Structural basis of translation inhibition by a valine tRNA-derived fragment

We analyzed three structures of Val-tRF-30S ribosomal complexes, which revealed the mechanism by which val-tRF inhibits protein translation. The mechanism includes the binding of val-tRNA to the decoding site, inhibiting the binding of tRNA-mRNA, and inhibiting the binding of aIF1A.


Introduction
Page 2, l59.Val-tRFs interact with 30S but also 70S ribosomes.Though, all the following experiments study their interactions with isolated small subunits only (see below, remark on p4).Please comment this choice.

Results and Discussion
Page 2, l84: Val-tRF are in vitro transcribed.Are their "real" counterparts modified in vivo?If yes would these modifications influence their binding capabilities?Page 3: the authors identified 30 different Val-tRF conformations, reflecting the high flexibility of the RNA.However, they decided to choose and solve only three structures over the 30, based on the (very) relative higher number of particles.The number of particles is still very low (<10000) and the global resolutions rather poor regarding the current standards.Do this reflect any biological significance of the three selected models over the other ones?Page 4, top.Could the interactions and differences between the various models described be due to the absence of the large subunit?Are the structures described on the 30S SSU compatible with 70S ribosomes?Indeed, Page 7, l272 the authors state that Val-tRF can interfere with 70S assembly while in the introduction the state that it binds to 70S.Page 4, l150: show the similarities with ASL binding on a figure "wet" control, using the same ribosomes and cryo-EM procedure.In other words, a map without Val-tRF is necessary here to check whether the h24, h44 and h45 helices are not altered.It could simply come from empty 30S particles that were not used in the final reconstructions.
Overall global resolutions are rather poor, which can be explained by the small amounts of particles and the dynamics of the 30S.However, what are the local resolutions of the Val-tRF described in the three complexes?If they are too low did the authors use rigid body fitting?What is the precision of their atomic models?Methods Do the authors use montioncor2 (line 344) and then patch motioncorrection in cryosparc (line 350)?Please explain Line 349 : Tilted data ?Please explain: did the authors used titled series?I am a bit confused by the particle numbers: Starting from 854284 particles the authors keep only 9742 + 5281 +6795 = 21818 particles to reconstruct the three major classes.That means that 97.5% are not used?How can the authors claim that only 2.5% of the particles are biologically relevant, without overinterpretation?The manuscript submitted by Wu et al. present structural analyses of a particular tRNA-derived RNA fragment originating from the 5´end of H. volcanii Val-tRNA in complex with the 30S ribosomal subunit from Sulfolobus acidocaldarius.This Val-tRNA-derived 26 residue long fragment (called Val-tRF) has previously been identified and functionally characterized by others.It has been suggested to inhibit translation, reduce peptide bond formation and compete with mRNA binding to archaeal ribosomes.The new cryo-EM structures presented herein provide unprecedented insight into the mode of action of this tRNA fragment.The authors show three different Val-tRF/30S subunit complexes that, however, all have in common that Val-tRF binds close to universally conserved 16S rRNA residues in the decoding center and furthermore destabilizes several helices in close proximity of the decoding site (h24, h44, h45).This is the first structure of a ribosome-targeted tRF and thus this report clearly advances the field of regulatory ncRNA research.In particular the authors could deepen our understanding of the mode of action of Val-tRF on the archaeal ribosome and provide structural explanations for the previously reported functional data on Val-tRF.
Response: We express our gratitude to the reviewers for acknowledging the importance of our structural investigations.Our goal is to decipher the structural foundation that underpins the outcomes of prior functional experiments through structural analysis.Our structures enable a deep understanding of molecular mechanisms, unveiling the manner in which Val-tRF competes with mRNA for binding and how Val-tRF obstructs the initiation of translation.
In order to further improve this submission, the authors need to address the following points: 1) Very recently the field of tRNA-derived fragments has witnessed a landmark paper on a unifying nomenclature of tRFs (Holmes et al., Nature Methods, 2023, Vol. 20; https://doi.org/10.1038/s41592-023-01813-2)It is thus important and necessary that the authors of this submission follow this novel nomenclature and they should (at least once in their manuscript) use the standardized naming tDR (for tRNA-derived RNA).
Response: We are grateful to the reviewers for reminding us to use the most up-to-date nomenclature for tRNA derived fragment.Accordingly, we used tDF-1:26-Val-GAC-1 for Val-tRF and have clarified this in the manuscript introduction section.
2) Abstract, lines 27-28: it is unclear if the authors mean Val-tRNA or Val-tRF in this sentence.I think the authors mean the latter.This needs to be clarified.
Response: We apologize for this typo.It should be Val-tRF in the sentence.
3) Fig. 2G and H legend: it states "Val-tRNA" but I am certain that the authors actually meant "Val-tRF" instead.This needs to be changed.
The manuscript "Structural basis of translation inhibition by valine tRNA-derived fragment" provides a structural explanation for the inhibitory function of the Val-tRF on protein synthesis described in previous papers.The results described offer interesting potential explanations for the functional role of tRFs during gene expression.Using cryo-electron microscopy, the authors generated structures of the small ribosomal subunit of Sulfolobus acidocaldarius in combination with Val-tRF and observed the formation of 3 major complexes with Val-tRF in different conformations.The proposed binding site of the tRNA fragment and its impact on the ribosomal structure suggest why Val-tRF is inhibitory for protein synthesis.Val-tRF is located at conserved RNA binding sites and thus blocks tRNA binding, aIF1A binding and destabilizes helices involved in the decoding of the mRNA/tRNA interactions.In addition, the 70S assembly is potentially disturbed in the presence of the tRNA fragment.Overall, the manuscript provides a valuable insight into the function of tRNA fragments and provide a mechanism of function.However, several points should be addressed to improve the clarity of the manuscript.
Response: We are grateful for the reviewer's positive feedback on the significance of the three structures We clarified the role of Val-tRF in gene expression by solving the structures of Val-tRF bound with 30S ribosome subunit.Our results are consistent with previous functional studies.
1.Where does the Val-tRNA sequence come from (Haloferax or Sulfolobus)?If they are different, can this affect the binding of the tRF?What about the differences between the Haloferax and Sulfolobus ribosomes-are the binding sites identical?
Response: The Val-tRF sequence originates from Haloferax volcanii, while the 30S subunit is derived from Sulfolobus acidocaldarius (Sac).We opted for the Sac 30S ribosome due to its relative stability and suitability for cryo-EM studies.Val-tRF binds to highly conserved components on the 30S ribosome, including the decoding center, h23, and the 30S neck helix.As depicted in Figure 1, residues such as G496 (in the decoding center), G659, A660 (in helix 23), and G895, G896 (in helix 28 on the 30S neck) are highly conserved and engage in direct interactions with Val-tRF.The binding sites of Val-tRF are consistent across Haloferax and Sulfolobus.
10.Is there any functional evidence that the 30S subunit is functional after the purification?Is there a possibility for quality control?
Response: We appreciate the reviewer's emphasis on the importance of verifying the activity of the 30S ribosome.We have supplemented our work with experiments on in vitro translation activity using the 30S subunit.
11.The authors state that the tRF destabilizes h44, h45 and h24.However, they show the helices in their structure with the tRF (4B).In 4C the steric clash between the tRF and the helices is depicted.How is the overall structure changed in respect to h24, h45, h44 when comparing the subunits with and without tRFs?According to 4C it overlaps considerably and where do these residues go and how are they rearranged to fit the tRF?Is there any information on the binding affinity?It is impressive that the tRF can rearrange rather large proportions of the helices.
Response: We are grateful to the reviewer for highlighting the significant conformational changes in h24, h25, and h44.Upon comparing the 30S structure in the presence and absence of Val-tRF, we observed notable overall structural conformational differences (Figure 2A), especially in the head domain of 30S subunit.Specifically, for h44, adjacent components, such as uS5 and the G374 loop of the 16S rRNA, appeared to shift away from h44 (Figure 2B).In the case of h24, it was found to be distorted, contributing to its increased flexibility (Figure 2C).Regarding h45, the clash between the linker connecting h44 and h45 with the acceptor stem strand was observed, leading to increased flexibility in this region (Figure 2D).The destabilizing conformational effects on h24, h44, and h45 induced by Val-tRF binding are depicted in Figure 2D.Val-tRFs interact with 30S but also 70S ribosomes.Though, all the following experiments study their interactions with isolated small subunits only (see below, remark on p4).Please comment this choice.
Response: The three high-resolution structures of the Val-tRF-30S ribosome complex we analyzed, along with other low-resolution structures, all indicate that Val-tRF is incompatible with the 70S ribosome.This suggests that the binding sites and conformations of Val-tRF when bound to the 70S ribosome may differ from those when it is bound to the 30S subunit.Additionally, it is also possible that the limited number of conformations of Val-tRF bound to the 30S we captured did not include any that are compatible with the 70S ribosome.

Results and Discussion
Page 2, l84: Val-tRF are in vitro transcribed.Are their "real" counterparts modified in vivo?If yes would these modifications influence their binding capabilities?
Response: We appreciate the reviewer pointing out the differences between in vitro transcribed Val-tRF and its in vivo real modified counterparts.Currently, specific details on the modification residues of D stem loop Val-tRF in Haloferax volcanii are not available in the literature.These modifications could play roles in ensuring the stability and functionality of Val-tRF in high salt concentrations and temperatures, characteristic of the habitats of Haloferax volcanii.This might also partly explain why we were able to observe such a diverse range of conformations in the complex of in vitro transcribed Val-tRF with the 30S ribosome.This could be a limitation of our current study.
Page 3: the authors identified 30 different Val-tRF conformations, reflecting the high flexibility of the RNA.However, they decided to choose and solve only three structures over the 30, based on the (very) relative higher number of particles.The number of particles is still very low (<10000) and the global resolutions rather poor regarding the current standards.Do this reflect any biological significance of the three selected models over the other ones?
Response: Using Val-tRF as a mask for 3D classification, we obtained structures of multiple distinct Val-tRF-30S complexes.As shown in the figure below, we selected nine structures that exhibited clear Val-tRF densities.The cryo-EM maps reveal that in these nine structures, Val-tRF is consistently located between the decoding center and h24.Furthermore, the binding of Val-tRF induces conformational flexibility in the 16S rRNA regions h24, h44, and h45.Based on the comparison of these structures, we consider Structures I and II to be representative of the Val-tRF-30S complex, with the main distinction from other structures being differences in the conformation of Val-tRF Figure 3. Nine structures of Val-tRF bound with Sac 30S ribosome.In all these structures, h24, h44 and h45 are disordered.Also, Val-tRF locates between decoding center and h23.
Page 4, top.Could the interactions and differences between the various models described be due to the absence of the large subunit?Are the structures described on the 30S SSU compatible with 70S ribosomes?Indeed, Page 7, l272 the authors state that Val-tRF can interfere with 70S assembly while in the introduction the state that it binds to 70S.
Response: A previous study has demonstrated that Val-tRF primarily binds to the small ribosomal subunit.Utilizing polysome gradient analyses and in vitro binding studies with Haloferax volcanii cell lysates, Val-tRF was shown to primarily co-migrate with the 30S subunit fraction (Gebetsberger, Archaea, 2012).Consequently, our structural studies have elucidated how Val-tRF binds to the 30S ribosome subunit to inhibit translation.As depicted in Figure 3, across 11 structures resolved by cryo-EM (9 structure in figure 3 and structure 1 and 2), Val-tRF is primarily situated between the decoding center and h23, although the conformation of Val-tRF varies.This variability in conformation can be attributed to two main factors.Firstly, there is a lack of sufficient internal base pairs to constrain its overall structure and conformation.Secondly, the in vitro transcribed Val-tRF may be missing modifications that are crucial for stabilizing its 3D structure.Considering that previous studies have indicated Val-tRF's ability to bind to the intact 70S ribosome, this suggests that the binding site on the 70S ribosome could be distinct from that on the 30S subunit.Page 5, l208 and so on.The description of 30S modifications induced by the binding of Val-tRF suffer from the absence of a real "wet" control, using the same ribosomes and cryo-EM procedure.In other words, a map without Val-tRF is necessary here to check whether the h24, h44 and h45 helices are not altered.It could simply come from empty 30S particles that were not used in the final reconstructions.
Response: We appreciate the reviewer's highlighting of the importance of a real wet control.In fact, the structure of EMD-34862 (Figure 4A) utilized 30S ribosomal subunits from the same ribosome preparation as those used to analyze the Val-tRF-30S complex.Additionally, during the electron microscopy data analysis of the Val-tRF-30S complex, no empty sub-classes lacking Val-tRF were identified.
Overall global resolutions are rather poor, which can be explained by the small amounts of particles and the dynamics of the 30S.However, what are the local resolutions of the Val-tRF described in the three complexes?If they are too low did the authors use rigid body fitting?What is the precision of their atomic models?
Response: The Val-tRNA residue 1-26 derived from previous crystal structure model were first docked in the EM map.The rigid body fitted model was subjected into phenix for real-space refinement.During Phenix cryo-EM real-space refinement, the atomic model is iteratively adjusted to improve its fit to the experimental cryo-EM density map.This process involves optimizing parameters such as atomic coordinates, atomic B-factors (temperature factors), and occupancy values to maximize agreement between the model and the experimental data.The refinement also considers the local resolution variation within the density map, allowing for more accurate fitting in regions of higher resolution.Figure 5A and B show the differences of initial rigid fitting and after real-space refinement.Response: We apologize for any confusion regarding the image processing.The images were processed using cryoSPARC.The *.ERR movies were first imported into cryoSPARC and subjected to "patch motion correction."The resulting .mrcfiles were then utilized for CTF correction and particle picking.We have revised the relevant section in the methods.
Line 349 : Tilted data ?Please explain: did the authors used titled series?
Response: Tilt series data is collected using a transmission electron microscope (TEM) equipped with a cryo-holder capable of tilting the specimen.We used cryoSPARC to process the titled data including "Import Tilt Series" "Motion Correction", "CTF Estimation" "Particle Picking" and "Volume Reconstruction".I am a bit confused by the particle numbers: Starting from 854284 particles the authors keep only 9742 + 5281 +6795 = 21818 particles to reconstruct the three major classes.That means that 97.5% are not used?How can the authors claim that only 2.5% of the particles are biologically relevant, without overinterpretation?
Response: We appreciate the reviewer for highlighting the limited number of particles used to solve the three reported structures.Approximately 97.5% of the particles were utilized to solve around 30 additional structures without the precise adjustment of the Val-tRF models (as shown in Figure 3).As illustrated in Figure 3, the majority of Val-tRF binding patterns observed in the other ~30 structures closely resemble those found in structures I and II.Therefore, we consider structures I and II to be the most robust models, effectively explaining how Val-tRF influences translation.Thank you for submitting your revised manuscript entitled "Structural basis of translation inhibition by a valine tRNA-derived fragment".We would be happy to publish your paper in Life Science Alliance pending final revisions necessary to meet our formatting guidelines.
Along with points mentioned below, please tend to the following: -please be sure that the authorship listing and order is correct -please upload your main and supplementary figures as single files -please add a Running Title and a Summary Blurb/Alternate Abstract to our system -please add ORCID ID for the secondary corresponding author --they should have received instructions on how to do so -please add a Category for your manuscript in our system -please add the Twitter handle of your host institute/organization as well as your own or/and one of the authors in our system -please remove your figures from the manuscript file -please incorporate any points from the Conclusion section into the Discussion; we only allow a Discussion section -please add an Author Contributions section to your main manuscript text -please add your main, supplementary figure, and table legends to the main manuscript text after the references section -please upload your Table in editable .docor Excel format -please add callouts for Figures 1A,B,D,E; 2G,H; 4C; S1A-C; S2A-C; S3A-E; S4A-L to your main manuscript text -in the Data Availability section, please say where the structures have been deposited If you are planning a press release on your work, please inform us immediately to allow informing our production team and scheduling a release date.
LSA now encourages authors to provide a 30-60 second video where the study is briefly explained.We will use these videos on social media to promote the published paper and the presenting author (for examples, see https://twitter.com/LSAjournal/timelines/1437405065917124608).Corresponding or first-authors are welcome to submit the video.Please submit only one video per manuscript.The video can be emailed to contact@life-science-alliance.orgTo upload the final version of your manuscript, please log in to your account: https://lsa.msubmit.net/cgi-bin/main.plexYou will be guided to complete the submission of your revised manuscript and to fill in all necessary information.Please get in touch in case you do not know or remember your login name.
To avoid unnecessary delays in the acceptance and publication of your paper, please read the following information carefully.

A. FINAL FILES:
These items are required for acceptance.
--An editable version of the final text (.DOC or .DOCX) is needed for copyediting (no PDFs).
--High-resolution figure, supplementary figure and video files uploaded as individual files: See our detailed guidelines for preparing your production-ready images, https://www.life-science-alliance.org/authors --Summary blurb (enter in submission system): A short text summarizing in a single sentence the study (max.200 characters including spaces).This text is used in conjunction with the titles of papers, hence should be informative and complementary to the title.It should describe the context and significance of the findings for a general readership; it should be written in the present tense and refer to the work in the third person.Author names should not be mentioned.

B. MANUSCRIPT ORGANIZATION AND FORMATTING:
Full guidelines are available on our Instructions for Authors page, https://www.life-science-alliance.org/authorsWe encourage our authors to provide original source data, particularly uncropped/-processed electrophoretic blots and spreadsheets for the main figures of the manuscript.If you would like to add source data, we would welcome one PDF/Excel-file per figure for this information.These files will be linked online as supplementary "Source Data" files.**Submission of a paper that does not conform to Life Science Alliance guidelines will delay the acceptance of your manuscript.****It is Life Science Alliance policy that if requested, original data images must be made available to the editors.Failure to provide original images upon request will result in unavoidable delays in publication.Please ensure that you have access to all original data images prior to final submission.****The license to publish form must be signed before your manuscript can be sent to production.A link to the electronic license to publish form will be available to the corresponding author only.Please take a moment to check your funder requirements.****Reviews, decision letters, and point-by-point responses associated with peer-review at Life Science Alliance will be published online, alongside the manuscript.If you do want to opt out of having the reviewer reports and your point-by-point responses displayed, please let us know immediately.**Thank you for your attention to these final processing requirements.Please revise and format the manuscript and upload materials within 7 days.
Thank you for this interesting contribution, we look forward to publishing your paper in Life Science Alliance.

Figure 2
Figure 2 Conformational flexibility of 30S ribosome caused by Val-tRF binding.(A) cryo-EM density showing the overall conformational between Sac 30S ribosome in the presence (yellow) and in the absence of Val-tRF (green).The 30S head domain exhibits different conformations between these two structures.(B) Flexibility of h44 by binding Val-tRF induced displacement of uS5 and G374 loop.(C) Val-tRF induced distortion of h24.(D) Val-tRF caused unstable conformation of h24, h44 and h45, therefore no apparent density attribute to these components.
Page 4, l150: show the similarities with ASL binding on a figure Response: The similarities between Val-tRF and ASL binding are illustrated in Figure 4 (see below).Furthermore, the comparable binding of ASL-mRNA and Val-tRF is shown in the main Figure 3A.page 16).

Figure 4
Figure 4 Comparison of tRNA ASL and Val-tRF's binding on 30S ribosome.

Figure 5
Figure 5 Cryo-EM density showing the fitting of Val-tRF.(A) Initial rigid fitting of Val-tRNA fragment derived from crystal structures were fitted in the density.(B) After phenix real-space refinement, Val-tRF were fitted into the map.Methods