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. 2020 Feb 5;117(7):3610–3620. doi: 10.1073/pnas.1904469117

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Copyright © 2020 the Author(s). Published by PNAS.

This open access article is distributed under Creative Commons Attribution-NonCommercial-NoDerivatives License 4.0 (CC BY-NC-ND).

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Fig. 2. — Three-color imaging of presteady-state tRNA selection. (A) Structural representation depicting the approximate locations of P-site tRNA (orange), aa-tRNA (white), and EF-Tu (red) during tRNA selection. The locations of Cy3 (green circle), LD650 (red circle), and LD750 (brown circle) are shown. (B, Upper) Fluorescence intensity trajectories of Cy3 (dark green), LD650 (red), and LD750 (brown) and corresponding (Lower) single-molecule trajectories of P-site tRNA(Cy3):aa-tRNA(LD650) FRET (purple) and aa-tRNA(LD650):EF-Tu(LD750) FRET (fuchsia) imaged at 25-ms time resolution during stopped-flow delivery of a dual-labeled ternary complex to P-site–labeled ribosomes at 15 mM Mg²⁺. (C) Population histograms for P-site tRNA(Cy3):aa-tRNA(LD650) FRET (Upper) and aa-tRNA(LD650):EF-Tu(LD750) FRET (Lower) during presteady-state tRNA selection without drug (Left), with GDPNP (Center), or with kirromycin (Right). (D) Structural representation depicting the approximate locations of L11 (blue), A-site tRNA (white), and EF-Tu (red) in complexes used to study the kinetics of EF-Tu and aa-tRNA from the perspective of the large subunit during tRNA selection. The locations of Cy3 (green circle), LD650 (red circle), and LD750 (brown circle) are shown. (E, Upper) Fluorescence intensity trajectories of Cy3B (dark green), LD650 (red), and LD750 (brown) and corresponding (Lower) single-molecule trajectories of L11(Cy3B):aa-tRNA(LD650) FRET (purple) and aa-tRNA(LD650):EF-Tu(LD750) FRET (fuchsia) imaged at 25-ms time resolution during stopped-flow delivery of a dual-labeled ternary complex to ribosomes with donor-labeled L11 at 15 mM Mg²⁺. (F) Population histograms for L11(Cy3B):aa-tRNA(LD650) FRET (Upper) and aa-tRNA(LD650):EF-Tu(LD750) FRET (Lower) during presteady-state tRNA selection without drug (Left), with GDPNP (Center), or with kirromycin (Right).