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

Most prokaryotes require Asp-tRNA^Asn for the synthesis of Asn-tRNA^Asn. This misacylated tKNA 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 non-permissive 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.