Molecular and General Genetics MGG

, Volume 252, Issue 6, pp 717–722 | Cite as

Genetic analysis of functional connectivity between substrate recognition domains ofEscherichia coli glutaminyl-tRNA synthetase

  • M. Kitabatake
  • H. Inokuchi
  • M. Ibba
  • K. W. Hong
  • D. Söll
Original Paper


It has previously been shown that the single mutation E222K in glutaminyl-tRNA synthetase (GlnRS) confers a temperature-sensitive phenotype onEscherichia coli. Here we report the isolation of a pseudorevertant of this mutation, E222K/C171G, which was subsequently employed to investigate the role of these residues in substrate discrimination. The three-dimensional structure of the tRNAGln: GlnRS:ATP ternary complex revealed that both E222 and C171 are close to regions of the protein involved in interactions with both the acceptor stem and the 3′ end of tRNAGln. The potential involvement of E222 and C171 in these interactions was confirmed by the observation that GlnRS-E222K was able to mischargesupF tRNATyr considerably more efficiently than the wild-type enzyme, whereas GlnRS-E222K/C171G could not. These differences in substrate specificity also extended to anticodon recognition, with the double mutant able to distinguishsupE tRNA CUA Gln from tRNA 2 Gln considerably more efficiently than GlnRS E222K. Furthermore, GlnRS-E222K was found to have a 15-fold higher Km for glutamine than the wild-type enzyme, whereas the double mutant only showed a 7-fold increase. These results indicate that the C171G mutation improves both substrate discrimination and recognition at three domains in GlnRS-E222K, confirming recent proposals that there are extensive interactions between the active site and regions of the enzyme involved in tRNA binding.

Key words

Glutaminyl-tRNA synthetase Temperature sensitivity Substrate specificity Aminoacylation Suppressor tRNA 


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Copyright information

© Springer-Verlag 1996

Authors and Affiliations

  • M. Kitabatake
    • 1
  • H. Inokuchi
    • 1
  • M. Ibba
    • 2
  • K. W. Hong
    • 2
  • D. Söll
    • 2
  1. 1.Department of Biophysics, Faculty of ScienceKyoto UniversityKyotoJapan
  2. 2.Department of Molecular Biology and BiochemistryYale UniversityNew HavenUSA

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