Functional consequences of T-stem mutations in E. coli tRNA^Thr_UGU in vitro and in vivo

Binding of T-stem variants of unmodified E. coli Thr-tRNA^Thr_UGU to E. coli EF-Tu•GTP. (A) Individual determinations of the dissociation rates of WT (•), 18 (▴), and 5 (▪) in buffer A at 0°C. The lines are single exponential fits with k_off = 23 × 10⁻⁴ sec⁻¹, 9 × 10⁻⁴ sec⁻¹, and 78 × 10⁻⁴ sec⁻¹, respectively. (B) Comparison of experimental ΔG° values for tRNA^Thr_UGU variants with multiple base-pair substitutions with ΔG° values calculated from the addition of ΔΔG° values determined for single base-pair substitutions. The best linear fit has a slope of 0.9. (C,D) Relationship between the ΔG° values for the E. coli EF-Tu–Thr tRNA^Thr complex and the T. thermophilus EF-Tu–Phe-tRNA^Phe complex (Schrader et al. 2009) and the identity of the 49–65 (C) and 51–63 (D) base pairs. In both cases, data are for single base-pair substitutions of the wild-type tRNA sequence. The best linear fits have slopes of 0.98 and 0.95 when the G51•U63 data point is excluded.