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At least 19 recordsLinked to original sources
PubMed1992

The first human genes for tRNA(ArgICG), tRNA(GlyUCC), and tRNA(ThrIGU) and more tRNA(Val) pseudogenes: expression and pre-tRNA maturation in HeLa cell-free extracts.

A functional tRNA(Val) gene, which codes for the major tRNA(ValIAC) isoacceptor species, and three new tRNA(Val) pseudogenes have been isolated from human genomic DNA. Two tRNA(Val) pseudogenes and a tRNA(Val) variant gene were found to be associated with tRNA genes encoding tRNA(ArgICG), tRNA(GlyUCC), and tRNA(ThrIGU), respectively, on distinct DNA fragments. All tRNA genes, including the pseudogenes, are actively transcribed in HeLa nuclear extract. Pre-tRNAs of tRNA(Val), tRNA(Arg), tRNA(Thr), and tRNA(Gly) genes are correctly processed to mature-sized tRNAs, whereas the three tRNA(Val) pseudogenes yield stable pre-tRNAs in vitro. These findings reveal that, together with the three known pseudogenes, half of the members of the human tRNA(Val) gene family are pseudogenes, all of which are active in homologous nuclear extracts in vitro and presumably also in vivo.

Base Sequence↗
PubMed1977

Hydrolytic action of aminoacyl-tRNA synthetases from baker's yeast. "Chemical proofreading" of Thr-tRNA Val by valyl-tRNA synthetase studied with modified tRNA Val and amino acid analogues.

The properties of native and of two modified tRNA Val species in the correction of misactivated threonine by valyl-tRNA synthetase have been studied. Whereas Thr-tRNA Val-C-C-A could not be isolated in the valyl-tRNA synthetase catalyzed reaction, Thr-tRNA Val-C-C-3'dA is isolable in up to 50% yield in this system and tRNA Val-C-C-3'NH2A is fully aminoacylated with threonine by the same enzyme. The hydrolysis of preformed Thr-tRNA Val-C-C-A by free valyl-tRNA synthetase is 30 times faster than the corresponding breakdown of Val-tRNA Val-C-C-A. This hydrolytic activity is also observed with Thr-tRNA Val-C-C-3'dA although the rate is reduce to that of the reaction of Val-tRNA Val-C-C-A. Modification of the threonine to O-methylthreonine, which is also a substrate for valyl-tRNA synthetase, leads to stabilization of the O-methylthreonyl-tRNA esters. The AMP/PP independent hydrolysis under aminoacylating conditions, which is a measure of the correction process, indicates that O-MeThr-tRNA Val-C-C-A is only very slowly corrected while the tRNA Val-C-C-3'dA and tRNA Val-C-C-3'NH2A esters are completely stable. Removal of the methoxy group of O-methylthreonine as in alpha-amino-butyric acid increases the rate of the hydrolytic reaction and once again alpha-Abu-tRNA Val-C-C-A and alpha-Abu-tRNA Val-C-C-3'dA are unstable under aminoacylating conditions and not isolable.

Amino Acyl-tRNA Synthetases↗
PubMed1975

The plant aminoacyl-tRNA synthetases. Purification and characterization of valyl-tRNA, tryptophanyl-tRNA and seryl-tRNA synthetases from yellow-lupin seeds.

Valyl-tRNA, tryptophanyl-tRNA, and seryl-tRNA synthetases from yellow lupin seeds Lupinus luteus were purified to homogeneity by ammonium sulfate fractionation, hydrophobic chromatography on aminohexyl-Sepharose column and affinity chromatography on tRNA-Sepharose column. Valyl-tRNA synthetase consists of one polypeptide chain of molecular weight 125000 as judged by Sephadex G-200 gel filtration and dodecylsulfate-polyacrylamide gel electrophoresis in the presence of reducing agent. Seryl-tRNA synthetase, Mr equals 110000, is composed of two 55000-Mr subunits. Tryptophanyl-tRNA synthetase exhibits molecular weight of 200000 on Sephadex G-200 and 37000 in dodecylsulfate-polyacrylamide gel electrophoresis. This indicates that tryptophanyl-tRNA synthetase consists of several subunits (probably four). Since the seryl-tRNA synthetase exhibits the same mobility on dodecylsulfate-polyacrylamide gels both in the presence and absence of reducing agent it is concluded that there is no covalent bond(s) between the subunits of the enzyme. There is also no covalent bond(s) between the subunits of tryptophanyl-tRNA synthetase. Effect of anti-sulfhydryl reagents, monovalent salts, pH and different buffers on activity of the three synthetases is described. Kinetic constants for the substrates of the synthetases are also given. dATP is a substrate for seryl-tRNA synthetase but not for valyl-tRNA and tryptophanyl-tRNA synthetases.

Amino Acyl-tRNA Synthetases↗
PubMed1979

Studies on tRNA adaptation, tRNA turnover, precursor tRNA and tRNA gene distribution in Bombyx mori by using two-dimensional polyacrylamide gel electrophoresis.

Eighteen out of twenty amino acids have been used for identifying tRNAs from the silkworm Bombyx mori L. fractionated on two-dimensional polyacrylamide gel electrophoresis. 43 spots out of 53 have been identified. This mapping confirms previous results and brings new answers to some questions on the regulation of tRNA biosynthesis. 1. In addition to quantitative adaptation of tRNAs to the composition of silk proteins (fibroin from the posterior silk gland, sericin from the middle part) and of iso-tRNAs from posterior silk gland to the major codons of fibroin mRNA, we also observe adaptation of tRNA from various tissues to the average amino acid content of proteins from fat body, gut, gonads and carcass of the silkworm. 2. In the silk gland, turnover rates of several tRNA species are similar. The selective accumulation of tRNAs needed for decoding fibroin and sericin mRNAs which takes place during the Vth larval instar, cannot be explained by the occurrence of a preferential degradation of some tRNA species. 3. Under given conditions for incubating silk glands, it is possible to obtain an accumulation of precursor tRNA species, which are enriched in pre-tRNAAla and pre-tRNAGly in the posterior silk gland and pre-tRNASer in the middle part. 4. The distribution of tRNA genes is not random. tRNA genes for glycine, alanine and serine are prominent. Selective transcription of batteries of iso-tRNA genes could explain our data.

Animals↗
PubMed1977

The plant aminoacyl-tRNA synthetases. Effect of sodium chloride on tRNA aminoacylation and aminoacyl-tRNA decomposition catalysed by aminoacyl-tRNA synthetases from yellow lupin seeds.

The initial velocity and the extent of aminoacylation are affected by sodium chloride in the lupin aminoacylation systems involving serine, isoleucine, lysine, leucine, phenylalanine and valine. Pyrophosphorolysis and enzymatic hydrolysis of [14C]Val-tRNA catalysed by lupin valyl-tRNA synthetase are inhibited by sodium chloride nearly to the same extent. Evidence is presented that when a limiting amount of synthetase is used, the equilibrium of the aminoacylation reaction in the lupin valine system is determined only by the rate of aminoacylation and non-enzymatic deacylation of aminoacyl-tRNA, the former but not the latter reaction being dependent on concentration of the enzyme and monovalent salt.

Amino Acyl-tRNA Synthetases↗
PubMed1975

Phenylalanyl-tRNA synthetase from baker's yeast: role of 3'-terminal adenosine of tRNA-Phe in enzyme-substrate interaction studied with 3'-modified tRNA-Phe species.

TRNA-Phe species from baker's yeast modified at the 3'-terminus in many cases are phenylalanylatable substrates. Out of several tRNA-Phe species possessing a modified 3'-end that cannot be phenylalanylated, only two, tRNA-Phe-C-C-2'dA and the tRNA-Phe-C-C-formycin-oxi-red, are strong competitive inhibitors for tRNA-Phe-C-C-A during phenylalanylation. In the ATP/PPi exchange, both these inhibitors reduce Vmax to about 25%; but whereas tRNA-Phe-C-C-2dA has no influence on KmATP and Km Phe during ATP/PPi exchange, tRNA-Phe-C-C-formycin-oxi-red reduces KmATP from 1430 muM, found in the absence of tRNA-Phe, to 230 muM, and Km-Phe, from 38 to 14 muM. The values found in the presence of tRNA-Phe-C-C-formycin-oxi-red during ATP/PPi exchange are identical with those determined in the phenylalanylation of tRNA-Phe-C-C-A. All other tRNA-Phe species carrying a modified 3'end that cannot be phenylalanylated exhibit a mixed competitive-noncompetitive inhibition in the phenylalanylation reaction. In the ATP/PPi exchange, they do not influence KmATP and KmPHE and only weakly, if at all, Vmax. The results show that the 3'adenosine of tRNA-Phe cannot solely be a passive acceptor for phenylalanine, but must in addition play an active role during enzyme-substrate interaction. The data can be consistently explained by the hypothesis that the 3'-adenosine of tRNA-Phe triggers a conformational change of the enzyme.

Adenosine↗
PubMed1978

Threonyl-tRNA, lysyl-tRNA and arginyl-tRNA synthetases from Baker's yeast. Substrate specificity with regard to ATP analogues.

Sixteen analogues of ATP have been tested in the aminoacylation reaction of threonyl-tRNA, lysyl-tRNA, and arginyl-tRNA synthetases from baker's yeast. Two compounds are substrates for threonyl-tRNA and for lysyl-tRNA synthetases and five compounds for arginyl-tRNA synthetase. There are six inhibitors for threonyl-tRNA, nine for lysyl-tRNA, and six for arginyl-tRNA synthetase. Their Km and Ki values have been determined. Thus positions 2, 6, 7, 8 and 9 of the purine moiety and 2' and 3' of the sugar moiety of the ATP molecule are important for catalytic action of these aminoacyl-tRNA synthetases. Remarkably arginyl-tRNA synthetase is the first aminoacyl-tRNA synthetase which tolerates bulky substituents at the sugar moiety of ATP. These data fit with the idea that synthetases of subunit structure need magnesium-ion-ATP complexes with an anti conformation as substrates whereas single-chain enzymes accept this substrate in the syn conformation.

Adenosine Triphosphate↗
PubMed1992

Complex formation between glutamyl-tRNA synthetase and glutamyl-tRNA reductase during the tRNA-dependent synthesis of 5-aminolevulinic acid in Chlamydomonas reinhardtii.

The formation of a stable complex between glutamyl-tRNA synthetase and the first enzyme of chlorophyll biosynthesis glutamyl-tRNA reductase was investigated in the green alga Chlamydomonas reinhardtii. Apparently homogenous enzymes, purified after previously established purification protocols were incubated in various combinations with ATP, glutamate, tRNA(Glu) and NADPH and formed complexes were isolated via glycerol gradient centrifugation. Stable complexes were detected only after the preincubation of glutamyl-tRNA synthetase, glutamyl-tRNA reductase with either glutamyl-tRNA or free tRNA(Glu), ATP and glutamate, indicating the obligatory requirement of aminoacylated tRNA(Glu) for complex formation. The further addition of NADPH resulting in the reduction of the tRNA-bound glutamate to glutamate 1-semialdehyde led to the dissociation of the complex. Once complexed to the two enzymes tRNA(Glu) was found to be partially protected from ribonuclease digestion. Escherichia coli, Bacillus subtilis and Synechocystis 6803 tRNA(Glu) were efficiently incorporated into the protein-RNA complex. The detected complexes provide the chloroplast with a potential channeling mechanism for Glu-tRNA(Glu) into chlorophyll synthesis in order to compete with the chloroplastic protein synthesis machinery.

Adenosine Triphosphate↗
PubMed1976

Valyl-tRNA, isoleucyl-tRNA and tyrosyl-tRNA synthetase from baker's yeast. Substrate specificity with regard to ATP analogs and mechanism of the aminoacylation reaction.

Nineteen analogs of ATP have been tested in the aminoacylation of valyl-tRNA, isoleucyl tRNA and tyrosyl-tRNA synthetases from baker's yeast. Four compounds are substrates for valyl tRNA and two for isoleucyl-tRNA synthetase, but there is no modified substrate for the tyrosyl tRNA synthetase. There is one inhibitor for valyl-tRNA synthetase, eight compounds inhibit isoleucyl-tRNA synthetase and two compounds inhibit tyrosyl-tRNA synthetase. Their Km and Ki and V values have been determined. The substrate specificity shows that positions 2, 6, 7, 8, 9, 2', and 3' of ATP are important for catalytic action of these aminoacyl-tRNA synthetases.

Adenosine Triphosphate↗
PubMed1992

Construction and characterization of an Escherichia coli mutant deficient in the metY gene encoding tRNA(f2Met): either tRNA(f1Met) or tRNA(f2Met) is required for cell growth.

The Escherichia coli metY gene, encoding tRNA(f2Met), was split by the kanamycin-resistance-encoding gene. The resulting mutant exhibited the same growth rate as the wild type, indicating that tRNA(f2Met) is not indispensable as is the case with the metZ gene encoding tRNA(f1Met) [Kenri et al., Gene 103 (191) 31-36]. beta-Galactosidase was produced efficiently from the start codon AUG of the intact lacZ gene or a trpA'::lac'Z fusion gene, in the metY mutant. The lac repressor from the lacI gene and the chimeric protein from a hupB'::lac'Z fusion gene, whose start codons are GUG, were also synthesized efficiently in the insertion mutant. These results provide evidence that tRNA(f2Met) is not essential for growth of E. coli and that the start codons, AUG and GUG, are both recognized by tRNA(f1Met), a major N-formyl methionine-specific tRNA, in the tRNA(f2Met)-depleted cells. We were unable to construct mutants deficient in both tRNA(f1Met) and tRNA(f2Met) by P1 phage-mediated transduction with the metY and metZ mutations. Moreover, the ampicillin-resistance marker of the pUC9 plasmid carrying metZ+ was not cured at 42 degrees C in host cells with the polAts and metY-metZ double mutations. These results indicate that either tRNA(f1Met) or tRNA(f2Met) is required for the growth of E. coli.

Blotting, Southern↗
PubMed1979

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.

Amino Acids↗
PubMed1992

Molecular mimicry in translational control of E. coli threonyl-tRNA synthetase gene. Competitive inhibition in tRNA aminoacylation and operator-repressor recognition switch using tRNA identity rules.

We previously showed that: (i) E.coli threonyl-tRNA synthetase (ThrRS) binds to the leader of its mRNA and represses translation by preventing ribosome binding to its loading site; (ii) the translational operator shares sequence and structure similarities with tRNA(Thr); (iii) it is possible to switch the specificity of the translational control from ThrRS to methionyl-tRNA synthetase (MetRS) by changing the CGU anticodon-like sequence to CAU, the tRNA(Met) anticodon. Here, we show that the wild type (CGU) and the mutated (CAU) operators act as competitive inhibitors of tRNA(Thr) and tRNA(fMet) for aminoacylation catalyzed by E.coli ThrRS and MetRS, respectively. The apparent Kd of the MetRS/CAU operator complex is one order magnitude higher than that of the ThrRS/CGU operator complex. Although ThrRS and MetRS shield the anticodon- and acceptor-like domains of their respective operators, the relative contribution of these two domains differs significantly. As in the threonine system, the interaction of MetRS with the CAU operator occludes ribosome binding to its loading site. The present data demonstrate that the anticodon-like sequence is one major determinant for the identity of the operator and the regulation specificity. It further shows that the tRNA-like operator obeys to tRNA identity rules.

Acylation↗
PubMed1976

On the interaction of seryl-tRNA synthetase with tRNA Ser. A contribution to the problem of synthetase-tRNA recognition.

By following the tryptophan fluorescence of yeast seryl-tRNA synthetase on addition of tRNA Ser it was observed that the number of binding sites for tRNA decreases from two to one with increasing temperature, ATP or KCl concentration. Concomitantly a considerable decrease of the apparent binding constant was observed. The variation in the number of binding sites is explained by the presence of at least one temperature and ionic strength sensitive binding site and one temperature and ionic strength independent binding site. Relaxation kinetic experiments revealed two binding processes: a fast one depending on tRNA concentration and ionic strength and a slow one, which appeared to be independent of tRNA concentration and ionic strength. Enzyme kinetic studies showed that the activity of seryl-tRNA synthetase strongly depends on the KCl concentration and exhibits a maximum at 0.2 M KCl. Based on the data from relaxation and enzyme kinetic experiments a model is suggested for the recognition process involving a first unspecific step where all tRNAs, cognate and non-cognate, are bound to the synthetase (scanning step). The identification of the cognate tRNA is then performed at the recognition site by a conformational transition of the tRNA . synthetase complex (identification step).

Amino Acyl-tRNA Synthetases↗
PubMedDate not supplied

[Modification of one tRNA recognition site of phenylalanyl-tRNA synthetase from E. coli MRE-600 with N-chlorambucilyl-phenylalanyl-tRNA].

Affinity labelling of phenylalanyl-tRNA synthetase from E. coli MRE-600 with N-chlorambucilyl-phenylalanyl-tRNA results in a binding of 1 mole of the reagent per 1 mole of the enzyme. Exhaustive alkylation of phenylalanyl-tRNA synthetase completely blocks the aminoacylation and partially inhibits the reaction of ATP--[32P]pyrophosphate exchange. Removal of the tRNA moiety of the reagent by hydrolysis of the ester bond N-chlorambucilyl-phenylalanine and terminal adenosine does not result in a restoration of ATP--[32P]pyrophosphate exchange and aminoacylation activity. The latter result may testify a chemical modification of amino acid residues essential for enzymatic activity. Possibility of blocking one of the two tRNA binding sites is discussed.

Adenosine Triphosphate↗
PubMed1979

Regulation of the biosynthesis of aminoacyl-tRNA synthetases and of tRNA in Escherichia coli. IV. Mutants with increased levels of leucyl- or seryl-tRNA synthetase.

Spontaneous revertants of a temperature-sensitive Escherichia coli strain harboring a thermolabile leucyl-tRNA synthetase and seryl-tRNA synthetase were selected for growth at 40 degrees C. Among these, strains were found with increased levels of both thermolabile synthetases. Two distinct genetic loci were found responsible for enzyme overproduction. leuR, located near xyl, causes elevated levels of leucyl-tRNA synthetase; while serR, located near leu, causes elevated levels of seryl-tRNA synthetase.

Amino Acyl-tRNA Synthetases↗
PubMed1979

Novel mechanism of post-transcriptional modification of tRNA. Insertion of bases of Q precursors into tRNA by a specific tRNA transglycosylase reaction.

The guanine insertion enzyme isolated from Escherichia coli (tRNA transglycosylase) catalyzed the incorporation of bases of Q (queuosine) precursors into E. coli undermodified tRNAAsn and tRNATyr. These bases of Q precursors were inserted in the first position of the anticodon of tRNASn and tRNATyr, replacing guanine originally located in that position. This is a novel type of post-transcriptional modification, inserting a modified base into the polynucleotide chain by cleavage of the N--C glycoside bond without breakage of the phosphodiester bond. One of the bases of Q precursors, 7-(aminomethyl)-7-deazaguanine, was found in the acid-soluble fraction of E. coli cells, supporting the conclusion that formation of Q, 7-(3,4-trans-4,5-cis-dihydroxy-1-cyclopenten-3-ylaminomethyl)-7-deazaguanosine, in tRNA in vivo actually proceeds by the tRNA transglycosylase reaction.

Escherichia coli↗
PubMed1979

Phenylalanyl-tRNA synthetase of Escherichia coli K 10. Multiple enzyme-aminoacyl-tRNA complexes as a consequence of substrate specificity.

The interaction between Phe-tRNA(Phe) or other acyl-tRNA derivatives thereof and phenylalanyl-tRNA synthetase of Escherichia coli K 10 has been investigated by nonequilibrium dialysis, by fluorescence titration in the presence of 2-p-toluidinylnaphthalene-6-sulfonate, by the kinetics of the aminoacylation of tRNA(Phe), and by the kinetics of the catalytic hydrolysis of Phe-tRNA(Phe). Phe-tRNA(Phe), or derivatives thereof, forms two types of complexes with the synthetase. One type involves the attachment of the phenylalanyl moiety to the phenylalanine-specific site of the enzyme, and the other type, to the tRNA(Phe)-specific binding site. They resemble alternative modes of a destabilized enzyme-product complex and are predicted on the basis of thermodynamic considerations. The two modes of binding of acyl-tRNA compete with each other. The attachment of Phe-tRNA(Phe) to the phenylalanine-specific site dominates. At equilibrium, this complex is present at a fourfold higher concentration than the other type of complex. The HNO2 deaminated Phe-tRNA(Phe) binds exclusively to the site specific for L-phenylalanine. On the contrary, Ile-tRNA(Phe) adds at 94.1% to the tRNA(Phe)-specific site. The association of Phe-tRNA(Phe) with this site leads to enzymatic hydrolysis into L-phenylalanine and tRNA(Phe). The complex involving the phenylalanine-specific site is hydrolytically unproductive. L-Phenylalanine acts as an activator of the hydrolysis by occupying the amino acid specific site and by shifting the equilibrium between the complexes toward the binding ot Phe-tRNA(Phe) at the tRNA(Phe)-specific site. The association of Phe-tRNA(Phe) at the phenylalanine-specific site does not interfere sterically with the binding of free tRNA(Phe). The sequential addition of free and aminoacylated tRNA(Phe) exhibits negative cooperativity. Such a mechanism could help to expel the product from the enzyme.

Amino Acyl-tRNA Synthetases↗