Single Molecule FRET Studies on Kinetics of Elongation Factor Tu Binding to the Ribosome during the tRNA Selection Process

Abstract

Elongation factor Tu (EF-Tu) ensures fidelity in protein synthesis by mediating the entry of cognate aminoacyl-tRNA (aa-tRNA) into the A-site of the ribosome via formation of an EF-Tu.GTP.aa-tRNA ternary complex (TC). In order to probe the kinetic details of EF-Tu interactions with both aminoacyl tRNA and the ribosome during the tRNA selection process, we have constructed, purified, and labeled an E. coli EF-Tu mutant at position 348 (E348C) with either a fluorescence quencher (QSY9) or a fluorescent dye (Cy3 or Cy5). This position of labeling allows monitoring of EF-Tu interactions with fluorescent derivatives of ribosomal protein L11 (labeled at position 87) and aa-tRNA (labeled in the dihydroU loop). These three positions form an almost equilateral triangle within the ribosome, at distances that are appropriate for sensitive monitoring by single molecule fluorescence resonance energy transfer (smFRET). Two kinetic steps, denoted as TC association (4.7±0.3×107 M−1s−1) and EF-Tu dissociation (12±1 s−1), are found with Cy3-Cy5 FRET pairs or Cy3-QSY9 pairs placed on L11/EF-Tu, tRNA/EF-Tu (A-site) or tRNA/tRNA (A-site/P-site) pairs. The reaction rates are almost independent of labeling strategy and agree with ensemble measurements. At 10 ms time resolution, the FRET between L11/EF-Tu and tRNA/EF-Tu (A-site) pairs showed only one EF-Tu bound conformational state that is detectable during the tRNA selection process, providing strong evidence that, at this time resolution, EF-Tu loses proximity essentially simultaneously with both L11 and aa-tRNA. Alternating-laser excitation experiments demonstrate that, following EF-Tu dissociation, a fraction of aa-tRNA remains stably bound to the ribosome, corresponding to aa-tRNA that has successfully accommodated into the A-site, while the remainder dissociates rapidly, presumably due to rejection via proofreading.Supported by NIH (GM080376, GM071014 and HG004364) and NIST (70NANB7H0711).