A dual-specificity aminoacyl-tRNA synthetase in the deep-rooted eukaryote Giardia lamblia

Cysteinyl-tRNA (Cys-tRNA) is essential for protein synthesis. In most organ isms the enzyme responsible for the formation of Cys-tRNA is cysteinyl-tRNA synthetase (CysRS). The only known exceptions are the euryarchaea Methanoc occus jannaschii and Methanobacterium thermoautotrophicum, which do not enc ode a CysRS. Deviating from the accepted concept of one aminoacyl-tRNA synt hetase 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 archa ea. Analysis of the preliminary genomic sequence of the primitive eukaryote Giardia lamblia indicated the presence of an archaeal prolyl-tRNA syntheta se (ProRS). Its proS gene was cloned and the gene product overexpressed in Escherichia coil. By using G. lamblia, M. jannaschii, or E, coil tRNA as su bstrate, this ProRS was able to form Cys-tRNA and Pro-tRNA in vitro. Cys-AM P formation, but not Pro-AMP synthesis, was tRNA-dependent. The in vitro da ta were confirmed in vivo, as the cloned G. lamblia proS gene was able to c omplement a temperature-sensitive E. coil cysS strain. Inhibition studies o f CysRS activity with proline analogs (thiaproline and 5'-O-[N-(L-prolyl)-s ulfamoyl]adenosine) in a Giardia S-100 extract predicted that the organism also contains a canonical CysRS. This prediction was confirmed by cloning a nd analysis of the corresponding cysS gene. Like a number of archaea, Giard ia contains two enzymes, ProCysRS and CysRS, for Cys-tRNA formation. In con trast, the purified Saccharomyces cerevisiae and E, coil 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 p rolyl- and alanyl-tRNA synthetases refine our understanding of the evolutio n and interaction of archaeal and eukaryal translation systems.