ABSTRACT
Aminoacyl-tRNA (transfer RNA) synthetases are essential components of the cellular translation machinery as they provide the ribosome with aminoacyl-tRNAs. Aminoacyl-tRNA synthesis is generally well understood. However, the mechanism of Cys-tRNACys formation in three methanogenic archaea ( Methanocaldococcus jannaschii, Methanothermobacter thermautotrophicus and Methanopyrus kandleri) is still unknown, since no recognizable gene for a canonical cysteinyl-tRNA synthetase could be identified in the genome sequences of these organisms. Here we review the different routes recently proposed for Cys-tRNACys formation and discuss its possible link with cysteine biosynthesis in these methanogenic archaea.
Subject(s)
Archaea/metabolism , Cysteine/biosynthesis , Genes, Archaeal , RNA, Transfer, Amino Acyl/chemistry , Binding Sites , Cysteine/chemistry , Methane/chemistry , Models, Biological , Models, Molecular , Phylogeny , Protein Biosynthesis , Protein Conformation , RNA/chemistryABSTRACT
Translation is the process by which ribosomes direct protein synthesis using the genetic information contained in messenger RNA (mRNA). Transfer RNAs (tRNAs) are charged with an amino acid and brought to the ribosome, where they are paired with the corresponding trinucleotide codon in mRNA. The amino acid is attached to the nascent polypeptide and the ribosome moves on to the next codon. Thus, the sequential pairing of codons in mRNA with tRNA anticodons determines the order of amino acids in a protein. It is therefore imperative for accurate translation that tRNAs are only coupled to amino acids corresponding to the RNA anticodon. This is mostly, but not exclusively, achieved by the direct attachment of the appropriate amino acid to the 3'-end of the corresponding tRNA by the aminoacyl-tRNA synthetases. To ensure the accurate translation of genetic information, the aminoacyl-tRNA synthetases must display an extremely high level of substrate specificity. Despite this highly conserved function, recent studies arising from the analysis of whole genomes have shown a significant degree of evolutionary diversity in aminoacyl-tRNA synthesis. For example, non-canonical routes have been identified for the synthesis of Asn-tRNA, Cys-tRNA, Gln-tRNA and Lys-tRNA. Characterization of non-canonical aminoacyl-tRNA synthesis has revealed an unexpected level of evolutionary divergence and has also provided new insights into the possible precursors of contemporary aminoacyl-tRNA synthetases.
Subject(s)
Evolution, Molecular , Genomics , RNA, Transfer, Amino Acyl/biosynthesis , RNA, Transfer, Amino Acyl/genetics , Amino Acyl-tRNA Synthetases/genetics , Amino Acyl-tRNA Synthetases/metabolism , Phylogeny , Protein Biosynthesis , RNA, Messenger/geneticsABSTRACT
The protein translation apparatus of Methanococcus jannaschii possesses the unusual enzyme prolyl-cysteinyl-tRNA synthetase (ProCysRS), a single enzyme that attaches two different amino acids, proline and cysteine, to their cognate tRNA species. Measurement of the ATP-PP(i) exchange reaction revealed that amino acid activation, the first reaction step, differs for the two amino acids. While Pro-AMP can be formed in the absence of tRNA, Cys-AMP synthesis is tRNA-dependent. Studies with purified tRNAs indicate that tRNA(Cys) promotes cysteine activation. The k(cat) values of wild-type ProCysRS for tRNA prolylation (0.09 s(-1)) and cysteinylation (0.02 s(-1)) demonstrate that both aminoacyl-tRNAs are synthesized with comparable rates, the cysteinyl-tRNA synthetase activity being only 4.5-fold lower than prolyl-tRNA synthetase activity. Kinetic analysis of ProCysRS mutant enzymes, generated by site-directed mutagenesis, shows glutamate at position 103 to be critical for proline binding, and proline at position 100 to be involved in cysteine binding. The proximity in ProCysRS of amino acid residues affecting binding of either cysteine or proline strongly suggests that structural elements of the two amino acid binding sites overlap.
