Affinity labelling of E. coli leucyl-tRNA synthetase with 3'-oxidized tRNA(Leu)

The E. coli leucyl-tRNA synthetase (E.C. 6.1.1.4) was specifically labelled with 3'-oxidized tRNA(Leu) (tRNA(oxLeu)). The procedure involves a Schiff's base formation and its subsequent reduction by sodium cyanoborohydride. Stoichiometric inactivation of aminoacylation was achieved with the incorporation of 1 mol of tRNA(oxLeu) per mol LeuRS. On the other hand, the amino acid activation activity of LeuRS-tRNA(ox) complex was partially inhibited. After extensive digestion of the complex by pancreatic ribonuclease, the amino acid activation activity was fully recovered, while the aminoacylation activity was not restored at all.