title: In vivo selection of lethal mutations reveals two functional domains in arginyl-tRNA synthetase.
abstract: Using random mutagenesis and a genetic screening in yeast, we isolated 26 mutations that inactivate Saccharomyces cerevisiae arginyl-tRNA synthetase (ArgRS). The mutations were identified and the kinetic parameters of the corresponding proteins were tested after purification of the expression products in Escherichia coli. The effects were interpreted in the light of the crystal structure of ArgRS. Eighteen functional residues were found around the arginine-binding pocket and eight others in the carboxy-terminal domain of the enzyme. Mutations of these residues all act by strongly impairing the rates of tRNA charging and arginine activation. Thus, ArgRS and tRNA(Arg) can be considered as a kind of ribonucleoprotein, where the tRNA, before being charged, is acting as a cofactor that activates the enzyme. Furthermore, by using different tRNA(Arg) isoacceptors and heterologous tRNA(Asp), we highlighted the crucial role of several residues of the carboxy-terminal domain in tRNA recognition and discrimination.
authors.affiliation: Unité Propre de Recherche 9002 Structure des Macromolécules Biologiques et Mécanismes de Reconnaissance, Institut de Biologie Moléculaire et Cellulaire, Centre National de la Recherche Scientifique, Strasbourg, France.
authors.forename: R
authors.initials: R
authors.lastname: Geslain
authors.forename: F
authors.initials: F
authors.lastname: Martin
authors.forename: B
authors.initials: B
authors.lastname: Delagoutte
authors.forename: J
authors.initials: J
authors.lastname: Cavarelli
authors.forename: J
authors.initials: J
authors.lastname: Gangloff
authors.forename: G
authors.initials: G
authors.lastname: Eriani
mesh_terms: D001126:Arginine-tRNA Ligase / Q000737:chemistry / Q000235:genetics*; D003001:Cloning, Molecular; D005656:Fungal Proteins / Q000096:biosynthesis / Q000235:genetics; D015966:Gene Expression Regulation, Fungal; D005800:Genes, Fungal / Q000235:genetics; D005804:Genes, Lethal / Q000235:genetics*; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics*; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics
chemical_list: D005656:Fungal Proteins; D010446:Peptide Fragments; D001126:Arginine-tRNA Ligase