A Single Amidotransferase Forms Asparaginyl-tRNA and Glutaminyl-tRNA in Chlamydia trachomatis

Gregory Raczniak,Hubert D Becker,Bokkee Min,Dieter Söll

doi:10.1074/jbc.m109494200

Abstract

Aminoacyl-tRNA is generally formed by aminoacyl-tRNA synthetases, a family of 20 enzymes essential for accurate protein synthesis. However, most bacteria generate one of the two amide aminoacyl-tRNAs, Asn-tRNA or Gln-tRNA, by transamidation of mischarged Asp-tRNA(Asn) or Glu-tRNA(Gln) catalyzed by a heterotrimeric amidotransferase (encoded by the gatA, gatB, and gatC genes). The Chlamydia trachomatis genome sequence reveals genes for 18 synthetases, whereas those for asparaginyl-tRNA synthetase and glutaminyl-tRNA synthetase are absent. Yet the genome harbors three gat genes in an operon-like arrangement (gatCAB). We reasoned that Chlamydia uses the gatCAB-encoded amidotransferase to generate both Asn-tRNA and Gln-tRNA. C. trachomatis aspartyl-tRNA synthetase and glutamyl-tRNA synthetase were shown to be non-discriminating synthetases that form the misacylated tRNA(Asn) and tRNA(Gln) species. A preparation of pure heterotrimeric recombinant C. trachomatis amidotransferase converted Asp-tRNA(Asn) and Glu-tRNA(Gln) into Asn-tRNA and Gln-tRNA, respectively. The enzyme used glutamine, asparagine, or ammonia as amide donors in the presence of either ATP or GTP. These results suggest that C. trachomatis employs the dual specificity gatCAB-encoded amidotransferase and 18 aminoacyl-tRNA synthetases to create the complete set of 20 aminoacyl-tRNAs.

Highlights

The codons of messenger RNA are paired on the ribosome with aminoacyl-tRNAs (AA-tRNAs)1 during the process of protein biosynthesis
All gatCAB-encoded bacterial AdTs studied to date are responsible only for in vivo synthesis of one of the two possible amide AA-tRNAs, whereas the other amide AA-tRNA is formed by direct aminoacylation by asparaginyl-tRNA synthetase (AsnRS) or glutaminyl-tRNA synthetase (GlnRS)
We report that pure C. trachomatis amidotransferase can amidate Chlamydia AsptRNAAsn and Glu-tRNAGln generated by the homologous aspartyl-tRNA synthetase (AspRS) and glutamyl-tRNA synthetase (GluRS) enzymes

Summary

EXPERIMENTAL PROCEDURES

General—C. trachomatis genomic DNA was a gift of L. After an 8-h incubation, cells were harvested by centrifugation at 4,000 ϫ g for 10 min at 4 °C and suspended in buffer A (20 mM Tris-HCl, pH 7.4, 20 mM MgCl2, 10 mM 2-mercaptoethanol) to a final volume of 12.5 ml. Formation of Aminoacyl-tRNA—For amidotransferase assays, a total of 5 nmol of unfractionated tRNA from an E. coli stain that expresses the C. trachomatis tRNAGln or tRNAGlu was charged with [14C]glutamate (50 ␮M, 260 mCi/mmol) by 4 ␮g of partially purified native or. C. trachomatis tRNAAsn or tRNAAsp (4 nmol of unfractionated tRNA from an E. coli strain expressing the tRNA or the purified tRNAAsp and tRNAAsn species) was charged with [14C]aspartate (50 ␮M, 213 mCi/mmol) by 4 ␮g of partially purified native or His6-tagged C. trachomatis AspRS. Localization of [14C]amino acids was confirmed by ninhydrin assay using 50 nmol of unlabeled standards

RESULTS

Relative purification

TABLE II Requirements of the amidation reaction