Coordinated conformational and compositional dynamics drive ribosome translocation

Abstract

During translation elongation, the ribosome compositional factors elongation factor G (EF-G; encoded by fusA) and tRNA alternately bind to the ribosome to direct protein synthesis and regulate the conformation of the ribosome. Here, we use single-molecule fluorescence with zero-mode waveguides to directly correlate ribosome conformation and composition during multiple rounds of elongation at high factor concentrations in Escherichia coli. Our results show that EF-G bound to GTP (EF-G-GTP) continuously samples both rotational states of the ribosome, binding with higher affinity to the rotated state. Upon successful accommodation into the rotated ribosome, the EF-G-ribosome complex evolves through several rate-limiting conformational changes and the hydrolysis of GTP, which results in a transition back to the nonrotated state and in turn drives translocation and facilitates release of both EF-G-GDP and E-site tRNA. These experiments highlight the power of tracking single-molecule conformation and composition simultaneously in real time.

Figure 1. Correlating conformation and composition with nonfluorescent FRET acceptor and ZMW

a, Position of the Cy3B FRET probe on the body domain of the 30S subunit with respect to the nonfluorescent FRET acceptor, BHQ, on the 50S subunit, reporting on the rotational state of the 30S subunit. The tRNA (in A, P, and E sites) and EF-G are not within FRET distance with the Cy3B and BHQ dye on the ribosome. b, Expected sequence of fluorescence signals using Cy3B and BHQ FRET to correlate conformational and compositional dynamics. Cy3B intensity (green) reports on the conformation of the ribosome, while Cy5 pulses (red) report on ligand occupancy on the ribosome. This allows the correlation of conformation and composition to extract state-specific ligand dynamics. c, Experimental setup. Pre-initiation complex with Cy3B-30S, fMet-tRNA^fMet and IF2-GTP are immobilized on the bottom of the ZMW well through a biotinylated mRNA. The reaction was started by delivering IF2-GTP, BHQ-50S, Lys-tRNA^Lys ternary complex, and either Cy5 labeled EF-G-GTP or Phe-tRNA^Phe ternary complex to the surface.

Figure 2. Ribosome conformation drives translocation and regulates tRNA dynamics

a, Representative trace of Cy3B and BHQ labeled ribosome elongating with Phe-(Cy5)tRNA^Phe. Delivery of reagents results in 50S subunit joining and the arrival of FRET between Cy3B and BHQ, followed by multiple cycles of low-high-low green intensities, each reporting on ribosome rotating and counter-rotating during one round of elongation. The arrival and departure of Phe-(Cy5)tRNA^Phe ternary complex are superimposed as red pulses, allowing correlation of tRNA arrival and departure with ribosome conformation. Brief sampling pulses of Phe-(Cy5)tRNA^Phe ternary complex are observed after arrival at the stop codon, as characterized by Uemura et al. b, Post-synchronization of the (Cy5)tRNA^Phe to ribosome rotating and counter-rotating shows that tRNA arrival is correlated with ribosome rotation and that tRNA departure is correlated with ribosome counter-rotation, as emphasized by the shaded areas. Ribosome conformational counter-rotation thus underlies translocation and E-site tRNA release. The number of molecules analyzed is n = 141.

Figure 3. EF-G regulates ribosome conformational dynamics

a, Representative trace of Cy3B and BHQ ribosome elongating superimposed with Cy5-EF-G occupancy signal showing EF-G correlating with the ribosome counter-rotation. A single cysteine mutant of EF-G (S73C) was labeled with Cy5-maleimide, with the labeling site distant from all EF-G functional domains. Each ribosome conformational transition from rotated to non-rotated state (high to low green intensity change) is correlated with a red pulse, corresponding to the arrival and rapid departure of Cy5-EF-G. Non-productive sampling events are observed to both ribosome conformations. b, The mean arrival times to the two conformations of the ribosome, at different concentrations of tRNA ternary complex (TC) and EF-G-GTP, showing the arrival times decrease with increasing EF-G concentration. The arrival times of EF-G-GTP to the rotated state are lower than to the non-rotated state, suggesting that EF-G-GTP binds with higher affinity to the rotated state. The arrival time to the non-rotated state at 500 nM EF-G and TC is only a lower estimate, due to the decreased non-rotated state lifetime from the increased TC concentration. From left to right, n = 139, n = 216, n = 126, n = 103. Error bars are standard error. c, Post-synchronization of ribosome counter-rotating with EF-G, at 30 frames per second (n = 106). EF-G arrives prior to the intersubunit conformation transition (as emphasized by the shaded area) and departs rapidly after.

Figure 4. Role of EF-G GTP hydrolysis

a, Representative trace of ribosome counter-rotating with EF-G-GTP. EF-G-GTP efficiently drives ribosome counter-rotation. b, Representative trace of ribosome counter-rotating with EF-G-GDPNP. EF-G-GDPNP drives ribosome counter-rotation less efficiently; only after multiple prolonged binding events does translocation occur. c, Mean dwell time and the number of EF-G binding events required for a successful ribosome counter-rotation for EF-G-GTP and EF-G-GDPNP. Error bars are standard error. c, The fraction of ribosomes that counter-rotated in the presence of EF-G with the GTP, GDPNP, or GDP, at 5 and 15 mM Mg²⁺, within the 5 minute observation window (normalized to GTP) with the arrival time of the counter-rotation. The data is fit to a single exponential for visualization. EF-G-GTP catalyzes ribosome counter-rotation efficiently, with most of the ribosomes counter-rotating within the first 50 s. The translocation efficiency of EF-G-GTP is the same at 5 and 15 mM Mg²⁺. EF-G-GDPNP catalyzes ribosome counter-rotation >50 fold less efficiently than EF-G-GTP. At 15 mM Mg²⁺, however, counter-rotation is only 25 fold less efficient with GDPNP. From top to bottom, n = 44, n = 143, n = 151, n = 76.

Figure 5. Conformational selection for EF-G

a, The arrival times for EF-G-GDPNP (200 nM) and EF-G-GDP (200 nM) to the two different ribosome conformations. b, Dwell times for EF-G-GDPNP and EF-G-GDP binding to the two different ribosome conformations. For both panels a and b, from left to right, n = 115, n = 371, n = 162, n = 135. Error bars are standard error. c, Post-synchronization of ribosome counter-rotation correlated with EF-G-GDPNP. For the ribosomes that successfully translocated by EF-G-GDPNP within the 5 minute observation window, EF-G binds to the ribosome for a prolonged period of time. After the ribosome counter-rotates, EF-G departs rapidly due to the decreased affinity of the ribosome’s non-rotated state with EF-G. Extended occupancy of EF-G-GDPNP on the ribosome surmounts the translocation energy barrier partially by locking the ribosome in an intermediate state of ratcheting, enabling thermal energy to eventually complete translocation at high concentrations of Mg²⁺. d, Post-synchronization of the ribosome rotation to Cy5-EF-G-GTP. There is a window when the ribosome rotates that EF-G does not bind, emphasized by the shaded area, which is when tRNA-EF-Tu-GTP binds and accommodates in the ribosome. Only post EF-Tu GTP hydrolysis and EF-Tu departure is EF-G able to bind to the A-site. The higher density of EF-G binding events in the rotated state further suggests that EF-G binds with higher affinity to the rotated state of the ribosome.

Figure 6. State-specific dynamics of EF-G to different ribosome conformations

a, Dwell-time distribution of EF-G binding to the non-rotated state is a single exponential decay, suggesting rapid binding and dissociation with a single rate-limiting step process. b, Dwell-time distribution for EF-G binding to the rotated state is a single exponential decay, also implying a single rate-limiting step process. c, EF-G dwell-time distribution for events that lead to successful ribosome counter-rotation. The probability density is best fit by a Poisson distribution with n = 3, implying a process with multiple rate-limiting steps. The inset shows the R² values for the Poisson fits with different n. d, e, Relative timing of EF-G binding and dissociation with ribosome counter-rotation. Post-synchronization to either EF-G binding or EF-G dissociation shows that EF-G binds before ribosome counter-rotation (~50 ms), and departs rapidly after (~10 ms). Partitioning of the EF-G dwell time pre- and post-ribosome counter-rotation reveals that after conformational transition, EF-G departs rapidly, with only a single rate-limiting step process. All the kinetically significant steps and conformational changes of EF-G occur prior the ribosome counter-rotation.

Figure 7. Perturbations of EF-G dynamics by antibiotics

a, Representative trace of Cy3B and BHQ ribosome elongating with 80 nM Cy5-EF-G-GTP, 80 nM Phe-tRNA^Phe ternary complex, and 80 nM Lys-tRNA^Lys ternary, in the presence of 100 µM spectinomycin. The EF-G dwell time distribution for a binding that led to a successful counter-rotation is a multi-step process same as observed without antibiotics. The distribution for sampling to rotated state shifted to be a multi-step distribution, indicative that these sampling events likely involve futile GTP hydrolysis. The distribution for sampling to the non-rotated state remains exponential. b, Mean number of EF-G binding events for a successful ribosome counter-rotation without drugs and in the presence of translocation inhibiting antibiotics, spectinomycin and viomycin. These futile sampling apparently involve GTP hydrolysis, enhancing the energetic cost of translation in the presence of these drugs. From left to right, n = 216, n = 161, n = 125. Error bars are standard error. c, Schematic of ribosome counter-rotating with EF-G-GTP, EF-G-GTP in the presence of translocation inhibiting antibiotics, and EF-G-GDPNP. Translocation with EF-G-GTP is efficient, with sampling events not hydrolyzing GTP. In the presence of antibiotics, multiple EF-G binding events are required, with the sampling events representing futile GTP hydrolysis cycles. With EF-G-GDPNP, multiple prolonged EF-G events are required to eventually translocate the ribosome.

Figure 8. Schematic of elongation

a, The two conformations of the ribosome (rotated and non-rotated) are regulated by peptide bond formation and EF-G GTP hydrolysis. The two conformations of the ribosome, in turn, allow the ribosome to selectively discriminate between binding of tRNA-EF-Tu-GTP or EF-G-GTP. b, The ribosome is separated into two global conformations, the “unlocked” state and the “locked” state. Upon peptide bond formation, the ribosome “unlocks” (with the rotation of the 30S body), releasing fluctuations of the L1 stalk between open and closed states, fluctuations of tRNAs between the classical and hybrid states, and spontaneous rotations in the 30S head domain. EF-G-GTP binding then stabilizes the L1 stalk in the closed state and tRNA in the hybrid state, as well as causing the head of the 30S subunit to rotate. GTP hydrolysis by EF-G unlocks mRNA movement, followed by translocation of tRNAs to the P and E sites (driven by back-rotation of the 30S body and head domains) and relocking of the ribosome and mRNA movement to preserve the reading frame. The E-site tRNA and EF-G-GDP departs rapidly, returning the ribosome to the original state.