A fast dynamic mode of the EF-G-bound ribosome

Abstract

A key intermediate in translocation is an 'unlocked state' of the pre-translocation ribosome in which the P-site tRNA adopts the P/E hybrid state, the L1 stalk domain closes and ribosomal subunits adopt a ratcheted configuration. Here, through two- and three-colour smFRET imaging from multiple structural perspectives, EF-G is shown to accelerate structural and kinetic pathways in the ribosome, leading to this transition. The EF-G-bound ribosome remains highly dynamic in nature, wherein, the unlocked state is transiently and reversibly formed. The P/E hybrid state is energetically favoured, but exchange with the classical P/P configuration persists; the L1 stalk adopts a fast dynamic mode characterized by rapid cycles of closure and opening. These data support a model in which P/E hybrid state formation, L1 stalk closure and subunit ratcheting are loosely coupled, independent processes that must converge to achieve the unlocked state. The highly dynamic nature of these motions, and their sensitivity to conformational and compositional changes in the ribosome, suggests that regulating the formation of this intermediate may present an effective avenue for translational control.

Figure 1

Structural models of the ribosome with fluorescent components. The distances shown are in agreement with those predicted by smFRET experiments. Structural models were constructed according to published procedures (Tung et al, 2002; Munro et al, 2009a). The P-site tRNA is in blue and the L1 stalk is in purple. (A) The locked ribosome configuration with the tRNA in the classical P site, and the L1 stalk in the open position, consistent with the low-FRET (0.1) state observed in smFRET trajectories acquired from complexes with labelled L1 and P-site tRNA. (B) The unlocked ribosome configuration stabilized by EF-G (shown in cyan) in which the P/E hybrid state is formed, the L1 stalk is closed and the subunits are ratcheted—consistent with the high-FRET (0.65) state observed with labelled L1 and P-site tRNA, and the low-FRET (0.25) state observed with labelled EF-G and P-site tRNA.

Figure 2

EF-G(GDPNP) binding at the A-site stabilizes the P/E hybrid state and lowers the activation barriers for L1 stalk motions. Here the dynamics of the EF-G-bound ribosome complex with P-site initiator tRNA^fMet are shown. Two unique labelling schemes were used for smFRET imaging: (A) labelled L1 and P-site tRNA in the presence of 10 μM EF-G and 2 mM GDPNP, and (B) labelled P-site tRNA in the presence of 0.1 μM labelled EF-G and 2 mM GDPNP. (Left panels) Cartoon models of the EF-G-bound complex indicating the dynamic elements and the sites of labelling. (Centre panels) Single-molecule fluorescence (Cy3, green; Cy5, red) and FRET (blue) trajectories. The idealization is overlaid in red on the FRET trace. (Right panels) Histograms indicating the occupancies in the observed FRET states as determined by idealization. Highlighted in red/pink are those states that relate to formation of the unlocked ribosome. ‘N' denotes the number of individual smFRET trajectories included in the analysis.

Figure 3

With tRNA^Phe in the P site, the complex has greater occupancy in the P/E hybrid and unlocked states. Here the dynamics of the EF-G-bound ribosome complex with P-site elongator tRNA^Phe shown. Two unique labelling schemes were used for smFRET imaging: (A) labelled L1 and P-site tRNA in the presence of 10 μM EF-G and 2 mM GDPNP, and (B) labelled P-site tRNA in the presence of 0.1 μM labelled EF-G and 2 mM GDPNP. (Left panels) Cartoon models of the EF-G-bound complex indicating the dynamic elements and the sites of labelling. (Centre panels) Single-molecule fluorescence (Cy3, green; Cy5, red) and FRET (blue) trajectories. The idealization is overlaid in red on the FRET trace. (Right panels) Histograms indicating the occupancies in the observed FRET states as determined by idealization. Highlighted in red/pink are those states that relate to formation of the unlocked ribosome.

Figure 4

Direct observation of dynamic mode switching observed in smFRET trajectories. Ribosome complexes with labelled L1 and either (A) P-site tRNA^fMet or (B) P-site tRNA^Phe show dramatic switches in the dynamic mode of the L1 stalk in the presence of intermediate EF-G concentrations (0.1–1 μM). These trajectories represent a minority of molecules (∼1–4%), consistent with slow EF-G binding and release relative to the observation window (∼30sec). FRET traces are shown in blue with idealization, estimated by HMM analysis (Qin, 2004), overlaid in red.

Figure 5

EF-G-mediated dynamic mode switching in the ribosome reveals the existence of two distinct low-FRET states. (A) Histograms indicating the distributions of the logarithm of the transition rate out of the low-FRET state (k_{low →}=k_{0.1 → 0.25}+k_{0.1 → 0.4}), calculated for each individual molecule (see Materials and methods section), as a function of EF-G concentration. At each concentration, including in the absence of EF-G, a bimodal distribution of rates is observed that represent slow (light grey) and fast (dark grey) dynamic modes of the ribosome. The populations in each mode were estimated by fitting the distributions to a double Gaussian function (R²>0.9) with mean values μ=−0.8 (s.d., σ=1.0; k_{low →}^slow≈0.5 per s) and μ=1.4 (σ=1.0; k_{low →}^fast≈4 per s) for the tRNA^fMet-bound complex (left panels), and μ=0.8 (σ=1.2; k_{low →}^slow≈2 per s) and μ=3.2 (σ=1.2; k_{low →}^fast⩾25 per s) for the tRNA^Phe-bound complex (right panels). In the case of tRNA^Phe, only a portion of the fast dynamic mode distribution was observed due to the short low-FRET state lifetime relative to the experimental time resolution. (B) Plots showing the change in integrated areas of the slow and fast dynamic modes for each system as a function of EF-G concentration. Fitting changes in integrated areas of slow and fast populations to hyperbolic functions provides an estimate of the apparent K_D (∼80 nM) for EF-G(GDPNP) binding to the ribosome (R²>0.9).

Figure 6

Three-colour smFRET displays uncoupled motions of P-site tRNA the L1 stalk on the EF-G-bound ribosome. smFRET trajectories acquired from ribosome complexes bearing Cy5.5-labelled L1, Cy3-labelled P-site tRNA, in the presence of 100 nM A647N-labelled EF-G(GDPNP). Fluorescence and FRET trajectories are shown for complexes containing (A) P-site tRNA^fMet or (B) P-site tRNA^Phe. Anti-correlated changes in A647N and Cy5.5 fluorescence, resulting of FRET from A647N to Cy5.5 in the unlocked configuration, verify the presence of both fluorophores.