Figure 4. Ribosome translates through a hairpin via two parallel pathways that bifurcate before EF-G binding and converge after hairpin opening.

(A) Cumulative density of τ_dwell, the time ribosome spends at each codon, is best fit by a mixture of two exponentials given by: $Cdf\left(t\right)=1-f_{\text{fast path}}\cdot e^{-k_{\text{fast path}}\cdot t}-f_{\text{slow path}}\cdot e^{-k_{\text{slow path}}\cdot t}$.

(B) Summary of fast and slow pathway rates, k_{fast path} and k_{slow path} obtained from fits to the cumulative distribution of τ_dwell at forces 3 pN (n = 551 events, 19 molecules), 5 pN (n = 256 events, 8 molecules), 10 pN (n = 776 events, 19 molecules) and 15 pN (n = 889 events, 24 molecules). Error bars represent 95% confidence intervals. Concentration of EF-G is 10 µM. k_{fast path} and k_{slow path}, remain constant at ~0.92 codon/s and ~0.15 codon/s respectively at various forces.

(C) Fraction of events going through the slow pathway, ƒ_{slow path}, as obtained from fitting shown in (A). ƒ_{slow path} increases from ~10 % at high force (> 10 pN) to ~30–50 % at low force (< 7 pN). Error bars represent 95% confidence intervals.

(D) Cumulative density of τ_unwinding, the time between EF-G binding and hairpin opening, is best fit by a mixture of two exponentials given by: $Cdf\left(t\right)=1-f_{\text{unwinding}}^{\text{fast}}\cdot e^{-k_{\text{unwinding}}^{\text{fast}}\cdot t}-f_{\text{unwinding}}^{\text{slow}}\cdot e^{-k_{\text{unwinding}}^{\text{slow}}\cdot t}$.

(E) Summary of bi-exponential rates $k_{\text{unwinding}}^{\text{fast}}$ and $k_{\text{unwinding}}^{\text{slow}}$ obtained from fits to the cumulative distribution of τ_unwinding at forces 5–8 pN (n = 62 events, 14 molecules), 7–9 pN (n = 46 events, 10 molecules), 12–13 pN (n = 80 events, 20 molecules) and 14–17 pN (n = 53 events, 22 molecules). These experiments were performed in passive mode, i.e., the force is maintained in a particular regime rather than being held constant. Concentration of EF-G is 10 nM. Error bars represent 95% confidence intervals.

(F) Fraction of events going through the slow unwinding pathway, $f_{\text{unwinding}}^{\text{slow}}$, increase from ~15% at high force (> 12 pN) to ~40% at low force (< 9 pN). Error bars represent 95% confidence intervals.

(G) Proposed kinetic scheme: first, the ribosome ‘senses’ the hairpin barrier and irreversibly switches into either a ‘fast’ state (shown in green) or a ‘slow’ state (shown in red) via rates $k_{\text{sensor}}^{\text{fast}}$ or $k_{\text{sensor}}^{\text{slow}}$ respectively. The ratio, $k_{\text{sensor}}^{\text{fast}}/k_{\text{sensor}}^{\text{slow}}$, fraction of translation events that go through either pathway as shown in (C) and (F). Then, the ribosome in either ‘fast’ or ‘slow’ state must undergo an intermediate transition ($k_{\text{intermediate}}^{\text{fast}}$ or $k_{\text{intermediate}}^{\text{slow}}$) that becomes rate limiting at low force and determines the overall rates shown in (B). It is possible that this intermediate transition is the rate of EF-G binding (see Discussion). Then, the ribosome unwinds the hairpin via rates $k_{\text{unwinding}}^{\text{fast}}$ and $k_{\text{unwinding}}^{\text{slow}}$ shown in (E). Finally, EF-G is released in a similar fashion in either pathway suggesting that the bifurcated pathways converged upon unwinding (Figure S6). A cartoon of the fleezers trajectory for both the fast and slow pathway is shown above and below the kinetic scheme. Notice that while τ_unwinding increases in the slow pathway, it does not increase enough to account for the total increase in τ_dwell.