Arginyl-tRNA synthetase with signature sequence KMSK from Bacillus stearothermophilus

Abstract

ArgRS (arginyl-tRNA synthetase) belongs to the class I aaRSs (aminoacyl-tRNA synthetases), though the majority of ArgRS species lack the canonical KMSK sequence characteristic of class I aaRSs. A DNA fragment of the ArgRS gene from Bacillus stearothermophilus was amplified using primers designed according to the conserved regions of known ArgRSs. Through analysis of the amplified DNA sequence and known tRNAArgs with a published genomic sequence of B. stearothermophilus, the gene encoding ArgRS (argS´) was amplified by PCR and the gene encoding tRNAArg (ACG) was synthesized. ArgRS contained 557 amino acid residues including the canonical KMKS sequence. Recombinant ArgRS and tRNAArg (ACG) were expressed in Escherichia coli. ArgRS purified by nickel-affinity chromatography had no ATPase activity. The kinetics of ArgRS and cross-recognition between ArgRSs and tRNAArgs from B. stearothermophilus and E. coli were studied. The activities of B. stearothermophilus ArgRS mutated at Lys382 and Lys385 of the KMSK sequence and at Gly136 upstream of the HIGH loop were determined. From the mutation results, we concluded that there was mutual compensation of Lys385 and Gly136 for the amino acid-activation activity of B. stearothermophilus ArgRS.