain that possessed a deletion of three genes of the ilv gene cluster, ilvD, A, and C, and in a strain with the same deletion that also carried the lambdadilvCB bacteriophage. It was observed that the branched-chain tRNA synthetase activities of both strains were considerably less than those of the normal strain during growth in unrestricted medium. Furthermore, during an isoleucine limitation, there was a further reduction in isoleucyl-tRNA synthetase activity and an absence of the isoleucine-mediated derepression of valyl-tRNA synthetase formation in both of these mutants, as compared with the normal strain. In addition, it was observed that these branched-chain synthetase activities were reduced in steady-state cultures of several ilvA point mutants. However, upon the introduction of the ilv operon to these ilvA mutants by use of lambda bacteriophage, there was a specific increase in the branched-chain synthetase activities to levels comparable to those of the normal strain. These results support our previous findings that the stability and repression control of synthesis of these synthetases require some product(s) missing in the ilvDAC deletion strain and strongly suggest this component is some form of the ilvA gene product, threonine deaminase.

Photobinding of 8-methoxypsoralen to transfer RNA and 5-fluorouracil-enriched transfer RNA

The photobinding of [3H]8MOP to tRNA upon irradiation at 365 nm in the absence of O2 was determined by gel filtration. The maximum photobinding was found to be ca. 4 mol of 8MOP er mol of tRNA and 5FU-tRNA, with an overall quantum yield of 2.3 X 10(-3). The photobinding kinetics for 8MOP-tRNA showed an apparent induction period or sigmoidal kinetic curve, indicating a specific initial photobinding site on tRNA which was identified as 4-thiouridine at position 8 from the 5'-end of Escherichia coli tRNA. Photobinding of 8MOP to 5FU-tRNA proceeded without an apparent induction period. 8MOP-tRNA and 8MOP-5FU-tRNA adducts were characterized by absorption, fluorescence, and CD spectroscopy. A modified procedure was also developed to analyze the nucleoside composition in modified 8MOP-tRNA and 8MOP-5FU-tRNA. The results showed that 8MOP photochemically added mainly to pyrimidine bases. The photobinding of 8MOP changed the conformation (secondary in particular) of tRNA and inhibited aminoacyl-tRNA synthetase activity.

TRNA sulfurtransferase: A member of the aminoacyl-tRNA synthetase complex in rat liver

Transfer RNA sulfurtransferase, tRNA methyltransferase, and aminoacyl-tRNA synthetase activity are associated in a complex in rat liver, which is excluded from Sephadex G-200 columns. The complex can also be isolated by subjecting cell supernatants to further centrifugation at 160,000 x g for 18 hours. The resulting pellet contains 70% of the total sulfurtransferase activity, and a 3-fold increase in specific activity is accomplished through pelleting. The data suggest that the enzymes of tRNA metabolism are organized in a large complex in rat liver.

Occurrence of a complex of aminoacyl-tRNA synthetases in lactating rat mammary gland

1. (1) Postmicrosomal particles were isolated from the cytosol of lactating rat mammary gland. These particles contained up to 40% of the cytosol protein, and about 40% of the total aminoacyl-tRNA synthetase activity of the cytosol. 2. (2) Sucrose density gradient centrifugation of these postmicrosomal particles showed that the activity of seventeen synthetases was distributed between one or other of two major gradient regions, corresponding to values of 20–28 S and 5–7 S. Arginyl and cysteinyl tRNA synthetases showed peaks in both major regions. 3. (3) A complex of eight aminoacyl-tRNA synthetases (those for lysine, leucine, isoleucine, methionine, glutamine, arginine, cysteine and valine) of molecular weight 3 · 10 6 was isolated from the postmicrosomal particles by gel filtration and chromatography on DEAE-cellulose.

Growth-dependent factors in the regulation of aminoacyl-tRNA synthetase activities of Tetrahymena pyriformis

Changes in phenylalanyl-tRNA synthetase ( l-phenylalanine : tRNA Phe ligase, EC 6.1.1.20) and leucyl-tRNA synthetase ( l-leucine : tRNA Leu ligase. EC 6.1.1.4) activities were studied during the growth cycle of Tetrahymena pyriformis. High levels of charged tRNA observed during exponential growth were associated with elevated aminoacyl-tRNA synthetase activities. Low levels of charged tRNA in the stationary phase culture were associated with decreased aminoacyl-tRNA synthethase activities together with a concomitant accumulation of factor(s) which inhibited the enzyme activities. The inhibitory factor(s) has been partially purified and evidence is presented to rule out RNA, RNAases, proteases and ATPases as the responsible inhibitory factor(s) of the aminoacyl-tRNA synthetases.

Detection and partial purification of rapidly sedimenting forms of aminoacyl-transfer ribonucleic acid synthetases from human placenta

The pattern of aggregation of aminoacyl-tRNA synthetases in extracts of human placenta is described. The sedimentation behavior of aminoacyl-tRNA synthetases in crude extracts of human placenta was analyzed by velocity gradient centrifugation. Varying proportions of seven aminoacyl-tRNA synthetase activities (arginyl-, glutaminyl-, glutamyl-, isoleucyl-, leucyl-, lysyl-, and methionyl-tRNA synthetases) sedimented as broad peaks at 17 to 21 S. Twelve other aminoacyl-tRNA synthetases sedimented at 5 to 9 S, a range more typical of individual aminoacyl-tRNA synthetases. The seven aminoacyl-tRNA synthetases which are aggregated in the placenta are the same enzymes which are most often reported to be aggregated in diverse mammalian species and tissues. All seven of the aggregated activities copurified as 16.2 to 18.8 S forms by ammonium sulfate precipitation, fractionation on alumina C γ and calcium phosphate gels, and chromatography on diethylaminoethyl (DEAE) cellulose. Before their gradient elution from DEAE-cellulose, the aggregated aminoacyl-tRNA synthetases could be stored with little loss of activity or change in sedimentation profile. Gradient elution of the aggregated activities from DEAE-cellulose columns resulted in accelerated loss of the activities during storage and in reduced sedimentation rates. Further reductions in sedimentation coefficients of aminoacyl-tRNA synthetases were observed after storage of fractions recovered from DEAE-cellulose columns.

Inhibition of amino acyl tRNA synthetase activity by copper complexes of two metal binding ligands [formula omitted] isatin beta-thiosemicarbazone and 8-hydroxyquinoline

Copper complexes of N- methyl isatin beta-thiosemicarbazone, 1-formyl isoquinoline thiosemicarbazone and thiosemicarbazide inhibit amino acyl tRNA synthetase activity. Copper complexes of 8-hydroxyquinoline and 8-mercaptoquinoline also inhibit. The 1 : 1 ligand-metal complex is significantly more active than the 2 : 1 complex. The free ligand alone and copper sulfate alone have little, if any, effect. These complexes have no effect on the ATP-PP i exchange reaction and do not cause deacylation of amino acyl tRNAs. This indicates that the process inhibited by these complexes is the amino acylation reaction. This is the first report that these copper binding ligands can inhibit enzymatic processes which involve nucleic acids but which are not viral, bacterial or mammalian cell polymerases.

The presence of transfer RNA in the axoplasm of the squid giant axon.

Previous work has revealed that 4S RNA is the primary species of RNA in the axoplasm from the giant axons of the squid and Myxicola. This study shows that axoplasmic 4S RNA from the squid giant axon has the functional properties of tRNA. Axoplasmic RNA was charged with amino acids by aminoacyl-tRNA synthetases prepared from squid brain. Tthe aminoacylation was prevented by incubating the RNA with RNase prior to running the reaction. The amino acid-RNA complex was labile at pH 9, which is characteristic of the acyl linkage between an amino acid and its tRNA. Aminoacyl-tRNA synthetase activity was also present in the axoplasm, primarily in the soluble fraction.

A semiautomated assay procedure for the determination of aminoacyl-tRNA synthetase activity

A semiautomated assay procedure for the determination of aminoacyl-tRNA synthetase activity is described. The assay system employs a Technicon AutoAnalyzer with a continuous filter paper attachment. A larger number of samples can be measured with greater accuracy, reproducibility, and more rapidly than the manual filter paper disk assay system.

Initial position of aminoacylation of individual Escherichia coli, yeast, and calf liver transfer RNAs

Transfer RNAs from Escherichia coli, yeast (Sacharomyces cerevisiae), and calf liver were subjected to controlled hydrolysis with venom exonuclease to remove 3'-terminal nucleotides, and then reconstructed successively with cytosine triphosphate (CTP) and 2'- or 3'-deoxyadenosine 5'-triphosphate in the presence of yeast CTP(ATP):tRNA nucleotidyltransferase. The modified tRNAs were purified by chromatography on DBAE-cellulose or acetylated DBAE-cellulose and then utilized in tRNA aminoacylation experiments in the presence of the homologous aminoacyl-tRNA synthetase activities. The E. coli, yeast, and calf liver aminoacyl-tRNA synthetases specific for alanine, glycine, histidine, lysine, serine, and threonine, as well as the E. coli and yeast prolyl-tRNA synthetases and the yeast glutaminyl-tRNA synthetase utilized only those homologous modified tRNAs terminating in 2'-deoxyadenosine (i.e., having an available 3'-OH group). This is interpreted as evidence that these aminoacyl-tRNA synthetases normally aminoacylate their unmodified cognate tRNAs on the 3'-OH group. The aminoacyl-tRNA synthetases from all three sources specific argining, isoleucine, leucine, phenylalanine, and valine, as well as the E. coli and yeast enzymes specific for methionine and the E. coli glutamyl-tRNA synthetase, used as substrates exclusively those tRNAs terminating in 3'-deoxyadenosine. Certain aminoacyl-tRNA synthetases, including the E. coli, yeast, and calf liver asparagine and tyrosine activating enzymes, the E. coli and yeast cysteinyl-tRNA synthetases, and the aspartyl-tRNA synthetase from yeast, utilized both isomeric tRNAs as substrates, although generally not at the same rate. While the calf liver aspartyl- and cysteinyl-tRNA synthetases utilized only the corresponding modified tRNA species terminating in 2'-deoxyadenosine, the use of a more concentrated enzyme preparation might well result in aminoacylation of the isomeric species. The one tRNA for which positional specificity does seem to have changed during evolution is tryptophan, whose E. coli aminoacyl-tRNA synthetase utilized predominantly the cognate tRNA terminating in 3'-deoxyadenosine, while the corresponding yeast and calf liver enzymes were found to utilize predominantly the isomeric tRNAs terminating in 2'-deoxyadenosine. The data presented indicate that while there is considerable diversity in the initial position of aminoacylation of individual tRNA isoacceptors derived from a single source, positional specificity has generally been conserved during the evolution from a prokaryotic to mammalian organism.

Subcellular distribution of aminoacyl-tRNA synthetases in various eukaryotic cells.

The total amount, size distribution and binding of aminoacyl-tRNA synthetases to ribosomes in a variety of mammalian and avian cells was studied under standard conditions of sample preparation and assay. Aminoacyl-tRNA synthetases appear to exist in three general forms; 'free' enzyme of about 4-9 S, one or more 'enzyme complexes' of about 18-25 S, and in association with ribosomes. The aminoacyl-tRNA synthetase activity for many individual amino acids was surprisingly similar in cell types chosen to be diverse with respect to differentiation state, transformation, and growth rate. Total activity for all amino acids varied about 4-fold, based on a constant volume of cells. Embryonic tissues had a comparatively high proportion of total synthetase activity associated with ribosomes, whereas this value was relatively low for mouse liver. Distinctive distribution patterns with common and variable features were observed for individual enzymes. The only aminoacyl-tRNA synthetases found not to be associated in significant amounts with either 18-25 S enzyme complexes or ribosomes in any of the cell types examined were the enzymes for alanine, histidine, and serine. All cell types evidenced 18-25-S synthetase activity for arginine, aspartic acid, glutamine, glutamic acid, isoleucine, leucine, lysine, methionine, proline, and valine, although in quite variable porportions of the total activity observed for these amino acids. For example, of the valyl-tRNA synthetase activity not associated with ribosomes, 35% and 100% were found to sediment at 18-25 S in Friend leukemia cells and mouse liver respectively. All cells had two easily distinguishable peaks of arginyl tRNA synthetase activity at 4-9S and 18-25S respectively; however, the relative proportion of enzyme activity in the peaks differed between cell types. Phenylalanyl-tRNA synthetase was not observed to occur in an 18-25-S complex in any of the cell types examined but was bound to ribosomes in variable but generally relatively high proportions. Numerous other specific differences are described. No underlying physiological or biochemical principle has been recognized to account for the specific distribution patterns observed. However, they may reflect variations in cellular architecture that may be related to regulation of protein synthesis.

Characterization of a unique enzyme complex composed of S-adenosyl-L-methionine-tRNA-methyltransferase and aminoacyl-tRNA synthetase activities.

S-adenosyl-L-methionine-tRNA methyltransferases of a murine leukemia cell line were found to exist in a high molecular weight enzyme complex. Aminoacyl-tRNA synthetase activity always co-chromatographed and co-sedimented with methyltransferase activity in evidence of a unique association of these two groups of enzymes. Molecular weight studies showed a probable molecular weight of 9 X 10(5) daltsons for the intact complex which dissociates to complexes of 6 X 10(5) and 3 X 10(5) daltons. The complexes contain discrete polypeptides of 25,000-90,000 daltons as determined from SDS-gel electrophoresis. High resolution fatty acid analysis showed that only very small amounts of saponifiable lipids were associated with the purified enzyme complex. Similarly very little protein-bound sugars was found within the complex indicating that neither lipids nor sugars were involved in the protein-protein interactions of the complex. Analysis of tRNA methylated in vitro indicated the presence of most methyltransferase activities in the purified complex. Of note was the absence from the complex of the methyltransferase responsible for the production of ribo Tp.

Wechselwirkungen zwischen Aminoacyl-tRNS Synthetasen (AAS) und Zellteilung bei einer temperatursensitiven filamentösen Mutante vonBacillus subtilis SB 19 III. Charakterisierung des Vorinkubationseffektes und Einfluß eines AAS-Inhibitors ausAgrostemma githago-Keimlingen

Further results on the correlations between the regulation of bacterial cell division and amino-acyl-tRNA synthetase are presented. Activity of aminoacyl-tRNA synthetases, extracted from a filamentous mutant of Bacillus subtilis SB 19, may be stimulated by preincubation of crude extracts. The mechanism of this stimulating effect has been studied by means of an inhibitor of amino-acyl-tRNA synthetases produced during the growth of Agrostemma githago-seedlings. According to preliminary results we suggest, this inhibitor can reduce the activity of subunits only, but not that of higher associates. Association of subunits to oligomers will be prevented by the inhibitor, too. Our results may be indicative of the assumption that the increase of enzyme activity during subunits with a low catalytic activity to functional oligomers. As to the verification of these hypotheses further work will still have to be done.

Subcellular localization of S-adenosyl-L-methionine:tRNA methyltransferases with aminoacyl-tRNA synthetases in human and mouse: normal and leukemic leukocytes.

The subcellular distributions of S-adenosyl-L-methionine:tRNA methyltransferases and aminoacyl-tRNA synthetases were investigated with the use of human and mouse normal and leukemic leukocyte cell lines. Differential centrifugation of homogenized cell suspensions produced three pelleted subcellular fractions (nuclear and membrane, microsomal, and postribosomal) and a supernatant fraction. Each fraction was assayed for both methyltransferase activity and synthetase activity. The largest amounts, 40-50%, of total methyltransferase and synthetase activities were localized in either the microsomal or the postribosomal fractions, depending on cell type. In addition, the highest specific activities of these two enzyme systems were found to be present in the microsomal and postribosomal fractions. The psotribosomal fraction from leukemic leukocytes had a methyltransferase specific activity higher than that of the microsomal fraction, while the same two fractions of normal leukocytes had approximately equal activities. Specific activities of aminoacyl-tRNA synthetases were found to be approximately equal for these two fractions, whether they were from normal or leukemic leukocytes. The activity of tRNA methyltransferases and synthetases within the postribosomal fraction of the cytoplasm suggests the existence of high-molecular-weight enzyme complexes for the modification as well as the aminoacylation of tRNA.