Aminoacyl-tRNA is generally formed by aminoacyltRNA 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 AsptRNA 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 glutamyltRNA 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.

Two types of aspartyl-tRNA synthetase exist: the discriminating enzyme (D-AspRS) forms only Asp-tRNA Asp , while the nondiscriminating one (ND-AspRS) also synthesizes Asp-tRNA Asn , a required intermediate in protein synthesis in many organisms (but not in Escherichia coli). On the basis of the E. coli trpA34 missense mutant transformed with heterologous ND-aspS genes, we developed a system with which to measure the in vivo formation of Asp-tRNA Asn and its acceptance by elongation factor EF-Tu. While large amounts of Asp-tRNA Asn are detrimental to E. coli, smaller amounts support protein synthesis and allow the formation of up to 38% of the wild-type level of missense-suppressed tryptophan synthetase.Aspartyl-tRNA synthetase (AspRS) exists in two different forms with respect to tRNA recognition (7). The discriminating enzyme (D-AspRS) recognizes only tRNA Asp , while the nondiscriminating one (ND-AspRS) also recognizes tRNA Asn and therefore forms both Asp-tRNA Asn and Asp-tRNA Asp . Most bacteria and archaea lack asparaginyl-tRNA synthetase and are unable to synthesize Asn-tRNA Asn by direct acylation of tRNA. These organisms rely on the ND-AspRS to produce the misacylated Asp-tRNA Asn , which is then converted by a tRNA-dependent amidotransferase to the correctly acylated Asn-tRNA Asn (1,4,5,19). Thus, the ND-AspRS is essential in organisms that form Asn-tRNA by transamidation.The primary sequence distinguishes two general groups of AspRS. There is a predominantly bacterial type of AspRS that is about 580 amino acids, in addition to a shorter archaealeukaryotic type of about 430 amino acids. In vitro data have made clear that discriminating and nondiscriminating enzymes exist in both groups (16,20). The determinants in the protein sequence responsible for tRNA discrimination are not known.The two AspRS types are usually separated in nature. Genome analyses of bacteria and archaea have revealed that the presence of the ND-AspRS is always accompanied by the occurrence of the heterotrimeric GatCAB amidotransferase, an enzyme capable of converting the misacylated Asp-tRNA Asn to Asn-tRNA Asn (2,5,19). Presumably, this is to avoid introducing the misacylated Asp-tRNA Asn into an organism's translational apparatus and potentially endangering protein synthesis. This reasoning is supported by the fact that the heterologous expression of ND-AspRS or ND-GluRS in Escherichia coli, which lacks GatCAB, is highly toxic to the cell, especially when the synthetase genes are overexpressed (15). However, some organisms (e.g., Deinococcus radiodurans and Thermus thermophilus) contain a D-AspRS in addition to an ND-AspRS and a GatCAB amidotransferase (1,3,5,9).We wanted to observe how E. coli copes with in vivo mischarging effected by the ND-AspRS, as this organism is unable to eliminate the toxic Asp-tRNA Asn . Therefore, we developed an approach that would, in fact, require E. coli to be dependent on the presence of mischarged Asp-tRNA Asn for growth. To this aim, we used missense suppression of a specific mutation in the trp...

Integral membrane proteins are predicted to play key roles in the biogenesis and function of nuclear pore complexes (NPCs). Revealing how the transport apparatus is assembled will be critical for understanding the mechanism of nucleocytoplasmic transport. We observed that expression of the carboxyl-terminal 200 amino acids of the nucleoporin Nup116p had no effect on wild-type yeast cells, but it rendered the nup116 null strain inviable at all temperatures and coincidentally resulted in the formation of nuclear membrane herniations at 23 degrees C. To identify factors related to NPC function, a genetic screen for high-copy suppressors of this lethal nup116-C phenotype was conducted. One gene (designated SNL1 for suppressor of nup116-C lethal) was identified whose expression was necessary and sufficient for rescuing growth. Snl1p has a predicted molecular mass of 18.3 kDa, a putative transmembrane domain, and limited sequence similarity to Pom152p, the only previously identified yeast NPC-associated integral membrane protein. By both indirect immunofluorescence microscopy and subcellular fractionation studies, Snl1p was localized to both the nuclear envelope and the endoplasmic reticulum. Membrane extraction and topology assays suggested that Snl1p was an integral membrane protein, with its carboxyl-terminal region exposed to the cytosol. With regard to genetic specificity, the nup116-C lethality was also suppressed by high-copy GLE2 and NIC96. Moreover, high-copy SNL1 suppressed the temperature sensitivity of gle2-1 and nic96-G3 mutant cells. The nic96-G3 allele was identified in a synthetic lethal genetic screen with a null allele of the closely related nucleoporin nup100. Gle2p physically associated with Nup116p in vitro, and the interaction required the N-terminal region of Nup116p. Therefore, genetic links between the role of Snl1p and at least three NPC-associated proteins were established. We suggest that Snl1p plays a stabilizing role in NPC structure and function.

Thermus thermophilus strain HB8 is known to have a heterodimeric aspartyl-tRNA(Asn) amidotransferase (Asp-AdT) capable of forming Asn-tRNA(Asn) [Becker, H.D. and Kern, D. (1998) Proc. Natl. Acad. Sci. USA 95, 12832-12837]. Here we show that, like other bacteria, T. thermophilus possesses the canonical set of amidotransferase (AdT) genes (gatA, gatB and gatC). We cloned and sequenced these genes, and constructed an artificial operon for overexpression in Escherichia coli of the thermophilic holoenzyme. The overproduced T. thermophilus AdT can generate Gln-tRNA(Gln) as well as Asn-tRNA(Asn). Thus, the T. thermophilus tRNA-dependent AdT is a dual-specific Asp/Glu-AdT resembling other bacterial AdTs. In addition, we observed that removal of the 44 carboxy-terminal amino acids of the GatA subunit only inhibits the Asp-AdT activity, leaving the Glu-AdT activity of the mutant AdT unaltered; this shows that Asp-AdT and Glu-AdT activities can be mechanistically separated.