Overproduction and domain structure of the glutamyl-tRNA synthetase of <i>Escherichia coli</i>

Brisson, A.; Brun, Yves V.; Bell, Alexander W.; Roy, Paul H.; Lapointe, Jacques

doi:10.1139/o89-065

Cited by 9 publications

(7 citation statements)

References 21 publications

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“…Glutamyl-tRNA synthetase (GluRS) puri®cation. Puri®cation of GluRS was carried out according to Brisson et al (11). The speci®c activities were comparable to those reported in 11.…”

Section: Methodssupporting

confidence: 62%

Solvation change and ion release during aminoacylation by aminoacyl-tRNA synthetases

Banerjee

Mandal

Saha

et al. 2003

Nucleic Acids Research

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Discrimination between cognate and non-cognate tRNAs by aminoacyl-tRNA synthetases occurs at several steps of the aminoacylation pathway. We have measured changes of solvation and counter-ion distribution at various steps of the aminoacylation pathway of glutamyl- and glutaminyl-tRNA synthetases. The decrease in the association constant with increasing KCl concentration is relatively small for cognate tRNA binding when compared to known DNA-protein interactions. The electro-neutral nature of the tRNA binding domain may be largely responsible for this low ion release stoichiometry, suggesting that a relatively large electrostatic component of the DNA-protein interaction free energy may have evolved for other purposes, such as, target search. Little change in solvation upon tRNA binding is seen. Non-cognate tRNA binding actually increases with increasing KCl concentration indicating that charge repulsion may be a significant component of binding free energy. Thus, electrostatic interactions may have been used to discriminate between cognate and non-cognate tRNAs in the binding step. The catalytic constant of glutaminyl-tRNA synthetase increases with increasing osmotic pressure indicating a water release of 8.4 +/- 1.4 mol/mol in the transition state, whereas little change is seen in the case of glutamyl-tRNA synthetase. We propose that the significant amount of water release in the transition state, in the case of glutaminyl-tRNA synthetase, is due to additional contact of the protein with the tRNA in the transition state.

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“…Glutamyl-tRNA synthetase (GluRS) puri®cation. Puri®cation of GluRS was carried out according to Brisson et al (11). The speci®c activities were comparable to those reported in 11.…”

Section: Methodssupporting

confidence: 62%

Solvation change and ion release during aminoacylation by aminoacyl-tRNA synthetases

Banerjee

Mandal

Saha

et al. 2003

Nucleic Acids Research

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“…D. S611. Purification and Characterization of E. coli GluRS and Its Fragments. GluRS was purified from the overproducing strain, E. coli DH5a (pLQ7612) (Brisson et al, 1989), by a rapid procedure, as previously described (Lin et al, 1992), with the following modification: 10 mM ZnCl2 was added to the growth medium. The homogeneity of the purified enzyme was verified by electrophoresis on polyacrylamide gel in the presence of SDS (SDS-PAGE), after denaturation and reduction of the protein (Laemmli, 1970).…”

Section: Methodsmentioning

confidence: 99%

The glutamyl-tRNA synthetase of Escherichia coli contains one atom of zinc essential for its native conformation and its catalytic activity

Liu¹,

Lin²,

Blochet³

et al. 1993

Biochemistry

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The glutamyl-tRNA synthetase of Escherichia coli contains one atom of zinc. This metal ion is strongly bound, as it is not removed by 8 M urea. Slow removal of the zinc at 4 degrees C in the presence of the specific chelating agent, 1,10-phenanthroline, is proportional to the loss of aminoacylation activity and to the presence of a more open conformer of the enzyme. This conformer migrates more slowly than the native enzyme during gel electrophoresis under nondenaturing conditions and binds tRNA(Glu). Infrared spectroscopy measurements show that it differs from the native enzyme by a lower alpha-helix content and a higher proportion of beta-sheet and unordered structures. ATP protects the enzyme against 1,10-phenanthroline-mediated zinc removal, suggesting that the zinc-binding region is closely associated with the catalytic site. Additional support for this conclusion comes from the presence of zinc in the 27-kDa N-terminal half of the enzyme and in a 10-kDa fragment. The latter is homologous to the tRNA acceptor helix binding domain of E. coli glutaminyl-tRNA synthetase. The presence of the conserved CYC motif in this domain of the zinc-containing glutamyl-tRNA synthetases of E. coli and Bacillus subtilis, and its absence in that of Thermus thermophilus and the E. coli glutaminyl-tRNA synthetase which do not contain zinc, suggest that the cysteines of this motif and the C- and H-rich 125CRHSHEHHX5C138 segment present in the 10-kDa zinc-binding fragment are involved in zinc binding by the glutamyl-tRNA synthetase of E. coli.

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“…We attempted to enhance the suppressor activity of the U36 allele by introducing a plasmid which leads to an approximately 100-fold over-expression of GIuRS [15]. CSH106 (lacZ(AAG461)) and CSH142F'100 were transformed with combinations of plasmids pMO10 (WT tRNA ~lu or tRNAG1uU36) and either pBR322 or pLQ7611DNruI (pBR322 containing the gltX gene encoding GluRS) and assayed for fl-galactosidase activity.…”

Section: Resultsmentioning

confidence: 99%

“…[10]. c% suppression is calculated as the activity in the laeZ mutant divided by the activity in the corresponding isogenic lacZ + strain multiplied by 100. aWild-type or U36 tRNA G~u alleles were combined either with pBR322 or with pLQ7611ANruI, which over-expresses GluRS by approximately 100 fold [15]. bfl-Galactosidase activity is expressed as Miller units [10].…”

Section: Discussionmentioning

confidence: 99%

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Effects of mutations at position 36 of tRNA^Glu on missense and nonsense suppression in Escherichia coli

Gregory¹,

Dahĺberg²

1995

FEBS Letters

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Mutations in the anticodon of tRNA Clu (UUC) were isolated or constructed and characterized for their ability to suppress cognate nonsense or missense mutations in vivo. The C36-to-A36 transversion mutation was isolated as an ochre and an amber suppressor, while the G36 transversion was selected as a CAG missense suppressor, tRNA G~u suppressors of an AAG missense mutation could not be isolated, and a 1536 transition mutation introduced into tRNA G~u in vitro conferred no suppressor phenotype. Over-expression of glutamyl-tRNA synthetase did not increase the activity of the U36 mutant tRNA clu, suggesting a defect at the level of translation rather than at the level of synthetase recognition.Key words: Suppressor tRNA; Translational efficiency; tRNAG~U; Anticodon a GAG(glu) to AAG(lys) missense mutation at the same position in lacZ were unsuccessful. A tRNA c~u allele containing a C-to-U transition at position 36 constructed by oligonucleotide-directed mutagenesis failed to suppress the same lacZ AAG missense mutation. The activity of the U36 mutant tRNA ~u, measured as fl-galactosidase activity, was not increased by over-expression of glutamyl-tRNA synthetase (GluRS). While defects in aminoacylation cannot be excluded, our observations are consistent with previous findings [6,7] and indicate that mutations at position 36 in the anticodon of tRNA Clu fail to produce efficient translational suppressors due primarily to a defect in codon recognition or tRNA-ribosome interaction. Materials and methods

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

Cited by 9 publications

References 21 publications

Solvation change and ion release during aminoacylation by aminoacyl-tRNA synthetases

Solvation change and ion release during aminoacylation by aminoacyl-tRNA synthetases

The glutamyl-tRNA synthetase of Escherichia coli contains one atom of zinc essential for its native conformation and its catalytic activity

Effects of mutations at position 36 of tRNA^Glu on missense and nonsense suppression in Escherichia coli

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

Cited by 9 publications

References 21 publications

Solvation change and ion release during aminoacylation by aminoacyl-tRNA synthetases

Solvation change and ion release during aminoacylation by aminoacyl-tRNA synthetases

The glutamyl-tRNA synthetase of Escherichia coli contains one atom of zinc essential for its native conformation and its catalytic activity

Effects of mutations at position 36 of tRNAGlu on missense and nonsense suppression in Escherichia coli

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Effects of mutations at position 36 of tRNA^Glu on missense and nonsense suppression in Escherichia coli