Through an exhaustive search for Escherichia coli aminoacyl-tRNA synthetase(s) responsible for the misacylation of yeast suppressor tRNA(Tyr), E. coli lysyl-tRNA synthetase was found to have a weak activity to aminoacylate yeast amber suppressor tRNA(Tyr) (CUA) with L-lysine. Since our protein-synthesizing system for site-specific incorporation of unnatural amino acids into proteins is based on the use of yeast suppressor tRNA(Tyr)/tyrosyl-tRNA synthetase (TyrRS) pair as the "carrier" of unusual amino acid in E. coli translation system, this misacylation must be repressed as low as possible. We have succeeded in effectively repressing the misacylation by changing several nucleotides in this tRNA by genetic engineering. This "optimized" tRNA together with our mutant TyrRS should serve as an efficient and faithful tool for site-specific incorporation of unnatural amino acids into proteins in a protein-synthesizing system in vitro or in vivo.