Structure-function relationship of arginyl-tRNA synthetase from Escherichia coli: isolation and characterization of the argS mutation MA5002.
The Escherichia coli K12 argS MA5002 mutant appears to have a functionally altered arginyl-tRNA synthetase (ArgRS). The gene coding for this enzyme was isolated from E. coli genomic DNA using the PCR procedure and inserted into a pUC18 multicopy vector. Sequencing revealed that it differs from the wildtype ArgRS structural gene only by one mutation: a replacement of a C by an A residue which results in substitution of an arginine by a serine at position 134, located two residues downstream from the HVGH consensus sequence. As compared to the genomic enzyme level, this recombinant vector, containing the mutated gene, produces in E. coli JM103, about 100 times as much modified ArgRS. This enzyme was obtained nearly pure after only two chromatographic steps; it exhibits a 4-6 times as low activity and a 5 times as high Km value for ATP as the wildtype enzyme in the aminoacylation and ATP-PPi reactions; Km values for arginine and tRNAArg remained unaltered. The position of this mutation and its effect on enzymatic properties suggest the implication of arginine 134 in ATP binding as well as in the activation catalytic process.
Eriani
G
G
Institut de Biologie Moléculaire et Cellulaire, Centre National de la Recherche Scientifique, Strasbourg, France.
Dirheimer
G
G
Gangloff
J
J
D000595:Amino Acid Sequence; D000604:Amino Acyl-tRNA Synthetases / Q000235:genetics*; D001126:Arginine-tRNA Ligase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D001483:Base Sequence; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics* / Q000302:isolation & purification; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D005798:Genes, Bacterial; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D015345:Oligonucleotide Probes; D016133:Polymerase Chain Reaction
D004269:DNA, Bacterial; D015345:Oligonucleotide Probes; D000604:Amino Acyl-tRNA Synthetases; D001126:Arginine-tRNA Ligase