The Interface between Escherichia coli Elongation Factor Tu and Aminoacyl-tRNA

Abstract

Nineteen of the highly conserved residues of Escherichia coli (E. coli) Elongation factor Tu (EF-Tu) that form the binding interface with aa-tRNA were mutated to alanine to better understand how modifying the thermodynamic properties of EF-Tu–tRNA interaction can affect the decoding properties of the ribosome. Comparison of ΔΔGo values for binding EF-Tu to aa-tRNA show that the majority of the interface residues stabilize the ternary complex and their thermodynamic contribution can depend on the tRNA species that is used. Experiments with a very tight binding mutation of tRNATyr indicate that interface amino acids distant from the tRNA mutation can contribute to the specificity. For nearly all of the mutations, the values of ΔΔGo were identical to those previously determined at the orthologous positions of Thermus thermophilus (T. thermophilus) EF-Tu indicating that the thermodynamic properties of the interface were conserved between distantly related bacteria. Measurement of the rate of GTP hydrolysis on programmed ribosomes revealed that nearly all of the interface mutations were able to function in ribosomal decoding. The only interface mutation with greatly impaired GTPase activity was R223A which is the only one that also forms a direct contact with the ribosome. Finally, the ability of the EF-Tu interface mutants to destabilize the EF-Tu–aa-tRNA interaction on the ribosome after GTP hydrolysis were evaluated by their ability to suppress the hyperstable T1 tRNATyr variant where EF-Tu release is sufficiently slow to limit the rate of peptide bond formation (kpep) . In general, interface mutations that destabilize EF-Tu binding are also able to stimulate kpep of T1 tRNATyr, suggesting that the thermodynamic properties of the EF-Tu–aa-tRNA interaction on the ribosome are quite similar to those found in the free ternary complex.

Structure of a novel antibacterial toxin that exploits elongation factor Tu to cleave specific transfer RNAs

“…Despite this discrepancy, we propose that EF-Ts stimulates the activity of each toxin by increasing steady-state GTP·EF-Tu·aa-tRNA substrate levels as described by Blanchard and colleagues ( 68 , 69 ). Though EF-Tu has nanomolar affinity for aa-tRNA at 0–4°C ( 70 ), these interactions are estimated to be 20- to 30-fold weaker at 37°C ( 71 , 72 ), indicating that the dissociation constants approach the micromolar range under the conditions used here to monitor in vitro tRNase activity. Coupled with the low affinity of CdiA-CT NC101 for EF-Tu, in vitro nuclease activity is presumably impeded by the low probability that EF-Tu, aa-tRNA and toxin all converge simultaneously.…”

Broad range of missense error frequencies in cellular proteins

“…We also found that the error frequency varies for different positions in EF-Tu. Positions with low error frequencies cluster at the aminoacyl-tRNA binding interface of EF-Tu (69), which leads us to speculate that the removal of aberrant EF-Tu molecules may depend on their impaired functional activity. For instance, the mutation R378A results in a 10-fold larger destabilization of the EF-Tu–aminoacyl-tRNA complex, compared to mutations at positions R59 and R283 (69).…”

Comparative secretomics reveals novel virulence-associated factors of Vibrio parahaemolyticus

“…In this study, two other translation-associated proteins, vps15 (EF-Tu) and vps 16 (EF-Ts), were also identified. These two proteins are important components of the multistep ribosomal decoding pathway that ensures rapid and accurate translation (Burnett et al, 2013 ; Yikilmaz et al, 2014 ). It has been reported that EF-Tu of Streptococcus pneumonia was a new virulence factor that binds human complement factors, aids in immune evasion and host tissue invasion (Sarkar et al, 2013 ).…”