Structural characterization of the ANTAR antiterminator domain bound to RNA

  1. James L Walshe
  2. Rezwan Siddiquee
  3. Karishma Patel
  4. Sandro F Ataide

  • Curated by eLife

    eLife logo

    Summary: The reviewers were excited by the structural data, and felt that the structure represents an important advance in our understanding of ANTAR domain proteins. Nonetheless, while the reviewers found the proposed model of ANTAR regulation to be interesting, they raised concerns about the limited evidence in support of this model. In addition to the suggestions in the individual reviews, the authors thought the model could be tested using mutagenesis together with an in vivo or in vitro reporter system, and/or by structural studies of nascent transcripts in transcription complexes with EutV.

Reviewed by

Read the full article

Listed in

Log in to save this article

Abstract

Regulated transcription termination provides an efficient and responsive means to control gene expression. In bacteria, rho-independent termination occurs through the formation of an intrinsic RNA terminator loop, which disrupts the RNA polymerase elongation complex, resulting in its dissociation from the DNA template. Bacteria have a number of pathways for overriding termination, one of which is the formation of mutually exclusive RNA motifs. ANTAR domains are a class of antiterminator that bind and stabilize dual hexaloop RNA motifs within the nascent RNA chain to prevent terminator loop formation. We have determined the structures of the dimeric ANTAR domain protein EutV, from Enterococcus faecialis, in the absence of and in complex with the dual hexaloop RNA target. The structures illustrate conformational changes that occur upon RNA binding and reveal that the molecular interactions between the ANTAR domains and RNA are restricted to a single hexaloop of the motif. An ANTAR domain dimer must contact each hexaloop of the dual hexaloop motif individually to prevent termination in eubacteria. Our findings thereby redefine the minimal ANTAR domain binding motif to a single hexaloop and revise the current model for ANTAR-mediated antitermination. These insights will inform and facilitate the discovery of novel ANTAR domain RNA targets.

Article activity feed

  1. Version published to 10.1093/nar/gkac074
  2. eLife

    Reviewer #4:

    PREreview of "Structural characterization of an RNA bound antiterminator protein reveal a successive binding mode" Authored by James L. Walshe et al. and posted on bioRxiv DOI: 10.1101/2020.09.27.315978

    Review authors in alphabetical order: Monica Granados, Katrina Murphy

    This review is the result of a virtual, live-streamed journal club organized and hosted by PREreview and eLife. The discussion was joined by ten people in total, including researchers and publishers from several regions of the world and the event organizing team.

    Overview and take-home message

    In this preprint, Walshe et al. use a structural approach to examine a bacteria's RNA-binding ANTAR protein, EutV, including how EutV's antitermination mechanism works to prevent transcription termination and thus regulate gene expression. In addition, the team examined how a single hexaloop with the conserved G4 is recognized in succession by conserved residues in the ANTAR domains, how conserved A1 helps with proper RNA folding, and how these interactions support RNA binding. Although this research is of interest in the field, there are some concerns that could be addressed in the next version. These are outlined below.

    Positive Feedback:

    -I appreciate the comment on how crucial it is to understand the system and structure of these proteins for therapeutic purposes. It helps exemplify the relevancy for people outside of this field.

    -I think it's interesting that there is potential for a new current model for ANTAR-mediated antitermination.

    -I found it interesting that the two domains of the dimer cannot bind to the P1 and P2 helices of the same RNA.

    -New data is used in this preprint and displayed openly in Supplementary Table 1.

    -This research is novel because it's the start of looking at specifics of the mechanisms ANTAR domain proteins use to prevent termination.

    -It will be interesting to look at bioinformatic analyses for the ANTAR domain across diverse bacterial strains. Especially in diverse ecological niches such as host-pathogen.

    -It would be interesting to look at the structure in the context of an RNA construct that includes the P1, P2, and all of the T-loop.

    -I am outside of this field of study, however, there are definitely a lot of details in this paper that it seems to be enough to reproduce. Though others possibly in the field have said, reproducibility is less likely in this type of work.

    -I'm outside of the field, but it is nice that they deposited the atomic sequences on a public repository. I wonder whether this is mandatory for acceptance?

    -Yes [the results are likely to lead to future research], now that there is more interest in mechanisms that ANTAR domain proteins use for antitermination.

    -Are these findings applicable for similar ANTAR proteins (homologues/orthologues) in other bacteria? What about more complex organisms?

    -Interesting topic!

    -First RNA bound!

    -Yes [I would recommend this manuscript to others and peer review], I think this is a promising manuscript.

    Major Concerns:

    -Lot of the details are included [in the preprint], lacking, however, is information in the method section about the modeling of the RNA using RNAComposer. It is mentioned in the results section, but not in the methods section.

    -It's not clear where the EMSA assay is used in the paper. It's mentioned in the methods section, but not anywhere else.

    -I think it would be helpful to see whether ANTAR mutants have anti-termination defects in a transcription reaction. Authors might consider being cautious talking about anti-termination without functional studies.

    Acknowledgments:

    We thank all participants for attending the live-streamed preprint journal club. We especially thank those that engaged in the discussion.

    Below are the names of participants who wanted to be recognized publicly for their contribution to the discussion:

    Aaron Frank | University of Michigan | Assistant Professor, Biophysics and Chemistry | Ann Arbor, MI Monica Granados | PREreview | Leadership Team | Ottawa, ON Katrina Murphy | PREreview | Project Manager | Portland, OR

  3. eLife

    Reviewer #3:

    General assessment:

    Antitermination (AT) is a widespread mechanism to regulate transcription and can be mediated by ANTAR domains which prevent the formation of the terminator hairpin by binding to and stabilising a dual hexaloop motif in the nascent RNA. In the submitted manuscript Walshe and coworkers address the molecular basis of this AT mechanism which is largely unknown. They report two crystal structures of the dimeric ANTAR protein EutV from E. faecialis, one of EutV alone and one in the presence of a 51 nt long RNA containing the dual hexaloop motif, and combine this structural data with biochemical and biophysical data.

    The study

    -Reveals structural rearrangements that occur upon RNA binding and provides molecular insights into the RNA binding mode

    -Shows for the first time that a Met residue is obligatory for RNA binding

    -Redefines the minimal ANTAR domain binding motif

    -Suggests a new model for ANTAR-mediated AT

    Thus, the study is a comprehensive work, the experiments are performed thoroughly, and the conclusions are supported by the data. The results are of interest to a broad audience, ranging from the field of transcription in all domains of life to protein:nucleic acid interactions in general.

    However, the authors should address the following concerns:

    1. p 5, lines 15-17: The interactions should be described more clearly, i.e. are the hydrogen bonds between main chain atoms or between side chains? Which atoms/functional groups are involved (e.g. carboxy group of sidechain of Glu161)

    2. p 8, line 1-2: The SEC-MALS data indicates that the sample is not homogeneous and the authors suggest that this might be a concentration-dependent effect. This hypothesis is, however, not supported by the data. First, there is no information provided about the concentration used in the SEC run . Second, the SEC run was carried out on a S200 column. The experiment should be repeated on a S75 column which has a better resolution in the range of interest. Furthermore, the SEC runs should be performed with different concentrations to check if the oligomerization is indeed concentration-dependent and it could be used to check if the oligomerization is reversible (i.e. by collecting the "dimeric" form and re-run the solution and see if there is an equilibrium). Finally, as the authors discuss the dimerization behavior/mechanism, they might check if/how phosphorylation influences the oligomerization. These tests are important as this sample was used for the SPR experiments. If the sample, however, is not homogeneous, interpretation of the data might be compromised due to a mixture of different oligomeric states so that concentrations are not correct or a 1:1 binding model cannot be sued (most probably, the concentration of EutV is higher in the SPR experiments than in the SEC run and if there is concentration-dependent oligomerization this might be a significant issue).

    3. p 8: the chronology of Fig. 2 does not correspond to the chronology of the panels mentioned in the text.

    4. p 11, line 20: the authors state that G4 makes the only base specific interaction between the protein and the RNA hairpins. However, the details of the interactions are discussed only later in the manuscript so that this conclusion cannot be drawn at this stage. Thus, the author should present the interaction analysis earlier or adapt their argumentation (maybe by pointing to Fig. 3).

    5. Fig. 3: The interaction network between RNA (bases) and the protein is a very important point in the manuscript. In order to emphasize that only one of the bases, G4, makes base-specific contacts is, most probably, thus responsible for sequence-specific read-out, a 2D representation of the interaction network should be provided as Figure Supplement. (e.g. using LigPlot)

    6. p. 14: alanine mutagenesis. In order to confirm the importance of G4 the authors might substitute the base by another base and repeat the SPR measurements. Moreover, the quality of the protein samples should be checked (and data should ideally be provided as supplemental material), i.e. is the samples homogeneous (see comment on SEC runs) and are the samples free of nucleic acid contamination (how is the A260/A280?)

    7. p. 14: EutV binding to P1 and P2 RNA tested by SPR: was the sample homogeneous ? (see comment above on SEC runs).

    8. p 14: The authors should comment on the differences in the CD spectra in the region around 220 nm.

    9. p 20, ,lines 14-23. G4 plays a critical role in sequence-specific recognition. This recognition mode is reminiscent of the mechanism an operon-specific transcription factor, RfaH, uses. Here, RNA polymerase pauses at a pause site and exposes the nontemplate strand, which forms a hairpin. This hairpin stabilizes the flipping-out of a base in the loop region and allows sequence-specific read-out. Similar to EutV, sequence-specific recognition relies on very few base-specific interaction. However, RfaH binds to DNA. Moreover, also the sigma factor uses a flipped-out residue for recognition, although applying a different mode of stabilization. Thus, a comparison of these recognition modes might be of interest.

    10. p. 22: revised AT mechanism: The proposed model is reasonable and fully supported by the data. Is there a possibility to check the role of the two hairpins in vivo? I.e. if there is a possibility/assay to distinguish between recruitment and AT efficiency, the proposed model could be tested.

  4. eLife

    Reviewer #2:

    In the manuscript, "Structural characterization of an RNA bound antiterminator protein reveals a successive binding mode," the authors present the solved crystal structure of Enterococcus faecalis EutV by itself as well as bound to its RNA substrate. In previous work, the RNA substrate was proposed to consist of a dual hairpin and the genetics strongly suggested that both hairpins of this feature were crucial to functional antitermination in vivo. The finding revealed by the crystal structure in this work is that the EutV dimer does not appear to bind both hairpins simultaneously. The structure shows one EutV chain binding a hairpin in one RNA molecule and the second binding a second hairpin in a separate RNA molecule. The orientation of the two ANTAR domains is such that it is not possible to bind one RNA molecule simultaneously. Based on their findings, the authors propose a model of successive antitermination in which EutV binds to the first hairpin as it is generated by RNA polymerase and then this somehow favors binding to the second hairpin overlapping the terminator sequence as soon as it is made to prevent terminator formation. My overall assessment is that this is potentially an important and interesting contribution to the fields of transcription termination/antitermination and RNA/protein structural biology. However, there are concerns with how conclusive the data is, how exactly the model can work, and a lack of experimental evidence for the model.

    Major Comments:

    1. One major concern about the structure is that it is of non-phosphorylated EutV bound to its RNA substrate. Two-component system regulators almost always undergo conformational changes upon phosphorylation and therefore I think it is still an open question whether the structure truly represents active EutV bound to RNA. Perhaps the ANTAR binding domains of the EutV dimer change orientation upon phosphorylation such that binding to both hairpins can occur.

    2. If binding does only occur with one hairpin, then why are two necessary for activation? If it is impossible for one dimer to bind both hairpins simultaneously, how does binding to the first hairpin help binding to the second? This is not clearly explained. Also, no experimental evidence is presented to support the model.

    3. Wording of the abstract does not well reflect the final model presented. The abstract makes it sound like the second hairpin is not important, which is not what is shown here or in the previous work. I think the authors should say a bit more about what the actual model is in the abstract to eliminate this misconception.

    4. Ramesh et al. (2012) observed that EutV bound the eutP UTR with a higher KD (less efficiently) when just the P1 loop was used in an EMSA assay compared to P1/P2. This study found the same KD, whether P1, P2, or both are used in a SPR assay. Could the difference in these findings be related to the different techniques or the fact that slightly different versions of the EutV protein were used?

  5. eLife

    Reviewer #1:

    This paper looks at the mechanism of transcription regulation by the ANTAR domain protein, EutV. ANTAR domain proteins are an evolutionarily widespread family of RNA-binding regulators in bacteria. EutV has been proposed to regulate expression of target genes by binding two RNA loops in a 5' UTR, leading to a change in the RNA structure that modulates premature transcription termination. The current study determines the structure of dimeric EutV bound to an RNA target with two binding sites. Surprisingly, the interactions between the ANTAR domains in each monomer and each of the two RNA loops are incompatible with simultaneous binding of one EutV dimer to both loops. Hence, the authors propose a model in which EutV is "handed off" from one loop to the other as the RNA is transcribed.

    The structural information regarding the interaction between the ANTAR domain and RNA is an important advance, although there is very little comparison to previous studies, including a study that identified many of the same residues as being required for RNA binding (reference 33). The evidence that a EutV dimer cannot bind both RNA loops simultaneously is strong, and inconsistent with a previously proposed model of regulation. However, other than the structure, there are no data that support the authors' proposed hand-off model. In fact, as it is drawn in Figure 6D, I don't think the model is possible based on the same structural constraints that prevent simultaneous binding of the EutV dimer to both RNA loops. Without further experiments, I don't think the authors can conclude much about the mechanism other than it being unlikely that a single EutV protein binds both RNA sites simultaneously.

    Major comments:

    1. Throughout the paper, there is insufficient description of previous work on ANTAR domain proteins. In particular, there is little comparison to published structural data, including modeled RNA-bound structures. There is also very little discussion of the mutagenesis in reference 33 that identified many of the same residues as being required for RNA binding. There is no doubt that the structural work in the current study represents a substantial advance over previous studies, but it is important to describe the similarities and differences to prior work.

    2. Discussion, second paragraph. The evidence for a conformational shift in EutV upon phosphorylation is weak. This hypothesis is based on structural modeling from a homologous protein that has only 37% sequence similarity.

    3. The structure does appear to rule out the possibility of EutV binding both RNA hexaloops simultaneously, but the hand-off model is still rather speculative, and not supported by any additional experimental data; binding of two EutV dimers to the same nascent RNA would seem just as likely. There is insufficient discussion of how the hand-off model fits with previous mutagenesis studies (e.g. reference 25), and no follow-up experiments designed to test the model. If EutV is unable to bind both hexaloops simultaneously due to spatial constraints, how is it able to transition from one hexaloop to the other, as depicted in Figure 6D? I would expect the same spatial constraints to apply.

  6. eLife

    Summary: The reviewers were excited by the structural data, and felt that the structure represents an important advance in our understanding of ANTAR domain proteins. Nonetheless, while the reviewers found the proposed model of ANTAR regulation to be interesting, they raised concerns about the limited evidence in support of this model. In addition to the suggestions in the individual reviews, the authors thought the model could be tested using mutagenesis together with an in vivo or in vitro reporter system, and/or by structural studies of nascent transcripts in transcription complexes with EutV.

  7. Version published to 10.1101/2020.09.27.315978v1 on bioRxiv