Understanding the Sequence Specificity of tRNA Binding to Elongation Factor Tu using tRNA Mutagenesis

Abstract

Measuring the binding affinities of 42 single base pair mutants in the acceptor and T-stems of S. cerevisiae tRNAPhe to T. thermophilus EF-Tu revealed that most of the specificity for tRNA occurs at the 49-65, 50-64, and 51-63 base pairs. Introducing the same mutations at the three positions into E. coli tRNALeuCAG resulted in similar changes in the binding affinity. Swapping the three pairs from several E. coli tRNAs into yeast tRNAPhe resulted in chimeras with EF-Tu binding affinities similar to the donor tRNA. Finally, analysis of double and triple base pair mutants of tRNAPhe shows that the thermodynamic contributions at the three sites are additive, permitting reasonably accurate prediction of the EF-Tu binding affinity for all E. coli tRNAs. Thus, it appears that the thermodynamic contributions of three base pairs in the T-stem primarily account for tRNA binding specificity to EF-Tu.

Tuning the affinity of aminoacyl-tRNA to elongation factor Tu for optimal decoding

“…Since nearly all the EF-Tu residues that contact aa-tRNA are conserved among bacteria, it is likely that the conclusions made here for E. coli are generally applicable. Indeed, the thermodynamic effects of T-stem tRNA mutations determined here using E. coli EF-Tu are virtually identical to those previously determined for T. thermophilus (10,15,16). However, as discussed elsewhere, the sequences of individual tRNA species vary considerably among bacteria because multiple T-stem sequences can have a similar EF-Tu affinity (16,39,40).…”

“…As previously observed with other tRNAs, (12,21,22) the unmodified WT tRNA Val GAC bound EF-Tu very similarly to native, fully modified tRNA Val GAC consistent with the absence of modifications in the EF-Tu binding site. The seven mutations showed the desired broad range of binding affinity to E. coli EF-Tu that is similar to the range in affinities observed for different tRNA bodies with T. thermophilus EF-Tu (10,16). T1 to T3 bound from 0.5 to 1.0 kcal∕mol tighter, W1 to W3 bound from 1.0 to 2.0 kcal∕mol weaker, and Ψ bound nearly the same as the WT tRNA Val .…”

“…Transcribed tRNAs were prepared as described (16) and stored in TE buffer (10 mM Tris-HCl [pH 7.6], 0.1 mM EDTA) at concentrations greater than 100 μM. Native E. coli tRNA Val was purified by hybridization using biotinylated DNA oligonucleotides designed in (47).…”

“…Aminoacylation of tRNAs with [ 3 H]-amino acids (Amersham) was performed as in (16). 3′-[ 32 P] labeling of tRNA and aminoacylation was performed as previously described(33) with typical aminoacylation yields of >70%.…”

“…The crystal structure of Thermus aquaticus EF-Tu•GTP bound to Saccharomyces cerevisiae Phe-tRNA Phe (13) reveals that the protein primarily forms extensive interactions with the helical phosphodiester backbone of the acceptor and T stems of tRNA Phe . Recent protein (14) and tRNA (15,16) mutagenesis experiments indicate that much of the specificity is the result of interactions made between three amino acids of EF-Tu and three adjacent base pairs in the T stem (16). Additional mutagenesis experiments indicate that the thermodynamic contribution of each of the three base pairs is independent of the others, making it possible to adjust the affinity of aa-tRNAs to EF-Tu in a predictable manner.…”