title: A dual-specificity aminoacyl-tRNA synthetase in the deep-rooted eukaryote Giardia lamblia.
abstract: Cysteinyl-tRNA (Cys-tRNA) is essential for protein synthesis. In most organisms the enzyme responsible for the formation of Cys-tRNA is cysteinyl-tRNA synthetase (CysRS). The only known exceptions are the euryarchaea Methanococcus jannaschii and Methanobacterium thermoautotrophicum, which do not encode a CysRS. Deviating from the accepted concept of one aminoacyl-tRNA synthetase per amino acid, these organisms employ prolyl-tRNA synthetase as the enzyme that carries out Cys-tRNA formation. To date this dual-specificity prolyl-cysteinyl-tRNA synthetase (ProCysRS) is only known to exist in archaea. Analysis of the preliminary genomic sequence of the primitive eukaryote Giardia lamblia indicated the presence of an archaeal prolyl-tRNA synthetase (ProRS). Its proS gene was cloned and the gene product overexpressed in Escherichia coli. By using G. lamblia, M. jannaschii, or E. coli tRNA as substrate, this ProRS was able to form Cys-tRNA and Pro-tRNA in vitro. Cys-AMP formation, but not Pro-AMP synthesis, was tRNA-dependent. The in vitro data were confirmed in vivo, as the cloned G. lamblia proS gene was able to complement a temperature-sensitive E. coli cysS strain. Inhibition studies of CysRS activity with proline analogs (thiaproline and 5'-O-[N-(l-prolyl)-sulfamoyl]adenosine) in a Giardia S-100 extract predicted that the organism also contains a canonical CysRS. This prediction was confirmed by cloning and analysis of the corresponding cysS gene. Like a number of archaea, Giardia contains two enzymes, ProCysRS and CysRS, for Cys-tRNA formation. In contrast, the purified Saccharomyces cerevisiae and E. coli ProRS enzymes were unable to form Cys-tRNA under these conditions. Thus, the dual specificity is restricted to the archaeal genre of ProRS. G. lamblia's archaeal-type prolyl- and alanyl-tRNA synthetases refine our understanding of the evolution and interaction of archaeal and eukaryal translation systems.
authors.affiliation: Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520-8114, USA.
authors.forename: S
authors.initials: S
authors.lastname: Bunjun
authors.forename: C
authors.initials: C
authors.lastname: Stathopoulos
authors.forename: D
authors.initials: D
authors.lastname: Graham
authors.forename: B
authors.initials: B
authors.lastname: Min
authors.forename: M
authors.initials: M
authors.lastname: Kitabatake
authors.forename: A L
authors.initials: AL
authors.lastname: Wang
authors.forename: C C
authors.initials: CC
authors.lastname: Wang
authors.forename: C P
authors.initials: CP
authors.lastname: Vivarès
authors.forename: L M
authors.initials: LM
authors.lastname: Weiss
authors.forename: D
authors.initials: D
authors.lastname: Söll
mesh_terms: D000604:Amino Acyl-tRNA Synthetases / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005816:Genetic Complementation Test; D016829:Giardia lamblia / Q000201:enzymology* / Q000235:genetics*; D007700:Kinetics; D008969:Molecular Sequence Data; D010802:Phylogeny; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D013379:Substrate Specificity
chemical_list: D000604:Amino Acyl-tRNA Synthetases; C100134:prolyl T RNA synthetase; C019975:cysteinyl-tRNA synthetase