The 51-63 base pair of tRNA confers specificity for binding by EF-Tu

Abstract

Elongation factor Tu (EF-Tu) exhibits significant specificity for the different elongator tRNA bodies in order to offset its variable affinity to the esterified amino acid. Three X-ray cocrystal structures reveal that while most of the contacts with the protein involve the phosphodiester backbone of tRNA, a single hydrogen bond is observed between the Glu390 and the amino group of a guanine in the 51-63 base pair in the T-stem of tRNA. Here we show that the Glu390Ala mutation of Thermus thermophilus EF-Tu selectively destabilizes binding of those tRNAs containing a guanine at either position 51 or 63 and that mutagenesis of the 51-63 base pair in several tRNAs modulates their binding affinities to EF-Tu. A comparison of Escherichia coli tRNA sequences suggests that this specificity mechanism is conserved across the bacterial domain. While this contact is an important specificity determinant, it is clear that others remain to be identified.

FIGURE 1.

The effect of the EF-Tu Glu390Ala mutation on the binding of cognate E. coli aa-tRNAs with (red) and without (blue) a guanosine in the 51–63 base pair. ΔΔG° is the ΔG° of wild-type EF-Tu minus the ΔG° for the Glu390Ala mutant protein measured in 0.5 M NH₄Cl, 20 mM MgCl₂, 50 mM HEPES (pH 7.0) at 4°C. Listed below the tRNA is the 51–63 base pair. The asterisk indicates where binding to the Glu390Ala protein was too weak to be accurately measured.

FIGURE 2.

Crystallographic details of Glu390 contacting the 51–63 base pair. (A) General location and (B) detail of the contact of T. aquaticus EF-Tu•GDPPNP bound to yeast Phe-tRNA^Phe (PDB:1TTT). (C) E. coli EF-Tu•GDPPNP complexed with kirromycin and yeast Phe-tRNA^Phe (PDB: 1OB2). (D) T. aquaticus EF-Tu•GDPNP bound to E. coli Cys-tRNA^Cys (PDB: 1B23). The distances are between the caboxylate oxygen of Glu390 nearest to the amino nitrogen.

FIGURE 3.

Cloverleaf representation of unmodified E. coli tRNA^Leu showing the effect of mutations of the 51–63 base pair on the binding to wild-type T. thermophilus EF-Tu and the Glu390Ala mutation. Binding experiments were performed in the same buffer as Figure 1.

FIGURE 4.

The correlation of the prevalence of guanosine in the 51–63 base pair in 129 bacterial tRNA isoacceptors with the position of the tRNA on the EF-Tu binding hierarchy. Listed in parentheses on the horizontal axis is the EF-Tu binding affinity of the tRNA body as determined for E. coli tRNAs (Asahara and Uhlenbeck 2002). Green (G51-U63), yellow (U51-G63), red (C51-G63), and blue (G51-C63).