Switching the amino acid specificity of an aminoacyl-tRNA 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 involves a recognition stage generally followed by a proofreading reaction. Therefore, to change the amino acid specificity of a synthetase in the aminoacylation reaction, it is necessary to alleviate the molecular barriers which contribute its editing function. In an attempt to accommodate a noncognate amino acid into the active site of a synthetase, we chose a pair of closely related enzymes. The current hypothesis designates glutaminyl-tRNA synthetase (GlnRS) as a late component of the protein synthesis machinery, emerging in the eukaryotic lineage by duplication of the gene for glutamyl-tRNA synthetase (GluRS). By introducing GluRS-specific features into the Rossmann dinucleotide-binding domain of human GlnRS, we constructed a mutant GlnRS which preferentially aminoacylates tRNA with glutamate instead of glutamine. Our data suggest that not only the transition state for aminoacyl-AMP formation but also the proofreading site of GlnRS are affected by that mutation.
Laboratoire d'Enzymologie et Biochimie Structurales, UPR 9063 du CNRS, Gif-sur-Yvette, France.
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Agou
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Quevillon
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Kerjan
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Mirande
D000215:Acylation; D000255:Adenosine Triphosphate / Q000378:metabolism; D000604:Amino Acyl-tRNA Synthetases / Q000235:genetics / Q000378:metabolism*; D005975:Glutamate-tRNA Ligase / Q000378:metabolism; D018698:Glutamic Acid / Q000378:metabolism; D005973:Glutamine / Q000235:genetics / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D016297:Mutagenesis, Site-Directed; D010710:Phosphates / Q000378:metabolism; D012352:RNA, Transfer, Glu / Q000378:metabolism; D013379:Substrate Specificity; D046249:Transfer RNA Aminoacylation / Q000235:genetics
D010710:Phosphates; D012352:RNA, Transfer, Glu; D005973:Glutamine; D018698:Glutamic Acid; D000255:Adenosine Triphosphate; D000604:Amino Acyl-tRNA Synthetases; D005975:Glutamate-tRNA Ligase; C020659:glutaminyl-tRNA synthetase