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. 2016 Jun 21;113(27):7527–7532. doi: 10.1073/pnas.1604790113

Fig. 5.

Fig. 5.

EVN/AVI inhibit accommodation of tRNA into the A site. (A) Comparison of the relative binding positions on the ribosome of AVI (red), EF-Tu (blue), A/T-tRNA (green) (36, 56), and A/A-tRNA (teal) (38). (B) Schematic diagram of smFRET measurements of tRNA selection. After delivery of EF-Tu⋅GTP⋅tRNA ternary complex containing cognate Phe-tRNAPhe(LD650) to the A site of E. coli 70S ribosomes containing tRNAiMet(Cy3) in the P site, tRNA motion can be tracked through the progression of FRET efficiencies from low (0.2) to intermediate (0.35) FRET during initial steps of selection to high (0.63) FRET upon A-site tRNA accommodation, which is inhibited by AVI/EVN. (C–E) Ensemble smFRET histograms showing the time course of aa-tRNA selection, imaged in the absence of drugs (C) or in the presence of 20 μM AVI (D) or 20 μM EVN (E). The histograms were postsynchronized by aligning each observed event to the first appearance of nonzero FRET states. (F–H) Transition density plots for the data shown in C–E, respectively. These 2D histograms juxtapose the FRET efficiencies immediately before and after FRET transitions. As indicated by arrows, EVN and AVI promote reversible transitions between high and intermediate FRET.