- A procedure for replacing residues 33-35 in the anticodon loop of yeast tRNATyr with any desired oligonucleotide has been developed. The three residues were removed by partial ribonuclease A digestion. An oligonucleotide was inserted into the gap in four steps by using RNA ligase, polynucleotide kinase, and pseT 1 polynucleotide kinase. The rate of aminoacylation of anticodon loop substituted tRNATyr by yeast tyrosyl-tRNA synthetase was found to depend upon the sequence of the oligonucleotide inserted. This suggests that the nucleotides in the anticodon loop of yeast tRNATyr are required for optimal aminoacylation. In addition, tRNATyr modified to have a phenylalanine anticodon was shown to be misacylated by yeast phenylalanyl-tRNA synthetase at a rate at least 10 times faster than unmodified tRNATyr. Thus, the anticodon is used by phenylalanyl-tRNA synthetase to distinguish between tRNAs.