The tRNA specificity of Thermus thermophilus EF-Tu

Abstract

By introducing a GAC anticodon, 21 different Escherichia coli tRNAs were misacylated with either phenylalanine or valine and assayed for their affinity to Thermus thermophilus elongation factor Tu (EF-Tu)*GTP by using a ribonuclease protection assay. The presence of a common esterified amino acid permits the thermodynamic contribution of each tRNA body to the overall affinity to be evaluated. The E. coli elongator tRNAs exhibit a wide range of binding affinities that varied from -11.7 kcal/mol for Val-tRNA(Glu) to -8.1 kcal/mol for Val-tRNA(Tyr), clearly establishing EF-Tu*GTP as a sequence-specific RNA-binding protein. Because the ionic strength dependence of k(off) varied among tRNAs, some of the affinity differences are the results of a different number of phosphate contacts formed between tRNA and protein. Because EF-Tu is known to contact only the phosphodiester backbone of tRNA, the observed specificity must be a consequence of an indirect readout mechanism.

Figure 1

Time courses of RNase protection by T. thermophilus EF-Tu⋅GTP for Val-tRNA^Gly (●), Val-tRNA^Pro (○), Val-tRNA^Arg (▴), and Val-tRNA^Gln (▵) in 0.5M NH₄Cl buffer at 2°C. Lines correspond to k_off = 0.018, 0.20, 0.33, and 1.6 min⁻¹, respectively.

Figure 2

(A) NH₄Cl concentration dependence of dissociation rates at 2°C with Val-tRNA^Gly (●), Val-tRNA^Pro (○), Val-tRNA^Arg (▴), and Val-tRNA^Gln (▵). (B) Similar data with Val-tRNA^fMet (●), Val-tRNA^Val (○), Val-tRNA^Leu (▴), Val-tRNA^Cys (▵), Val-tRNA^Thr (■), and Val-tRNA^Ala (□). Slopes of plots and K_D values at 0.5 M NH₄Cl (vertical dashed line) are given in Table 2.

Figure 3

NH₄Cl concentration dependence of dissociation rates at 2°C with Val-tRNA^Tyr (●), Phe-tRNA^Tyr (○), and Val-tRNA^Lys (▴), Phe-tRNA^Lys (▵), Val-tRNA^Met (■), and Phe-tRNA^Met (□). Slopes of plots and K_D values at 0.5 M NH₄Cl (vertical dashed line) are given in Tables 2 and 3.

Figure 4

ΔG^o of binding Val-tRNAs to T. thermophilus EF-Tu⋅GTP in 0.5 M NH₄Cl buffer at 2°C presented in order of affinity. For amino acids marked by asterisks (*), ΔG^o values of Phe-tRNAs are converted to those of Val-tRNAs by using ΔΔG (Val-Phe) = 0.52 kcal/mol.

Figure 5

Acceptor and T arm sequences of E. coli tRNAs that contact EF-Tu. All residues (except position 73) form normal base pairs except when the residue is boxed where GU or AC pairs are formed. Potentially important AU and GC pairs are highlighted in dark and light gray, respectively. The tRNA isoacceptor number (39) is given in parentheses.