A dual-specificity aminoacyl-tRNA synthetase in the deep-rooted eukaryote <em>Giardia lamblia</em>

S Bunjun

C Stathopoulos

D Graham

B Min

^{Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520-8114;}^{Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, CT 06511;}^{Department of Microbiology, University of Illinois at Urbana-Champaign, Urbana, IL 61801;}^{Department of Pharmaceutical Chemistry, University of California, San Francisco, CA 94143-0446;}^{Laboratoire de Parasitologie Moleculaire et Cellulaire, Université Blaise Pascal, 63177 Aubiere Cedex, France; and}^{Department of Pathology, Albert Einstein College of Medicine, Bronx, NY 10461}

^{S.B. and C.S. contributed equally to the work.}

^{To whom reprint requests should be addressed at: Department of Molecular Biophysics and Biochemistry, Yale University, P.O. Box 208114, 266 Whitney Avenue, New Haven, CT 06520-8114. E-mail:}ude.elay.mehc.anrt@llos.

Contributed by Dieter Söll

Accepted 2000 Sep 15.

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.

Abstract

Aminoacyl-tRNA synthetases (AARSs) are essential for the faithful translation of the genetic code. They ensure the fidelity of protein synthesis by correctly acylating a tRNA species with its cognate amino acid (1). This crucial family of enzymes is divided into two distinct classes (I and II) based on characteristic signature motifs. These and other conserved structural features allow facile recognition of orthologous enzymes in many organisms by sequence similarity searches of the available databases. They also allow comparison between the AARSs and thus classification into subtypes with respect to their phylogenetic origin (2). These enzymes have exquisite specificity for their substrates (amino acid and tRNA), a process mediated in some cases by intricate editing mechanisms (3–5). It is commonly accepted that each cell contains 20 such enzymes, one for each canonical amino acid. This assumption was supported by the description of 20 AARSs found in some bacteria (e.g., E. coli) and in the eukaryotic cytoplasm. However, recent discoveries arising from functional genomics studies in bacteria and archaea have overturned this concept and revealed that most organisms do not use a full complement of 20 canonical AARSs (6–8). The major exception is the route to Gln-tRNA or Asn-tRNA formation, which in many organisms involves an amidation of a mischarged Glu-tRNA or Asp-tRNA (8–10). Most surprisingly, it was demonstrated in vitro and in vivo that the ProRS of M. jannaschii, M. thermoautotrophicum, and Methanococcus maripaludis is able to catalyze the formation of cysteinyl-tRNA (Cys-tRNA; ref. 7). The fact that the two former organisms lack a canonical cysteinyl-tRNA synthetase (CysRS) implies that these organisms use the dual-specificity prolyl-cysteinyl-tRNA synthetase (ProCysRS) for the synthesis of both Cys-tRNA and Pro-tRNA required for protein synthesis. The fact that a single AARS is capable of and required for supplying two different aminoacyl-tRNAs for protein synthesis challenged the accepted view of these enzymes (1).

As we showed earlier, the genome of the lower eukaryote Giardia lamblia genome contains genes encoding the archaeal genre of ProRS and alanyl-tRNA synthetase (AlaRS; ref. 2). G. lamblia is a parasitic, amitochondrial protist that diverged early in the eukaryotic lineage (11, 12). By comparing genes from this organism with homologous eukaryotic and archaeal genes it may be possible to infer the events that led to the formation of the modern eukaryal cell type. The parasite G. lamblia is found in every region of the United States and throughout the world and infects about 200 million people annually worldwide (13). It has become recognized as one of the most common causes of waterborne disease in humans in the United States, causing diarrhea, abdominal cramps, and nausea. Further knowledge of the essential enzymes of protein synthesis may reveal new targets for development of antiparasitic agents. Here we report the existence of the dual-specificity ProCysRS and its activity in G. lamblia.

Acknowledgments

We thank M. Ibba and C. R. Woese for constructive discussions. We are indebted to S. Cusack for a sample of 5′-O-[N-(l-prolyl)-sulfamoyl]adenosine, and to H. D. Becker and T. Li for experimental advice. This work was supported by grants from the National Institute of General Medical Sciences and from National Aeronautics and Space Administration. M.K. was a postdoctoral fellow of the Japanese Ministry for Education.

Acknowledgments

Abbreviations

AARSs	aminoacyl-tRNA synthetases
Cys-tRNA	cysteinyl-tRNA
CysRS	cysteinyl-tRNA synthetase
ProCysRS	prolyl-cysteinyl-tRNA synthetase
ProRS	prolyl-tRNA synthetase
AlaRS	alanyl-tRNA synthetase

Abbreviations

Footnotes

Data deposition: The sequences reported in this paper have been deposited in the GenBank database (accession nos. {"type":"entrez-nucleotide","attrs":{"text":"AF245445","term_id":"10798906","term_text":"AF245445"}}AF245445, {"type":"entrez-nucleotide","attrs":{"text":"AF299082","term_id":"11528338","term_text":"AF299082"}}AF299082, and {"type":"entrez-nucleotide","attrs":{"text":"AF245446","term_id":"10800404","term_text":"AF245446"}}AF245446).

Article published online before print: Proc. Natl. Acad. Sci. USA, 10.1073/pnas.230444397.

Article and publication date are at www.pnas.org/cgi/doi/10.1073/pnas.230444397

Footnotes

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