ABSTRACT
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.
Subject(s)
Nitrogenous Group Transferases/metabolism , RNA, Transfer, Amino Acyl/metabolism , Thermus thermophilus/enzymology , Amino Acid Sequence , Cloning, Molecular , Escherichia coli/genetics , Genes, Bacterial , Molecular Sequence Data , Nitrogenous Group Transferases/chemistry , Nitrogenous Group Transferases/genetics , Protein Structure, Quaternary , RNA, Bacterial/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sequence Deletion , Substrate Specificity , Thermus thermophilus/geneticsABSTRACT
Accurate aminoacyl-tRNA synthesis is essential for faithful translation of the genetic code and consequently has been intensively studied for over three decades. Until recently, the study of aminoacyl-tRNA synthesis in archaea had received little attention. However, as in so many areas of molecular biology, the advent of archaeal genome sequencing has now drawn researchers to this field. Investigations with archaea have already led to the discovery of novel pathways and enzymes for the synthesis of numerous aminoacyl-tRNAs. The most surprising of these findings has been a transamidation pathway for the synthesis of asparaginyl-tRNA and a novel lysyl-tRNA synthetase. In addition, seryl- and phenylalanyl-tRNA synthetases that are only marginally related to known examples outside the archaea have been characterized, and the mechanism of cysteinyl-tRNA formation in Methanococcus jannaschii and Methanobacterium thermoautotrophicum is still unknown. These results have revealed completely unexpected levels of complexity and diversity, questioning the notion that aminoacyl-tRNA synthesis is one of the most conserved functions in gene expression. It has now become clear that the distribution of the various mechanisms of aminoacyl-tRNA synthesis in extant organisms has been determined by numerous gene transfer events, indicating that, while the process of protein biosynthesis is orthologous, its constituents are not.
Subject(s)
Amino Acyl-tRNA Synthetases/physiology , Archaea/enzymology , Archaeal Proteins/physiology , Amino Acyl-tRNA Synthetases/genetics , Amino Acyl-tRNA Synthetases/metabolism , Archaea/genetics , Archaeal Proteins/genetics , Euryarchaeota/enzymology , Evolution, Molecular , Gene Expression Regulation, Archaeal , Lysine-tRNA Ligase/genetics , Lysine-tRNA Ligase/physiology , Phenylalanine-tRNA Ligase/genetics , Phenylalanine-tRNA Ligase/physiology , Phylogeny , RNA, Archaeal/genetics , RNA, Transfer/genetics , RNA, Transfer/metabolism , Selenocysteine/metabolism , Serine-tRNA Ligase/genetics , Serine-tRNA Ligase/physiologyABSTRACT
Asparaginyl-tRNA (Asn-tRNA) and glutaminyl-tRNA (Gln-tRNA) are essential components of protein synthesis. They can be formed by direct acylation by asparaginyl-tRNA synthetase (AsnRS) or glutaminyl-tRNA synthetase (GlnRS). The alternative route involves transamidation of incorrectly charged tRNA. Examination of the preliminary genomic sequence of the radiation-resistant bacterium Deinococcus radiodurans suggests the presence of both direct and indirect routes of Asn-tRNA and Gln-tRNA formation. Biochemical experiments demonstrate the presence of AsnRS and GlnRS, as well as glutamyl-tRNA synthetase (GluRS), a discriminating and a nondiscriminating aspartyl-tRNA synthetase (AspRS). Moreover, both Gln-tRNA and Asn-tRNA transamidation activities are present. Surprisingly, they are catalyzed by a single enzyme encoded by three ORFs orthologous to Bacillus subtilis gatCAB. However, the transamidation route to Gln-tRNA formation is idled by the inability of the discriminating D. radiodurans GluRS to produce the required mischarged Glu-tRNAGln substrate. The presence of apparently redundant complete routes to Asn-tRNA formation, combined with the absence from the D. radiodurans genome of genes encoding tRNA-independent asparagine synthetase and the lack of this enzyme in D. radiodurans extracts, suggests that the gatCAB genes may be responsible for biosynthesis of asparagine in this asparagine prototroph.
Subject(s)
Amino Acyl-tRNA Synthetases/metabolism , Asparagine/biosynthesis , Gram-Positive Cocci/enzymology , Nitrogenous Group Transferases/metabolism , Acylation , Cloning, Molecular , DNA, Bacterial/chemistry , DNA, Bacterial/genetics , Genome, Bacterial , Glutamate-tRNA Ligase/metabolism , Gram-Positive Cocci/genetics , Kinetics , Models, Chemical , Open Reading FramesABSTRACT
The mechanism of aminoacyl-tRNA synthesis differs substantially between Archaea, Bacteria and Eukarya. Sequencing of archaeal genomes has suggested that the asparaginyl-, cysteinyl-, glutaminyl- and lysyl-tRNA synthetases are absent from a number of organisms in this kingdom. The absence of the asparaginyl- and glutaminyl-tRNA synthetases is in agreement with the observation that Asn-tRNA and Gln-tRNA are synthesized by tRNA-dependent transamidation of Asp-tRNA and Glu-tRNA respectively in the archaeon Haloferax volcanii. Biochemical and genetic studies have now shown that while the cysteinyl- and lysyl-tRNA synthetases are present, the enzymes responsible for these activities are unique to Archaea.
