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J Lapointe

Publications and source records attributed to J Lapointe.

At least 37 records · Page 2Linked to original sources
PubMed1986

Glutamyl-tRNA synthetase of Escherichia coli. Isolation and primary structure of the gltX gene and homology with other aminoacyl-tRNA synthetases.

The gltX gene encoding the glutamyl-tRNA synthetase of Escherichia coli and adjacent regulatory regions was isolated and sequenced. The structural gene encodes a protein of 471 amino acids whose molecular weight is 53,810. The codon usage is that of genes highly expressed in E. coli. The amino acid sequence deduced from the nucleotide sequence of the gltX gene was confirmed by mass spectrometry of large peptides derived from the glutamyl-tRNA synthetase. The observed peptides confirm 73% of the predicted sequence, including the NH2-terminal and the COOH-terminal segments. Sequence homology between the glutamyl-tRNA synthetase and other aminoacyl-tRNA synthetases of E. coli was found in four segments. Three of them are aligned in the same order in all the synthetases where they are present, but the intersegment spacings are not constant; these ordered segments may come from a progenitor to which other domains were added. Starting from the NH2-end, the first two segments are part of a longer region of homology with the glutaminyl-tRNA synthetase, without need for gaps; its size, about 100 amino acids, is typical of a single folding domain. In the first segment, containing sequences homologous to the HIGH consensus, the homology is consistent with the following evolutionary linkage: gltX----glnS----metS----ileS and tyrS.

Amino Acid Sequence↗
PubMed1986

Non-linear kinetics of glutamyl-tRNA synthesis catalyzed by high molecular weight complexes from rat brain neuronal cells but not from glial cells.

High molecular weight complexes of aminoacyl-tRNA synthetases isolated from rat brain catalyze the formation of glutamyl-tRNA with an initial lag time of the order of 1 min, as previously reported for the formation of glutamyl-tRNA and glutaminyl-tRNA catalyzed by similar complexes from bovine brain (Vadeboncoeur and Lapointe, Eur. J. Biochem., 109 (1980) 581-587). To determine the type(s) of brain cell(s) where this phenomenon occurs, we have studied the kinetics of glutamyl-tRNA formation catalyzed by high molecular weight complexes of aminoacyl-tRNA synthetases isolated from neuronal and from glial cells, either transformed (Neuro-2A and C6), or from primary cultures, or isolated from rat brain. The delay in the formation of glutamyl-tRNA was observed only in the case of neuronal cells isolated from rat brain, whereas a delay in the formation of glutaminyl-tRNA was also seen in these cells, as well as in neuronal cells in primary culture and in synaptosomes. The kinetics of formation of aspartyl-tRNA and valyl-tRNA catalyzed by high molecular weight complexes from all these cells was linear.

Acylation↗
PubMed1986

A single glutamyl-tRNA synthetase aminoacylates tRNAGlu and tRNAGln in Bacillus subtilis and efficiently misacylates Escherichia coli tRNAGln1 in vitro.

In the presence or absence of its regulatory factor, the monomeric glutamyl-tRNA synthetase from Bacillus subtilis can aminoacylate in vitro with glutamate both tRNAGlu and tRNAGln from B. subtilis and tRNAGln1 but not tRNAGln2 or tRNAGlu from Escherichia coli. The Km and Vmax values of the enzyme for its substrates in these homologous or heterologous aminoacylation reactions are very similar. This enzyme is the only aminoacyl-tRNA synthetase reported to aminoacylate with normal kinetic parameters two tRNA species coding for different amino acids and to misacylate at a high rate a heterologous tRNA under normal aminoacylation conditions. The exceptional lack of specificity of this enzyme for its tRNAGlu and tRNAGln substrates, together with structural and catalytic peculiarities shared with the E. coli glutamyl- and glutaminyl-tRNA synthetases, suggests the existence of a close evolutionary linkage between the aminoacyl-tRNA synthetases specific for glutamate and those specific for glutamine. A comparison of the primary structures of the three tRNAs efficiently charged by the B. subtilis glutamyl-tRNA synthetase with those of E. coli tRNAGlu and tRNAGln2 suggests that this enzyme interacts with the G64-C50 or G64-U50 in the T psi stem of its tRNA substrates.

Amino Acyl-tRNA Synthetases↗
PubMed1986

The so-called tRNAGlu1 of Escherichia coli is a stable denatured conformer of the major isoacceptor tRNAGlu2.

A single peak of tRNAGlu is obtained upon chromatography of unfractionated tRNA from Escherichia coli on DEAE-Sephadex A-50 if this tRNA was previously renatured, whereas two peaks of tRNAGlu are resolved if the sample chromatographed is a mixture of native (renatured) and denatured tRNA. Higher resolution analysis of native E. coli tRNA by RPC-5 chromatography showed that most of the tRNAGlu is present in one peak, eluted shortly after a minor peak containing about or less than 5% of the total amount of tRNAGlu; these two peaks were also observed with commercially available tRNAGlu purified from E. coli. When denatured, the tRNAGlu present in each of these two peaks was eluted from the RPC-5 column at a much lower salt concentration. The properties of the denatured conformers obtained from native tRNAGlu present in the major and minor peaks, and the variation, with growth conditions of E. coli, in the relative amount of tRNAGlu in the minor peak suggest that the tRNAGlu present in the minor peak is an undermodified form of the tRNAGlu present in the major peak. This tRNAGluUUC (or tRNAGluSUC when modified in the anticodon) would then be the only tRNA species acceptor of glutamate in E. coli.

Carbon Radioisotopes↗
PubMed1985

Dimeric tRNA gene arrangement in Schizosaccharomyces pombe allows increased expression of the downstream gene.

Three Schizosaccharomyces pombe dimeric tRNA genes, consisting of a tRNASer gene encoding a minor species with an intervening sequence followed by a tRNAMeti gene, have been described [Mao et al. (1980) Cell 21, 509-516; Hottinger et al. (1982) Mol. Gen. Genet. 188, 219-224; Willis et al. (1984) EMBO J. 3, 1573-1580]. We have examined the reason for the dimeric structure by comparing the transcriptional efficiencies and competitive abilities of the genes subcloned from the dimeric arrangement. Both of the subcloned genes are active in vivo in Saccharomyces cerevisiae, but only the tRNASer gene is efficiently transcribed in vitro. The tRNASer gene competes efficiently for transcription factors, while the tRNAMeti gene does so only weakly. Thus, it appears that the dimeric arrangement is required to support expression of the tRNAMeti gene. S. pombe genes encoding major species of tRNASer are transcribed considerably less efficiently than are the minor genes from the dimers, so coupling of the tRNAMeti gene to the minor species genes should lead to efficient production of tRNAMeti.

Cloning, Molecular↗
PubMed1979

Glutamyl transfer ribonucleic acid synthetase of Escherichia coli. Study of the interactions with its substrates.

The binding of the various substrates to Escherichia coli glutamyl-tRNA synthetase has been investigated by using as experimental approaches the binding study under equilibrium conditions and the substrate-induced protection of the enzyme against its thermal inactivation. The results show that ATP and tRNAGlu bind to the free enzyme, whereas glutamate binds only to an enzyme form to which glutamate-accepting tRNAGlu is associated. By use of modified E. coli tRNAsGlu and heterologous tRNAsGlu, a correlation could be established between the ability of tRNAGlu to be aminoacylated by glutamyl-tRNA synthetase and its abilities to promote the [32P]PPi-ATP isotope exchange and the binding of glutamate to the synthetase. These results give a possible explanation for the inability of blutamyl-tRNA synthetase to catalyze the isotope exchange in the absence of amino acid accepting tRNAGlu and for the failure to detect an enzyme-adenylate complex for this synthetase by using the usual approaches. One binding site was detected for each substrate. The specificity of the interaction of the various substrates has been further investigated. Concerning ATP, inhibition studies of the aminoacylation reaction by various analogues showed the existence of a synergistic effect between the adenine and the ribose residues for the interaction of adenosine. The primary recognition of ATP involves the N-1 and the 6-amino group of adenine as well as the 2'-OH group of ribose. This first interaction is then strengthened by the phosphate groups- Inhibition studies by various analogues of glutamate showed a strong decrease in the affinity of this substrate for the synthetase after substitution of the alpha- or gamma-carboxyl groups. The enzyme exhibits a marked tendency to complex tRNAs of other specificities even in the presence of tRNAGlu. MgCl2 and spermidine favor the specific interactions. The influence of monovalent ions and of pH on the interaction between glutamyl-tRNA synthetase and tRNAGlu is similar to those reported for other synthetases not requiring their cognate tRNA to bind the amino acid. Finally, contrary to that reported for other monomeric synthetases, no dimerization of glutamyl-tRNA synthetase occurs during the catalytic process.

Adenosine Triphosphate↗
PubMed1979

The glutamyl-tRNA synthetase of Escherichia coli: substrate-induced protection against its thermal inactivation.

The substrates-induced protection against the heat-inactivation of the glutamyl-tRNA synthetase has been investigated. tRNAGlu and ATP protect efficiently the enzyme, whereas glutamate does not. In the presence of tRNAGlu, glutamate induces an additional protection to that given by the tRNAGlu alone. A weak synergism was observed between ATP and tRNAGlu, whereas no synergism was detected between ATP and glutamate. These results suggest that tRNAGlu and ATP, but not glutamate are able to bind to the free enzyme form; glutamate binds only to the Enzyme.tRNAGlu and to the Enzyme.tRNAGlu.ATP complexes. The presence of the three substrates induces a higher stabilization of the enzyme than that expected from the protection observed for the various other substrates combinations, suggesting the existence of a marked synergism between the three substrates against the heat-inactivation of the enzyme. The protection constants determined from this study are similar to the dissociation constants determined by direct binding experiments and to the Km values determined kinetically.

Adenosine Triphosphate↗
PubMed1979

The monomeric glutamyl-tRNA synthetase of Escherichia coli. Purification and relation between its structural and catalytic properties.

The glutamyl-tRNA synthetase has been purified to homogeneity from Escherichia coli with a yield of about 50%. It is a monomer with a molecular weight of 56,000 and has the same kinetic properties as those of the alpha chain of the dimeric alphabeta-glutamyl-tRNA synthetase described previously (Lapointe, J., and Söll, D. (1972) J. Biol. Chem. 247, 4966-4974). It is the smallest amino-acyl-tRNA synthetase purified from E. coli and contains no important sequence repetition. It is also the only monomeric aminoacyl-tRNA synthetase reported so far to contain no major sequence duplication. Considering its structural and mechanistic similarities with the glutaminyl- and the arginyl-tRNA synthetases of E. coli, we propose the existence of a relation between the true monomeric character of the glutamyl-tRNA synthetase (as opposed to monomers with sequence duplications) and its requirement for tRNA in the activation of glutamate. A single sulfhydryl group of the native enzyme reacts with 5,5'-dithiobis(2-nitrobenzoic acid) causing no loss of enzymatic activity, whereas four such groups per enzyme react in the presence of 4 M guanidine HCl.

Amino Acids↗
PubMed1979

The twenty aminoacyl-tRNA synthetases from Escherichia coli. General separation procedure, and comparison of the influence of pH and divalent cations on their catalytic activities.

A general separation procedure of the twenty E. coli aminoacyl-tRNA synthetases including either a 105 000 g centrifugation or a poly-ethyleneglycol-dextran two-phases partition fractionation, and chromatographies on DEAE-cellulose, phosphocellulose and hydroxyapatite is described. The specific activities of the synthetases have been determined after each chromatographic step and compared to their respective activities in the 105 000 g supernatant. Some aminoacyl-tRNA synthetases were obtained at 80 per cent purity. The presence of phenylmethylsulfonyl fluoride does not significantly modify either the elution patterns of the synthetases during the various chromatographic steps or their specific activities. Thus, contrarily to enzymes from various eukaryotic organisms no significant inactivation of the E. coli aminoacyl-tRNA synthetases occurs via proteolytic processes during the purification procedure. The effects of various factors: pH, magnesium, and other bivalent cations including spermidine, were tested on the aminoacylation and the [32P] PPi-ATP isotope-exchange reactions, and the optimal aminoacylation and isotope-exchange conditions determined for 18 of the 20 E. coli aminoacyl-tRNA synthetases.

Amino Acyl-tRNA Synthetases↗
PubMed1975

Thermosensitive mutants of Escherichia coli K-12 altered in the catalytic Subunit and in a Regulatory factor of the glutamy-transfer ribonucleic acid synthetase.

The glutamyl-transfer ribonucleic acid synthetase (GluRS) of a partial revertants (ts plus or minus) of the thermosensitive (ts) mutant strain JP1449 (LOcus gltx) and of a ts mutant strain EM111-ts1 with a lesion in or near the locus gltx have been studied to find the relation between these two genetic loci known to influence the GluRS activity in vitro and the presence of a catalytic subunit and of a regulatory subunit in the GluRS purified from Escherichia coli K-12. The ts character of strain JP1449-18ts plus or minus is co-transduced with the marker dsdA at the same frequency as is the ts character of strain JP1449. Its purified GluRS is very thermolabile and its Km for glutamate is higher than that of a wild-type GluRS. These results indicate that the locus gltX is in the structural gene for the catalytic subunit of this enzyme. The location of the mutation causing the partial ts reversion in strain JP1449-18ts plus or minus is discussed. The GluRS purified from the ts mutant strain EM111-ts1 has the same stability as the wild-type enzyme, but its Km forglutamate increases with the temperature, suggesting that the locus gltE codes for a regulatory factor, possibly for the polypeptide chain that is co-purified with the catalytic subunit.

Acylation↗
PubMed1975

Derepressed levels of glutamate synthase and glutamine synthetase in Escherichia coli mutants altered in glutamyl-transfer ribonucleic acid synthetase.

The levels of glutamate synthase and of glutamine synthetase are both derepressed 10-fold in strain JP1449 of Escherichia coli carrying a thermosensitive mutation in the glutamyl-transfer ribonucleic acid (tRNA) synthetase and growing exponentially but at a reduced rate at a partially restrictive temperature, compared with the levels in strain AB347 isogenic with strain JP1449 except for this thermosensitive mutation and the marker aro. These two enzymes catalyze one of the two pathways for glutamate biosynthesis in E. coli, the other being defined by the glutamate dehydrogenase. We observed a correlation between the percentage of charged tRNAGlu and the level of glutamate synthase in various mutants reported to have an altered glutamyl-tRNA synthetase activity. These results suggest that a glutamyl-tRNA might be involved in the repression of the biosynthesis of the glutamate synthase and of the glutamine synthetase and would couple the regulation of the biosynthesis of these two enzymes, which can work in tandem to synthesize glutamate when the ammonia concentration is low in E. coli but whose structural genes are quite distant from each other. No derepression of the level of the glutamate dehydrogenase was observed in mutant strain JP1449 under the conditions where the levels of the glutamine synthetase and of the glutamate synthase were derepressed. This result indicates that the two pathways for glutamate biosynthesis in E. coli are under different regulatory controls. The glutamate has been reported to be probably the key regulatory element of the biosynthesis of the glutamate dehydrogenase. Our results indicate that the cell has chosen the level of glutamyl-tRNA as a more sensitive probe to regulate the biosynthesis of the enzymes of the other pathway, which must be energized at a low ammonia concentration.

Amino Acyl-tRNA Synthetases↗