Abstract
Directional translocation of the ribosome through the mRNA open reading frame is a critical determinant of translational fidelity. This process entails a complex interplay of large-scale conformational changes within the actively translating particle, which together coordinate the movement of tRNA and mRNA substrates with respect to the large and small ribosomal subunits. Using pre–steady state, single-molecule fluorescence resonance energy transfer imaging, we tracked the nature and timing of these conformational events within the Escherichia coli ribosome from five structural perspectives. Our investigations revealed direct evidence of structurally and kinetically distinct late intermediates during substrate movement, whose resolution determines the rate of translocation. These steps involve intramolecular events within the EF-G–GDP–bound ribosome, including exaggerated, reversible fluctuations of the small-subunit head domain, which ultimately facilitate peptidyl-tRNA's movement into its final post-translocation position.
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Acknowledgements
We thank R. Green (Johns Hopkins University) for providing the S13-knockout strain and P. Schultz (Scripps Research Institute) for providing the L5-knockout strain. We also thank D. Terry of the Blanchard laboratory for assistance with three-color FRET data acquisition and analysis. This work was supported by the US National Institutes of Health (2R01GM079238 and 5R01GM098859 to S.C.B.).
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M.R.W. prepared dye-labeled ribosomes and EF-G. R.B.A. and M.R.W. prepared dye-labeled tRNAs. J.L.A. and M.R.W. performed the smFRET imaging. J.L.A. analyzed the smFRET results. J.L.A. and M.R.W. made the figures. S.C.B., M.R.W. and J.L.A. designed the study. All authors discussed the results and contributed to the writing of the manuscript.
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S.C.B. and R.B.A. have an equity interest in Lumidyne Technologies.
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Supplementary Figure 1 Kinetic analysis of the S13-L1 FRET translocation signal.
(a,b) Representative traces of (a) initiator and (b) elongator complexes imaged during translocation. The EF-G injection, low-FRET arrival (Δtlow; POST formation), (productive) intermediate-FRET arrival (Δtarr) and intermediate-FRET dwell (Δtint) times are indicated. (c) Cumulative distributions of the waiting times to irreversible transitions from the high-FRET (rotated, PRE) state to the stable low-FRET (unrotated, POST) state. The distributions are shown for 1 mM GTP and varying concentrations of EF-G (0.25 μM EF-G, squares; 1 μM EF-G, circles; 10 μM EF-G, triangles; 10 μM EF-G with 2mM GDPNP, inverted triangles). The EF-G(GTP) distributions were fit by double exponentials (black curves). The EF-G(GDPNP) distribution was fit to a single exponential (red curve). (d–f) Waiting time distributions acquired across a range of EF-G concentrations were fit to double (Δtlow and Δtarr) or single exponentials (Δtint). The fast component rates of (d) low-FRET and (e) intermediate-FRET arrival displayed an EF-G concentration dependence that was well described by hyperbolic functions (blue – initiator; red – elongator), whereas the slow component rates did not vary with EF-G concentration (dashed lines). (f) The rates of exit from the intermediate-FRET state into the low-FRET, POST state were independent of EF-G concentration. Error bars represent standard deviations from 3 independent data sets. Fast component percentages from the double exponential fits as well as hyperbolic and linear fit parameters are indicated.
Supplementary Figure 2 Kinetic analysis of the tRNA-tRNA FRET translocation signal.
(a,b) Representative traces of (a) initiator and (b) elongator complexes imaged during translocation. The EF-G injection, (productive) high-FRET state arrival (ΔtHF arr), high-FRET state dwell (ΔtHF) and FRET loss (ΔtFRET loss; E-site tRNA departure) times are indicated. (c–e) Waiting time distributions acquired across a range of EF-G concentrations were fit to double (ΔtHF arr and ΔtFRET loss) or single exponentials (ΔtHF). (c) The fast component rates of high-FRET arrival displayed an EF-G concentration dependence that was well described by hyperbolic functions (blue – initiator; red – elongator), whereas the slow component rates did not vary with EF-G concentration (dashed lines). (d) The rates corresponding to the high-FRET state lifetime were independent of EF-G concentration. (e) The fast component rates of high-FRET loss displayed an EF-G concentration dependence that was well described by hyperbolic functions (blue – initiator; red – elongator), whereas the slow component rates did not vary with EF-G concentration (dashed lines). Error bars represent standard deviations from 3 independent data sets. Fast component percentages from the double exponential fits as well as hyperbolic and linear fit parameters are indicated.
Supplementary Figure 3 Kinetic analysis of the S13–A-site tRNA FRET translocation signal.
(a,b) Representative traces of (a) initiator and (b) elongator complexes imaged during translocation. The EF-G injection and waiting times to FRET rise (Δtrise; POST formation) are indicated. (c) Waiting time distributions acquired across a range of EF-G concentrations were fit to double exponentials (Δtrise). The fast component rates of FRET rise displayed an EF-G concentration dependence that was well described by hyperbolic functions (blue – initiator; red – elongator), whereas the slow component rates did not vary with EF-G concentration (dashed lines). Error bars represent standard deviations from 3 independent data sets. Fast component percentages from the double exponential fits as well as hyperbolic and linear fit parameters are indicated.
Supplementary Figure 4 Kinetic analysis of the S13-L5 FRET translocation signal.
(a,b) Representative traces of (a) initiator and (b) elongator complexes imaged during translocation. The EF-G injection and high-FRET arrival (Δtrise; POST formation) times are indicated. (c) Waiting time distributions acquired across a range of EF-G concentrations were fit to double exponentials (Δthigh). The fast component rates of high-FRET arrival displayed an EF-G concentration dependence that was well described by hyperbolic functions (blue – initiator; red – elongator), whereas the slow component rates did not vary with EF-G concentration (dashed lines). Error bars represent standard deviations from 3 independent data sets. Fast component percentages from the double exponential fits as well as hyperbolic and linear fit parameters are indicated.
Supplementary Figure 5 Antibiotic effects on early events in translocation.
The effects of the translocation inhibitors hygromycin B (HygB), viomycin (Vio) and spectinomycin (Spc) on the action of saturating EF-G(GTP) (10 μM EF-G; 1 mM GTP) is displayed for elongator complexes labeled on S13-L1, tRNA-tRNA, S13-A-site tRNA and S13-L5. To more clearly demonstrate drug activity on EF-G-bound complexes, traces initially in the FRET states corresponding to the rotated/hybrid conformation were selected. (a) HygB (400 μM) inhibits formation of the translocation intermediate in both the S13-L1 and S13-L5 signals. The dynamic FRET increase observed in the tRNA-tRNA signal, and the slight FRET increase observed in the S13-A-site tRNA signal suggest that hygromycin B allows EF-G to stabilize the A/P hybrid state, but inhibits formation of the head-swiveled, fully tRNA-compacted intermediate observed during uninhibited translocation. (b) Viomycin (200 μM) inhibits all transitions from the rotated, hybrid tRNA state to the intermediate or POST conformations. (c) Spectinomycin (3 mM) blocks the formation of the intermediate conformation identified in this study. However, it stabilizes what appears to be another intermediate conformation (INT1; Pan, D. et al., Mol Cell. 25, 519–529, 2007). The S13-L1 and S13-L5 panels suggest that Spc only allows a detectable, but limited amount of head swivel, while the tRNA-tRNA and S13-A-site tRNA signals suggest that the drug does not inhibit tRNA motions (compaction).
Supplementary Figure 6 Postsynchronized population FRET histograms of the translocation reaction coordinate from the perspective of motion.
Population FRET histograms showing the response of translocating elongator PRE complexes in response to the addition of EF-G(GTP) (10 μM; 1 mM) when site-specifically labeled as described in Figures 2 and 3 at (a) ribosomal proteins S13 and L1, (b) deacyl- and peptidyl-tRNA and (c) ribosomal protein S13 and peptidyl-tRNA, where each data set was post-synchronized to the final FRET state observed during translocation.
Supplementary Figure 7 Exaggerated motions of the small-subunit head domain are observed from two distinct structural perspectives.
(a) Schematic of the translocation reaction coordinate monitored by an smFRET labeling strategy that reports on P-site tRNA (orange) movement relative to the small subunit head domain protein S13 (light grey). The sites of labeling of donor and acceptor fluorophores are indicated with green and red circles, respectively. FRET values differ between initiator and elongator complexes due to different sites of P-site tRNA labeling (Online Methods). Traces initially in the FRET states corresponding to the rotated/hybrid conformation were selected. (b) Following EF-G(GTP) delivery, complexes transition from high-FRET (0.59 ± 0.07 – initiator, not shown; 0.80 ± 0.06 – elongator, shown) to lower FRET states, followed by FRET loss (E-site tRNA departure). (c) Fusidic acid (FA) stabilizes an intermediate-FRET state (0.50 ± 0.08 – initiator, not shown; 0.64 ± 0.08 – elongator, shown) and is followed by FRET loss. Inspection of individual S13-P-site tRNA traces revealed a low-FRET ‘dip’ from the FA-stabilized intermediate prior to FRET loss. To more clearly demonstrate the order and timing of this apparent additional FRET state, population histograms of (d) initiator and (e) elongator traces were post-synchronized to intermediate-FRET following the appearance of the FRET dip. Representative traces demonstrate fluctuations between the intermediate-FRET (FA-stabilized, INT2) and low-FRET states (INT3; ~0.40 and ~0.55 for initiator and elongator, respectively) prior to FRET loss. As highlighted in Figure 6, similar results are observed with the S13-L5 (f) initiator and (g) elongator complexes. Dashed lines specify the FRET state assignments.
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Wasserman, M., Alejo, J., Altman, R. et al. Multiperspective smFRET reveals rate-determining late intermediates of ribosomal translocation. Nat Struct Mol Biol 23, 333–341 (2016). https://doi.org/10.1038/nsmb.3177
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DOI: https://doi.org/10.1038/nsmb.3177
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