SWITCHING THE AMINO-ACID SPECIFICITY OF AN AMINOACYL-TRANSFER-RNA SYNTHETASE

The accuracy of protein synthesis essentially rests on aminoacyl-tRNA synthetases that ensure the correct attachment of an amino acid to the cognate tRNA molecule. The selection of the amino acid substrate invo lves a recognition stage generally followed by a proofreading reaction . Therefore, to change the amino acid specificity of a synthetase in t he aminoacylation reaction, it is necessary to alleviate the molecular barriers which contribute its editing function. In an attempt to acco mmodate a noncognate amino acid into the active site of a synthetase, we chose a pair of closely related enzymes. The current hypothesis des ignates glutaminyl-tRNA synthetase (GlnRS) as a late component of the protein synthesis machinery, emerging in the eukaryotic lineage by dup lication of the gene for glutamyl-tRNA synthetase (GluRS). By introduc ing GluRS-specific features into the Rossmann dinucleotide-binding dom ain of human GlnRS, we constructed a mutant GlnRS which preferentially aminoacylates tRNA with glutamate instead of glutamine. Our data sugg est that not only the transition state for aminoacyl-AMP formation but also the proofreading site of GlnRS are affected by that mutation.