A tRNA body with high affinity for EF-Tu hastens ribosomal incorporation of unnatural amino acids

Effects of EF-Tu concentration on the kinetics of dipeptide synthesis from fMet-tRNA_i^fMet and Ala-tRNA^AlaB (A,B) or Phe-tRNA^AlaB (C,D). Left: Time course (normalized) of f[³H]Met-Ala (A) or f[³H]Met-Phe (C) dipeptide formation at different EF-Tu concentrations in the reaction mixture. Right: (B,D) Hyperbolic fitting of the dependence of the fast phase fraction of the dipeptide formation (A,C) on EF-Tu concentrations. K_d for the binding of Ala-tRNA^AlaB and Phe-tRNA^AlaB to EF-Tu:GTP were estimated as 0.22 and 0.056 µM, respectively, from the fit. Experiments were done in LS3 buffer at 37°C (see Materials and Methods).