Bokkee Min

Transfer RNA-dependent amino acid biosynthesis: An essential route to asparagine formation

Biochemical experiments and genomic sequence analysis showed that Deinococcus radiodurans and Thermus thermophilus do not possess asparagine synthetase (encoded by asnA or asnB), the enzyme forming asparagine from aspartate. Instead these organisms derive asparagine from asparaginyl-tRNA, which is made from aspartate in the tRNA-dependent transamidation pathway [Becker, H. D. & Kern, D. (1998) Proc. Natl. Acad. Sci. USA 95, 12832–12837; and Curnow, A. W., Tumbula, D. L., Pelaschier, J. T., Min, B. & Söll, D. (1998) Proc. Natl. Acad. Sci. USA 95, 12838–12843]. A genetic knockout disrupting this pathway deprives D. radiodurans of the ability to synthesize asparagine and confers asparagine auxotrophy. The organisms capacity to make asparagine could be restored by transformation with Escherichia coli asnB. This result demonstrates that in Deinococcus, the only route to asparagine is via asparaginyl-tRNA. Analysis of the completed genomes of many bacteria reveal that, barring the existence of an unknown pathway of asparagine biosynthesis, a wide spectrum of bacteria rely on the tRNA-dependent transamidation pathway as the sole route to asparagine.

The heterotrimeric Thermus thermophilus Asp-tRNAAsn amidotransferase can also generate Gln-tRNAGln

Thermus thermophilus strain HB8 is known to have a heterodimeric aspartyl‐tRNAAsn amidotransferase (Asp‐AdT) capable of forming Asn‐tRNAAsn [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‐tRNAGln as well as Asn‐tRNAAsn. 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.

Protein Synthesis in Escherichia coli with Mischarged tRNA

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.

In vivo formation of glutamyl-tRNAGln in Escherichia coli by heterologous glutamyl-tRNA synthetases

Two types of glutamyl‐tRNA synthetase exist: the discriminating enzyme (D‐GluRS) forms only Glu‐tRNAGlu, while the non‐discriminating one (ND‐GluRS) also synthesizes Glu‐tRNAGln, a required intermediate in protein synthesis in many organisms (but not in Escherichia coli). Testing the capacity to complement a thermosensitive E. coli gltX mutant and to suppress an E. coli trpA49 missense mutant we examined the properties of heterologous gltX genes. We demonstrate that while Acidithiobacillus ferrooxidans GluRS1 and Bacillus subtilis Q373R GluRS form Glu‐tRNAGlu, A. ferrooxidans and Helicobacter pylori GluRS2 form Glu‐tRNAGln in E. coli in vivo.

Recognition of One tRNA by Two Classes of Aminoacyl-tRNA Synthetase

Lysyl-tRNA synthetases are unique amongst the aminoacyl-tRNA synthetases in being composed of two unrelated families. In most bacteria and all eukarya, the known lysyl-tRNA synthetases are subclass He-type aminoacyl-tRNA synthetases whereas some archaea and bacteria have been shown to contain an unrelated class I-type lysyl-tRNA synthetase. We have now examined substrate recognition by a bacterial (from Borrelia burgdorferi) and an archaeal (from Methanococcus maripaludis) class I lysyl-tRNA synthetase. The genes encoding both enzymes were able to rescue an Escherichia coli strain deficient in lysyl-tRNA synthetase, indicating their ability to functionally substitute for class II lysyl-tRNA synthetases in vivo. In vitro characterization revealed lysine activation and recognition to be tRNA-dependent, a phenomenon previously reported for other class I aminoacyl-tRNA synthetases. More detailed examination of tRNA recognition has shown that class I lysyl-tRNA synthetases recognize the same elements in tRNALys as their class II counterparts; specifically, the discriminator base (N73) and the anticodon serve as recognition elements. The implications of these results for the evolution of Lys-tRNALys synthesis and their possible indications of a more ancient origin for tRNA then aminoacyl-tRNA synthetases will be discussed.