The ribosome derives the energy to translocate and unwind mRNA from EF-G binding

Domain: physics
Published: 2025-12-16
Editorial outcome: major_revision

Abstract
The GTPase EF-G catalyzes translocation of mRNA and tRNAs relative to the ribosome and helps maintain the reading frame during protein synthesis. Which events directly require EF-G-mediated GTP hydrolysis during translocation are still debated. Using high-resolution optical tweezers endowed with single-molecule fluorescence detection, we simultaneously monitored binding of fluorescently-labeled EF-G to ribosomes and either mRNA unwinding or mRNA translocation relative to the body domain of the small ribosomal subunit. Using EF-G mutants and GTP analogs, we find that neither mRNA unwinding nor translocation require GTP hydrolysis and that these are independent events that may or may not temporally coincide. We propose that “tight binding” of EF-G to the ribosome triggers mRNA unwinding and translocation of mRNA relative to the 30S body domain and that while GTP hydrolysis kinetically accelerates translocation, it is thermodynamically required only to liberate the tightly bound EF-G from the ribosome.

Introduction
During the elongation phase of protein synthesis, the ribosome translocates mRNA in single-codon (3 nt) steps1,2. This coordinated translocation of the mRNA and tRNAs is catalyzed by the GTPase elongation factor EF-G and guided by conformational changes of the large and small ribosomal subunits following peptide bond formation3–10. Specifically, the 30S subunit rotates with respect to the 50S subunit, which moves the tRNAs on the 50S subunit resulting in hybrid tRNA states11. Next, forward rotation or “swivel” of the 30S head domain, accompanied by a partial reversion of inter-subunit rotation, moves the mRNA and tRNAs relative to the body domain to produce chimeric hybrid tRNA states12,13. This step is followed by the reverse 30S head rotation without carrying back the mRNA and tRNAs, which together with full reverse inter-subunit rotation, completes the mRNA/tRNA movement and resets the ribosome to the classical post-translocation state.

Translocation involves directional mRNA movement in precise codon steps and unwinding of mRNA secondary structures2,7,14. In the absence of an external energy source, these processes are endergonic (ΔG>0) and will not occur spontaneously. To drive these processes, the ribosome must couple them with exergonic reactions, which could include peptidyl transfer between P- and A-site tRNAs and EF-G-mediated GTP hydrolysis. After GTP hydrolysis, EF-G retains the cleaved γ-phosphate for some time15,16 before releasing it as inorganic phosphate (Pi), which contributes to the free energy available from hydrolysis. Two roles for GTP hydrolysis and Pi release by EF-G during translocation have been described. In the first role, the energy from GTP hydrolysis and Pi release is suggested to be directly converted to mechanical work4,15,17,18. In the second role, this energy is instead used for reducing the affinity of EF-G to the ribosome, allowing for EF-G release and the resetting of the translational cycle9,19–25. Recently, three independent cryo-electron microscopy (cryo-EM) studies in the presence18,23 and absence22 of inhibitors investigated the structures of ribosomal translocation intermediates. These structures show distinct modes of binding of EF-G to the ribosome over the course of translocation, differing in the conformational state of the ribosome and in the presence or absence of the γ-phosphate of GTP. Specifically, Pi release is seen concomitant with conformational changes that move the mRNA relative to the 30S body domain. Strictly speaking, it is not possible to establish a causal relationship between these two correlated events from structural snapshots. Indeed, the three studies did not agree on when GTP hydrolysis and Pi release are required during translocation.

Ensemble kinetics experiments have indicated that GTP hydrolysis occurs early in the process of translocation4,26. Importantly, ribosomes incubated with EF-G and non-hydrolyzable GTP analogs are able to perform single-turnover translocation, albeit at slower rates and with lower efficiency than with GTP4,16,21,24,27,28. Likewise, the antibiotic sparsomycin induces single-turnover mRNA unwinding and 30S translocation in the absence of EF-G altogether14,29. Moreover, multi-turnover translation can also occur in the absence of EF-G, albeit at very slow rates and under special conditions (e.g. addition of thiol-modifying reagents)30,31, indicating that the peptidyl transfer energy suffices to drive translocation, at least on unstructured mRNA. GTP hydrolysis and Pi release by EF-G further contribute to the overall free energy balance and may be essential for multi-turnover translocation and unwinding of mRNAs that contain stable secondary structures. However, the fundamental question of whether GTP hydrolysis and Pi release are required in advance to drive the energetically uphill mechanical events (e.g., mRNA unwinding) during each cycle of ribosomal translocation is still a matter of debate17,24.

Here, we investigate the requirement for EF-G-mediated GTP hydrolysis and Pi release during translocation by using high-resolution dual-trap optical tweezers endowed with single-molecule fluorescence detection capabilities (“fleezers”). This instrument enables simultaneous monitoring of binding of fluorescent EF-G to the ribosome and ribosomal unwinding of a hairpin. By perturbing the system with EF-G mutants, a ribosomal protein mutant, or GTP analogs, we show that neither GTP hydrolysis nor Pi release is required for the endergonic hairpin unwinding step of translocation. Furthermore, by simultaneously monitoring hairpin unwinding and changes in Förster resonance energy transfer (FRET) between the 30S head and body domains, we find that forward 30S head rotation (a process that involves movement of the mRNA and tRNAs relative to the 30S body domain) can temporally coincide with unwinding. However, using an alternative fleezers assay that simultaneously monitors fluorescently-labeled EF-G binding to the ribosome and direct mRNA translocation relative to the 30S body, we find that translocation can, under GTPase perturbations, be uncoupled from hairpin unwinding and occur after it. Importantly, this alternative assay also shows that mRNA translocation relative to the 30S body does not require GTP hydrolysis, although it is kinetically accelerated by it. We conclude that EF-G-mediated GTP hydrolysis is not required in ribosomal translocation until after mRNA translocation relative to the 30S body has occurred. We propose that the energy derived from binding of EF-G to the ribosome and intrinsic ribosome conformational changes drive the early endergonic events of the translation cycle, including hairpin unwinding and mRNA translocation relative to the 30S body. We argue that Pi release provides instead the drop in free energy required to dissociate EF-G and to initiate reverse 30S head rotation, thus completing the translocation cycle.

Results
Simultaneous monitoring of hairpin unwinding and EF-G binding
To investigate the requirement for EF-G-mediated GTP hydrolysis in mRNA unwinding, we used an optical tweezers assay in which an mRNA hairpin is tethered between two optically trapped polystyrene beads using DNA handles (Fig. 1a). During each translocation cycle, the ribosome unwinds the hairpin by one codon (3 bp) measured by the optical traps, while the binding of Cy3-labeled EF-G to the ribosome is co-temporally monitored by the confocal single-molecule fluorescence detection capability of the fleezers instrument32 (Fig. 1b, Supplementary Fig. 1). Using this assay, we measured the time elapsed between EF-G binding and hairpin unwinding (τunwinding) and the time between unwinding and EF-G release (τrelease) (Fig. 1c). Under moderately high tether pulling forces (~14 pN) and in the presence of 1 mM GTP, the observed distributions for these parameters yielded an overall characteristic time estimate of 72±45 ms for τunwinding and 380±230 ms for τrelease, similar to those found previously32 (Fig. 1c, top green bar, Fig. 1d, e, magenta dots, see Methods). The distributions appear bi-exponential, indicating that unwinding and release follow bifurcated paths, the causes of which are unknown.

It is not known how perturbing EF-G-mediated GTP hydrolysis and Pi release affect τunwinding and τrelease. The answer will reveal when these chemical events are required in the translocation cycle. For example, if GTP hydrolysis is slowed and τunwinding is lengthened as a result, then hydrolysis is likely required for hairpin unwinding and occurs before or concurrently with it. Alternatively, if only τrelease is lengthened, then hydrolysis must not be required for hairpin unwinding but must be required instead for EF-G unbinding. (Supplementary Fig. 2).

Hairpin unwinding by the ribosome does not require EF-G-mediated GTP hydrolysis or Pi release
We first tested whether GTP hydrolysis is required for hairpin unwinding by using a translation solution containing fluorescently labeled WT EF-G and a mixture of GTP and a GTP analog. Using only the analog and no GTP is unfeasible for this multi-turnover assay, because the other essential elongation factor, EF-Tu, also requires GTP.

Total EF-G dwell times (τunwinding + τrelease) for a mixture of 1 mM GTP and 0.5 mM of the non-hydrolyzable GDPNP varied from hundreds of milliseconds to tens of seconds (Supplementary Fig. 3a), in contrast to WT EF-G dwell times which are typically under 500 ms. Importantly, the distribution of τunwinding in the presence of the mixture of GTP and GDPNP shows no significant change compared to that of GTP alone, whereas τrelease is lengthened considerably (Fig. 1d, e, blue dots). Compared to the GTP-only condition (Fig. 1e, magenta dots, dashed line), the distribution of τrelease for the GTP+GDPNP mixture contains a slow population likely associated with GDPNP events (Fig. 1c, third row, Fig. 1e, blue dots, dashed line). This population has a characteristic τrelease fit value of 8.5±1.5 s (Fig. 1c), over an order of magnitude longer than that of the fast (GTP) population.

Similarly, using a mixture of 1 mM GTP and 0.5 mM of slowly-hydrolyzable GTPγS increased the total EF-G dwell time, although to a lesser extent than with GDPNP (Supplementary Fig. 3b). Again, τunwinding for the GTP+GTPγS mixture remains unchanged compared to the GTP-only condition, whereas the distribution of τrelease contains a slow population with a characteristic time of 1.8±0.2 s, almost an order of magnitude longer than that of GTP (Fig. 1c, bottom row, Fig. 1d, e, purple dots, dashed line).

GDPNP is essentially non-hydrolyzable at our experimental timescale and practically limits translocation to a single turnover4,24. However, when using a GTP+GDPNP mixture, we observed processive multi-turnover translocation with interspersed normal and lengthened EF-G binding events that are productive (Supplementary Fig. 3a). We rule out the possibility of exchange of EF-G·GDPNP for EF-G·GTP on the ribosome since we do not detect unbinding and re-binding of EF-G during the long dwell times. This observation leaves only two possibilities to explain the multi-turnover unwinding displaying lengthened but finite EF-G dwell times: either GDPNP is eventually exchanged for GTP on the ribosome-bound EF-G, allowing translocation to resume after only a delay, or EF-G·GDPNP is eventually released from the ribosome after productive stepping without any hydrolysis. As mentioned above, the τrelease distribution for the GTP+GDPNP condition consists of a fast (GTP) and a slow (GDPNP) population. If nucleotide exchange occurs, we expect that altering the GDPNP:GTP ratio would change not only the slow population fraction in the τrelease distribution, but also the characteristic release time for this population (which would be limited by a ratio-dependent rate of exchange of GDPNP and GTP). By contrast, if the long release times are due entirely to EF-G·GDPNP (without exchange), altering the ratio should only change the slow fraction but not its characteristic time. To distinguish between these possibilities, we performed a titration of GDPNP:GTP ratio while keeping the total nucleotide concentration constant. Increasing this ratio from 1:9 to 3:1 increased both the characteristic time (from 2.5±1.9 s to 17±5.6 s) and the fraction estimate (from 0.11±0.1 to 0.45±0.1) of the slow population for τrelease (Fig. 1f, Supplementary Fig. 3c). This result supports the hypothesis that EF-G can exchange its bound nucleotide while remaining bound to the ribosome. While nucleotide exchange by ribosome-bound EF-G was proposed previously27, our data suggest that such exchange is very slow (seconds timescale). Whether EF-Tu·GTP·aa-tRNA can also exchange its GTP for GDPNP under these conditions remains to be examined.

To complement the experiments performed with GTP analogs, we also perturbed GTP hydrolysis by using the H92A mutant of EF-G which is highly deficient in both GTP hydrolysis and Pi release33. In the presence of GTP, τunwinding for this mutant remained unchanged relative to WT while τrelease increased dramatically (~20 s), occasionally exceeding 50 s (Supplementary Fig. 4b, c). Nonetheless, we observe multi-turnover translocation with this mutant.

If τunwinding is insensitive to perturbed GTP hydrolysis, it is expected to also be insensitive to perturbed Pi release. We therefore tested the H92Q mutant of EF-G which has been reported to exhibit a 25-fold reduced rate of Pi release compared to wild type (WT) without significantly affecting the rate of GTP hydrolysis33. We observed greatly lengthened H92Q EF-G dwell times that extended to multiple seconds (Supplementary Fig. 5). As expected, fitting the distributions of τunwinding and τrelease for H92Q EF-G clearly revealed an unchanged τunwinding compared to WT EF-G, but a significantly lengthened τrelease (2.0±0.4 s) (Fig. 1c, second row, Fig. 1d, e, green dots). Furthermore, the distribution of τrelease for the H92Q mutant, unlike that of the WT, is well described by a single-exponential fit (Fig. 1e, solid line), suggesting that Pi release in these conditions has become the overall rate-limiting step of EF-G unbinding from the ribosome.

To further verify that Pi release is not required for hairpin unwinding, we tested reconstituted ribosomes containing the V67D mutant of ribosomal protein L7/L12, which was shown to slow down Pi release from EF-G34. As in the case of H92Q EF-G, we found that using WT EF-G with V67D ribosomes has no effect on τunwinding but lengthens τrelease (Supplementary Fig. 4a, c).

Taken together, the observed insensitivity of τunwinding to perturbed GTP hydrolysis and Pi release clearly indicates that hairpin unwinding by the ribosome does not require GTP hydrolysis or Pi release by EF-G. On the other hand, the sensitivity of τrelease indicates that GTP hydrolysis and Pi release are required for EF-G release from the ribosome following productive translocation.

Unproductive EF-G binding events do not require GTP hydrolysis
In the hairpin unwinding fleezers assay, EF-G binding is sometimes followed by unbinding without a hairpin unwinding step. These “unproductive” binding events occur randomly throughout the molecular trajectories of the ribosomes (Supplementary Fig. 6a), indicating that they represent a natural sampling behavior of competent translating ribosomes. The mean dwell time of unproductive events (τunproductive) is 300±80 ms for WT EF-G with GTP (Supplementary Fig. 6b, magenta dots, Supplementary Fig. 6c, top green bar).

Interestingly, τunproductive is not lengthened by perturbed GTP hydrolysis and Pi release (Supplementary Fig. 6b, blue, green, and purple dots, Supplementary Fig. 6c, bottom three bars), unlike the total dwell time for productive events (τunwinding + τrelease). This insensitivity indicates that the release of EF-G in unproductive events, in contrast to that of productive ones, does not require GTP hydrolysis. In turn, this observation suggests that during a productive event, after an initial “loose” and reversible binding, EF-G switches into a “tight” binding mode (that will require GTP hydrolysis and Pi release to unbind), whereas during unproductive events EF-G remains loosely bound and can unbind without GTP hydrolysis. Indeed, the existence of multiple modes of EF-G binding to the ribosome has been described by structural18,22,23 and functional35 studies. While we cannot rule out that the initial EF-G binding mode could be different in these two types of events, for example, by transient binding of inactive or misfolded EF-G in the case of the unproductive events, the existence of reversible binding of EF-G·GTP is supported by single-molecule and bulk measurements8,36.

Hairpin unwinding and forward 30S head rotation are temporally correlated
What is the conformational event responsible for unwinding of the mRNA hairpin during EF-G dwell on the ribosome? It has been previously shown that slowing down reverse 30S head rotation does not delay hairpin unwinding32, indicating that unwinding occurs before this rotation. Could forward 30S head rotation be responsible for hairpin unwinding?

To address this question, we first assessed the temporal relationship between hairpin unwinding and forward or reverse head rotation by reconstituting ribosomes with Atto550 and Atto647N fluorophores attached to proteins S12 and S19 on the body and head domains of the 30S, respectively. FRET between these positions reports on the state of 30S head rotation (low FRET corresponding to the rotated state) as shown in bulk stopped-flow experiments24,37 (Supplementary Fig. 7a, b). We performed the hairpin fleezers assay with non-labeled EF-G and doubly-labeled ribosomes to simultaneously monitor multi-turnover hairpin unwinding and single-molecule FRET (Fig. 2a). The trajectories reveal that in most cases, hairpin unwinding occurs around the time that a change from high to low FRET is observed, as expected if forward head rotation coincides with hairpin unwinding (Fig. 2b, c). Although shot noise does not allow the precise timing of FRET change for individual steps, event averaging (n=396) around the unwinding time clearly shows the FRET change to correlate with unwinding (Fig. 2d). Average FRET starts dropping a few milliseconds before unwinding, reaches a minimum around the time of unwinding, and then slowly recovers over a few hundred milliseconds. Fitting to a two-sided exponential yields rate estimates for forward (115±30 s−1) and reverse (5.3±0.4 s−1) head rotation, in general agreement with bulk results24,36,37 (Supplementary Fig. 7c, d).

The observed temporal correlation under unperturbed conditions raises the possibility that hairpin unwinding results from forward head rotation. However, as shown next, such a causal relationship likely does not exist.

Hairpin unwinding does not result from mRNA translocation relative to the 30S body domain
To better estimate when hairpin opening occurs relative to the movement of mRNA with respect to the 30S body (which likely corresponds to forward head rotation), we implemented an alternative fleezers assay that monitors when the movement of mRNA relative to the 30S body domain occurs with respect to EF-G binding. In this assay, DNA handles are used to tether the 5’ end of a hairpin-less mRNA to one bead and the biotinylated protein S16 of the ribosome7 to the other bead (Fig. 3a, d). The applied tension in this geometry assists ribosome translocation along the mRNA, and movement of mRNA relative to the 30S body in each translocation cycle increases the tether extension by one codon (3 nt). We obtained translocation traces in this novel assisting-force geometry and simultaneously monitored Cy3-labeled EF-G binding (Fig. 3g, Supplementary Fig. 8a). We refer to the time between EF-G binding and mRNA stepping (relative to the 30S body) in this assay as τpre, and the time between the step and EF-G unbinding as τpost to distinguish them from τunwinding and τrelease in the hairpin unwinding assay (Fig. 3b, c, e, f).

In the presence of WT EF-G and GTP, the mean total dwell times of EF-G in the assisting force assay are similar to those observed in the hairpin unwinding assay (τunwinding + τrelease = 445±70 ms, τpre + τpost = 308±55 ms, Supplementary Fig. 9, top row). Furthermore, τpre is marginally longer than τunwinding (τpre = 151±45 ms, τunwinding = 72±14, Fig. 3h, top row, left cyan and green bars), suggesting that hairpin unwinding coincides with or is shortly followed by mRNA-body translocation under unperturbed conditions. In the presence of 0.2 mM fusidic acid, an antibiotic that has been shown to slow down only late stages of translocation including reverse 30S head rotation23,38, we observed lengthening of τpost but not τpre (Fig. 3h, second row, Supplementary Fig. 8b), consistent with the idea that the tether extension change in the assisting force assay corresponds to forward, and not reverse, head rotation.

Similar to the hairpin assay, we subjected ribosomes in the assisting force assay to conditions that impede GTP hydrolysis and/or Pi release. Notably, under all of the four perturbed conditions tested, we observed lengthening of τpre and, more severely, τpost on the order of seconds (Fig. 3h, bottom four rows, Supplementary Fig. 8c-f): in the presence of WT EF-G with 0.25 mM GTP mixed with an excess (0.5 mM) of either GDPNP or GTPγS, or in the presence of H92Q EF-G mutant with 1 mM GTP, τpre was lengthened to 1–3 s and τpost was lengthened to 2–7 s. More dramatically, the use of H92A EF-G with 1 mM GTP lengthened τpre and τpost to more than 10 s and 20 s, respectively. The ribosome trajectories in this assay exhibit forward and backward steps in rapid succession during τpre before displaying a decisive step forward (Supplementary Fig. 8c-f), suggesting reversible mRNA stepping attempts before eventual irreversible mRNA translocation relative to the 30S body occurs. Mean total EF-G dwell times (τpre + τpost) in the assisting force assay under GTPase perturbed conditions are longer compared to those in the hairpin unwinding assay (τunwinding + τrelease), likely due to differences in experimental conditions including the analog concentration ratios used (Supplementary Fig. 9, bottom four rows).

The assisting force assay shows that the time between EF-G binding and mRNA translocation relative to the 30S body (τpre) is sensitive to the use of EF-G mutants and GTP analogs, whereas the hairpin assay shows that hairpin unwinding time (τunwinding) is insensitive to those perturbations. Together, these results strongly suggest that hairpin unwinding and mRNA translocation relative to the 30S body are not the same event despite their temporal correlation under normal conditions, and that they can be uncoupled such that unwinding precedes mRNA-body translocation when using EF-G mutants and GTP analogs. The observation that mRNA-body translocation is delayed under GTPase perturbations and hairpin unwinding is not, indicates that translocation is not responsible for driving hairpin unwinding.

mRNA translocation relative to the 30S body is accelerated by GTP hydrolysis but does not require it
Consistent with the established notion that GTP hydrolysis accelerates mRNA translocation, the assisting-force assay results (Fig. 3h) show that both τpre and τpost are lengthened by GTPase perturbations. In particular, this observation mirrors previous bulk single-turnover studies24,36 in which GTPase perturbations delayed both forward and reverse 30S head rotations, corresponding to mRNA-body translocation and EF-G release here, respectively.

The lengthening of both τpre and τpost by GTPase perturbations can be rationalized by a model in which GTP hydrolysis, which normally occurs rapidly upon ribosome-EF-G binding, and possibly Pi release, kinetically accelerate mRNA-body translocation without being thermodynamically required for it, while dissociation of EF-G from the ribosome requires GTP hydrolysis and Pi release, and under these conditions, these processes could occur after mRNA-body translocation. Alternative models in which GTP hydrolysis is strictly required for mRNA-body translocation predict a lengthened τpre, but since in those models GTP hydrolysis has already occurred by the time of mRNA-body translocation, they cannot satisfactorily explain why the subsequent dissociation of EF-G (τpost) is also delayed. Predictions of each model for τpre and τpost under GTPase perturbations are described in Supplementary Fig. 10. Our observations in the assisting force assay best match the model in which GTP hydrolysis and Pi release catalyze but do not thermodynamically fuel, and are not required for, mRNA translocation relative to the 30S body.

Methods
Buffers and reagents
Synthesis of DNA handles. DNA handles (2.5 kb) were generated by PCR using a forward primer modified to contain a 5’ biotin (for bead attachment in hairpin unwinding assays) or digoxigenin (assisting force assays), and a reverse primer containing either an internal 3-carbon spacer (iSpC3) to generate a 21-nt 5’ overhang (for mRNA 5’ annealing), a string of inverted bases to generate a 22-nt 3’ overhang (for mRNA 3’ annealing), or a 5’ biotin (to attach to ribosomes in assisting force assays). PCR products were cleaned up (Qiagen). The digoxigenin-biotin handle was pre-incubated with 10x excess of free streptavidin (Sigma) prior to deposition.

Preparation of mRNA and tRNA. The V50 mRNA which encodes 50 valine codons in the 5’ strand of its hairpin32 (for unwinding assays) and a hairpin-less version of it lacking the 3’ strand of the hairpin (for assisting force assays) were synthesized from EcoRI-linearized plasmid templates using MEGAscript T7 Transcription Kit (Invitrogen) at 37°C for 4 h, purified by polyacrylamide gel electrophoresis separation followed by phenol-chloroform extraction, ethanol precipitation, and G25 desalting, and stored at -80°C. Total tRNA (from Escherichia coli MRE600, Roche) was deacylated by heat treatment and purified via phenol-chloroform extraction and ethanol precipitation32. For experiments with total tRNA, charging was performed at the time of translation mix preparation (see below). For experiments with valine tRNA only, 10 units of valine-specific tRNA (Sigma) were charged using DEAE-purified S100 extract32.

Translation mix. Typically, the translation mix (250 µL) consisted of GTP (Promega), GDPNP (Sigma), and GTPγS (Roche) at concentrations indicated in the text, 1 mM ATP, 420 μM valine, 12 mM creatine phosphate (CP), 0.4 U/μL RNase Out (Invitrogen), 2 μM valine tRNA (in total tRNA), 3.9 μg/mL creatine kinase (CK), 3 μg/mL myokinase (MK), 0.08 μM nucleoside diphosphate kinase (NDPK), 1 μg/mL pyrophosphatase (PP), 1 μM val-tRNA synthetase, 4 μM EF-Tu, and either 10 nM Cy3-labeled EF-G (WT or a mutant as indicated) or 1 μM unlabeled EF-G, in 1x TLC buffer augmented with an oxygen scavenging system (see below) to improve tether and fluorophore lifetimes. The CP, CK, MK, NDP, and PP constitute a phosphate regeneration system. For assisting force assays, a simpler translation mix (250 µL) was used that lacked the regeneration system and only contained 2 µM EF-Tu·GTP·val-tRNAval ternary complex pre-formed as described previously49 at 50 µM in 10 µL, plus EF-G, GTP (or analogs), and the buffer with oxygen scavenging system.

For experiments with labeled EF-G, the oxygen scavenging system consisted of 2 mM Trolox, 0.8% glucose, 0.3 mg/mL glucose oxidase (Sigma), 0.04 mg/mL catalase (Sigma), and 0.5 U/μL RNase Out. For FRET experiments, the mix contained 2 mM Trolox, 0.8% glucose, 0.6 mg/mL glucose oxidase, 0.2 mg/mL catalase, and 1 U/μL RNase Out. Additionally, for FRET experiments, the HEPES concentration was raised to 200 mM for a stronger buffer, and βME was omitted to improve acceptor fluorophore behavior. Due to the acidification of the translation mix by the oxygen scavenging system, the mix was made fresh every 4 h. The mix was passed through a 0.22 μm filter prior to addition to the optical tweezers chamber.

Preparation and labeling of ribosomes and proteins. The preparation of E. coli MRE600 tight-couple 70S ribosomes50, S16-biotinylated ribosomes7, and DEAE-purified S-100 extracts51 was performed as previously described. FRET reporter ribosomes were prepared by in vitro reconstitution24 using fluorophores Atto550 and Atto647N (Attotec). For V67D L7/L12 ribosomes, MRE600 50S subunits were depleted of L12 by NH4Cl/Ethanol treatment34, reconstituted with mutant L12 by incubating at 37°C for 30 min, and associated with natural 30S subunits as described52. Histidine-tagged IF-1, IF-2, IF-3, Val-tRNA synthetase, EF-Tu, and EF-G (unlabeled) were prepared as described32. For labeled EF-G, the single-cysteine variant EF-G (S73C)8 was purified and labeled with Cy3 as described32. The EF-G mutants (H92Q and H92A) were generated from this variant using site-directed mutagenesis (QuickChange, Agilent) and labeled in the same way. The V67D L12 mutant was generated by site-directed mutagenesis from the pSV281 plasmid encoding L1253 and cloned into pET21b excluding any tags. The untagged mutant protein was then expressed in E. coli BLR (DE3) cells essentially as described54 and purified by ion-exchange and size exclusion chromatography using Resource Q, Superdex75, and Resource S columns (Pharmacia Biotech). All proteins were stored in 25 mM Tris·HCl pH 7.5, 60 mM NH4Cl, 10 mM MgCl2 and 5 mM βME at -80°C.

Preparation and deposition of stalled ribosome complexes. Ribosome initiation complex formation was carried out as described previously32 for natural, biotinylated, L7/12 mutant (V67D), or FRET ribosomes, except that a longer incubation time (30–40 min) was used for the FRET ribosomes to improve the yield. Subsequent translocation and stalling of ribosomes at the Lys9 codon was also performed in bulk as described32 to select for active ribosomes and to allow annealing of the 5’ handle (containing biotin or digoxigenin for the unwinding and assisting force assays, respectively). Stalled complexes were flash-frozen and stored at -80°C. For the unwinding assays, a 2 μL stalled complex aliquot was mixed with 1 μL of a 0.1% suspension of 1 μm streptavidin-coated polystyrene beads (Bangs Labs); separately, 1 μL of 100 nM 3’ overhang handle was mixed with 1 μL of the same beads. For assisting force assays, a 2 μL stalled complex aliquot was mixed with 1 μL of a 0.1% suspension of 1 μm anti-digoxigenin antibody-coated beads55, and separately, 1 μL of 100 nM biotin-digoxigenin handle (pre-incubated with streptavidin) was mixed with 1 μL of the same beads. After deposition by incubation at room temperature for 10–20 min, the beads were suspended in 1 mL of 1x TLC buffer (40 mM HEPES pH 7.5, 60 mM NH4Cl, 10 mM Mg(OAc)2, 6 mM βME) and injected in separate channels of the fluidic chamber.

Optical tweezers measurements. Tethers were formed inside the fluidic chamber by placing a stalled-ribosome bead in one optical trap and a handle bead in another. Tethering was made through hybridization of mRNA and the 3’ handle in the unwinding assays, and by biotin-streptavidin binding in the assisting force assay. For unwinding assays, correct tethers containing a single stalled ribosome-mRNA complex were identified by their force-extension unfolding signature32, and for assisting force assays, correct tethers were simply identified by their contour length obtained from force-extension curves. The translation mix was then delivered via a shunt to the tethered stalled ribosomes, while the tether was held at relatively high forces (13.5–14.5 pN for unwinding assays and 9–12 pN for assisting force assays) by manually adjusting the trap distances as needed. These forces were chosen to be sufficiently high to provide single-codon resolution, and in the case of the unwinding assay, also far below the critical force2,32,48 of the hairpin so that hairpin unwinding would not occur spontaneously without the participation of the active ribosome (Supplementary Fig. 1c).

Extension and fluorescence measurements. Extension and fluorescence data were typically collected at 1.33 kHz and 1 kHz, respectively. The instrument was controlled using LabView and the data were visualized and analyzed in MATLAB56. Extension was converted to contour length (and number of codons) using extensible worm-like chain parameters extracted from tether force-extension curves. For experiments with labeled EF-G, transition times for extension (steps) were detected using Hidden Markov Model (HMM) fitting, and those for fluorescence (EF-G binding events) were detected using a combination of HMM and Pruned Exact Linear Time fitting32. Only well-resolved single steps that occurred during well-resolved binding events were included for analysis of productive events, and only well-resolved binding events that did not overlap with steps were included for analysis of unproductive events. The observation of very long (>40 s) fluorescence lifetimes for some of the conditions tested, e.g. the EF-G (H92A) mutant or GDPNP used at high ratios, demonstrated that fluorophore lifetimes do not limit the validity of typical observed event times (a few seconds or less) in these assays. For FRET experiments, FRET ratio was calculated after applying low-pass and median filters to normalized background-corrected donor and acceptor fluorescence signals. A contrast-adjusted “false color” plot, in which the red and green color values correspond to FRET and 1-FRET, was made for better visualization. For event averaging, multiple events were aligned by their unwinding time, and their FRET ratios and extension changes were median-averaged. For estimation of forward and reverse head rotation rates (kf and kr), a two-sided five-parameter exponential function, FRET(t) = h + (l-h) e-kf(t-m), t ≥ m; FRET(t) = l + (h-l) e-kr(m-t), t < m (1) was fitted to the FRET average using the least squares method, where h and l are the high and low average FRET values, respectively, and m the center time (Supplementary Fig. 7c).

Fitting of distributions. For conditions with higher throughput, fitting of EF-G dwell time was done using the maximum likelihood estimation (MLE) method, and characteristic time estimates and 95% confidence intervals are reported. The distribution of τrelease for H92Q fit well to a single exponential distribution, Psingle(t) = ksingle e-ksinglet (2) with the characteristic time <τ> = 1/ksingle. The distribution of τrelease for all other conditions tested was best fit to a bi-exponential distribution, Pbi(t) = f1 k1 e-k1t + (1-f1) k2 e-k2t (3) with individual characteristic times <τ1> = 1/k1 and <τ2> = 1/k2, and the overall characteristic time <τ> = f/k1 + (1-f)/k2 where reported. The distribution of τunwinding was fit to a bi-exponential distribution (Eq. 3) for all conditions tested. Note that for experiments performed with a mixture of GTP and GTP analogs, f for τrelease represents the slow population likely associated with the analog. Results for fits for τunwinding are shown in Supplemental Table 1, results for fits for τrelease are shown in Supplemental Table 2, and results for fits for τunproductive are shown in Supplemental Table 3. The evaluation of statistical significance for different conditions, compared to the WT condition, is shown in Supplemental Table 4. The tests were chosen to assess significance despite the limited sample sizes obtained. In the assisting force assay, compared to the unwinding assay, the stepping signal is smaller (3 versus 6 nt), and the lower signal is exacerbated by noise from the mechanical dynamics arising from ribosome tethering in this assay. These limit the number of data points that can be reliably analyzed for precise time measurements and further compromise the throughput. For all conditions with lower throughput (Supplemental Table 5), or when comparing hairpin unwinding and assisting force assay results (in Fig. 3 and Supplementary Fig. 9), sample means and their standard errors are used instead of fit values for data analysis and visualization.

Statistics and reproducibility
All data sets were reproducible under repeated rounds of data collection. Data from all rounds under each condition were combined to arrive at the final set for that condition. Data for different conditions were collected at a random order and were interleaved to minimize systematic errors. No statistical method was used to predetermine sample size. Sample sizes were sufficient for demonstrating statistically significant differences between GTPase-perturbation groups and the control group (WT EF-G with GTP) as shown in Figs. 1 and 3 and by the statistical tests shown in Supplementary Table 4. Some data points were excluded based on pre-established criteria, including a low signal-to-noise ratio in the optical tweezers or the fluorescence channel, or the presence of obvious experimental artifacts that precluded reliable measurements. No blinding was implemented. However, data from all groups were analyzed via the same pre-defined procedure.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The paper by Van Patten et al. presents experiments that simultaneously monitor several steps of tRNA–mRNA translocation on the ribosome using high‐resolution optical tweezers with fluorescence detection, a method validated in their previous work. This technique enables the reconstruction of the order of events, including mRNA unwinding relative to EF-G binding, mRNA unwinding versus 30S head rotation, and mRNA translocation versus EF-G binding.\nWhile the experiments appear robust overall, aside from some technical issues noted below, the authors do not fully address the questions they initially pose. For example, they begin by asking about the GTP requirement for translocation but then discuss their data in terms of the ground-state thermodynamics of tRNA movement—specifically, whether the energy from GTP hydrolysis directly drives translocation by stabilizing the ground state of reaction intermediates. The distinction between the “mere acceleration” of translocation and a “requirement” for GTP hydrolysis is difficult to follow, as translocation can proceed spontaneously, even in the absence of GTP or EF-G. Thus, it is unclear why “requirement” is interpreted in terms of ground-state stabilization while “acceleration” is (correctly) associated with lowering the transition state barrier.\nThe literature referenced in the manuscript consistently indicates that GTP hydrolysis and subsequent Pi release reduce the transition state barrier for mRNA translocation and promote EF-G release from the ribosome. Earlier kinetic studies suggested that Pi release, rather than GTP hydrolysis per se, is the key step, and recent cryo-EM structures (refs. 18 and 22) demonstrate how Pi release is coupled to tRNA translocation. Most models interpret the effects of Pi release in terms of a reduced transition state barrier, with little evidence supporting the idea that GTP hydrolysis stabilizes or destabilizes the ground state or “drives energetically uphill events” (c.f. p. 2). Power-stroke models, which the authors mention, focus on rate acceleration and, additionally, propose that conformational changes following Pi release prevent backward tRNA movement. There is no doubt that EF-G binding plays an important role in translocation, but the contribution of GTP hydrolysis and Pi release is essential, as they bring the rate of translocation to physiologically relevant levels. When these literature findings are compared with the data presented in Fig. 3—where replacing GTP with a non-hydrolysable analog increases both τ(Pre) (the time to translocation) and τ(Release and Post) (due to impaired EF-G release)—there is no apparent contradiction. Instead, the results are entirely consistent with established models. This section would benefit from revision to clarify this point and avoid suggesting inconsistencies where none exist.\nA second concern relates to the authors' claims regarding the role of GTP hydrolysis and Pi release in stabilizing the ground state of the post-translocation complex. The paper does not provide direct evidence for this statement, as it relies solely on rate measurements, making it difficult to distinguish between ground-state stabilization/destabilization and changes in the transition state barrier. An It is well established that without GTP hydrolysis, EF-G forms a stable complex with the ribosome, however this could reflect a high transition state barrier for EF-G dissociation rather than a true ground state stabilization. Additionally, it is well established that many tRNA pairs on the ribosome exhibit a favorable thermodynamic gradient for forward translocation. However, whether the tRNA(Val) used in this study shares this property remains unclear, and the authors may wish to experimentally assess whether the forward direction is indeed thermodynamically favorable.\nThere is also a potential issue with the energy profile presented in Fig. 4. The model begins with a pre-translocation complex (two tRNAs in the A and P sites) and ends with a complex containing only a tRNA in the P site—a state expected to be destabilized due to the loss of A-site interactions. This raises the question of why the final state (N+1) appears to have a lower energy than the pre-translocation state (N) after EF-G dissociation in the presence of GTP. A more detailed discussion of this aspect would improve clarity. Additionally, performing single-codon translocation experiments with tRNAs that thermodynamically favor backward translocation could provide further insight into the role of GTP hydrolysis.\nThe section on mRNA helix unwinding is also somewhat unclear. The data suggest that unwinding occurs early and is independent of both GTP hydrolysis and Pi release, aligning with the authors’ previous work in Molecular Cell. However, the overlap with their earlier findings and the novelty of these results are not clearly delineated, and the final mechanistic interpretation of unwinding remains somewhat speculative. Similarly, the observation that EF-G can bind and unbind from the ribosome before productive engagement has been reported in several smFRET studies and is already incorporated into most translocation models. Its inclusion in this study appears to serve completeness rather than introduce novel mechanistic insight.\nIn addition, the terminology used to describe conformational changes is confusing. The authors refer to “30S head rotation” without clarifying whether this term is equivalent to “swiveling” or how it relates to the formation of the chimeric state—a key intermediate in translocation, as shown by the Noller group. Cryo-EM studies (refs. 18 and 22) indicate that tRNA movement into the chimeric state requires Pi release. Notably, there is no disagreement between these two papers; rather, both differ from ref. 23 (as discussed in refs. 18 and 22). Given these findings, the order of events in Fig. 4 (where complete forward head rotation is shown before Pi release) appears to be reversed. Since Pi release is not measured directly in this study, the rationale for this ordering remains unclear. The discussion also relies on findings with the EF-G H92Q mutant, which is based on prior work of a different lab suggesting that this mutant retains GTPase activity but has reduced Pi release (ref. 33). However, concerns arise from the fact that the supposedly GTPase-deficient H92A mutant still shows significant GTPase activity (ref 33), possibly due to contamination, casting doubt on the reliability of these findings. It would be important for the authors to validate that the H92Q mutant is indeed GTPase-active but specifically deficient in Pi release.\nFinally, several technical issues warrant attention. The quality of the smFRET and stopped-flow data for 30S head rotation does not appear sufficient to support strong conclusions. In Fig. 2C (middle panel), the donor and acceptor traces exhibit little anticorrelation, making it difficult to confidently interpret the FRET signal. Similarly, in Supp. Fig. 7, the trace is very noisy, and the final signal does not return to the start value, as would be expected for complete forward and reverse 30S head rotations. The EF-G binding trajectories in Supp. Fig. 8 also appear unusually noisy. Additionally, it remains unclear how EF-Tu behaves in experiments using non-hydrolysable GTP analogs—does it also exchange nucleotides, similarly to EF-G, and if so, how does this affect the experiments?\nOverall, while the experiments are valuable and the results broadly align with existing models, the Introduction and Discussion need further clarification and focusing. Addressing the concerns outlined above—particularly the interpretation of GTP hydrolysis and Pi release, the sequence of conformational changes, and the technical quality of key data—would significantly strengthen the manuscript.", "reviewer_2": "The manuscript of Van Patten et al. is an important piece of biophysics that explores the mechanism by which the ribosome unwinds mRNA structure during translocation. This has been an area of debate, in particular the role of GTP hydrolysis in the translocation step and in the unwinding of RNA secondary structures during translocation. Bustamante and co-workers have previously performed elegant optical trapping and correlated fluorescence-force (fleezer) experiments to outline the general dynamics and energetics of translation elongation through a long hairpin. Here they expand these approaches using the fleezer assay correlated with hairpin unwinding using a variety of GTP concentrations and analog mixtures as well as mutant EF-Gs that change hydrolysis or phosphate release. The authors study the effect of GTP hydrolysis and phosphate release from EF-G on the unwinding of an mRNA hairpin during elongation using a combined optical tweezers/fluorescence microscopy technique (fleezers) by using GTP analogues and EF-G mutants. These first experiments beautifully correlate EF-G binding events with hairpin unwinding in 3 nt steps (shown previously). Their results show that GTP hydrolysis or phosphate release does not have any effect on hairpin unwinding but does affect the departure of EF-G from the ribosome and return of the ribosome to the post-translocation state. Their results suggest that tight EF-G binding leads to hairpin unwinding and subsequent mRNA movement based on previous structures and the fact that they see futile “loose” EF-G binding events that are not dependent on GTP hydrolysis. In a parallel set of fleezer experiments, they use doubly-labeled 30S subunits to correlate hairpin unfolding with head-body relative movement and here see a correlation with FRET changes (head rotation) and unwinding. Finally, the perform a distinct force measurement, pulling on the 30S subunit directly on an unstructured mRNA where translocation extends the tether, and correlate these events (under force) with EF-G binding under different conditions as above for the hairpin unwinding.\nThe results here clearly support their model whereby GTP hydrolysis is required only to release a tightly bound EF-G from the ribosome, where the translocation/unwinding etc is driven by the free energy of EF-G binding to the ribosome. This is an important work in the biophysics of translation and absolutely deserves publication in Nature Communications. For the most part it is clearly written and presented and the data are rigorously analyzed. I have some suggestions that should hopefully help the authors improve the presentation.\n1. For the head to body FRET experiments, S12 to S19, the data are very noisy. This probably a highly dynamic conformation, as also shown by Fig sup 7 bulk FRET. I do not believe single-molecule FRET data has ever been presented, so I would have liked to see the behavior of this signal in TIRF under a range of conditions. Have the authors measured any FRET, which would allow the reader to better understand the Fleezer experiment.\n2. Some of the figures are hard to follow, in particular the schematic in Figure 2. Its hard to see the double dye position. Maybe the authors could improve this figure, and also add a structure figure to supplemental data to show the dye positions in the non-rotated and rotated head states.\n3. Fig 4 schematic free energy diagram is critical, and I wonder how the relative magnitudes for the free energies were calculated. There is no scale on the z-axis, but there is the free energy of peptide bond formation and GTP hydrolysis (where did these values come from? The PT value seems too small to me.\n4. One key aspect of the model is the idea of EF-G “tight binding”. It would be nice if the authors could expound more on this state in the text and what is the free energy of this binding interaction. Do they have data that at least put bounds on the difference from the weak (sampling) and “tight” state in terms of lifetime/free energy?\nLove this work--Jody Puglisi", "reviewer_3": "In this paper, Van Patten et al. use single-molecule approaches to investigate the role of GTP turnover by EF-G in driving ribosome translocation during translation. Using a hairpin unwinding assay with fluorescent EF-G, they show that slowing GTP hydrolysis/ Pi release elongate EF-G binding after, but not before, each codon unwinding event. Using an assisting-force translocation assay with fluorescent EF-G, they show that slowing GTP hydrolysis/ Pi release elongates EF-G binding both before and after a one-codon translocation. They conclude unwinding and translocation are distinct events, associated with a “tightening” isomerization event and head rotation, respectively; neither event relies on GTP hydrolysis of Pi release. They propose that the energy from GTP is instead used for undoing the “tightening” isomerization, as needed to proceed to the next cycle. The single-molecule Fleezers experiments are intricate, and the data quality are excellent. Overall, I find that the claims made are relatively well-justified by the data provided, and think that the impact will be well-appreciated in the translation and single-molecule fields. However, I do have some concerns, listed below, about some assay details and more generally about statistics from the very small data sets acquired. I recommend publication after addressing these issues in revision.\nMajor concerns\n1. The arguments in this work depend heavily on the priors that GDPNP is nonhydrolyzable and that GTPγS is slowly hydrolysable by EF-G. These are likely true assumptions, but it would strengthen the work to test them directly using the exact constructs tested in the single-molecule assays. A commercially available phosphate detection assay (e.g. Malachite green) would suffice.\n2. Fig. 2 data are not particularly convincing. If noise is a limiting factor (line 224-225), and averaging was used to improve baselines, then why were only N=27 steps analyzed? I do not understand how the 20 ms estimate was made. Furthermore, there are essentially no claims made from the data (even the temporal correlation claim is later negated). Getting this experiment to work would strongly improve the impact of this paper. However, if revisiting this experiment to find conditions with cleaner FRET signal is out of the question, then an alternative option is to remove the figure from the manuscript entirely (lines 85-88 similarly).\n3. Fig. 3: I am concerned as to whether or not translation is occurring here. Please provide more raw data in the main figure space. Fig. S8 only shows single steps -- was there any indication of processive translation in these experiments? If not, and they are indeed only single steps, what evidence is there that the observed steps are actually coupled with translation (was a peptide bond made)? If peptide synthesis is not occurring, I am not convinced that the results are meaningful. Peptidyl transfer is repeatedly identified as a key part of the overall thermodynamics in this system.\n4. In both assay types used in this work (hairpin and assisting force), the applied force contributes to the kinetics measured. The authors should investigate whether the trends they observe are independent of applied force by testing more than one force level. Particularly since a goal of the work is to investigate underlying driving forces.\nMinor concerns\n1. Fig. 1b, why do ~half the steps not have an associated fluorescence peak? Is the ribosome stepping without EF-G? Are a subset of the EF-G molecules unlabeled?\n2. Please provide details in main/methods for how errors on characteristic times were determined. Are they 95% CI from bootstrapping the CDF fit? Simple CI from fitted residuals may underestimate error in CDF fitting.\n3. Towards the point above, the N event reported throughout this work are low. Typically 60-90 events, of which less than half contribute to the interpretable phase of the bi-exponential fits. Some are ~N=20. I appreciate the difficulty/labor of these complex experiments, and am reluctant to request higher N even though I think it would strengthen the work. Minimally, please explain assay limitations in the main or methods, and justify your acceptance of these very small sample sizes..\n4. Fig. 1c, why is the sample mean reported, rather than 1/k2? The fast rate constant is interpreted as the GTP rate for the mixed nucleotide experiments, and k2 should be within error for all experiments with GTP as such. It is unclear why “WT” matches a bi-exponential, and what the slow phase may represent. Please explain.\n5. Fig. 2 may be difficult to read for individuals who are red-green colorblind\n6. Methods: Translation mix lists valine – specific components. Is this true? Synthesizing poly-valine? Is this important? Please provide more details.\n7. Supp. Table 1: Only one of the three GFPNP conditions ran has its fit reported. Table should be the same size as Supp. Table 2."}, "author_response": "We thank the reviewer for their detailed comments and helpful suggestions. We have addressed individual remarks below. Descriptions of updates made to the manuscript are indicated in boldface below. All changes in the manuscript DOCX file itself have been made with ‘Track Changes’ enabled.\nWhile the experiments appear robust overall, aside from some technical issues noted below, the authors do not fully address the questions they initially pose. For example, they begin by asking about the GTP requirement for translocation but then discuss their data in terms of the ground-state thermodynamics of tRNA movement—specifically, whether the energy from GTP hydrolysis directly drive translocation by stabilizing the ground state of reaction intermediates. The distinction between the “mere acceleration” of translocation and a “requirement” for GTP hydrolysis is difficult to follow, as translocation can proceed spontaneously, even in the absence of GTP or EF-G. Thus, it is unclear why “requirement” is interpreted in terms of ground-state stabilization while “acceleration” is (correctly) associated with lowering the transition state barrier.\nWe feel there may have been a misunderstanding and we appreciate the opportunity to clarify the questions addressed in the paper. In Introduction, we asked the “fundamental question of whether the energy from GTP hydrolysis and Pi release are required to drive the energetically uphill events during ribosomal translocation.” (paragraph 3, last sentence.) This question directly aims at the energetic contribution of GTP hydrolysis/Pi release; specifically, at what time is this contribution required during translocation? The energetic requirement is best interpreted in terms of an energy landscape, where free energy differences between intermediates determine whether transitions between them are thermodynamically favorable. For example, if GTP hydrolysis was energetically required for mRNA unwinding, transition to the unwound state would be unfavorable (could not occur irreversibly) unless in the presence of GTP hydrolysis. The energetic requirement is distinct from kinetic effects, where an already favorable transition can be accelerated (by lowering the transition barrier) or not, depending on the presence of GTP for example. Also, as the reviewer mentioned, and as we also stated in Introduction (paragraph 3, line 9), “translocation can proceed spontaneously, even in the absence of GTP or EF-G, albeit at very slow rates and under special conditions” but this has only been observed on unstructured mRNA. While this observation indicates that the free energy released as a result of peptidyl transfer (estimated 3-4kcal/mol by Liu et al, ref 7) is sufficient to favor unidirectional translocation on unstructured mRNA, the presence of stable hairpins changes the energy balance significantly. We have updated the Introduction and Discussion text to clarify these points. As the updated text indicates, hairpins can be as stable as ~9kcal/mol per codon, so the energy landscape in the presence of stable hairpins may be unfavorable overall unless the GTP hydrolysis contribution (~12kcal/mol) is also supplied. Importantly, however, this does not automatically mean that prior GTP hydrolysis is directly required for unwinding and translocation in each translocation cycle. Rather, in principle, another exergonic transition (such as EF-G tight binding) can first supply the energy to unwind the hairpin, with GTP hydrolysis being required only later to reverse the transition and reset the system. Indeed, our results support exactly this type of scenario, which we discuss in the manuscript.\nThe literature referenced in the manuscript consistently indicates that GTP hydrolysis and subsequent Pi release reduce the transition state barrier for mRNA translocation and promote EF-G release from the ribosome. Earlier kinetic studies suggested that Pi release, rather than GTP hydrolysis per se, is the key step, and recent cryo-EM structures (refs. 18 and 22) demonstrate how Pi release is coupled to tRNA translocation. Most models interpret the effects of Pi release in terms of a reduced transition state barrier, with little evidence supporting the idea that GTP hydrolysis stabilizes or destabilizes the ground state or “drives energetically uphill events” (c.f. p. 2). Power-stroke models, which the authors mention, focus on rate acceleration and, additionally, propose that conformational changes following Pi release prevent backward tRNA movement. There is no doubt that EF-G binding plays an important role in translocation, but the contribution of GTP hydrolysis and Pi release is essential, as they bring the rate of translocation to physiologically relevant levels. When these literature findings are compared with the data presented in Fig. 3—where replacing GTP with a non-hydrolysable analog increases both τ(Pre) (the time to translocation) and τ(Release and Post) (due to impaired EF-G release)—there is no apparent contradiction. Instead, the results are entirely consistent with established models. This section would benefit from revision to clarify this point and avoid suggesting inconsistencies where none exist.\nThere are inconsistencies with some previous interpretations in the sense that kinetic results had been interpreted by some investigators (e.g., in refs 4,15,17,18,35,39,40) to mean that the energy of GTP hydrolysis or Pi release is what drives the mechanical translocation of mRNA (also necessarily its unwinding if base-paired), summarized as “Converting GTP hydrolysis into motion” (title of ref 17). We did not intend to dispute the important and well-known kinetic role of EF-G-mediated GTP hydrolysis in accelerating translocation. Rather, our results clarify and emphasize the separation between kinetic and energetic contributions of GTP hydrolysis to specific events during translocation. This is because, unlike most previous studies, we can directly probe mechanical work performed by individual ribosomes during translocation.\nTranslocation involves multiple intermediates with transition states between them. While GTP hydrolysis/Pi release accelerates the pathway as a whole, it is important to determine which intermediates and transitions are specifically affected. And the effect on each intermediate could be energetic (stability of the intermediate), kinetic (height of barrier to that intermediate), or both. In this manuscript, we particularly examined the effect on unwinding of mRNA and on mRNA-body translocation. We found that unwinding is not affected at all (kinetically or thermodynamically); furthermore, mRNA-body translocation still occurs without hydrolysis, meaning it is still thermodynamically favorable although kinetically slow. This is inconsistent with the view that mRNA translocation and unwinding are driven primarily by GTP hydrolysis/Pi release energy.\nTo avoid suggesting inconsistencies where they do not exist, we have amended the text in the last section of Results, and also changed the title of that section to emphasize the well-known kinetics aspect. We have also added a note in Discussion (paragraph “Our findings enable ..”) to highlight the distinction between causal and temporal relationships. We are grateful to the reviewer for the clarifying suggestion.\nWe will address the cryo-EM structure interpretations in response to another comment further below.\nA second concern relates to the authors' claims regarding the role of GTP hydrolysis and Pi release in stabilizing the ground state of the post-translocation complex. The paper does not provide direct evidence for this statement, as it relies solely on rate measurements, making it difficult to distinguish between ground-state stabilization/destabilization and changes in the transition state barrier. An It is well established that without GTP hydrolysis, EF-G forms a stable complex with the ribosome, however this could reflect a high transition state barrier for EF-G dissociation rather than a true ground state stabilization. Additionally, it is well established that many tRNA pairs on the ribosome exhibit a favorable thermodynamic gradient for forward translocation. However, whether the tRNA(Val) used in this study shares this property remains unclear, and the authors may wish to experimentally assess whether the forward direction is indeed thermodynamically favorable.\nOur depiction of the stabilization of the post-translocation state due to GTP hydrolysis and Pi release is based on thermodynamic principles rather than direct observation: when the GTP hydrolysis reaction (GTP + H2O -> GDP + Pi) is coupled to translocation, the ΔG between the pre- and post-translocation states of the system with hydrolysis will differ from that without hydrolysis by precisely the amount of the free energy change of the hydrolysis reaction. (It is always the free energy of the entire system of reactants and products that is considered.) We have updated the Discussion paragraph (“The energy expenditure of ..”) which we hope better clarifies this point.\nWe agree with the reviewer that based solely on our results, lengthening of EF-G release time could in principle be kinetic (not thermodynamic). However, the bulk studies showing stable ribosome-EF-G binding in the absence of GTP hydrolysis (e.g., in complex with GTP analogs) as mentioned by the reviewer, provide direct evidence for this claim, as they are performed under equilibrium or near-equilibrium where a small dissociation constant (stable complex) corresponds to thermodynamically unfavorable unbinding.\nDifferent tRNAs have indeed been shown to have different propensities for forward translocation, and it has been suggested that the spontaneous factor-free translocation observed for poly-U mRNA (poly-Phe) may not be reproducible in other contexts. Although we wish to study the effect of different tRNAs, not just valine tRNA, in future studies, we note that the conclusions of this manuscript are unaffected by any tRNA-specific difference. In fact, a smaller degree of favorability or even unfavorability of translocation without EF-G (black path in Fig 4b) as alluded to by the reviewer, would strengthen our argument. We further expand on this point in response to the next comment.\nThere is also a potential issue with the energy profile presented in Fig. 4. The model begins with a pre-translocation complex (two tRNAs in the A and P sites) and ends with a complex containing only a tRNA in the P site—a state expected to be destabilized due to the loss of A-site interactions. This raises the question of why the final state (N+1) appears to have a lower energy than the pre-translocation state (N) after EF-G dissociation in the presence of GTP. A more detailed discussion of this aspect would improve clarity. Additionally, performing single-codon translocation experiments with tRNAs that thermodynamically favor backward translocation could provide further insight into the role of GTP hydrolysis.\nWe thank the reviewer for highlighting this issue. Indeed, the pre- and post-translocation states depicted in Fig 4 only span the translocation phase of the elongation cycle, and do not represent the same state in successive elongation cycles. Their ΔG is therefore not that of the entire elongation cycle which is on average equal to ΔG . We have thus replaced ΔG with ΔG in Fig 4b peptidyltransfer peptidyltransfer intrinsic which allows it to have any value. Because of the loss of A-site interactions as the reviewer mentioned, ΔG could be less negative than ΔG (closer to zero or even positive for some tRNAs; see previous response). We have also amended the corresponding text in the Discussion paragraph (“Accordingly, a conceivable ..”) to improve clarity on this point.\nWe note that the pre-translocation complex follows peptidyl transfer and corresponds to the hybrid-state conformation. Thus, we do not start with two tRNAs in the A and P sites in Fig 4, but rather with one tRNA in the A/P state and the other in the P/E state. The P/E tRNA is very weakly bound, so there would be no large loss of binding energy when it moves to the E site. Therefore, the main energy event is movement of the A/P tRNA to the P site (P/P state). Its CCA end does not change much on the 50S subunit, so the change in binding energy is essentially that of the anticodon step loop (ASL). The ASL moves from the 30S A site to the 30S P site, which are similarly strong binding sites. In fact, it is believed that the P/P state is stronger than the A/P state. Together, these considerations would agree qualitatively with a negative total free energy change depicted in Fig. 4.\nIf anything, taking into account the tRNA binding energy differences and having a diminished (or even positive) overall ΔG between pre and post states in the uncatalyzed (black) path in Fig 4b would bolster, rather than weaken, the two major qualitative takeaways from our proposed landscape: (1) the proposal that EF-G tight binding drives mRNA unwinding and mRNA-body translocation will be bolstered, because the role of GTP hydrolysis is eliminated by our results and the other source of energy (now denoted ΔG ) can now be more easily excluded, and (2) the idea that EF-G-ribosome binding is intrinsic thermodynamically trapped unless rescued by GTP hydrolysis is also bolstered, because now there is no source of energy other that GTP hydrolysis to help favor EF-G release.\nTherefore, we believe that while performing translocation with other tRNAs will be informative, they are not essential for the arguments presented in the manuscript.\nThe section on mRNA helix unwinding is also somewhat unclear. The data suggest that unwinding occurs early and is independent of both GTP hydrolysis and Pi release, aligning with the authors’ previous work in Molecular Cell. However, the overlap with their earlier findings and the novelty of these results are not clearly delineated, and the final mechanistic interpretation of unwinding remains somewhat speculative. Similarly, the observation that EF-G can bind and unbind from the ribosome before productive engagement has been reported in several smFRET studies and is already incorporated into most translocation models. Its inclusion in this study appears to serve completeness rather than introduce novel mechanistic insight.\nThe unwinding results are entirely novel and form a major contribution of this work. Our previous study published in Molecular Cell only employed WT conditions with GTP and did not address the role of GTP hydrolysis. The antibiotic fusidic acid was employed in that study to prevent EF-G release, which lengthened τ as expected. The present study is the first to directly examine the effect of GTP release hydrolysis and Pi release on single-molecule unwinding kinetics, and we used various perturbations including the use of GTP analogs and EF-G and ribosome mutants in the unwinding assay. While the mechanism of unwinding remains speculative, the results are nevertheless highly significant as they show that the ribosome-EF-G complex can readily and rapidly unwind a stable hairpin (a significant amount of mechanical work) regardless of whether GTP can be hydrolyzed. We re-worded the Results text (first section, second paragraph) to highlight that the effect of GTP hydrolysis on τ and unwinding τ was previously unknown. release\nIt is true that unproductive EF-G binding has been reported, as we also cite in the manuscript. Here we have reported what we observed in our experimental traces. Furthermore, observation of unproductive binding and its insensitivity to GTPase perturbations are significant because they support our interpretation that for the ribosome to commit to translocation, EF-G must undergo a tight binding transition beyond the initial loose form of binding. Since tight binding can perform work (is exergonic), the insight from these observations is that hairpin unwinding (which requires work) can be driven by the free energy available from the EF-G tight binding.\nIn addition, the terminology used to describe conformational changes is confusing. The authors refer to “30S head rotation” without clarifying whether this term is equivalent to “swiveling” or how it relates to the formation of the chimeric state—a key intermediate in translocation, as shown by the Noller group.\nAs suggested, we have added clarification in the Introduction (first paragraph, tracked changes) to note that 30S head rotation refers to the same motion known as “swiveling” and that it produces the chimeric hybrid tRNA states.\nCryo-EM studies (refs. 18 and 22) indicate that tRNA movement into the chimeric state requires Pi release. Notably, there is no disagreement between these two papers; rather, both differ from ref. 23 (as discussed in refs. 18 and 22). Given these findings, the order of events in Fig. 4 (where complete forward head rotation is shown before Pi release) appears to be reversed. Since Pi release is not measured directly in this study, the rationale for this ordering remains unclear.\nWe believe that the structural data in refs 18, 22, and 23 are not conclusive whatsoever regarding whether 30S head rotation (chimeric hybrid state formation) requires Pi release. The set of ribosome configurations observed in these three studies are essentially the same. In all three, a head-non-rotated Pi-retaining state and a head-rotated Pi-released state are observed. Since two correlated changes (head rotation and loss of Pi) are seen at the same time, it is not possible to establish temporal precedence and therefore it is not possible in these studies to establish a causal connection. Indeed, Petrychenko et al (ref 18) interpreted the results to mean Pi release triggers the rotation, Carbone et al (ref 23) interpreted the opposite order, while Rundlet et al (ref 22) took a more agnostic position. With our powerful single-molecule assays, we have direct evidence that abolishing GTP hydrolysis and Pi release does not abolish completion of mRNA-body translocation (corresponding to head rotation). This result, which is consistent with recent stopped-flow experiments by Rexroad et al (ref 24), is presented and elaborated on in the final section of Results and in the Discussion. It indicates that at least under GTPase-perturbed conditions, head rotation happens before/without GTP hydrolysis and Pi release. Whether the same order also holds under normal conditions is not known; we have therefore remained cautious in Fig 4 and indicated “Pi release (if delayed)” (rather than just “Pi release”) on the dashed line separating states III and IV. Also, the red gradient bar at the bottom of Fig 4a marks the likely timeline of “GTP hydrolysis and Pi release (normally fast if unperturbed).” As the reviewer mentioned, we do not monitor Pi directly, and we do not claim to time this event independently from GTP hydrolysis using our results, except that it can occur only after hydrolysis. To further emphasize this, we now mention in Discussion (paragraph “Our findings enable ..”) to indicate that Pi release may temporally precede head rotation when not delayed. We thank the reviewer for bringing this issue to our attention.\nThe discussion also relies on findings with the EF-G H92Q mutant, which is based on prior work of a different lab suggesting that this mutant retains GTPase activity but has reduced Pi release (ref. 33). However, concerns arise from the fact that the supposedly GTPase-deficient H92A mutant still shows significant GTPase activity (ref 33), possibly due to contamination, casting doubt on the reliability of these findings. It would be important for the authors to validate that the H92Q mutant is indeed GTPase-active but specifically deficient in Pi release.\nWe can only speculate on why the estimated GTPase activity for H92A was higher in Koripella et al (ref 33) compared to earlier studies (e.g., Cunha et al, ref 39) but the possibility of WT contamination does not necessarily negate the findings regarding the other mutant H92Q which clearly appears defective. As the reviewer noted earlier, we have no direct measure of GTP hydrolysis or Pi release in our assay. In our interpretations, we have accordingly treated the two chemical events together, one simply following the other. Therefore, our conclusions do not rely on the validity of measurements by Koripella et al (ref 33). This is particularly the case since we observe qualitatively similar results for H92Q and H92A in our assays. Had we seen drastically different results, then the contrast between H92A and H92Q would have been more relevant.\nFinally, several technical issues warrant attention. The quality of the smFRET and stopped-flow data for 30S head rotation does not appear sufficient to support strong conclusions. In Fig. 2C (middle panel), the donor and acceptor traces exhibit little anticorrelation, making it difficult to confidently interpret the FRET signal. Similarly, in Supp. Fig. 7, the trace is very noisy, and the final signal does not return to the start value, as would be expected for complete forward and reverse 30S head rotations. The EF-G binding trajectories in Supp. Fig. 8 also appear unusually noisy. Additionally, it remains unclear how EF-Tu behaves in experiments using non-hydrolysable GTP analogs—does it also exchange nucleotides, similarly to EF-G, and if so, how does this affect the experiments?\nOur smFRET assay is a tour de force experiment that involves co-temporally monitoring two single-molecule channels, the smFRET channel and the optical tweezers channel, during active translation for the first time. The low signal to noise is in part due to the small FRET change (only ~0.3) and the short-lived low-FRET state. Despite noisy traces, we see clear anticorrelation between donor and acceptor emissions, as shown in Fig 2c where not only the green donor signal increases near unwinding steps but the red acceptor signal also decreases correspondingly. The correlation coefficient for the section shown is -0.7. Importantly, the conclusion we draw from the smFRET assay is merely that there is a temporal correlation between unwinding and forward head rotation (FRET decrease) under normal conditions. We believe the smFRET results strongly support such a correlation. To strengthen this claim, we have now added a large number of new data for the event averaging shown in Fig 2d, from n=27 in the original manuscript to n=~400, which shows FRET decrease near the unwinding steps in a much more convincing manner. We have also updated Supp Fig 7 with additional panels. We believe the new analysis strengthens our work and we thank the reviewer for the constructive criticism.\nWhile the raw data from the stopped-flow measurements shown in Supp Fig 7 (now panel b) are naturally noisy, they fully agree with previously published results (refs 24 and 37) and show a clear trend (an initial rapid decrease followed by a slow recovery) with kinetic parameters that match those measured previously. This result is included for completeness in our study to show that our Atto dyes produce the same behavior as the Alexa dyes used in the previous works. The interpretation of data and the kinetic parameters (rate and amplitude of forward and reverse head rotation curves) had been done in the cited references and we did not feel obliged to repeat those for this control experiment, as the original authors of those publications are also co-authors in this paper.\nThe assisting force data (in Supp Fig 8) are noisier compared to the unwinding assay because of smaller step size (3 vs 6 nt) and larger dynamics due to ribosome tethering. However, we can still see clear steps and EF-G binding events. In the new Fig 3 (panel g), we have added a trajectory with multiple steps to demonstrate that processive translocation by exact 3nt (codon) steps is taking place. We have also added a multi-step panel to Supp Fig 8 for the GDPNP condition, clearly showing (long) EF-G binding events.\nAs mentioned in Methods, EF-Tu was pre-incubated with GTP and loaded with tRNA to form a ternary complex before being added to the translation mix containing the GTP analogs. We expect the long half-life of ternary complex to limit the effect of the analogs on EF-Tu, but we cannot rule out that this factor may have also been affected. If EF-Tu activity is affected by the analogs, the time between consecutive unwinding steps (outside of the EF-G-Cy3 binding dwell time) would be expected to increase, for which we have seen no evidence. Importantly, since we focus on the EF-G dwell time in our analysis of translocation, such an increase would not affect our measurements or our conclusions regarding translocation. We have now noted the possibility that EF-Tu may be affected in Results (paragraph “GDPNP is ..”).\nOverall, while the experiments are valuable and the results broadly align with existing models, the Introduction and Discussion need further clarification and focusing. Addressing the concerns outlined above—particularly the interpretation of GTP hydrolysis and Pi release, the sequence of conformational changes, and the technical quality of key data—would significantly strengthen the manuscript.\nWe thank the reviewer again for all the feedback. We have made several changes in the Introduction and Discussion sections of the manuscript that we hope have helped with clarity and focus."}, {"round": 2, "reviewer_comments": {"reviewer_1": "In their revised manuscript, the authors have improved the technical quality of the data in Fig. 2d, increasing the number of events from n = 27 in the original manuscript to ~400, and they have introduced a few additional explanations. However, they did little to revise the text in response to my critical comments. I therefore reiterate my main concerns here, in slightly different words, to make my points absolutely clear.\n1. Novelty of the conclusions\nThe novelty is limited to the claim that mRNA unwinding is independent of GTP hydrolysis or Pi release. While this is possible, it is not sufficient to justify the energy diagram presented in Fig. 4. Moreover, it remains unclear whether the pulling force applied by optical tweezers destabilizes RNA structures to the point where unwinding becomes independent of external factors. This possibility is not adequately addressed. The other central messages—that translocation can occur without GTP hydrolysis and that the effect of GTP hydrolysis is kinetic—are not new; they have been reported repeatedly over the past 30 years. As such, the manuscript does not resolve any of the existing controversies.\n2. Placement of Pi release in the energy diagram\nThe most critical issue is the order of steps in the authors’ energy diagram. By placing Pi release after translocation, the authors implicitly assign Pi release as the trigger for EF-G dissociation. Their own data provide no direct evidence for this placement, and it contradicts the bulk of the literature. In particular, the cited cryo-EM structures (Petrychenko et al. and Carbone et al.) consistently show EF-G in the GDP–Pi state before tRNAs reach the chimeric hybrid state, and in the GDP state once the hybrid state is reached. This strongly argues against positioning Pi release after tRNA movement, as the authors do in Fig. 4. (The Rundlet et al. study [ref. 23] is not informative, as their assignment of a GDP–Pi intermediate as GTP is a misinterpretation of cryo-EM density.)\nAdditional kinetic studies from the Rodnina and Cooperman groups, which measured Pi release directly and compared it with defined steps of translocation, are in full agreement with the cryo-EM studies. These data are either ignored or downplayed by the authors, despite being directly relevant. Correctly placing Pi release at the step of tRNA movement (consistent with the measured kinetic effect) would yield a model fully consistent with existing cryo-EM data and with the prevailing loaded-spring GTPase model, in which Pi release triggers conformational rearrangements of EF-G that bias tRNA movement and accelerate translocation. In my opinion, the model in Fig. 4 must be revised accordingly: Pi release should be placed before tRNA movement to the chimeric states, and the corresponding energy barrier for translocation should be lowered.\n3. EF-G H92Q mutant\nThe interpretation of the results obtained with the EF-G H92Q mutant hinges entirely on its biochemical activity. Does this mutant, unlike other H92 variants, hydrolyze GTP but fail in Pi release? This must be tested explicitly by assaying GTP hydrolysis and Pi release. Without such validation, the interpretation of the mutant data is not reliable.\n4. Misrepresentation of the literature\nThe manuscript misrepresents earlier models for how GTP hydrolysis and Pi release drive translocation. While terms such as “power stroke” or “lever arm” have been used, these models (originating from multiple groups, not only Rodnina et al., as the citations suggest) were never meant to imply changes in the ground state. Rather, they consistently explain the kinetic effects of GTP hydrolysis in terms of lowering the transition barrier. This is clearly visible in the energy diagrams in the cited studies. By writing in the Introduction that “the energy of GTP hydrolysis and Pi release is directly converted to mechanical work,” the authors misrepresent the intent of prior work.\n5. Abstract statement on thermodynamic requirement\nGiven the problems outlined above regarding the placement of Pi release, I question the validity of the Abstract’s statement that “…while GTP hydrolysis kinetically accelerated translocation, it is thermodynamically required only to liberate the tightly bound EF-G from the ribosome.” The overall trajectory may differ from the one the authors propose, and in fact alternative models consistent with this possibility have already been suggested in the literature (e.g., ref. 36).\n6. Description of the tRNA trajectory\nA remaining issue concerns the Introduction’s description of tRNA translocation. The authors state that swiveling of the 30S head domain moves the tRNA and mRNA into chimeric hybrid states, which is correct. However, they omit that this step is accompanied by a backward movement of the 30S body domain. This feature is well established from cryo-EM structures (refs. 18, 22) and stopped-flow data, and it is an essential element of the translocation mechanism. It should be explicitly included.", "reviewer_2": "The authors have nicely and rigorously addressed my concerns, and also those of the other reviewer. The manuscript is now acceptable for publication in Nature Comm.", "reviewer_3": "The authors have addressed all my questions and suggestions appropriately. This is a wonderful manuscript, and I recommend it for publication."}, "author_response": "We thank all the reviewers for the constructive criticism. Author responses to the remaining points are written in blue. Descriptions of changes to the manuscript are in boldface. All new changes in the manuscript DOCX file have been tracked.\nIn their revised manuscript, the authors have improved the technical quality of the data in Fig. 2d, increasing the number of events from n = 27 in the original manuscript to ~400, and they have introduced a few additional explanations. However, they did little to revise the text in response to my critical comments. I therefore reiterate my main concerns here, in slightly different words, to make my points absolutely clear.\n1. Novelty of the conclusions\nThe novelty is limited to the claim that mRNA unwinding is independent of GTP hydrolysis or Pi release. While this is possible, it is not sufficient to justify the energy diagram presented in Fig. 4.\nWe respectfully disagree with the assessment that the novelty of this work is limited to the results of the unwinding assay. While this section of our results was perhaps the most unexpected, the other two major sets of experiments (the smFRET head rotation assay and the assisting-force mRNA-body translocation assay) yielded important results that corroborated our proposal regarding the energy requirement and its origin during translocation. First, the smFRET results directly and clearly measured for the first time the temporal correlation between 30S head rotation (mRNA-body movement) and mRNA unwinding during processive translocation under conditions in which GTP hydrolysis is not perturbed. Second, the assisting force assay results enabled us to distinguish mRNA-body movement from the mRNA unwinding step as separate events that, although correlated under normal GTP hydrolysis conditions, can become uncoupled under GTPase perturbation conditions. Third, the combination of these results yielded the conclusions that the unwinding step is independent of GTP hydrolysis both energetically and kinetically, and that mRNA-body translocation depends on GTP hydrolysis only kinetically but not thermodynamically. Together, these results constitute the main contribution of our work, results that have been obtained through the design of unprecedented single-molecule experiments, and that allowed us to dissect individual endergonic steps in the translocation pathway and to assign their relative free energies and barriers. The diagram presented in Fig 4 reflects this analysis and is intended to illustrate qualitatively the type of energy landscape that is consistent with the experimental findings. We have now edited all three panels of the Fig 4 and their captions for clarity; we have explicitly mentioned in the caption to panel b: “A qualitative model, consistent with experimental results, of the energy landscape of translocation under normal conditions (in red) and compared qualitatively with non-canonical conditions (without GTP hydrolysis or without EF-G altogether, in green and black, respectively).”\nMoreover, it remains unclear whether the pulling force applied by optical tweezers destabilizes RNA structures to the point where unwinding becomes independent of external factors. This possibility is not adequately addressed.\nWe had already addressed this point explicitly with references in the penultimate paragraph in Discussion: “Since the optical tweezers pulling force alone is insufficient to unwind the codon duplex without the ribosome’s assistance [refs 2, 32, 48], an exergonic change of state in the ribosome complex upon EF-G binding must be involved [..]” Our previous work has shown that the force applied by the optical tweezers certainly does not destabilize the hairpin to the point where unwinding becomes independent of external factors. The need for mechanical work performed by the ribosome to unwind mRNA is the foundational basis of this assay, which was established over a decade ago and thoroughly investigated under a range of forces (see refs 2, 32, 48). We have now amended the Methods text (under “optical tweezers measurements”) to clarify this point: “These forces were chosen to be sufficiently high to provide single-codon resolution, and in the case of the unwinding assay, also far below the critical force [refs 2,32,48] of the hairpin so that hairpin unwinding would not occur spontaneously without the participation of the active ribosome (Supp. Fig. 1c).”\nWe have also included a new panel in Supp Fig 1 (and also copied below) showing an example trajectory in which a hairpin held under an applied force of ~14 pN in front of an inactive ribosome remains folded for more than 10 minutes after which we terminate the experiment. As a figure directly derived from experimental data is worth more than the proverbial thousand words, if our results were due to an artefact of the force applied to the hairpin rather than to the activity of the ribosome, we would still be gathering the data.\nThe other central messages—that translocation can occur without GTP hydrolysis and that the effect of GTP hydrolysis is kinetic—are not new; they have been reported repeatedly over the past 30 years. As such, the manuscript does not resolve any of the existing controversies.\nOur work is the first to demonstrate: (1) the temporal coincidence of hairpin unwinding and head rotation (by the smFRET assay), (2) the breakdown of this correlation under GTPase perturbation (by the assisting-force translocation assay), (3) that the specific steps of mRNA unwinding and mRNA-body movement within a translocation cycle do not require GTP hydrolysis, and (4) that GTP hydrolysis can, nonetheless, have a kinetic (catalytic) effect on the mRNA-body movement. These results are completely novel and represent a significant advance over what had been established previously in the field, namely that, as a whole, “translocation can occur without GTP hydrolysis and that the effect of GTP hydrolysis is kinetic.” We have now amended the text in the third paragraph of Discussion, explaining our argument regarding the relevance of GTP hydrolysis energy (or lack thereof) in mRNA/tRNA movement: “Importantly, even if an event such as mRNA-body movement is accelerated as a result of GTPase activity, it is not necessarily the case that the energy of GTP hydrolysis is used in that acceleration. Rather, EF-G (after hydrolysis) can act as a catalyst, and acceleration will result simply from its preferential binding to, and stabilization of the transition state immediately preceding the event [new ref 41]. Clearly, the energy of this binding is unrelated to any energy released up to this event by hydrolysis. Thus, the kinetic acceleration observed in the presence of GTP hydrolysis does not imply that it utilized the energy resulting from hydrolysis.”\n2. Placement of Pi release in the energy diagram\nThe most critical issue is the order of steps in the authors’ energy diagram. By placing Pi release after translocation, the authors implicitly assign Pi release as the trigger for EF-G dissociation. Their own data provide no direct evidence for this placement, and it contradicts the bulk of the literature. In particular, the cited cryo-EM structures (Petrychenko et al. and Carbone et al.) consistently show EF-G in the GDP–Pi state before tRNAs reach the chimeric hybrid state, and in the GDP state once the hybrid state is reached. This strongly argues against positioning Pi release after tRNA movement, as the authors do in Fig. 4. (The Rundlet et al. study [ref. 23] is not informative, as their assignment of a GDP–Pi intermediate as GTP is a misinterpretation of cryo-EM density.)\nAs we label in Fig 4b, and as our previous response to the reviewer’s original remarks indicated (remarks already reflected in the revised text in the second paragraph of Discussion), we placed the Pi release strictly after mRNA-body translocation only when GTP hydrolysis is perturbed by EF-G mutants or GTP analogs (labeled “if delayed” in Fig 4). On the other hand, in the same figure, we clearly label the occurrence of Pi release under normal conditions as possibly taking place any time from as early in the pathway as before mRNA-body translocation, to a variable time after the latter. This would be in line with the branched kinetic pathway put forth originally by the Rodnina group (ref 15), where it was proposed that Pi release precedes tRNA movement in one branch and follows it in the other. In the new version of the paper, we now make this point explicit in Fig 4a and in its caption: “Note that under normal conditions, hydrolysis and Pi release can occur before or after mRNA-body translocation (bottom, red gradient), but they are strictly required only to progress to state IV which is followed by EF-G dissociation (red arrow).”\nFurthermore, we are in disagreement with the reviewer on the conclusiveness of cryoEM results regarding the timing or causality of Pi release relative to mRNA-body translocation (i.e., head rotation and formation of chimeric hybrid states). The cryoEM snapshots in which two changes occur together (Pi release and head rotation) do not establish temporal or causal order at all. We have amended the text in the second paragraph of Introduction to highlight this caveat: “Specifically, P release is seen concomitant with conformational changes that move the i mRNA relative to the 30S body domain. Strictly speaking, it is not possible to establish a causal relationship between these two correlated events from structural snapshots. Indeed, the three studies did not agree on when GTP hydrolysis and P are required during i translocation.”\nWe have directly examined causation by perturbing Pi release, and the scheme in Fig 4 simply reflects our results. This type of causality relations has been made possible by the use, for the first time, of correlated single-molecule force and fluorescence spectroscopies.\nAdditional kinetic studies from the Rodnina and Cooperman groups, which measured Pi release directly and compared it with defined steps of translocation, are in full agreement with the cryo-EM studies. These data are either ignored or downplayed by the authors, despite being directly relevant. Correctly placing Pi release at the step of tRNA movement (consistent with the measured kinetic effect) would yield a model fully consistent with existing cryo-EM data and with the prevailing loaded-spring GTPase model, in which Pi release triggers conformational rearrangements of EF-G that bias tRNA movement and accelerate translocation. In my opinion, the model in Fig. 4 must be revised accordingly: Pi release should be placed before tRNA movement to the chimeric states, and the corresponding energy barrier for translocation should be lowered.\nAs stated above, in our model, GTP hydrolysis and Pi release can occur before mRNA-body movement (or equivalently, chimeric hybrid state formation). The energy barrier to this movement is lowered by GTP hydrolysis, as already indicated in Fig 4b (see the difference between green and red landscapes at the boundary between states II and III). Following the reviewer’s suggestion, we have now magnified this difference in Fig 4b to further highlight the kinetic effect. However, our results clearly demonstrate that Pi release can also occur after mRNA-body movement.\n3. EF-G H92Q mutant\nThe interpretation of the results obtained with the EF-G H92Q mutant hinges entirely on its biochemical activity. Does this mutant, unlike other H92 variants, hydrolyze GTP but fail in Pi release? This must be tested explicitly by assaying GTP hydrolysis and Pi release. Without such validation, the interpretation of the mutant data is not reliable.\nWe addressed the reviewer’s original comment regarding EF-G H92Q mutant previously. Our interpretation does not hinge at all on whether H92Q can hydrolyze GTP (and is only defective in Pi release) or if it is mainly defective in GTP hydrolysis. This is partly because our results for H92Q and the hydrolysis-deficient H92A mutant are qualitatively similar, thus precluding us from making any specific statement about Pi release. Please see Supp Fig 10, where we explain that H92Q defect in GTP hydrolysis would be also consistent with our proposed model (in which hydrolysis and Pi release are not strictly required for mRNA-body translocation).\nFurthermore, the reviewer previously questioned the validity of the findings of Koripella et al (ref 33) on H92Q based on apparent contradiction between Koripella’s characterization of another mutant, H92A, and the prior characterization by Rodnina’s group of that same mutant (ref 39). The two groups reported different values for the absolute GTPase activity for the H92A mutation (~28 s-1 and undetected, respectively) which can be due to different experimental details and does not mean that the qualitative results obtained for this and other mutants studied by Koripella are wholly invalid. The reviewer had previously suggested that Koripella’s H92A results may be due to contamination with wild-type EF-G, implying that such a situation could invalidate any similar characterization by the Koripella’s group. This is an experimentally unsupported statement, and we point out that while it is possible for apparent hydrolysis to result from wild-type EF-G contamination, it is not easy to rationalize how WT contamination could result in reduced Pi release in H92Q. Incidentally, Koripella’s work was published in Scientific Reports in 2015 and has not since been challenged publicly.\n4. Misrepresentation of the literature\nThe manuscript misrepresents earlier models for how GTP hydrolysis and Pi release drive translocation. While terms such as “power stroke” or “lever arm” have been used, these models (originating from multiple groups, not only Rodnina et al., as the citations suggest) were never meant to imply changes in the ground state. Rather, they consistently explain the kinetic effects of GTP hydrolysis in terms of lowering the transition barrier. This is clearly visible in the energy diagrams in the cited studies. By writing in the Introduction that “the energy of GTP hydrolysis and Pi release is directly converted to mechanical work,” the authors misrepresent the intent of prior work.\nWe wish to re-emphasize a distinction that may be helpful here. To say that the effect of GTP hydrolysis is an overall kinetic effect over-simplifies the nature of the process. Each translocation cycle is a multi-step process, not a single step. The kinetic effect of hydrolysis will be different depending on what part of the cycle is being considered. What may appear to be a kinetic effect on the pathway as a whole, could result from changes both in some transition states (maxima in the reaction pathway) as well as some ground-state intermediates (minima) and not others. What we have shown is a kinetic effect of GTPase activity pinpointed precisely to the mRNA-body movement (transition from state II to state III in Fig 4), whereas the other endergonic event, hairpin unwinding (transition from I to II), is not affected at all. The ability to distinguish which events are affected kinetically by GTP hydrolysis has become possible through the use of our correlated single-molecule force and fluorescence spectroscopy experiments, and reflect the novelty of our results.\nIn the Introduction and Discussion, we specifically referred to works in which the authors have explicitly attributed the source of energy for tRNA movement to GTP hydrolysis: For example, Rodnina et al (ref 4) state: “The function of EF-G is to translate chemical energy, derived from GTP hydrolysis, into directional molecular movement on the ribosome, that is, to function as a motor protein.” Savelsbergh et al (ref 15) state: “These results indicate that the energy of GTP hydrolysis is utilized to promote the ribosome rearrangement [..]” Rodnina et al (ref 17) state: “[..] EF-G uses the energy of GTP hydrolysis for a power stroke-like motion acting on the ribosome and the tRNAs.” Petrychenko et al (ref 18) state: “Thus, the present PRE–EF-G–GDP–Pi and the CHI–EF-G–GDP structures represent key intermediates at the onset of translocation that show how the chemical energy from GTP hydrolysis and Pi release translates into the forward movement of the mRNA–tRNA complex.” Chen et al (ref 35) state: “Our results suggest that EF-G serves as a force-generating motor via a power stroke in the early steps of translocation, supporting the proposal that energy released from GTP hydrolysis triggers EF-G conformational changes to promote translocation.” Cunha et al (ref 39) state: “Thus, part of the energy of GTP hydrolysis is used to drive a conformational change of the ribosome that controls translocation [..]” Holtkamp et al (ref 40) state: “the energy of EF-G–GTP binding alone is not sufficient to promote rapid movement on the 30S subunit. Rather, the [step] which entails both 30S translocation and tRNA movement on the 50S subunit from the PRE A/P2 state into the early POST–G state, is driven by GTP hydrolysis, which couples conformational rearrangements of EF-G to the engagement of domain 4 with the 30S codon-anticodon complex.”\nIt is worth noting that a power stroke mechanism would necessitate a lowering of both the transition state energy as well as the ground (final) state energy due to the power stroke force applied along the reaction coordinate. Therefore, in the literature when GTP hydrolysis is attributed as the energy source for a power stroke model, it naturally follows that GTP hydrolysis causes a lowered ground state. In the third paragraph of Discussion, we have therefore now explicitly clarified that EF-G.GTP binding can catalyze translocation without the need for contribution of GTP hydrolysis energy: “Previous observations of rapid GTP hydrolysis by EF-G combined with results of kinetic fluorescence measurements with EF-G mutants and GTP analogs have led to a GTP hydrolysis-driven power-stroke model for translocation [refs 4,15,17,18,35,39,40]. This model posits that EF-G conformational changes, fueled by the energy of GTP hydrolysis, drive the mechanical movement of mRNA and tRNAs relative to the 30S body domain. However, the fact that hydrolysis normally occurs before an event during translocation does not necessarily imply that it is energetically required for the event. GTP hydrolysis presumably precedes both hairpin unwinding and mRNA-body translocation, but our results indicate that it is not required thermodynamically to drive either of these endergonic events. Importantly, even if an event such as mRNA-body movement is accelerated as a result of GTPase activity, it is not necessarily the case that the energy of GTP hydrolysis is used in that acceleration. Rather, EF-G (after hydrolysis) can act as a catalyst, and acceleration will result simply from its preferential binding to, and stabilization of the transition state immediately preceding the event [new ref 41]. Clearly, the energy of this binding is unrelated to any energy released up to this event by hydrolysis. Thus, the kinetic acceleration observed in the presence of GTP hydrolysis does not imply that it utilized the energy resulting from hydrolysis.”\nWe have also amended the text in the second paragraph of Introduction to avoid the impression of mutual exclusivity: “Two roles for GTP hydrolysis and P release by EF-G during translocation have been described. In the first role, the energy from GTP hydrolysis and P release is suggested to be directly converted to mechanical work [refs 4,15,17,18]. In the second role, this energy is instead used for reducing the affinity of EF-G to the ribosome, allowing for EF-G release and the resetting of the translational cycle [refs 9,19-25].”\n5. Abstract statement on thermodynamic requirement\nGiven the problems outlined above regarding the placement of Pi release, I question the validity of the Abstract’s statement that “…while GTP hydrolysis kinetically accelerated translocation, it is thermodynamically required only to liberate the tightly bound EF-G from the ribosome.” The overall trajectory may differ from the one the authors propose, and in fact alternative models consistent with this possibility have already been suggested in the literature (e.g., ref. 36).\nWe are aware of alternative models proposed in the literature (e.g. ref 36). However, as stated above, our work pertains to specific individual steps in the translocation pathway, namely the endergonic steps of mRNA unwinding and complete accurate mRNA-body movement.\n6. Description of the tRNA trajectory\nA remaining issue concerns the Introduction’s description of tRNA translocation. The authors state that swiveling of the 30S head domain moves the tRNA and mRNA into chimeric hybrid states, which is correct. However, they omit that this step is accompanied by a backward movement of the 30S body domain. This feature is well established from cryo-EM structures (refs. 18, 22) and stopped-flow data, and it is an essential element of the translocation mechanism. It should be explicitly included.\nFirst paragraph of Introduction has been changed to include this feature: “Next, forward rotation or “swivel” of the 30S head domain, accompanied by a partial reversion of inter-subunit rotation, moves the mRNA and tRNAs relative to the body domain to produce chimeric hybrid tRNA states [refs 12,13]. This step is followed by the reverse 30S head rotation without carrying back the mRNA and tRNAs, which together with full reverse inter-subunit rotation, completes the mRNA/tRNA movement and resets the ribosome to the classical post-translocation state.”"}]

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