Post-termination Ribosome Intermediate Acts as the Gateway to Ribosome Recycling.

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Cell Rep. Author manuscript; available in PMC 2017 August 14. Published in final edited form as: Cell Rep. 2017 July 05; 20(1): 161–172. doi:10.1016/j.celrep.2017.06.028.

Post-termination ribosome intermediate acts as the gateway to ribosome recycling Arjun Prabhakar1,2, Mark C. Capece1,3, Alexey Petrov1, Junhong Choi1,4, and Joseph D. Puglisi1,5 1Department

of Structural Biology, Stanford University School of Medicine, Stanford, California

94305, USA.

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2Program

in Biophysics, Stanford University, Stanford, California 94305, USA.

3Department

of Chemistry, Stanford University, Stanford, California 94305, USA.

4Department

of Applied Physics, Stanford University, Stanford, California 94305, USA.

Summary

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During termination of translation, the nascent peptide is first released from the ribosome, which must be subsequently disassembled into subunits via a process known as ribosome recycling. In bacteria, termination and recycling are mediated by translation factors RF, RRF, EF-G, and IF3, but their precise roles have remained unclear. Here, we use single-molecule fluorescence to track the conformation and composition of the ribosome in real-time during termination and recycling. Our results show that peptide release by RF induces a rotated ribosomal conformation. RRF binds to this rotated intermediate to form the substrate for EF-G that in turn catalyzes GTP-dependent subunit disassembly. After the 50S subunit departs, IF3 releases the deacylated tRNA from the 30S subunit, thus preventing reassembly of the 70S ribosome. Our findings reveal the post-termination rotated state as the crucial intermediate in the transition from termination to recycling.

eTOC Blurb

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Correspondence: [email protected]. 5Lead Contact Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Supplemental information Supplemental information includes Supplemental Experimental Procedures, seven figures, and one table. Author Contributions A. Prabhakar and J.D.P. conceived of and designed the experiments. A. Prabhakar performed all the experiments and data analysis. A. Prabhakar interpreted the data. A. Petrov and J. C. assisted in reagent preparation. A. Prabhakar and M.C.C. wrote the manuscript with input from A. Petrov, J.C., and J.D.P.

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Ribosome intersubunit conformation plays a critical role in ribosome recycling after translation termination. Prabhakar et al. use single-molecule techniques to temporally resolve the posttermination intersubunit rotation that promotes factor-mediated disassembly of the ribosome. The observed ribosome conformational dynamics clarified the roles of the protein factors and timings of recycling events.

Keywords

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Ribosome; translation; termination; stop codon; release factor; recycling; RRF; EF-G; IF3; singlemolecule fluorescence

Introduction

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Completely synthesized proteins are released from the ribosome during the termination stage of translation. The ribosome is then disassembled for use in the next iteration of translation through a process called recycling. Termination is signaled by the presence of a stop codon (UAG, UGA, UAA) in the aminoacyl site (A site) of the ribosome, which triggers the recruitment of release factors (RFs) (Youngman et al., 2008). There are two classes of RFs. In bacteria, class I RFs, RF1 and RF2, recognize the UAG and UGA codons, respectively, and both recognize the UAA codon. Upon stop codon recognition, the class I RF engages the peptidyl transferase center (PTC) of the ribosome to release the nascent peptide through hydrolysis of the ester bond linking the peptide to the tRNA in the peptidyl site (P site) of the ribosome. Hydrolysis is accomplished through specific interactions between the PTC and the universally conserved GGQ motif of class I RFs, which has been shown structurally and biochemically to be essential for peptide hydrolysis (Frolova et al., 1999; Laurberg et al., 2008; Zavialov et al., 2002). The class II RF in bacteria, RF3, is a GTPase that accelerates the dissociation of class I RFs after peptide release (Freistroffer et al., 1997), but its mechanism remains unclear.

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Nonetheless, RF3 is nonessential for cell viability (Grentzmann et al., 1994; O’Connor, 2015), and only a subset of bacterial species have RF3 in their genomes (Margus et al., 2007). This absence suggests that RF3 is not part of a conserved mechanism of termination and that it might merely enhance termination and recycling efficiency (Koutmou et al., 2014; McDonald and Green, 2012).

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After termination and peptide release, the 70S complex bearing a deacylated P-site tRNA and mRNA is disassembled into its individual components through a recycling mechanism involving ribosome recycling factor (RRF), elongation factor EF-G and initiation factor IF3. RRF and EF-G have been proposed to work in concert when bound to the ribosome, requiring GTP hydrolysis by EF-G to catalyze the dissociation of the 50S subunit (Zavialov et al., 2005). The role of IF3, as well as the timings of subunit and tRNA dissociation, may follow one of two competing models. The first (Hirokawa et al., 2005) proposes that EF-G functions through a translocation-like mechanism by moving RRF into the P site of the ribosome, causing the P-site tRNA to dissociate first, followed by spontaneous dissociation of subunits and mRNA. IF3 then binds to the 30S subunit to prevent 50S subunit reassociation. Alternatively (Borg et al., 2016; Karimi et al., 1999; Peske et al., 2005; Zavialov et al., 2005) the 50S subunit may be first removed by RRF and EF-G. The rest of the complex would then be disassembled by IF3-induced P-site tRNA departure, followed by mRNA dissociation from the 30S subunit. A direct measurement of the timing of these events is required to elucidate the mechanism of ribosome recycling.


The terminating ribosome adopts conformational states that resemble those that occur during elongation. During elongation, peptide bond formation drives a 3–10° counterclockwise rotation of the 30S subunit with respect to the 50S subunit (Agirrezabala et al., 2008; Frank and Agrawal, 2000; Valle et al., 2003), which results in what is defined as the rotated conformation. It was proposed that the hydrolysis of the peptide from the P-site tRNA triggers a similar conformational change in the ribosome that activates RF3 to trigger dissociation of RF1 and RF2 (Zavialov et al., 2002). Cryo-electron microscopy (Cryo-EM) structures showed differences in ribosome intersubunit rotations (Gao et al., 2007) when bound to different classes of release factors. In complex with RF1 or RF2, the ribosome exhibited the non-rotated conformation resembling the post-translocation ribosome, whereas the RF3-bound ribosome was in a rotated-state conformation, reported as a 10° counterclockwise rotation. These conformational differences of ribosomes were further demonstrated in crystal structures of class I RF-bound (Korostelev et al., 2008; Laurberg et al., 2008; Weixlbaumer et al., 2008) and RF3-bound (Jin et al., 2011; Zhou et al., 2012) ribosomes. Structural data showed the RRF-bound 70S complex also in the rotated state (Dunkle et al., 2011; Gao et al., 2005). These observations hint that ribosome conformation is modulated by the factors and may play a key role in the mechanisms of termination and recycling. The dynamic interplay between factor binding and ribosome conformation during termination and recycling remains unclear. To elucidate the mechanisms of termination and recycling, we employed single-molecule fluorescence approaches to track the multiple steps of termination and recycling in real-time. We probed the interactions of class I RFs, EF-G, RRF, and IF3 with single translating ribosomes to correlate ligand composition with the conformational state of the ribosome, as


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monitored by Förster Resonance Energy Transfer (FRET). By connecting these states with ligand binding events, we revealed ribosome conformational dynamics occurring after termination that are central to the mechanism of ribosome recycling.

Results Real-time tracking of termination using fluorescently-labeled class I release factors

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We monitored the entire process of translation from initiation to recycling using our previously described single-molecule system that utilizes a modified zero-mode waveguide (ZMW)-based real-time sequencer (RS) (Chen et al., 2014) (Figure 1A). In this established system, E. coli 30S and 50S ribosomal subunits are site-specifically labeled with Cy3B fluorophore and BHQ-2 quencher, respectively, to detect intersubunit conformational changes during translation (Chen et al., 2012). To track termination events, we prepared and characterized fluorescently-labeled RF1 (Sternberg et al., 2009) and RF2 (Figure 1B). In detail, we delivered Cy5-labeled RF1 or RF2 with BHQ-2-labeled 50S subunits, aminoacyltRNA ternary complexes (Phe-tRNAPhe – EF-Tu – GTP) (Phe-TC), and EF-G to immobilized 30S pre-initiation complexes (PICs) consisting of Cy3B–labeled 30S subunit, initiator fMet-tRNAfMet, GTP-bound initiation factor IF2, and 5’-biotinylated mRNA with a AUG-UUC-stop (MF-stop) codon sequence in the open reading frame. The stop codon was either UAG or UAA for RF1 experiments and UGA or UAA for RF2 experiments. In the experiments presented here, we did not include RF3, as it is not essential for termination. We will address the role of RF3 in the context of our termination and recycling data in the Discussion.


By using the signals from dye-labeled RFs, we observed translation initiation, elongation, and termination (Figure 1C). Translation begins with 50S subunit joining to 30S PIC, yielding the non-rotated 70S ribosome, shown by the quenching of green Cy3B signal. Initiation is followed by a single round of elongation, whereby ternary complex binding and peptide bond formation drive subunits into the rotated state, and translocation by EF-G drives rotation back into the non-rotated state, as shown by the increase and decrease in Cy3B intensity, correspondingly. The above-described ribosome behavior agrees with previously reported ribosomal conformational rearrangements during translation in our system (Aitken and Puglisi, 2010; Chen et al., 2013; Marshall et al., 2008; Petrov et al., 2012). No binding signal of Cy5-RF1 or Cy5-RF2 was observed during the initiation and elongation stages when a sense codon is presented in the A site of the ribosome. Once translocation places the stop codon of the MF-stop mRNA into the A site, it is ready to be recognized by its cognate class I RF. Cy5-RF1 binding signal at this stage was only observed in experiments with MF-UAG and MF-UAA mRNAs, and Cy5-RF2 binding signal was only observed in those with MF-UGA and MF-UAA, demonstrating the specificity of RF1 and RF2 for their cognate stop codons. The association rates of Cy5-RF1 and Cy5-RF2 with pre-termination 70S complexes (kpreRF,on) were determined from a linear fit to observed rates (kobs) calculated from a single exponential fit of the arrival time distribution (Figure S1). The rates for Cy5-RF1 and Cy5RF2 were in the ranges of 15–17 µM−1s−1 and 10–16 µM−1s−1 (Table S1), respectively, which are similar to previously determined binding kinetics of RF1 (Hetrick et al., 2009). Cell Rep. Author manuscript; available in PMC 2017 August 14.

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Departure of Cy5-RF1 and Cy5-RF2 correlated with ribosome rotation, revealing a posttermination rotated-state intermediate that is characterized by the absence of factors (Figure 1C). Both Cy5-RFs retained affinity for this intermediate, as we observed frequent rebinding of the factors to the post-termination ribosome, suggesting that the stop codon still resides in the A site after termination. Cy5-RF rebinding events were correlated with reversible ribosome rotations, as the ribosome transitions to the non-rotated state when bound to either Cy5-RF. The RF-free rotated-state intermediate also precedes a 50S subunit dissociation event, shown by the dequenching of Cy3B signal back to the starting intensity, which is the first sign of ribosome disassembly.

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Post-termination ribosome rotation occurred after RF departure in 89±2% and 93±2% of RF1- and RF2-bound ribosomes, respectively; RF departure was not observed in the remaining molecules, censored by the end of our acquisition movie. This transition from the non-rotated to rotated state conformation of the 70S ribosome resembles peptidyl transfer during elongation. To compare the new rotated state after termination with the rotated state during elongation (Aitken and Puglisi, 2010), we quantified their FRET efficiencies using total internal reflection fluorescence microscopy (TIRFM). The measured FRET efficiencies for both the post-termination and elongation rotated states were 0.29±0.08 (Figure S2), comparable to previously reported FRET efficiencies (Marshall et al., 2008). Based on the limited conformational information of FRET, we postulate that the post-termination rotated state is similar to the pre-translocation rotated state adopted by the ribosome during elongation.


Transition from the non-rotated to rotated conformation of the 70S ribosome was also previously observed upon treatment of 70S elongation complexes containing peptidyl-tRNA with the drug puromycin (Ermolenko et al., 2007; Marshall et al., 2008; Valle et al., 2003). Puromycin binds to the PTC and activates peptidyl transfer from P-site tRNA to puromycin, leading to peptide release upon puromycin-peptide dissociation. Likewise, puromycin treatment of pre-termination 70S complexes also results in this transition from non-rotated into rotated state of the ribosome (Figure S3A, S3B). To test whether the transition to the rotated state after class I RF departure also requires hydrolysis of the peptidyl-tRNA, we repeated the termination assay using a Cy5-labeled mutant RF2 with a single-alanine mutation in the GGQ motif to GAQ (RF2 G251A), which reduces peptide hydrolysis activity of RF2 by 20,000-fold (Zavialov et al., 2002). With Cy5-(GAQ)RF2, the transition to the rotated state is observed in only 3±1% of the ribosomes that bound to RF2 (Figure S4), which is 33-fold lower compared to that of the functional Cy5-RF2. The remaining ribosomes show multiple Cy5-(GAQ)RF2 binding events that do not produce the posttermination rotated state. These experiments show that the post-termination rotated state formation requires nascent peptide release. As mentioned previously, class I RFs can rebind to the post-termination ribosome. RF rebinding events were observed in 77±6% and 66±7% of terminated ribosomes with RF1 and RF2, respectively; the remaining fraction of ribosomes underwent 50S subunit dissociation from the rotated state before RF2 rebinding can occur, which is explained in the next section. RF rebinding events occurred simultaneously with ribosome transitions between the rotated and non-rotated states, generating anticorrelated Cy3B and Cy5


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fluorescence intensities (Figure 1C). Although this anticorrelation can be misinterpreted as a signature of FRET between the 30S subunit and RF, structural modeling predicts the distances between the helix 44 labeling position of the 30S subunit and the labeling positions of the RFs to be 130 Å and 110 Å for Cy5-RF1 and Cy5-RF2, respectively (Dorywalska et al., 2005; Korostelev et al., 2008), which are well beyond the detectable range of FRET. To confirm that changes in Cy3B intensity is due to ribosome rotation and not due to FRET between the ribosome and RFs, we replaced the Cy5-RFs with unlabeled wild-type RF1 or RF2 and still observed the Cy3B intensity changes (Figure S5), confirming the coupling of post-termination ribosome rotations to RF occupancy. The dwell times of these additional post-termination rotated and non-rotated states were measured at different concentrations of RF1 and RF2 to probe the effect of factor binding on ribosome conformation. The mean dwell time of the rotated states is dependent on the RF concentration, whereas the mean dwell time of the non-rotated state is independent of the RF concentration. These dwell times indicate that RF association with the post-termination ribosome mediates ribosome intersubunit conformation. Peptide release by puromycin treatment of pre-termination ribosomes in the presence of dysfunctional Cy5-(GAQ)RF2 resulted in a similar coupling of Cy5-(GAQ)RF2 occupancy and ribosome conformation (Figure S6). This observation further identified peptide release as the prerequisite for posttermination ribosome rotation and that the catalytic activity of RF2 is dispensable for these changes. Spontaneous dissociation of the 50S subunit from the post-termination rotated state


Post-termination 70S complexes can dissociate to 50S and 30S subunits even in the absence of additional recycling components (RRF and IF3). 100% of 50S subunit dissociation events occurred from the rotated state of the post-termination ribosome (Figure 1C). When pretermination 70S complexes were treated with puromycin in the earlier-described experiment, 50S subunit dissociation was observed even in the absence of RF (Figure S3A, S3B). Thus, spontaneous 50S subunit dissociation occurs from the RF-unbound post-termination rotated state. RF rebinding events drive ribosomes to the non-rotated state, inhibiting 50S subunit dissociation. From our findings, we propose two competing pathways from the posttermination rotated state, as schematized in Figure 2A. The observed rate of disappearance of this post-termination rotated state (kobs) can be written as the sum of the rates of RF association rate to the post-termination rotated state (kpostRF,on) and spontaneous 50S subunit dissociation rate from the rotated state (k50S,off) (Figure 2A). The observed rate constant kobs was determined by measuring the dwell times of the post-termination rotated states (Figure 2B, Figure S3B) and fitting the ensemble distribution of these dwell times to a single exponential to obtain a first-order rate constant (Figure 2C, Figure S3C). The linear fit to these kobs values yielded the slope as the RF2 association rate (kpostRF,on = 2.8±0.2 µM−1s−1) and the y-intercept as the 50S dissociation rate (k50S,off = 0.022±0.001 s−1) (Figure 2D). To confirm the 50S subunit dissociation rate in the absence of RF2, we determined the rate of 50S subunit dissociation from the post-termination rotated state induced by puromycin (k50S,off,puromycin) by fitting the dwell time distribution of this rotated state to a single-exponential function. This rate was calculated to be 0.020±0.003 s−1, which is comparable to the earlier determined spontaneous 50S subunit dissociation rate from the RF2 termination experiments. Using a similar approach on the dwell times of the RF2-


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bound non-rotated state, we determined the dissociation rate of RF2 from the posttermination ribosome (kRF,off) to be 0.157±0.038 s−1. These kinetic parameters describe a relatively-unstable post-termination 70S particle with 50-seconds lifetime in the absence of recycling factors. This disassembly rate, however, is much slower than the 2.8±0.2 µM−1s−1 RF2 post-termination association rate at cellular RF2 concentrations of greater than 1 µM (Adamski et al., 1994). The presence of RF2 significantly reduces the probability of 50S subunit dissociation, thus additional protein factors are necessary to drive ribosome recycling in vivo.

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50S subunit dissociation in the absence of RRF and IF3 is reversible, and the 50S reassociation rate (k50S,on) was measured at 0.75±0.07 µM−1s−1. Importantly, reassociation put the two subunits in a rotated conformation (Figure S7A) similar to the states observed during initiation (Marshall et al., 2009). RFs can bind to the reassembled ribosome, again resulting in a 70S non-rotated state, strongly suggesting that the stop codon still resides in the A site. Thus, the reading frame must be maintained by deacylated tRNA remaining in the P site, and additional factors are required to complete disassembly of the translation complex during recycling. RRF and EF-G promote 50S subunit dissociation

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We next probed the effects of RRF and EF-G on post-termination events in the presence of 10 nM Cy5-RF2. The same experimental setup (Figure 1A) as described before was used with the addition of RRF to the delivery mixture of BHQ-2–50S, EF-G, Phe-TC, and Cy5RF2. In the absence of RRF at 50 nM EF-G, the majority (66%) of the ribosomes underwent RF2 rebinding events. With increasing addition of RRF, this fraction of RF2-bound posttermination non-rotated ribosomes decreased to 9±2% at physiological RRF concentration (20 µM) (Figure 3A). Since both class I RFs and RRF bind to the ribosomal A site, class I RFs and RRF likely compete for binding to the post-termination rotated-state ribosome, such that RRF association stabilizes the rotated state while class I RFs induce the transition to the non-rotated state. This hypothesis agrees with reported crystal structures of post-termination ribosomes in the rotated conformation when bound to RRF (Dunkle et al., 2011; Gao et al., 2005) and non-rotated conformation when bound to RF2 (Korostelev et al., 2008). The fraction of ribosomes with RF2 rebinding also decreases with increasing concentration of EF-G (Figure 3A, 3B) due to EF-G accelerating the 50S subunit dissociation step, which is explained in the next paragraph. Thus, RRF and EF-G cooperatively overcome the inhibitory effects of RF2 rebinding to promote ribosome recycling by binding to the rotated state.

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To characterize the rate of subunit disassembly in the presence of RRF and EF-G, we measured the dwell time of the last rotated state before 50S subunit dissociation. This subunit disassembly time in the absence of RRF was 14.6±2.1 s at 10 nM Cy5-RF2 and 50 nM EF-G (Figure 3C). Although the addition of RRF blocked RF2 rebinding events, increasing RRF alone had almost no effect on the subunit disassembly time at 50 nM EF-G. Even at ribosome-saturating concentrations of RRF (20 µM), the disassembly time was insignificantly reduced to 13.3±0.9 s. However, with increasing EF-G concentrations, we observed faster subunit disassembly at higher RRF concentrations. Subunit disassembly time was reduced to 1.5±0.2 s at near-physiological concentrations (20 µM RRF, 5 µM EF-G).


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The lack of RRF concentration dependence at low EF-G concentrations indicates that subunit disassembly was rate-limited by EF-G binding. As a result, increasing both RRF and EF-G concentrations also inhibit class I RF rebinding by accelerating the subunit disassembly process. At constant RRF concentration, the subunit disassembly time first decreased and then increased with increasing EF-G concentration (Figure 3D). The inhibitory effect at high EF-G and low RRF concentrations may suggest that EF-G binding before RRF binding does not lead to successful subunit dissociation. This inhibition decreases at higher RRF concentration, where RRF binding presumably precedes EF-G binding. Thus, only a sequential binding of RRF and EF-G, in that order, leads to successful catalysis of 50S subunit dissociation. EF-G catalyzes subunit disassembly through GTP hydrolysis

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To track the role of EF-G in recycling directly, we used Cy5-labeled EF-G, wild-type RF2, and RRF in the same single-molecule fluorescence setup. As previously observed, transient EF-G binding occurs during elongation and is correlated to the transition from the rotated to non-rotated state in translocation through GTP hydrolysis (Figure 4A) (Chen et al., 2013). After termination by RF2, we observed rapid EF-G binding events, represented by short Cy5 pulses, to the post-termination rotated states (Figure 4A and 4C). Some of these Cy5 pulses were one frame long (33ms) with varying fluorescence intensities, indicating that some Cy5EF-G binding events had durations less than 33ms. By estimating these events as being exactly one frame, we calculated an upper limit for the mean Cy5-EF-G dwell time to be 50–60ms (Figure 4E). The time intervals between the EF-G binding events, or EF-G arrival times, were fit to a single exponential to obtain association rates. In the presence of ribosome-saturating concentrations of RRF (20 µM), EF-G binds with an association rate (kG2) of 2.10±0.09 µM−1s−1 (Figure 4A). At 20 µM RRF and 50 nM Cy5-EF-G, 59±5% of the ribosomes undergoing subunit disassembly had a correlated Cy5-EF-G pulse, representing the coupling of EF-G binding and catalysis to subunit disassembly during recycling (Figure 4B). The remaining uncorrelated fraction most likely represents spontaneous 50S subunit dissociation, given the slow catalyzed dissociation rate at 20 µM RRF and 50nM EF-G (Figure 3C). The fraction of subunit disassembly events correlated with Cy5-EF-G pulses increased to 78±5% at 200nM Cy5-EF-G, further corroborating this rationale. In the absence of RRF, we observed rapid Cy5-EF-G sampling events on the posttermination rotated state, consistent with earlier experiments that EF-G can bind to the 70S termination complex without RRF. The association rate of EF-G without RRF was approximately 9-fold faster than that with 20 µM RRF (kG1 = 18.6±0.8 µM−1s−1, Figure 4C). In absence of RRF and at 50 nM Cy5-EF-G, there were far fewer ribosomes going through subunit disassembly, and of those only 8.5±3.7% correlated with Cy5-EF-G pulses. Overall, this suggests that EF-G cannot efficiently catalyze subunit disassembly without RRF. To test whether these EF-G binding events result in GTP hydrolysis, we tracked the behavior of Cy5-EF-G in the presence of GDPNP, a non-hydrolyzable analog of GTP (Figure 4D). In these experiments, Cy5-EF-G-GDPNP and RF2 were delivered to a 70S pre-termination complex. Inhibiting GTP hydrolysis caused at least a 500-fold increase in the occupancy time of EF-G, as measured by Cy5 lifetime, both in the presence and absence of 20 µM RRF


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(Figure 4E). This result indicates that GTP-bound EF-G has high affinity for the rotatedstate ribosome and is agnostic to RRF. GTP hydrolysis generates GDP-bound EF-G, which has low affinity for the rotated-state ribosome and quickly dissociates to produce the previously-described rapid Cy5 pulses. Thus, EF-G hydrolyzes GTP upon binding to both the RRF-bound and empty post-termination rotated-state ribosome, but only GTP hydrolysis with EF-G and RRF bound to the ribosome results in productive 50S subunit departure. IF3 dissociates P-site tRNA from the 30S complex after subunit splitting

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Experiments with RF2, RRF, and EF-G indicated that 30S post-termination complexes likely retain the P-site tRNA that maintains the A-site stop codon in proper frame. Even in the presence of RRF and EF-G, we observed that after the first 50S subunit dissociates, another one can reassociate with the 30S complex from the pool of 50S subunits in solution (Figure S7A). Moreover, Cy5-RF2 can bind to this new 70S complex and induce a ribosome conformational change to the non-rotated state. The fact that RF2 still binds with a long residence time to this reformed 70S complex indicated that the stop codon is still in the A site, with the P-site tRNA maintaining reading frame. To track the occupancy of the P-site tRNA directly, we delivered Cy5.5-labeled Phe-tRNAPhe along with 10 nM Cy5-RF2, 50 nM EF-G, and 1 µM RRF to the ribosome translating the MF-UGA sequence. From the persistence of the Cy5.5 signal throughout the experiment, we observed that the P-site tRNA stays bound to the 30S subunit after 50S subunit dissociation (Figure 5A, 5C).


Previous studies have shown that IF3 prevents re-formation of a 70S ribosome after recycling (Hirokawa et al., 2005; Peske et al., 2005). To reproduce this finding, we added 1 µM IF3 to our single-molecule experiment. In the presence of 1 µM IF3 and 1 µM RRF, 50S subunit reassociation was blocked. Instead, the Cy3B signal was lost rapidly (18.8±1.8 s) after 50S subunit departure (Figure S7B), reporting that the 30S subunit has dissociated from the immobilized mRNA. We then tracked the P-site tRNA occupancy using the Cy5.5labeled Phe-tRNAPhe in the IF3 delivery experiment and observed a two-fold reduction in Cy5.5 fluorescence lifetime (Figure 5C), and the Cy5.5 signal is lost between the time points of 50S and 30S subunit dissociation events (Figure 5B). Out of the measured time intervals between the three dissociation events (50S, tRNA, 30S), only the dwell time between 50S subunit dissociation and tRNA dissociation was dependent on IF3 concentration, showing a two-fold decrease when the IF3 concentration was doubled (Figure 5D). This correlation between tRNA departure rate and IF3 concentration indicates that IF3 induces dissociation of the deacylated tRNA from the 30S complex after the 50S subunit is recycled independently. After tRNA dissociation, the 30S subunit dissociates immediately from the mRNA (k30S = 0.514±0.017 s−1). This approximately 2-second mean residency time for the 30S subunit after tRNA departure is much shorter than its residency time in the absence of IF3 (longer than the 5-min observation window. Overall, these results show that IF3 induces tRNA departure from the 30S subunit, thereby completing the ribosome recycling process.

Discussion Here we have applied real-time single-molecule methods to track the conformational and compositional dynamics of bacterial translation termination and recycling. We observe


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specific recognition of stop codons in the A-site by class I RFs, and, indirectly, nascent peptide release through hydrolysis of the peptidyl-tRNA. After the class I RF releases the peptide on the P-site tRNA, it dissociates, resulting in a coupled counterclockwise rotation of the 30S subunit to form the rotated state. This post-termination rotated state is a key ribosome intermediate at the interface of termination and recycling. In the absence of recycling factors, this rotated-state intermediate undergoes two competing processes: (1) RF1 and RF2 can rebind to the ribosome, stabilizing the non-rotated conformation and blocking subunit disassembly or (2) the 50S subunit can slowly dissociate from the rotated state, but can later rejoin in the rotated state. Neither process is irreversible, thus complete and rapid recycling requires RRF, EF-G, and IF3. In the presence of RRF, the posttermination resampling of RF1 and RF2 is blocked and the rotated state is favored. While RRF and EF-G co-occupy the rotated-state ribosome, GTP hydrolysis by EF-G leads to rapid splitting of the 70S particle to individual subunits. IF3 then rapidly removes P-site tRNA, with subsequent loss of reading frame. The final process is thus made irreversible, shunting subunits to another round of translation.


Our results highlight the importance of ribosomal intersubunit conformation during termination and recycling. Termination experiments with Cy5-RF showed that peptide release leads to formation of the rotated state in the absence of bound protein factors. A similar conformational change occurs during elongation when the nascent peptide is transferred from P-site tRNA to A-site tRNA, as evidenced by similar FRET value distributions for the elongation and termination rotated states (Figure S2). In both elongation and termination, deacylation of P-site tRNA introduces conformational fluctuations of the tRNA between the P/P classical state and P/E hybrid state (Blanchard et al., 2004; Sternberg et al., 2009). Although it is still debated whether the ribosome intersubunit conformation fluctuates similarly after peptide transfer (Aitken and Puglisi, 2010; Chen et al., 2011; Chen et al., 2013; Cornish et al., 2008; Fei et al., 2008; Julian et al., 2008; Marshall et al., 2008; Munro et al., 2010), EF-G binds to stabilize the rotated state before translocation. During termination, we observe the post-peptide release ribosome in the rotated state after class I RF dissociation, but in contrast to elongation, RF rebinding brings the ribosome back to the non-rotated state, agreeing with past structural data (Korostelev et al., 2008). We propose that peptide release has altered the energetics of conformational exchange distinctively from the energetic landscape of the elongating ribosome during peptidyl transfer involving two tRNAs, such that RF binding drives transition to the non-rotated state. These transitions are absent when the peptide cannot be released by dysfunctional mutant class I RFs, but peptide release by puromycin restores the ability of mutant RFs to induce conformational changes. Furthermore, this post-termination rotated state is susceptible to uncatalyzed 50S subunit dissociation (k50S,off = 0.022±0.001 s−1). This reduction in intersubunit affinity upon posttermination ribosome rotation is possibly due to the disruption of key intersubunit bridges from subunit rotation (Liu and Fredrick, 2016; Yusupov et al., 2001). The labile posttermination rotated state, which is only present after successful peptide release, is the substrate for recycling by RRF and EF-G. Thus, this intermediate serves as the gateway to ribosome recycling. In our experiments, we did not observe a class I RF-bound post-termination rotated state, which suggests that the factor has low affinity to the post-termination rotated state. Since Cell Rep. Author manuscript; available in PMC 2017 August 14.

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RF3 was identified in the past to bind to the rotated state (Gao et al., 2007; Jin et al., 2011; Zhou et al., 2012), it was proposed that RF3 may actively induce ribosome rotation to accelerate class I RF dissociation (Gao et al., 2007; Koutmou et al., 2014; Peske et al., 2014; Shi and Joseph, 2016). The coupling we observe between ribosome conformation and class I RF occupancy supports this proposed model. The class I RF-bound non-rotated state greatly inhibits 50S subunit dissociation from the post-termination ribosome. The kinetic parameters determined here (Figure 2) quantitatively illustrate this problem as the rapid class I RF association rate to the post-termination ribosome suggests that the class I RF is almost always bound to prevent subunit disassembly when present at cellular concentrations (>1 µM) (Adamski et al., 1994).

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RRF changes the conformational equilibrium of the post-termination 70S ribosome to favor recycling. RRF binds to the rotated state to prevent RF-induced inhibition of subunit disassembly. The overlapping binding sites of class I RFs and RRF in the A site allows RRF to compete directly against RF-induced formation of non-rotated state, thus promoting 50S subunit dissociation from the rotated state. The competition between these factors have been noted in past biochemical experiments (Pavlov et al., 1997), but our results elucidate its mechanistic implication.

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The final key role of RRF is to form the correct substrate on the ribosome for EF-G to catalyze subunit dissociation through GTP hydrolysis. Our results from both wild-type EF-G and Cy5-EF-G experiments are consistent with the model that EF-G can bind to both the RRF-bound and RRF-free 70S ribosomes (Seo et al., 2004), but only EF-G binding to the RRF-70S complex leads to successful subunit disassembly. This sequential binding mechanism was proposed recently (Borg et al., 2016). This post-termination 70S state with both RRF and EF-G bound was structurally resolved recently using time-resolved Cryo-EM (Fu et al., 2016). Compared to translocation, the recycling efficiency of EF-G is poor at low RRF concentrations, leading to futile binding events of EF-G without subunit splitting. At high EF-G concentrations, these futile EF-G binding events outcompete RRF binding, leading to significant slowdown of the recycling process. As a result, it is important that RRF is present at high concentrations in the cell to bind to the ribosome before EF-G in order to achieve efficient splitting of the post-termination complex.

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IF3 is essential to ensure the irreversibility of recycling by accelerating dissociation of P-site tRNA from the 30S subunit. In the absence of IF3, 50S subunit and RF rebinding were observed. Thus, we confirmed that subunit disassembly is reversible and the P-site tRNA is still bound to the 30S subunit after 50S subunit departure. The dissociation of P-site tRNA from the 30S subunit is instead accelerated by IF3 after 50S subunit dissociation. tRNA departure leaves an unstable 30S–mRNA complex that disassembles spontaneously. Realtime tracking of events during recycling has provided an unambiguous description of the steps in the recycling pathway (Figure 6, Table 1). By tracking ribosome states during termination and recycling in real-time, we have unraveled the mechanism of ribosome recycling. Our data disagree with the first model of recycling that states that RRF and EF-G split the subunits through a translocation-like


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mechanism that dissociates the P-site tRNA first (Hirokawa et al., 2005). Instead, we have direct evidence that shows that tRNA only departs through the action of IF3 following 50S subunit dissociation by RRF and EF-G. These findings support the second model of recycling, whereby IF3 is required for disassembly of the 30S complex by inducing tRNA dissociation (Karimi et al., 1999).

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In summary, we have described an in vitro assay that can track all four stages of translation in real-time. Past bulk kinetics approaches have been limited to individual steps of termination or recycling in isolation, yet these processes are intermingled and thus must be studied together. Our single-molecule translation assay delineated ribosome conformational dynamics introduced during termination and showed how these dynamics are tuned by interactions with both termination and recycling factors to control progression from termination to recycling through the rotated-state intermediate. These time-resolved experiments also delineated timings of key molecular events of ribosome recycling and related them to the roles of protein factors. By elucidating the interplay between ribosome conformation and factor binding during termination and recycling, we have gained mechanistic insights into fundamental aspects of translation control.

Experimental Procedures Reagents and buffers for single-molecule experiments Preparation of Escherichia coli ribosomes, translation factors (IF2, EF-Tu, EF-G, EF-Ts), tRNAs and biotinylated mRNAs are described in Supplemental Experimental Procedures.

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Wild-type RF1, RF2, RRF, and IF3 were all purified by overexpressing them in E. coli BL21(DE3) cells transformed with pET-24b vectors (EMD Millipore) containing the corresponding gene from E.coli MG1655 K12 strain followed by a C-terminal six-histidine (6×His) affinity tag. Cells were lysed using sonication, and the lysate clarified by centrifugation was loaded onto a 5-ml HiTrap Ni2+ column (GE Healthcare). The fractions containing protein were pooled together and purified on a size-exclusion column (Superdex 200 26/60, GE Healthcare). Purification of Cy5-labeled RF1 and RF2 are described in Supplemental Experimental Procedures. All single-molecule experiments were conducted in a Tris-based polymix buffer consisting of 50 mM Tris-acetate (pH 7.5), 100 mM potassium chloride, 5 mM ammonium acetate, 0.5 mM calcium acetate, 5 mM magnesium acetate, 0.5 mM EDTA, 5 mM putrescine-HCl, and 1 mM spermidine. Preparation of the reaction mixtures for the different single-molecule experiments are described in detail in the Supplemental Experimental Procedures.

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RS instrumentation and data analysis Single-molecule intersubunit FRET and factor occupancy experiments were conducted using a commercial PacBio RS sequencer that was modified to allow the collection of singlemolecule fluorescence intensities from individual ZMW wells about 130 nm in diameter in 4 different dye channels corresponding to Cy3, Cy3.5, Cy5, and Cy5.5 fluorescence. The RS sequencer has two lasers for dye excitation at 532 nm and 632 nm. In all the Cy5-RF1, Cy5RF2, and Cy5-EF-G-GDPNP experiments, data was collected at 10 frames per second (100


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ms exposure time) for 5 min using energy flux settings of the green laser at 0.60 mW/mm2 and red laser at 0.10 mW/mm2. For all the Cy5-EF-G-GTP experiments, to better visualize the observed short Cy5 pulses in Cy5-EF-G-GTP experiments, the frame rate of these experiments was increased to 30 frames per second. So in all of the Cy5-EF-G-GTP experiments, data was collected at 30 frames per second (33 ms exposure time) for 5 min using energy flux settings of the green laser at 0.72 mW/mm2 and red laser at 0.24 mW/ mm2. For TIRFM instrumentation and data analysis, see Supplemental Experimental Procedures.

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Data analyses for all experiments were conducted with MATLAB (MathWorks) scripts written in-house (Chen et al., 2014). Briefly, traces from either the ZMW wells or immobilized complexes on TIRFM slide were automatically selected based on fluorescence intensity, fluorescence lifetime and the changes in intensity. Filtered traces exhibiting intersubunit FRET or single-molecule binding signals were then manually curated for further data analysis. The FRET and ligand occupancy states were assigned as previously described (Chen et al., 2013) based on a hidden Markov model based approach and visually corrected. The kinetics of state transitions were quantified by measuring the dwell times of the states. The ensemble distributions of these dwell times in the form of cumulative distribution plots were fit to single exponentials to obtain first-order rate constants (using curve-fitting tool on MATLAB). The bimolecular rate constants of ligand binding events were determined by measuring the slope of the linear fit to plot of first-order rate constants as a function of ligand concentration.

Supplementary Material Refer to Web version on PubMed Central for supplementary material.

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Acknowledgments This work was supported by U.S. NIH grants GM51266 and GM099687 to J.D.P., and NIH Molecular Biophysics Training Grant (T32-GM008294) and Stanford Interdisciplinary Graduate Fellowship to A. Prabhakar.

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Highlights •

Peptide release from the ribosome yields a post-termination rotated state intermediate

•

RRF binds to this intermediate to block off-pathway RF rebinding events

•

EF-G binds to RRF-bound rotated ribosome to catalyze 50S subunit dissociation

•

IF3 binds to remaining 30S complex to eject P-site tRNA for complete disassembly


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Author Manuscript Author Manuscript Author Manuscript Figure 1. Experimental setup

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(A) In all experiments, 30S preinitiation complexes (PIC) containing Cy3B-30S, fMettRNAfMet, and IF2-GTP is immobilized on the surface of the ZMW wells through biotinylated mRNAs. The reaction is started by delivery of BHQ-2–50S, Phe-TC, EF-G, and Cy5-RF1 or Cy5-RF2. (B) Structure of RF2-bound 70S ribosome (Korostelev et al., 2008) showing the C-terminal fluorescent labeling position (C367). (C) Bottom: representative trace of Cy3B- and BHQ-2-labeled ribosome translating in presence of Phe-TC, EF-G, and Cy5-RF. Top: schematic of translation, with ribosome states linked to fluorescence signals in the trace. Delivery of reagents results in initiation through 50S subunit joining (shown by quenching of green Cy3B signal), followed by one round of elongation (reported by a cycle of low and high Cy3B intensities), then binding of Cy5-RF


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during termination (shown by red Cy5 signal, see also Figure S1). Departure of Cy5-RF after peptide release correlates with ribosome rotation, presenting a post-termination rotatedstate intermediate (highlighted in grey). Cy5-RF can rebind to this intermediate to induce ribosome rotation back to non-rotated state. RRF and EF-G bind to this post-termination rotated ribosome to catalyze 50S subunit dissociation. Boxed: mRNA sequences used in these experiments.


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Author Manuscript Author Manuscript Figure 2. RF rebinding inhibits 70S ribosome disassembly after termination

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(A) Schematic describing the two competing pathways from the post-termination rotatedstate intermediate in the absence of any recycling factors. The observed rate of disappearance (kobs) of this intermediate is equal to the sum of the rates of the two competing pathways: RF rebinding (kpostRF,on[RF]) and spontaneous 50S subunit dissociation (k50S,off). (B) Representative trace highlighting the post-termination rotated states. (C) Post-synchronization of post-termination rotated state dwell times depict an exponential decay of post-termination rotated state population. This distribution was fit to a singleexponential equation to obtain kobs. (D) Plot of kobs as a function of [RF2]. The slope and y-intercept of the least-squares linear fit to the plot represent the RF rebinding rate (kpostRF,on) and 50S subunit dissociation rate (k50S,off), respectively. Error bars represent standard deviation.

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Author Manuscript Author Manuscript Author Manuscript Figure 3. RRF and EF-G block RF rebinding and catalyze subunit disassembly

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(A) Percentage of ribosomes showing RF rebinding at 0.05 and 1 µM EF-G as a function of [RRF]. (B) Percentage of ribosomes showing RF rebinding at zero, 1, and 20 µM RRF as a function of [EF-G]. (C) Subunit disassembly time at 0.05, and 1 µM EF-G as a function of [RRF]. Subunit disassembly time is defined as the mean dwell time of the post-termination rotated state before 50S subunit dissociation. Error bars represent S.E.M. (D) Subunit disassembly time at 1 and 20 µM RRF plotted as a function of [EF-G]. Error bars represent S.E.M.


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Author Manuscript Author Manuscript Figure 4. EF-G binds to RRF-bound ribosome to catalyze subunit disassembly by GTP hydrolysis

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(A) Representative trace of 50 nM Cy5-EF-G-GTP experiment in the presence of 20 µM RRF (and 10 nM wildtype RF2, 50 nM Phe TC, and 100 nM BHQ-2) showing Cy5-EF-G pulses during elongation and recycling. During recycling, Cy5-EF-G binds to the posttermination rotated state and is correlated with 50S subunit dissociation (boxed). (B) Post-synchronization of subunit disassembly signal and Cy5-EF-G(GTP) occupancy in the presence of RRF as boxed in panel (A). (C) Representative trace showing multiple rapid pulses of unproductive Cy5-EF-G(GTP) binding to the post-termination rotated state in the absence of RRF. (D) Representative trace showing long occupancy of Cy5-EF-G(GDPNP) to the rotated state both in presence and absence of RRF. FRET is observed between Cy5-EF-G and Cy3B-30S. (E) Mean dwell time of Cy5-EF-G pulses in the presence or absence of 20 µM RRF and different guanine nucleotides (G-ntd): GTP or GDPNP. Error bars represent S.E.M.

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Author Manuscript Author Manuscript Figure 5. IF3 binds to 30S–tRNA-mRNA complex to induce tRNA departure, followed by 30S subunit dissociation


(A) Representative trace showing that the Cy5.5-tRNAPhe does not depart in the presence of 1 µM RRF and 500nM EF-G without IF3. Cy5.5-tRNAPhe presence is necessary for the observed reversibility of 50S subunit dissociation and Cy5-RF2 rebinding. See also Figure S7A. (B) Mean lifetime of Cy5.5 fluorescence decreases in the presence of 1 µM IF3. Error bars represent S.E.M. (C) Representative trace showing that BHQ-2–50S subunit dissociation is followed by sequential departures of the Cy5.5-tRNAPhe and Cy3B-30S subunit in the presence of 1 µM RRF, 500 nM EF-G, and 1 µM IF3. Inset: Delay between 50S subunit dissociation and tRNA departure (blue line) and delay between tRNA departure and 30S subunit dissociation (orange line) are highlighted. See also Figure S7B. (D) Mean tRNA departure time, defined as the delay time between 50S subunit dissociation and tRNA departure (blue), is dependent on IF3 concentration. Mean 30S subunit departure time, defined as the delay time between tRNA departure and 30S subunit dissociation (orange), is independent of IF3 concentration. Error bars represent S.E.M.


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Figure 6. The complete model of ribosome recycling

Post-termination rotated-state 70S intermediate is the substrate for RRF and EF-G to bind and catalyze subunit disassembly. Although EF-G can bind to both RRF-free and RRFbound 70S complex, RRF and EF-G must bind sequentially to catalyze subunit disassembly. After 50S subunit dissociation, IF3 binds to the remaining 30S complex to induce P-site tRNA dissociation, followed by spontaneous dissociation of 30S subunit from the mRNA. The numerical values of the kinetic parameters here are presented in Table 1

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Table 1

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Kinetic parameters of termination and recycling measured in this study Rate constant

Definition

Value

kpreRF,on

RF2 association rate to pre-termination 70S complex

15.6±1.0 µM−1 s−1

kpostRF,on

RF2 association rate to post-termination 70S complex

2.8±0.2 µM−1 s−1

kRF,off

RF2 dissociation rate from post-termination 70S complex

0.157±0.038 s−1

k50S,off

Uncatalyzed 50S subunit dissociation rate from 70S complex

0.022±0.001 s−1

k50S,on

50S subunit rebinding rate to 30S complex

0.75±0.07 µM−1s−1

kG1

EF-G association rate to RRF-free post-termination 70S complex

18.6±0.8 µM−1s−1

kG2

EF-G association rate to RRF-bound post-termination 70S complex

2.10±0.09 µM−1s−1

k30S

30S subunit dissociation rate from mRNA

0.514±0.017 s−1


NMD: At the crossroads between translation termination and ribosome recycling.

Translocation as continuous movement through the ribosome.

Remembering the ribosome.

[Connecting isolated congenital asplenia to the ribosome].

The Modular Adaptive Ribosome.

Perspectives on the ribosome.

New insights into the enzymatic role of EF-G in ribosome recycling.

A Possible Role of the Full-Length Nascent Protein in Post-Translational Ribosome Recycling.

Trajectories of the ribosome as a Brownian nanomachine.

The ABCs of the ribosome.

Ribosome rescue, nearing the end.

Ribosome dynamics during decoding.

The conserved GTPase HflX is a ribosome splitting factor that binds to the E-site of the bacterial ribosome.

Orthogonal ribosome bio-firewall.

Purification, crystallization and preliminary crystallographic analysis of a ribosome-recycling factor from Thermoanaerobacter tengcongensis (TteRRF).

Ribosome changes during translation.

The ribosome can prevent aggregation of partially folded protein intermediates: studies using the Escherichia coli ribosome.

Phosphorylation controls binding of acidic proteins to the ribosome.

The binding of berenil to Escherichia coli ribosome.

Antimicrobial peptides targeting bacterial ribosome.

Ribosome rescue systems in bacteria.

Ribosome function determined by fluorescence.

Structural insights into ribosome translocation.

Exploring Internal Ribosome Entry Sites as Therapeutic Targets.