When contemporary aminoacyl-tRNA synthetases invent their cognate amino acid metabolism (original) (raw)
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Proceedings of The National Academy of Sciences, 2003
Faithful protein synthesis relies on a family of essential enzymes called aminoacyl-tRNA synthetases, assembled in a piecewise fashion. Analysis of the completed archaeal genomes reveals that all archaea that possess asparaginyl-tRNA synthetase (AsnRS) also display a second ORF encoding an AsnRS truncated from its anticodon binding-domain (AsnRS2). We show herein that Pyrococcus abyssi AsnRS2, in contrast to AsnRS, does not
Aminoacyl-tRNA synthesis in archaea: different but not unique
Molecular Microbiology, 2003
Accurate aminoacyl-tRNA synthesis is essential for correct translation of the genetic code in all organisms. Whereas many aspects of this process are conserved, others display a surprisingly high level of divergence from the canonical Escherichia coli model system. These differences are most pronounced in archaea where novel mechanisms have recently been described for aminoacylating tRNAs with asparagine, cysteine, glutamine and lysine. Whereas these mechanisms were initially assumed to be uniquely archaeal, both the alternative asparagine and lysine pathways have subsequently been demonstrated in numerous bacteria. Similarly, studies of the means by which archaea insert the rare amino acid selenocysteine in response to UGA stop codons have helped provide a better understanding of both archaeal and eukaryal selenoprotein synthesis. Most recently a new co-translationally inserted amino acid, pyrrolysine, has been found in archaea although again there is some suggestion that it may also be present in bacteria. Thus, whereas archaea contain a preponderance of non-canonical aminoacyl-tRNA synthesis systems most are also found elsewhere albeit less frequently.
Archaeal Aminoacyl-tRNA Synthesis: Diversity Replaces Dogma
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.
A single tRNA base pair mediates bacterial tRNA-dependent biosynthesis of asparagine
Nucleic Acids Research, 2006
In many prokaryotes and in organelles asparagine and glutamine are formed by a tRNA-dependent amidotransferase (AdT) that catalyzes amidation of aspartate and glutamate, respectively, mischarged on tRNA Asn and tRNA Gln . These pathways supply the deficiency of the organism in asparaginyl-and glutaminyl-tRNA synthtetases and provide the translational machinery with Asn-tRNA Asn and Gln-tRNA Gln . So far, nothing is known about the structural elements that confer to tRNA the role of a specific cofactor in the formation of the cognate amino acid. We show herein, using aspartylated tRNA Asn and tRNA Asp variants, that amidation of Asp acylating tRNA Asn is promoted by the base pair U 1 -A 72 whereas the G 1 -C 72 pair and presence of the supernumerary nucleotide U 20A in the D-loop of tRNA Asp prevent amidation. We predict, based on comparison of tRNA Gln and tRNA Glu sequence alignments from bacteria using the AdT-dependent pathway to form Gln-tRNA Gln , that the same combination of nucleotides also rules specific tRNAdependent formation of Gln. In contrast, we show that the tRNA-dependent conversion of Asp into Asn by archaeal AdT is mainly mediated by nucleotides G 46 and U 47 of the variable region. In the light of these results we propose that bacterial and archaeal AdTs use kingdom-specific signals to catalyze the tRNA-dependent formations of Asn and Gln.
Proceedings of the National Academy of Sciences of the United States of America, 2015
Many prokaryotes lack a tRNA synthetase to attach asparagine to its cognate tRNA(Asn), and instead synthesize asparagine from tRNA(Asn)-bound aspartate. This conversion involves two enzymes: a nondiscriminating aspartyl-tRNA synthetase (ND-AspRS) that forms Asp-tRNA(Asn), and a heterotrimeric amidotransferase GatCAB that amidates Asp-tRNA(Asn) to form Asn-tRNA(Asn) for use in protein synthesis. ND-AspRS, GatCAB, and tRNA(Asn) may assemble in an ∼400-kDa complex, known as the Asn-transamidosome, which couples the two steps of asparagine biosynthesis in space and time to yield Asn-tRNA(Asn). We report the 3.7-Å resolution crystal structure of the Pseudomonas aeruginosa Asn-transamidosome, which represents the most common machinery for asparagine biosynthesis in bacteria. We show that, in contrast to a previously described archaeal-type transamidosome, a bacteria-specific GAD domain of ND-AspRS provokes a principally new architecture of the complex. Both tRNA(Asn) molecules in the tran...
A Non‐Discriminating Aspartyl‐tRNA Synthetase from Halobacterium salinarum
RNA Biology, 2006
The tRNA-dependent transamidation pathway is the essential route for Asn-tRNA Asn formation in organisms that lack an asparaginyl-tRNA synthetase. This pathway relies on a nondiscriminating aspartyl-tRNA synthetase (ND-AspRS encoded by aspS), an enzyme with relaxed tRNA specificity, to form Asp-tRNA Asn . The misacylated tRNA is then converted to Asn-tRNA Asn by the action of an Asp-tRNA Asn amidotransferase. Here we show that Asn-tRNA Asn formation in the extreme halophile Halobacterium salinarum also occurs by this transamidation mechanism, and we explore the property of the haloarchaeal AspRS to aspartylate tRNA Asn in vivo and in vitro. Transformation of the E. coli trpA34 strain with the H. salinarum aspS and tRNA Asn genes led to restoration of tryptophan prototrophy by missense suppression of the trpA34 mutant with heterologously in vivo formed Asp-tRNA Asn . The haloarchaeal AspRS works well at low and high (0.1-3 M) salt concentrations but it is unable to use Escherichia coli tRNA as substrate. We show that mutations of two amino acids (H26 and P84) located in the AspRS anticodon binding domain limit the specificity of this nondiscriminating enzyme towards tRNA Asn . Thus, as was observed in an archaeal discriminating AspRS and a bacterial ND-AspRS, amino acids in these positions influence the enzyme's tRNA selection.
Functional Association between Three Archaeal Aminoacyl-tRNA Synthetases
Journal of Biological Chemistry, 2006
Aminoacyl-tRNA synthetases (aaRSs) are responsible for attaching amino acids to their cognate tRNAs during protein synthesis. In eukaryotes aaRSs are commonly found in multienzyme complexes, although the role of these complexes is still not completely clear. Associations between aaRSs have also been reported in archaea, including a complex between prolyl-(ProRS) and leucyl-tRNA synthetases (LeuRS) in Methanothermobacter thermautotrophicus that enhances tRNA Pro aminoacylation. Yeast two-hybrid screens suggested that lysyl-tRNA synthetase (LysRS) also associates with LeuRS in M. thermautotrophicus.