Glutamine—tRNA ligase

About: Glutamine—tRNA ligase is a research topic. Over the lifetime, 6 publications have been published within this topic receiving 152 citations. The topic is also known as: Gln-tRNA-synth & Glutamine-tRNA ligase.

Discrimination among tRNAs intermediate in glutamate and glutamine acceptor identity.

Abstract: The set of nucleotides in Escherichia coli tRNA(Gln) which facilitate aminoacylation by glutaminyl-tRNA synthetase (GlnRS) has been defined [Hayase et al (1992), EMBO J 11, 4159-4165] To determine whether the glutamine "identity set" is sufficient to confer acceptance on a noncognate tRNA, we constructed tRNA(Glu) mutants with the set of glutamine recognition elements These mutants were examined for aminoacylation in vitro with GlnRS and also with glutamyl-tRNA synthetase (GluRS) to correlate gains in glutamine acceptance with losses of glutamate acceptance Incorporating glutamine recognition elements in only the acceptor stem or anticodon loop of tRNA(Glu) improved the specificity constant (kcat/KM) for aminoacylation by GlnRS However, the introduction of all defined glutamine recognition elements in tRNA(Glu) resulted in a substrate with a specificity constant 100-fold below that for aminoacylation of tRNA(Gln) Including the tertiary framework of tRNA(Gln) (in addition to the glutamine recognition elements) in the tRNA(Glu) context further improved aminoacylation by GlnRS, but the specificity was still reduced compared with that of tRNA(Gln) The increase in glutamine acceptance was correlated for all mutants with a decrease in glutamate acceptance, indicating that GluRS also recognizes acceptor stem and anticodon sequences in cognate tRNA The inability to completely convert tRNA(Glu) to glutamine acceptance with these mutations suggests that tRNA(Glu) contains antideterminants to glutamine identity The analysis of these mutants with both enzymes revealed that there is a strong element of discrimination between glutamate and glutamine tRNAs associated with the anticodon To test this dependence, mutants of both tRNAs were made to effect anticodon switches to the possible glutamate and glutamine isoacceptors The kinetic evaluation of the anticodon switch mutants suggests that overlap in anticodon recognition is avoided through specificity for the third anticodon position coupled with divergent preferences for the wobble base

Functional communication in the recognition of tRNA by Escherichia coli glutaminyl-tRNA synthetase.

Abstract: Wild-type Escherichia coli glutaminyl-tRNA synthetase (GlnRS; EC 6.1.1.18) poorly aminoacylates opal suppressors (GLN) derived from tRNA(Gln). Mutations in glnS (the gene encoding GlnRS) that compensate for impaired aminoacylation were isolated by genetic selection. Two glnS mutants were obtained by using opal suppressors differing in the nucleotides composing the base pair at 3.70: glnS113 with an Asp-235-->Asn change selected with GLNA3U70 (GLN carrying G3-->A and C70-->U changes), and glnS114 with a Gln-318-->Arg change selected with GLNU70 (GLN carrying a C70-->U change). The Asp-235-->Asn change was identified previously by genetic selection. Additional mutants were isolated by site-directed mutagenesis followed by genetic selection; the mutant enzymes have single amino acid changes (Lys-317-->Arg and Gln-318-->Lys). A number of mutants with no phenotype also were obtained randomly. In vitro aminoacylation of a tRNA(Gln) transcript by GlnRS enzymes with Lys-317-->Arg, Gln-318-->Lys, or Gln-318-->Arg changes shows that the enzyme's kinetic parameters are not greatly affected by the mutations. However, aminoacylation of a tRNA(Gln) transcript with an opal (UCA) anticodon shows that the specificity constants (kcat/Km) for the mutant enzymes were 5-10 times above that of the wild-type GlnRS. Interactions between Lys-317 and Gln-318 with the inside of the L-shaped tRNA and with the side chain of Gln-234 provide a connection between the acceptor end-binding and anticodon-binding domains of GlnRS. The GlnRS mutants isolated suggest that perturbation of the interactions with the inside of the tRNA L shape results in relaxed anticodon recognition.

Selection of a minimal glutaminyl-tRNA synthetase and the evolution of class I synthetases

Abstract: The evolution of the aminoacyl-tRNA synthetases is intriguing in light of their elaborate relationship with tRNAs and their significance in the decoding process. Based on sequence motifs and structure determination, these enzymes have been assigned to two classes. The crystal structure of Escherichia coli glutaminyl-tRNA synthetase (GlnRS), a class I enzyme, complexed to tRNA(Gln) and ATP has been described. It is shown here that a 'minimal' GlnRS, i.e. a GlnRS from which domains interacting with the acceptor-end and the anticodon of the tRNA have been deleted, has enzymatic activity and can charge a tRNA(Tyr)-derived amber suppressor (supF) with glutamine. The catalytic core of GlnRS, which is structurally conserved in other class I synthetases, is therefore sufficient for the aminoacylation of tRNA substrates. Some of these truncated enzymes have lost their ability to discriminate against non-cognate tRNAs, implying a more specific role of the acceptor-end-binding domain in the recognition of tRNAs. Our results indicate that the catalytic and substrate recognition properties are carried by distinct domains of GlnRS, and support the notion that class I aminoacyl-tRNA synthetases evolved from a common ancestor, jointly with tRNAs and the genetic code, by the addition of non-catalytic domains conferring new recognition specificities.