Protein engineering of tyrosyl-tRNA synthetase: the charging of tRNA

Abstract
Protein engineering has been used to identify residues of the tyrosyl-tRNA synthetase from Bacillus stearothermophilus that are in contact with the tRNA Tyr . By using improved techniques in oligonucleotide-directed mutagenesis, forty lysine, arginine, or histidine residues on the surface of the enzyme were altered to either asparagine or glutamine. With an in vivo genetic complementation test, only thirteen mutants were found that seriously affect the overall activity of the enzyme. Detailed kinetics on the purified enzymes revealed that four of these mutants had a lesion at the level of the activation of tyrosine, and nine at the level of tRNA charging. Three of the mutants in tRNA charging lie in the N-terminal domain of the enzyme which is responsible for tyrosine activation, and the six others in the disordered C-terminal domain which is necessary for tRNA binding. This indicates that the tRNA spans both domains of the enzyme. The construction of heterodimers allows us to suggest a model for tRNA binding in which the acceptor stem of the tRNA binds to the N-terminal domain of one subunit, and other regions of tRNA Tyr such as the anticodon arm or extra loop bind to the C-terminal domain of the other subunit.