Author response:
The following is the authors’ response to the original reviews.
eLife Assessment
This is a useful study presenting solid data indicating that the bacterial GTPases EngA and ObgE enable single-step reconstitution of functional 50S ribosomal subunits under near-physiological conditions. The study elegantly bridges the gap between the non-physiological aspects of the previous two-step reconstitution method and the extract-dependent iSAT system to enable ribosome assembly under translation-compatible conditions; however, it is limited by reliance on rRNA and proteins extracted from native ribosomes and does not achieve a true bottom-up reconstruction from all synthetic components. The evidence is incomplete in not characterizing the spectrum of reporter polypeptides produced and not comparing their rate and yield of synthesis from reconstituted ribosomes to that obtained with pure native ribosomes; and the impact of the study is limited by not including reporters to examine the fidelity of initiation, elongation or termination achieved with the reconstituted ribosomes.
As described below, based on the comments from the public reviewers, we have summarized at the end of the Discussion how this study contributes toward true bottom-up reconstruction from fully synthetic components, as well as the aspects that will require further development. In addition, we have newly provided data characterizing the reporter polypeptides from multiple perspectives, demonstrating that the assembled ribosomes do not exhibit issues such as reduced fidelity (Fig. 6, 7, Supplementary Data 2, 3). We believe that these data adequately address the limitations that were pointed out in the eLife Assessment.
Public Reviews:
Reviewer #1 (Public review):
This study presents evidence that the addition of the two GTPases EngA and ObgE to reactions comprised of rRNAs and total ribosomal proteins purified from native bacterial ribosomes can bypass the requirements for non-physiological temperature shifts and Mg+2 ion concentrations for in vitro reconstitution of functional E. coli ribosomes.
Strengths:
This advance allows ribosome reconstitution in a fully reconstituted protein synthesis system containing individually purified recombinant translation factors, with the reconstituted ribosomes substituting for native purified ribosomes to support protein synthesis. This work potentially represents an important development in the long-term effort to produce synthetic cells.
Weaknesses:
While much of the evidence is solid, the analysis is incomplete in certain respects that detract from the scientific quality and significance of the findings:
(1) The authors do not describe how the native ribosomal proteins (RPs) were purified, and it is unclear whether all subassemblies of RPs have been disrupted in the purification procedure. If not, additional chaperones might be required beyond the two GTPases described here for functional ribosome assembly from individual RPs.
Native ribosomal proteins (RPs) were prepared from native ribosomes, according to the well-established protocol described by Dr. Knud H. Nierhaus [Nierhaus, K. H. Reconstitution of ribosomes in Ribosomes and protein synthesis: A Practical Approach (Spedding G. eds.) 161-189, IRL Press at Oxford University Press, New York (1990)]. In this method, ribosome proteins are subjected to dialysis in 6 M urea buffer, a strong denaturing condition that may completely disrupt ribosomal structure and dissociate all ribosomal protein subassemblies. To make this point clear, we described the detailed ribosomal protein (RP) preparation procedure in the manuscript, rather than merely referring to the book.
In addition, we would like to clarify one point related to this comment. The focus of the present study is to show that the presence of two factors is required for single-step ribosome reconstitution under translation-compatible, cell-free conditions. We do not intend to claim that these two factors are absolutely sufficient for ribosome reconstitution. Hence, we have revised the manuscript to more explicitly state what this work does and does not conclude.
(2) Reconstitution studies in the past have succeeded by using all recombinant, individually purified RPs, which would clearly address the issue in the preceding comment and also eliminate the possibility that an unknown ribosome assembly factor that co-purifies with native ribosomes has been added to the reconstitution reactions along with the RPs.
As noted in the response to the Comment (1), the focus of the present study is the requirement of the two factors for functional ribosome assembly. Therefore, we consider that it is not necessary to completely exclude the possibility that unknown ribosome assembly factors are present in the RP preparation. Nevertheless, we agree that it is important to clarify what factors, if any, are co-present in the RP fraction. To address this, we performed proteomic analysis of the TP70 preparation (Supplementary Data 3) and stated the possibility of other factors’ inclusion.
We also agree that additional, as-yet-unidentified components, including factors involved in rRNA modification, could plausibly further improve assembly efficiency. We also consider that such studies may contribute to extending the system to the use of in vitro-transcribed rRNA and fully recombinant ribosomal proteins, which could be essentially a next step of this study. We noted the possibility of as-yet-unidentified components and the future perspectives in the Discussion.
(3) They never compared the efficiency of the reconstituted ribosomes to native ribosomes added to the "PURE" in vitro protein synthesis system, making it unclear what proportion of the reconstituted ribosomes are functional, and how protein yield per mRNA molecule compares to that given by the PURE system programmed with purified native ribosomes.
According to this suggestion, we measured the sfGFP synthesis rate from the increase in fluorescence over time under conditions where the template mRNA is in excess, and compared this rate directly between reconstituted and native ribosomes. We consider that this comparison provides insight into what fraction of ribosomes reconstituted in our system are functionally active (Fig. 6).
As noted in the provisional responses, quantifying protein yield per mRNA molecule is substantially more challenging. The translation system is complex, and the apparent yield per mRNA can vary depending on factors such as differences in polysome formation efficiency. In addition, the PURE system is a coupled transcription–translation setup that starts from DNA templates, which further complicates rigorous normalization on a per-mRNA basis. Because the main focus of this study is to determine how many functionally active ribosomes can be reconstituted under translation-compatible conditions, we addressed this comment by just carrying out the experiment comparing sfGFP synthesis rate.
(4) They also have not examined the synthesized GFP protein by SDS-PAGE to determine what proportion is full-length.
We have added an affinity tag to the sfGFP reporter, and then, purified the synthesized products from the reaction mixture and analyzed it by SDS–PAGE (Fig. 7a).
(5) The previous development of the PURE system included examinations of the synthesis of multiple proteins, one of which was an enzyme whose specific activity could be compared to that of the native enzyme. This would be a significant improvement to the current study. They could also have programmed the translation reactions containing reconstituted ribosomes with (i) total native mRNA and compared the products in SDS-PAGE to those obtained with the control PURE system containing native ribosomes; (ii) with specifc reporter mRNAs designed to examine dependence on a Shine-Dalgarno sequence and the impact of an in-frame stop codon in prematurely terminating translation to assess the fidelity of initiation and termination events; and (iii) an mRNA with a programmed frameshift site to assess elongation fidelity displayed by their reconstituted ribosomes.
Following the recommendation, we selected DHFR as an enzymatically active protein and used it as a reporter, confirming that it exhibited enzymatic activity comparable to that observed when synthesized by native ribosomes (Fig. 7c). In addition, MS analysis of the purified sfGFP used for SDS-PAGE analysis showed that nearly all peptide fragments were detected, covering almost the entire sequence from the initiator amino acid to the amino acid immediately preceding the stop codon (Fig. 7b, Supplementary Data 2. These results suggest that protein synthesis by the newly assembled ribosomes proceeds smoothly from initiation to termination, with no apparent problem in fidelity, and therefore indicate that functional ribosomes were successfully reconstituted.
Reviewer #2 (Public review):
This study presents a significant advance in the field of in vitro ribosome assembly by demonstrating that the bacterial GTPases EngA and ObgE enable single-step reconstitution of functional 50S ribosomal subunits under near-physiological conditions-specifically at 37 {degree sign}C and with total Mg2+ concentrations below 10 mM.
This achievement directly addresses a long-standing limitation of the traditional two-step in vitro assembly protocol (Nierhaus & Dohme, PNAS 1974), which requires non-physiological temperatures (44-50 {degree sign}C), and high Mg2+ concentrations (~20 mM). Inspired by the integrated Synthesis, Assembly, and Translation (iSAT) platform (Jewett et al., Mol Syst Biol 2013), leveraging E. coli S150 crude extract, which supplies essential assembly factors, the authors hypothesize that specific ribosome biogenesis factors-particularly GTPases present in such extracts-may be responsible for enabling assembly under mild conditions. Through systematic screening, they identify EngA and ObgE as the minimal pair sufficient to replace the need for temperature and Mg2+ shifts when using phenol-extracted (i.e., mature, modified) rRNA and purified TP70 proteins.
However, several important concerns remain:
(1) Dependence on Native rRNA Limits Generalizability
The current system relies on rRNA extracted from native ribosomes via phenol, which retains natural post-transcriptional modifications. As the authors note (lines 302-304), attempts to assemble active 50S subunits using in vitro transcribed rRNA, even in the presence of EngA and ObgE, failed. This contrasts with iSAT, where in vitro transcribed rRNA can yield functional (though reduced-activity, ~20% of native) ribosomes, presumably due to the presence of rRNA modification enzymes and additional chaperones in the S150 extract. Thus, while this study successfully isolates two key GTPase factors that mimic part of iSAT's functionality, it does not fully recapitulate iSAT's capacity for de novo assembly from unmodified RNA. The manuscript should clarify that the in vitro assembly demonstrated here is contingent on using native rRNA and does not yet achieve true bottom-up reconstruction from synthetic parts. Moreover, given iSAT's success with transcribed rRNA, could a similar systematic omission approach (e.g., adding individual factors) help identify the additional components required to support unmodified rRNA folding?
We fully recognize the reviewer’s point that our current system has not yet achieved a true bottom-up reconstruction. Although we intended to state this clearly in the manuscript, the fact that this concern remains indicates that our description was not sufficiently explicit. We therefore added the paragraph to ensure that this limitation is clearly communicated to readers.
(2) Imprecise Use of "Physiological Mg2+ Concentration"
The abstract states that assembly occurs at "physiological Mg2+ concentration" (<10 mM). However, while this total Mg2+ level aligns with optimized in vitro translation buffers (e.g., in PURE or iSAT systems), it exceeds estimates of free cytosolic [Mg2+] in E. coli (~1-2 mM). The authors should clarify that they refer to total Mg2+ concentrations compatible with cell-free protein synthesis, not necessarily intracellular free ion levels, to avoid misleading readers about true physiological relevance.
We agree that this is a very reasonable point and revised the manuscript to clarify that we are referring to the total Mg2+ concentration compatible with cell-free protein synthesis, rather than the intracellular free Mg2+ level under physiological conditions. We also changed the term “physiological” to “near-physiological” to avoid the misunderstanding.
In summary, this work elegantly bridges the gap between the two-step method and the extract-dependent iSAT system by identifying two defined GTPases that capture a core functionality of cellular extracts: enabling ribosome assembly under translation-compatible conditions. However, the reliance on native rRNA underscores that additional factors - likely present in iSAT's S150 extract - are still needed for full de novo reconstitution from unmodified transcripts. Future work combining the precision of this defined system with the completeness of iSAT may ultimately realize truly autonomous synthetic ribosome biogenesis.
Recommendations for the authors:
Reviewing Editor Comments:
Recommendations for improvement:
(1) Assess the length distribution of GFP polypeptides being produced using SDS-PAGE.
SDS-PAGE was performed according to the comment 4 of the Reviewer #1 (Fig. 7b). Please refer to our response addressing the comment.
(2) Compare the rate and yield of GFP synthesized per mRNA using their reconstituted ribosomes to that obtained with pure native ribosomes.
The efficiency of the reconstituted ribosomes was compared to native ribosomes according to the comment 3 of the Reviewer #1 (Fig. 6). Please refer to our response addressing the comment.
(3) Expand the panel of reporter mRNAs being examined to compare the fidelity of initiation, elongation or termination achieved with reconstituted ribosomes to that obtained using native ribosomes.
DHFR synthesis was addressed and also MS analysis of synthesized sfGFP was performed according to the comment 5 of the Reviewer #1 (Fig. 7b, c). Please refer to our response addressing the comment.
(4) Revise the manuscript to clarify that the in vitro assembly demonstrated here is contingent on using native rRNA and thus does not achieve a true bottom-up reconstruction from synthetic parts.
We added to the Discussion a paragraph summarizing the findings of this study, limitations, and future perspectives according to the comment 1 and 2 of the Reviewer #1 and the comment 1 of the Reviewer #2. Please refer to our responses addressing these comments.
(5) Revise the manuscript to clarify that they are referring to total Mg2+ concentrations compatible with cell-free protein synthesis, not necessarily intracellular free ion levels, to avoid misleading readers about the physiological relevance of the reconstitution.
We revised the manuscript to clarify this point according to the comment 2 of the Reviewer #2. Please refer to our response addressing the comment.
(6) Revise the text to fully describe how the native ribosomal proteins (RPs) were purified and indicate whether all subassemblies of RPs were disrupted in the purification procedure.
We revised the Methods section to clarify how the native RPs were purified and that all subassemblies of RPs were disrupted according to the comment 1 of the Reviewer #1.
(7) Revise the text to indicate that achieving ribosome reconstitutions using all recombinant, individually purified RPs is required to achieve a true bottom-up reconstruction from all synthetic components.
As with our response to the comment 4, we have added the point at the end of the Discussion as a future perspective toward true bottom-up reconstruction from all synthetic components.
(8) Consider conducting a similar systematic omission approach (e.g., adding individual factors) to help identify the additional components required to support unmodified rRNA folding.
As with our response to the comment 4 and 7, we have added the point at the end of the Discussion as a future perspective toward identification of additional essential factors for true bottom-up reconstruction.
Reviewer #1 (Recommendations for the authors):
(1) Assessing the spectrum of GFP polypeptides being produced by SDS-PAGE and comparing the rate and yield of GFP produced to that obtained with pure native ribosomes would seem to be essential additional measurements needed to bolster the evidence supporting the main conclusions of the work.
SDS-PAGE and MS analysis of the synthesized sfGFP were performed (Fig. 7a, b). Comparison of the assembled ribosomes and native ones were also performed (Fig. 6).
(2) Examining translation of other reporter mRNAs designed to compare the fidelity of initiation, elongation or termination achieved with reconstituted ribosomes to that produced by native ribosomes in the PURE system would be required to elevate the scientific quality of the work and its significance to the field.
DHFR synthesis and its activity measurement were performed (Fig. 7c). Also, MS analysis of the purified sfGFP showed that nearly all peptide fragments were detected, covering almost the entire sequence from the initiator amino acid to the amino acid immediately preceding the stop codon (Fig. 7b). We consider that these findings indicate that there is no apparent problem with fidelity.
