Aspartyl-tRNA synthetase requires a conserved proline in the anticodon-binding loop for tRNA(Asn) recognition in vivo

Most prokaryotes require Asp-tRNAAsn for the synthesis of Asn-tRNA(Asn). This misacylated tRNA species is synthesized by a non-discriminating aspartyl-tRNA synthetase (AspRS) that acylates both tRNA(Asp) and tRNA(Asn) with aspartate. In contrast, a discriminating AspRS forms only Asp-tRNA(Asp). Here we show that a conserved proline (position 77) in the L1 loop of the non-discriminating Deinococcus radiodurans AspRS2 is required for tRNA(Asn) recognition in vivo. Escherichia coli trpA34 was transformed with DNA from a library of D. radiodurans aspS2 genes with a randomized codon 77 and then subjected to in vivo selection for Asp-tRNA(Asn) formation by growth in minimal medium. Only proline codons were found at position 77 in the aspS2 genes isolated from 21 of the resulting viable colonies. However, when the aspS temperature-sensitive E. coli strain CS89 was transformed with the same DNA library and then screened for Asp-tRNA(Asp) formation in vivo by growth at the nonpermissive temperature, codons for seven other amino acids besides proline were identified at position 77 in the isolates examined. Thus, replacement of proline 77 by cysteine, isoleucine, leucine, lysine, phenylalanine, serine, or valine resulted in mutant D. radiodurans AspRS2 enzymes still capable of forming Asp-tRNA(Asp) but unable to recognize tRNA(Asn). This strongly suggests that proline 77 is responsible for the non-discriminatory tRNA recognition properties of this enzyme

Genomics and the evolution of aminoacyl-tRNA synthesis.

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.</jats:p