However, in the presence of GTP the fraction of RF3 molecules that enter the engaged state may be too small to capture. This scenario is realistic at the concentrations of factors in the cell where RF3 is much more abundant than RF1 Schmidt et al. The smFRET data presented here for a simple model system present a starting point to study dynamics of more natural termination complexes containing long peptide nascent chains. While model termination systems are fully functional in all steps of termination and the rate of GTP hydrolysis by RF3 is similar with the fM-Stop and fMFTI-Stop termination contexts Zavialov and Ehrenberg, , the length of the nascent peptide and the nature of the P-site tRNA may attenuate the ribosome dynamics.
While currently such complexes are biochemically too heterogeneous to study, further development of smFRET techniques toward multicolor detection and better time resolution may provide a tool to decipher the dynamics of these heterogeneous assemblies. The preparation and functional characterization of ribosomes labeled with Cy3 at protein L11 and double-labeled at S6-Cy5 and L9-Cy3 was carried out as described Adio et al.
A single cysteine was introduced at position T of L1 and the protein purified as described in Fei et al. The RF2 construct was cloned from the E. Native cysteines were replaced by serine or alanine based on the sequence conservation analysis performed using the Consurf database. RF1 and RF2 were purified and in vitro methylated as described Kuhlenkoetter et al. Labeling was performed for 2 hr at RT and quenched by addition of a fold molar excess of 2-mercaptoethanol over dye.
The following sequences were used: mMetStop. Ribosome complexes were applied to the surface and immobilized through the mRNA-biotin:neutravidin interaction.
Fluorescence time courses for donor Cy3 and acceptor Cy5 were extracted as described Adio et al. Time traces for further analysis were selected from the dataset by choosing only those traces that contained single photobleaching steps for Cy3 and Cy5 as recommended in [ Fei et al.
All trajectories were smoothed over three data points. Time trajectories with only one transition per trace and with the FRET changes of less than 0. In experiments measuring the residence time of labeled release factors, FRET traces are synchronized to the beginning of the FRET event reporting on the binding of the factor to the ribosome. One-dimensional histograms at the right side of the contour plots summarize FRET values of the first 10—30 time frames 0.
Dwell time histograms were fitted to either one- or two-exponential function. Rates k were calculated by taking the inverse of dwell times. PreHC was prepared as described Peske et al. Pyruvate kinase 0. After centrifugation 30 min, 16, g , the amount of released f[ 3 H]Met in the supernatant was quantified by radioactive counting. All data generated or analysed during this study are included in the manuscript and supporting files.
In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.
The first decision letter after peer review is shown below. Thank you for submitting your work entitled "Dynamics of ribosomes and release factors during translation termination in E. Your article has been evaluated by a Senior Editor and three reviewers, one of whom is a member of our Board of Reviewing Editors.
We have received comments from three experts in the field and these reviewers have discussed their independent views to reach a decision. Based on these discussions and the individual reviews below, we regret to inform you that your work will not be considered further for publication in eLife. While all three reviewers found your data on the function of bacterial termination factors RFs interesting, and all appreciated the major effort to develop the story, the consensus view was that: 1 there were substantive experimental issues that still need to be addressed and 2 even if these concerns are addressed, the manuscript will lack a cohesive narrative that would be of broad interest to the readers of eLife.
The reviews are included but the critical points that the reviewers focused on were:. Is RF2 fully methylated? Is the site of modification a different site than used for RF1 problematic data not provided? Are complexes fully saturated or would incomplete binding explain some of the heterogeneity i. This latter possibility seems critical to establish since RF2 does seem to bind more weakly. The biphasic nature of many of the dwell time histograms are suggestive of such compositional heterogeneity.
This manuscript by Adio et al. Several new findings are described that refute the older model of RF3 function, particularly the effect of nucleotide binding and hydrolysis, though that particular point has been addressed by the Rodnina lab previously. The data for RF2 appear to contradict previous structural work though the experiments here were performed on tRNAs lacking a true peptide in the exit tunnel.
Finally, the fact that ribosomal subunit rotation and factor binding and dissociation are not kinetically coupled in a straightforward manner makes it difficult to propose a clear mechanistic model.
RF2 dissociates quickly from either pre or post termination complexes even without RF3. This finding is new and important. However, the figures seem to depict a dipeptidyl-tRNA. This should be clarified in the text, figures, and figure captions.
Is this allowed rotation specific to the use of this minimal substrate fMet-tRNA? What would happen with a longer peptidyl-tRNA that extends into the exit tunnel? This is an important question because this finding suggests contrary to the literature that the hybrid tRNA states and rotated state of the ribosome can be separated.
This paper is an attempt to use single-molecule FRET to dissect the events during termination of bacterial translation. Termination in bacteria involves class I release factors RF1 and RF2 that recognize the stop codon in the A site of the ribosome and trigger peptide release.
A second GTPase class II release factor RF3 binds to the ribosome and accelerates dissociation of release factors from the ribosome, setting the stage for recycling the final stage of translation. In particular, there have been competing models.
A model proposed by Ehrenberg's lab suggested that the ribosome functions as a GTP exchange factor for RF3 that is closely related to its mechanism. This paper expands on recent work by both the Rodnina and Green labs to show a number of things.
Firstly, it suggests that RF1 and RF2 do not function in precisely the same way. RF2 is less stably bound and does not require the action of RF3 to dissociate. Finally, the paper shows that the temporal order of dissociation of RF1 and RF3 is random. Rather the complex favors a form that facilitates dissociation of both. This is a nice study that is a clear advance on previous papers and helps clarify some of the confusion in this area. The difference between RF1 and RF2 is surprising.
This is never actually addressed. A larger question they might have asked is why not all bacteria have RF3, and whether this is related to a possible role of RF3 in quality control shown by the Green lab. In summary, the paper deals with details of the mechanism of translational termination in bacteria that although important, will be of interest to only a handful of people even in the ribosome field. My own feeling is that it is not clear that it belongs in a general interest journal like eLife.
The manuscript by Adio et al. Specifically, Adio et al. The first two of these are open questions in the field and the third remains controversial, with two different models represented in the current literature. Given this, the answers to these questions would undoubtedly be of importance to the field and, in principle, the manuscript by Adio, et al. However, as described in greater detail below, important controls are missing and, in several key instances, it is not clear that the data support the conclusions.
In some ways, it almost seems as if the work presented here is a work in progress that is not yet finished and ready for publication. Thus, the authors would need to address these concerns before the appropriateness of this work for publication in eLife could be properly assessed.
As the authors point out, this is a very surprising result. As such, it raises many important concerns that could be easily addressed by controls:. Given that the Ehrenberg group has shown that the affinity of RF2 for termination complexes and the catalytic activity of RF2 on termination complexes are both dependent on this post-translational modification Pavlov, et al.
Given the results that the authors have obtained with their fluorophore-labeled RF2 construct, it seems to me that controls demonstrating that both the affinity and the catalytic activity of the authors' fluorophore-labeled RF2 construct are unchanged relative to unlabeled RF2 must be shown.
It is also important to specify whether this comparison is being made to the unlabeled, single-cysteine mutant RF2 construct or to the unlabeled, fully wildtype RF2 construct. What is the affinity of the unlabeled RF3 for these complexes? Is the concentration of unlabeled RF3 that the authors use for these experiments high enough such that the complexes are saturated?
How dependent are the interpretation of these data and the conclusions that are drawn on the complexes being saturated with unlabeled RF3? It seems like the extremely low, 10 nM concentrations of fluoropore-labeled RF2 and the possibility that, at any one time, the termination complexes are only partially occupied with RF3 would generate compositional heterogeneity that would make the data hard to interpret. The authors should address these questions through controls e.
Particularly careful attention should be paid to the unlabeled RF2 and RF3 experiments, since the authors have discovered that RF2 binds to termination complexes with a very low affinity such that the termination complexes may not be saturated with RF2 at the RF2 concentrations that are used for these experiments.
Such a scenario would again result in compositional heterogeneity that would make interpretation of the unlabeled RF2 and RF3 experiments difficult and, in addition, would challenge the appropriateness of comparing these results with the results of the RF1 and RF3 experiments in which the termination complexes are more likely to be saturated with RF1 or at least have lower compositional heterogeneity due to the higher affinity of RF1 for termination complexes.
The authors have assigned this FRET state as corresponding to the rotated state of the ribosome and have made no distinction between this rotated state of the ribosome and the rotated state of the ribosome that is observed in other contexts e. Given that it is based on a single FRET measurement on a single construct, how confident can the authors really be about this assignment?
How do the authors' observation that RF2 has a low affinity for, and binds only transiently to, termination complexes play into this? Is it possible that sampling of the rotated state of the ribosome only happens under conditions in which RF2 has dissociated from the termination complex due to the low affinity this relates to the concerns regarding whether the complexes are actually saturated with RF2? Unless the authors can present arguments or controls to eliminate these alternative interpretations or, better yet, provide additional, independent data that RF2-bound termination complexes can occupy the rotated state, I don't think the assignment of this FRET state is supported by the data that has been presented here.
In each case, the authors should analyze the individual trajectories to determine and report whether a particular experiment exhibits static or dynamic heterogeneity and what the most likely origin of that heterogeneity is. The authors should be particularly attentive to static heterogeneity, which may be indicative of compositional heterogeneity arising from termination complexes that may not be saturated by a particular factor. Additionally, the amplitudes and rate constants presented in Supplementary file 1 do not have standard deviations.
Thus, it is not clear that the majority of the experiments were repeated and, if they were repeated, it is not clear why the authors have not performed and reported the statistical analyses necessary for assessing the reproducibility of the results and the validity of the interpretations. Thank you for submitting your article "Dynamics of ribosomes and release factors during translation termination in E. Your article has been evaluated by James Manley Senior Editor and three reviewers, one of whom is a member of our Board of Reviewing Editors.
The reviewers have opted to remain anonymous. The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.
We have received comments from three reviewers two of them new since the previous version. As you will read in the detailed comments, all three reviewers appreciated the substantial amount of work contained in the manuscript and the many interesting insights derived from the data.
However, the reviewers remain concerned about the physiological relevance of the short peptidyl-tRNA ribosome complexes being studied. The authors offer cryoEM evidence that these tRNAs do readily sample the rotated state, especially at higher temperatures, and indeed, reviewer 2 suggests that there is additional literature supporting this point that should be clearly cited.
Despite these arguments, the authors should acknowledge that these ribosome complexes carrying short peptidyl-tRNAs may not fully reflect the behavior of longer, more physiologically relevant, peptidyl-tRNAs where the classic ribosome configurations are more stabilized. As such, the authors should acknowledge that the detailed examinations here provide a starting point for defining the complex molecular events of termination rather than a definitive description.
In addition to these general concerns, reviewer 2 had numerous concerns about the statistical analysis throughout the study in particular, whether the FRET histograms of the data subsets actually look like the FRET histograms of the total population. Reviewers 2 and 3 felt that conclusions were generally overstated given the limitations of the data specifics are detailed in the reviews. Despite these limitations, all three reviewers felt that this manuscript contained important insights for the ribosome field, first on the relative differences in behavior between RF1 and RF2 on the ribosome which is undoubtedly relevant to their different in vivo roles , and second, on the dynamics of RF1 and RF2 on the ribosome, and how they are impacted by the GTPase RF3.
Finally, all three reviewers felt that the critical insights of the manuscript were often lost in the dense writing style, the overstatement of conclusions, and the lack of clarity in relating the work to some previous studies.
At this stage, eLife will consider the manuscript for publication if the reviewer issues can be thoughtfully and completely addressed, both at the level of de-compressing the manuscript, clarifying the critical statistics, and more cautiously stating the conclusions.
This revision of the manuscript by Adio et al. All three reviewers were concerned about the peptidyl-tRNA migrating to the rotated state and the implications of this for the termination mechanism — the authors argue that this has been observed previously with relatively short peptidyl-tRNAs they note in particular in cryoEM structures by Fischer et al.
There were broad concerns related to the modification state of the RFs whether fully methylated , the impact of the fluorescent labels on their behavior, the saturation of factors in various experiments, and the statistics of the analysis. The authors have systematically addressed each of these concerns to my satisfaction. These studies lead to several new findings that are important for defining the roles of these critical factors in translation termination, and indeed in defining the roles of such factors more broadly in biology.
The most important findings are:. These differences are interesting in light of the auxiliary roles played by RF2 in quality control mechanisms in bacteria both post-peptidyl QC as characterized by Zaher et al. While the data don't tell us why these factors behave differently, they provide a biophysical basis for thinking about their distinct in vivo functions.
It might be useful for the authors to compare the rates that they observe for RF1 departure as promoted by RF3 to previous studies determined by fluorescence Koutmou et al. Again, the differences here relative to RF2 which does not depend on RF3 function are interesting. These data are extremely dense, and include differences in behavior related to the type of GTP analog used as previously reported in biochemical and structural studies.
Overall, I feel the manuscript contains a substantial amount of important data on the dynamics and function of termination factors on the ribosome during translation termination.
These data fit nicely with earlier studies by the same group detailing the critical role of the RF3-GTP cycle during these same steps and extended here. The challenge for the manuscript remains that it is extremely dense and the main points are often lost in the detailed discussions of complex experiments.
As just one example, the FRET distribution plots are layered with color blue, pink and red, which all look very similar , to give the dimension of dynamics — which is useful and important, but nevertheless overwhelming. I broadly support publication of this work in eLife but would ask that the authors take one more pass to increase the accessibility of their main conclusions.
Perhaps the problem is this: there are two stories here 1 the details of the functional cycle of RF1 and RF3 on the ribosome and 2 the distinctions in behavior between RF1 and RF2 on the ribosome. Yes, these are related stories, but presented together, the reader struggles to figure out whether to pay attention to commonalities or differences. The authors present a multitude of experiments describing the impacts of RF1 or RF2 binding on the conformational dynamics of various ribosome complexes, the rates of peptide release and the effects of RF3 on these various processes.
Included in these investigations are direct measurements of factor binding interactions with the ribosome via FRET measurements as well as GTP hydrolysis studies to address an open question in the field about the role of GTP hydrolysis in the release mechanism and to refute reports that RF3-GDP is the physiological substrate for the ribosome in termination.
There is no doubt that the synthesis of all of these data required tremendous effort both technically and intellectually. Although I did not see the original manuscript, it would appear that the addition of a second structural perspective on the classical-hybrid equilibrium shown in Figure 3B, E increase one's confidence in the interpretation that acylated tRNAs can indeed achieve hybrid-like configurations see more about this below.
Although respectful of the amount of work that went into the present manuscript, my overarching conclusion is that it is exceedingly complicated. The salient physiologically relevant conclusions from the study are hard to grasp. The integration of ensemble and single-molecule experiments is of course extremely helpful at times as it provides confidence and grounding, but the number of experimental systems examined and the speculative conclusions made are dizzying, making it hard to keep track of key considerations.
Do histograms of the small subset of dynamic molecules mirror the ensemble? Error bars on the individual measurements seem to be lacking throughout. The underlying basis of the static and dynamic populations is not clearly explained and should be clarified.
As written, the manuscript seems to imply that this is expected from the biochemical system. But this is not clear to me where this notion comes from. The easier explanation is that this arises from rapid fluorophore photobleaching prior to evident conformational changes — in this context, I was unable to find the photobleaching rates for the distinct systems examined in the manuscript but it appears to be rapid i. Each of these concerns seem more or less consistent with the reviewer comments provided during initial review of the manuscript.
My sense is that these considerations are likely to render the manuscript challenging to distill for the general reader. One of the points raised by the initial reviewers is that significant complexities in the interpretation of the data presented arise from the use of ribosome complexes bearing short peptide mimics fMET-Phe, fMET, NAc-Phe , which allows the ribosome to fluctuate between classical and hybrid states in the absence and presence of RFs.
Although the authors choose to reference their own cryo-EM work indicating that the small subunit spontaneously and reversibly rotates at elevated temperatures, multiple studies prior and subsequent to their chosen reference have indicated hybrid-like states can be achieved with acylated-tRNA in the P site and that such hybrid-like configurations are sensitive to the nature and length of the nascent peptide see Cornish et al.
Referring to such states as "Hybrid" is inappropriate as structural data indicate that this includes A76 interactions with the C region. The average reader is likely to be confused by this potentially non-physiological aspect of the termination studies presented. As the initial reviewers suggested, had the study been performed with longer nascent chains, as is expected for the physiological substrate of release factors during translation termination, this complexity could likely have been entirely avoided.
The fact that it is difficult to prepare such complexes as the authors indicate in their response to reviewers is fully appreciated, but a focused study on the propensity of the chosen complexes to achieve hybrid configurations and the nuanced impacts of such dynamics on release factor binding could be seen as an appropriate prerequisite for the present studies and their interpretation of the results presented.
For example, one of the key takeaways from the present study from the perspective of the general reader is that RF1 and RF2 perform differently in termination based on their measured off rates. Yet, such differences may simply arise from small distinctions in the binding energies of RF1 and RF2 to the ribosomes, which reveal themselves as potentially meaningful in the context of the non-physiological substrates examined.
Such binding idiosyncrasies may be further exacerbated by the GGQ mutation that is used to prevent peptide release as seems to be evident when comparing Figure 1—figure supplement 2C and E. What is the argument that these distinctions are physiologically important? Focused studies on the binding differences of RF1 and RF2 concrete measurements of affinity, on and off rates, beyond the information that is presented and clarification as to why such differences are important in the context of RF3 functions seems relevant to discuss but is presently lacking.
As written it implies that the prior studies were just wrong and the studies by Koutmou and Peske are just right. How are the authors so sure that this is the case? Was the prior work retracted due to a technical error? It is written in the Introduction, "thus the lifetime of the apo-RF3 complex would be too short to assume a tentative physiological role".
The work cited seems to make a thermodynamic rather than a kinetic argument and the statement as written seems to create a false context. The authors should include references to Cornish et al. The authors want to interpret differences between RF1 and RF2 'contour' plots that are post-synchronized as differences in binding and unbinding, but direct information about binding and unbinding is not present in these data.
These types of plots may indicate differences in dynamics within the bound complexes and this needs to be clarified. While I have no doubt that there may be binding and unbinding information underlying the differences observed, the language used in this section is too strong. The authors should also try to put sufficient information in the figure legends to enable one to discern the relevant information about the experiment but looking at the figure legend alone.
From the legend of Figure 1—figure supplement 3 it is impossible to know what ribosome complex was being examined where does the FRET come from without going back to the main text. It also states that "suggesting the two processes are not tightly coupled, consistent with previous notions based on cryo-EM […] and smFRET work" While subtle, as stated this sentence implies that the cryoEM work came first and the smFRET work came later.
But the opposite is true. Thus, the orientation of RF3 in the complex differs from that formed in the absence of RF1" While such conclusions may indeed be correct, statistical analyses on one dimensional histograms comprised of biological repeats are needed to make this conclusion without a second structural perspective to support this interpretation. Here, it seems too strong to make such statements without direct evidence of RF1 and RF3 binding.
The authors took advantage of different labeling strategies to look at ribosome, tRNA and release factor conformations at different stages of the termination process. These were used to draw two main conclusions: 1 Unlike RF1, RF2 can dissociate efficiently from the ribosome in the absence of RF3 although RF3 helps ; 2 The order of binding and dissociation events appear to be stochastic and unlike other translational GTPases, GTP hydrolysis by RF3 serves as a rescue mechanism for the factor.
Overall the paper was well written and the experiments appear to have been well executed. I was not however convinced that the paper offered significant new insights into the mechanism of translation termination.
Furthermore the authors played down the effect of RF3 on RF2 the fold change in termination rates under substoichiometric conditions is similar to that observed for RF1. Whether these differences also apply to other peptidyl tRNAs was not explored. As for the second main point of the paper, the Ruben group has also looked at the dynamics of the tRNAs and ribosome during recycling albeit not to the same extent in this paper and similar observations were made. As for the presentation of the data, I felt that the results were over interpreted and the main conclusion was to a certain extent based on conjecture.
For instance, the idea that the process of recycling is stochastic is based on the observation that many of the rates being similar RF1, RF3 dissociation for example , but these experiments were conducted in the absence of peptide release either using post hydrolysis complex or pre-hydrolysis one with GAQ RF1. It's unclear whether peptide release plays a role in the conformational changes.
This is relevant, as under normal conditions release factors must bind in the presence of a peptidyl-tRNA and promote peptide release before it is recycled. At the end, I was left wondering how the different assays recapitulate what happens under normal conditions. Another major point is the authors' assertion that GTP hydrolysis by RF3 merely serves to rescue the factor from nonproductive binding events.
The data in Figure 6A then suggests that RF3 is as likely to bind in a nonproductive fashion as to a productive one. Given the overall contribution of the paper to the field together with the less than ideal interpretation of the data, my overall enthusiasm of the paper was tempered.
We changed the figures to depict the formyl group by a different symbol than that representing an amino acid. The cryo-EM work by Fischer et al. These findings further support our conclusion that RF3 changes the conformational dynamics of the PreHC. Concerning the experiments with PreHC carrying a longer peptidyl chain, those are well beyond the scope of this paper for several reasons.
First, fMet-tRNA fMet has proven to be a good analog of peptidyltRNA with respect to studies of translation termination and several groups working on termination use it as a model system Sternberg et al. Second, the whole set of new experiments with a longer peptidyl-tRNA presents a significant technical challenge, because it requires preparation of homogeneous FRET-labeled ribosome complexes and validation of their biochemical and photophysical performance.
In the absence of RF1, RF3 can rapidly dissociate from some ribosomes, but these experiments do not show whether RF3 dissociates from the N or R state of the ribosome Figure 3.
There is no contradiction between the data sets, as smFRET and ensemble measurements show different parts of the mechanism. The confusion comes primarily from the RF3 dissociation experiments carried out with non-hydrolysable analogs, which do not behave as authentic GTP in our experiments. If dynamic fluctuations are necessary for the rapid dissociation of RF3, and this is induced only by an authentic GTP-like conformation of RF3, then non-hydrolysable analogs are not suitable to study this question.
These considerations motivated us to remove Figure 6G, H from the main text to the Figure 6—figure supplement 4. The text is modified to describe the potential problems of non-hydrolysable analogs. The results of cryo-EM studies Fischer et al. We agree that this was not sufficiently explained in the manuscript and added the pertinent discussion in the revised manuscript. This is an interesting question which is entirely beyond the scope of this paper. It is likely that bacteria that do not express RF3 compensate by having more RF2, or release RF1 with the help of other, yet uncharacterized factors e.
On this point we politely disagree with the reviewer. We think that it is important to get the mechanism of translation termination right and make essential corrections to the inaccurate models that made their way to the textbooks. On a broader scale, this work shows that simple mechanistic models are not suitable to describe the dynamics of complex machineries, which may be an important lesson not only for the ribosome field but also for others dealing with macromolecular ensembles.
The factors are fully methylated. This is now shown in Figure 2—figure supplement 2 and stated in the respective text. The labeled position is C, we apologize for the misprint. The activity of the factor is not changed Figure 2—figure supplement 1.
The concentration of RF3 is fold over the saturation concentration determined from the RF3-dependence of RF1 turnover rate see Figure 2—figure supplement 1D and Zavialov et al. Despite this very large excess, it is difficult to entirely exclude a minor fraction of ribosomes that do not have RF3 at the moment when RF2 arrives. We would have easily recognized such complexes, because they are mostly static and favor the N state Supplementary file 1.
If some of the complexes have been taken for analysis, we would slightly underestimate the effect of RF3, which would make the observed trend even stronger. The ribosomes that do not have either RF1 or RF2 would show the rotation distribution of the complexes with RF3 alone.
If some of the complexes lack RF1 or RF2, the effect would be underestimated, which does not change the trend, but makes it even stronger. It has been used to study subunit rotation on free ribosomes, diverse ribosome complexes with a number of different tRNAs and with and without elongation factors. Noller et al. Clegg and Noller have tested all potential photophysical effects Hickerson et al. Termination factors cannot affect the reporter directly, because they bind from a different side of the ribosome, and there is no indication for the existence of some yet unobserved indirect effects.
The available structures do not reveal anything unusual in the S6-L9 region upon termination factor binding; furthermore they demonstrate that the N state favors by RF1 or RF2 is the same N state as in the absence of the factors and likewise the R state formed with RF3 is similar to that formed during translocation.
Thus, there is absolutely nothing to indicate that the two states we observe here are grossly different from the well-characterized N and R states observed so far with a wide variety of ribosome complexes.
The law of parsimony does not leave us any other choice but to conclude that the N and R states formed in the presence of termination factors are the same as sampled by all other ribosome complexes studies so far.
We analyze each trace individually and we separate traces into static no transitions and dynamic traces Supplementary file 1. We report as indicated in Supplementary file 1 transition rates of dynamic traces only. There is no kinetic heterogeneity in the static traces. The decay rate of static traces has little biologic meaning as it is dominated by the photobleaching rate of the FRET dyes. The compositional heterogeneity is addressed in our replies above. In some cases, the heterogeneity has a biological meaning, which is discussed in the text.
A notion on the non-canonical roles of RF2 has been included in the Introduction, first paragraph. The comparison with the rates reported in Koutmou et al. As stated in the text, we find that the data are in very good agreement given the different methods used to determine the dissociation rates. We agree that the section on the role of GTP hydrolysis is complex. Also reviewer 2 commented that a combination of smFRET and ensemble kinetics is difficult to follow.
To answer this criticism, we have restructured the text and removed the ensemble kinetics data. We do not think that the short peptidyl substrates play a role here, as the key experiments on RF3 dissociation are carried out with PostHC where the peptide is released anyway. Furthermore, there is no indication that the puromycin treatment produces complexes that are different from those obtained with RF1, see the extended comparison between these complexes in Figure 4.
Generally, we are surprised about the fundamental criticism concerning the short peptidyl-tRNAs as substrates, as this is a standard used by many in the termination field, e. Koutmou et al. They find that GTP hydrolysis rates are similar with MFTI and fMet which suggests that there is no significant difference in the interaction of termination factors with ribosomes carrying a short or a slightly longer peptide. Making the complexes with an authentic peptidyl-tRNA is not feasible, because the stop codons are read-through at the in vitro translation conditions in the absence of release factors and in the presence of large excesses of ternary complexes even at high accuracy conditions.
In order to make the representation of smFRET data less complex, we simplified the color scheme and now use only two colors red and gray to distinguish static from dynamic complexes. We realized that as presented, this value apparently leads to confusion see reply 1 to comments of reviewer 2.
These values are still given in Supplementary file 1 where it is perhaps easier to understand. To make the writing style less dense, we extended the description of the experiments. We hope that the general decompression of the paper helps to understand the results. The histograms displayed in the manuscript represent the entire population of molecules, rather than just a subset of dynamic traces. These values are still available in Supplementary file 1.
The definition of static and dynamic traces is provided in the legend to Figure 1 and Supplementary file 1. Supplementary file 1 provides the detailed statistical analysis including the standard deviations of all measurements. A release factor RF refers to a type of translation factor that triggers translation termination. Release factors fall into two classes; Class I release factors that bind the ribosome in response to the presence of a stop codon within the ribosomal A-site acceptor site.
This binding event triggers release of the nascent polypeptide by the ribosome, a so-called peptidyl release reaction. Class II release factors are GTP-binding proteins that serve to enhance the efficiency of translation termination, either by promoting release of the Class I factor from the ribosome after peptidyl release as in the case of bacterial release factors or by forming a complex with the Class I RF prior to stop codon recognition as Skip to main content Skip to table of contents.
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