[6] Glutamyl-tRNA synthetase from Escherichia coli

Lapointe, Jacques; Levasseur, S.; Kern, Daniel

doi:10.1016/s0076-6879(85)13009-0

Cited by 22 publications

(29 citation statements)

References 25 publications

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“…The specific activity of GluRS in extracts of untransformed DH5a cells was 1.25 units/mg protein, a value similar to that previously reported for another strain of E. coli (Lapointe et al 1985). For the sake of comparison, we call this value a relative specific activity of 1 ( Table 2).…”

Section: Overproduction Of Glurssupporting

confidence: 77%

“…(1984) with some modifications: cells were first grown at 32°C up to an A,,, value of 0.1, during which the plasmid is present at one copy per cell. This was followed by a 55-min thermal shock at 43"C, which allows the overproduction Partial proteolysis of GluRS GluRS was purified as described previously (Lapointe et al 1985), dialyzed against 50 mM Hepes-NaOH, pH 8.0, containing 10 mM 2-mercaptoethanol, 0.2 mM dithiothreitol, and 50% glycerol, and kept at a concentration of about 3 mg/mL at -20°C. Proteases (thermolysin, V8 protease, papain, elastase, chymotrypsin, plasmin, and trypsin) were dissolved at a concentration of 500 pg/mL in 100 mM Tris-HCI, pH 7.5, containing 1 mM 2-mercaptoethanol (Cassio and Waller 1971), and kept at -20°C.…”

Section: Growth Conditionsmentioning

confidence: 99%

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Overproduction and domain structure of the glutamyl-tRNA synthetase of Escherichia coli

Brisson¹,

Brun²,

Bell³

et al. 1989

Biochem. Cell Biol.

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The charging of glutamate on tRNA(Glu) is catalyzed by glutamyl-tRNA synthetase, a monomer of 53.8 kilodaltons in Escherichia coli. To obtain the large amounts of enzyme necessary for the identification of structural domains, we have inserted the structural gene gltX in the conditional runaway-replication plasmid pOU61, which led to a 350-fold overproduction of glutamyl-tRNA synthetase. Partial proteolysis of this enzyme revealed the existence of preferential sites of attack that, according to their N-terminal sequences, delimit regions of 12.9, 2.3, 12.1, and 26.5 kilodaltons from the N- to C-terminal of the enzyme. Their sizes suggest that the 2.3-kilodalton fragment is a hinge structure, and that those of 12.9, 12.1, and 26.5 kilodaltons are domain structures. The 12.9-kilodalton domain of the glutamyl-tRNA synthetase of E. coli is the only long region of this enzyme displaying a good amino acid sequence similarity with the glutaminyl-tRNA synthetase of Escherichia coli.

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Section: Overproduction Of Glurssupporting

confidence: 77%

Section: Growth Conditionsmentioning

confidence: 99%

Overproduction and domain structure of the glutamyl-tRNA synthetase of Escherichia coli

Brisson¹,

Brun²,

Bell³

et al. 1989

Biochem. Cell Biol.

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“…The tRNA aminoacylation activities were measured at 37 uC in 50 mM HEPES-KOH, pH 7.2, 16 mM MgCl 2 , 2 mM ATP, 3 mM DTT, 330 mM unfractionated tRNA from E. coli, and 200 mM [ 14 C]glutamine or [ 14 C]asparagine, for GlnRS and AsnRS activity measurements, respectively, using the filter paper assay described by Lapointe et al (1985). As a control, the same measurements were done with E. coli DH5a crude extracts prepared by a similar procedure.…”

Section: Methodsmentioning

confidence: 99%

Natural insertion of the bro-1 β-lactamase gene into the gatCAB operon affects Moraxella catarrhalis aspartyl-tRNAAsn amidotransferase activity

2012

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Only about half of bacterial species use an asparaginyl-tRNA synthetase (AsnRS) to attach Asn to its cognate tRNA Asn . Other bacteria, including the human pathogen Moraxella catarrhalis, a causative agent of otitis media, lack a gene encoding AsnRS, and form Asn-tRNA Asn by an indirect pathway catalysed by two enzymes: first, a non-discriminating aspartyl-tRNA synthetase (ND-AspRS) catalyses the formation of aspartyl-tRNA Asn (Asp-tRNA Asn ); then, a tRNAdependent amidotransferase (GatCAB) transamidates this 'incorrect' product into Asn-tRNA Asn . As M. catarrhalis has a Gln-tRNA synthetase, its GatCAB functions as an Asp-tRNA Asn amidotransferase. This pathogen rapidly evolved to about 90 % ampicillin resistance worldwide by insertion of a bro-1 b-lactamase gene within the gatCAB operon. Comparison of the GatCAB subunits from bro-1 b-lactamase-positive and bro-negative strains showed that the laterally transferred bro-1 gene, inserted into the gatCAB operon, affected the C-terminal sequence of GatA. The identity between the C-terminal sequences of GatA wt (residues 479-491) and of GatA BRO-1 (residues 479-492) was about 36 %, whereas the rest of the GatA sequence was relatively conserved. The characterization of these two distinct GatCABs as well as the hybrid GatCAB containing GatA(1-478) wt (479-492) BRO-1 and truncated GatCAB enzymes of M. catarrhalis showed that the substitution in GatA wt of residues 479-492 of GatA BRO-1 causes increased specificity for glutamine, and decreased specificity for Asp-tRNA Asn in the transamidation reaction. We conclude that the bro gene insertion has altered the kinetic parameters of Asp-tRNA Asn amidotransferase, and we propose a model for gatA evolution after the insertion of bro-1 at the carboxyl end of gatA.

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“…First, the tRNA is aminoacylated with the appropriate 14 C-labeled amino acid by a proper aaRS. The aminoacylation reaction can be monitored as previously described [20]. At the end of the reaction, the mixture is phenol (citrate-buffered, pH 4.5)/chloroform extracted and then ethanol precipitated.…”

Section: [ 14 C]-amino Acid/tlc Based Assaymentioning

confidence: 99%

“…The use of [ 14 C]-aa allows for a straightforward method for determining the amount of substrate aa-tRNA formed in the initial aminoacylation reaction [20] as well as a means of monitoring the deacylation of the tRNA over the course of the reaction [23]. The subsequent ethanol precipitation and deacylation steps make it a labor and time consuming assay to carry out for studying a tRNA-dependent modifying enzyme, especially when taking multiple time points.…”

Section: [ 14 C]-amino Acid/tlc Based Assaymentioning

confidence: 99%

Assays for transfer RNA-dependent amino acid biosynthesis

Sheppard

Akochy

Söll

2008

Methods

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Selenocysteinyl-tRNA Sec , cysteinyl-tRNA Cys , glutaminyl-tRNA Gln , and asparaginyl-tRNA Asn in many organisms are formed in an indirect pathway in which a non-cognate amino acid is first attached to the tRNA. This non-cognate amino acid is then converted to the cognate amino acid by a tRNAdependent modifying enzyme. The in vitro characterization of these modifying enzymes is challenging due to the fact the substrate, aminoacyl-tRNA, is labile and requires a prior enzymatic step to be synthesized. The need to separate product aa-tRNA from unreacted substrate is typically a labor-and time-intensive task; this adds another impediment in the investigation of these enzymes. Here we review four different approaches for studying these tRNA-dependent amino acid modifications. In addition, we describe in detail a [ 32 P]/nuclease P1 assay for glutaminyl-tRNA Gln and asparaginyl-tRNA Asn formation which is sensitive, enables monitoring of the aminoacyl state of the tRNA, and is less time consuming than some of the other techniques. This [ 32 P]/nuclease P1 method should be adaptable to studying tRNA-dependent selenocysteine and cysteine synthesis.

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[6] Glutamyl-tRNA synthetase from Escherichia coli

Cited by 22 publications

References 25 publications

Overproduction and domain structure of the glutamyl-tRNA synthetase of Escherichia coli

Overproduction and domain structure of the glutamyl-tRNA synthetase of Escherichia coli

Natural insertion of the bro-1 β-lactamase gene into the gatCAB operon affects Moraxella catarrhalis aspartyl-tRNAAsn amidotransferase activity

Assays for transfer RNA-dependent amino acid biosynthesis

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