Peer review of: Stringent control of the RNA-dependent RNA polymerase translocation revealed by multiple (2020)

Reviewers' comments: 

Reviewer #1 (Remarks to the Author): 

Synthesis of nucleic acids is a critical process for all life forms and is performed by nucleic acid 
polymerases that processively catalyse multiple rounds of template directed nucleotide addition. This 
manuscript reveals important new details of the nucleotide addition cycles (NAC) based on high 
resolution crystal structures of the enterovirus 71 RNA-dependent RNA polymerase (RdRP), which is 
active for several NACs in the crystal. 

Previous studies from the Gong lab on the same system have described six steps in the NAC, including 
both open and closed pre-translocation states of the active site, post-translocated states after 
pyrophosphate release and an intermediate state, where the product is partially translocated. The 
manuscript by Wang et al takes this work further and describes several X-ray structures that capture 
how product and especially the template translocation occurs, an aspect that has so far been poorly 
understood. In particular, using mutants in RdRP conserved motif G, which allow trapping of a state 
where the template has partially translocated, they show that motif G constitutes a hurdle to be 
overcome during translocation. They suggest that motif G’s role is to prevent sloppiness in template 
positioning and thus promote efficient catalysis. In addition, they reveal an intermediate in the 
pyrophosphate induced reverse translocation of the product and discuss the implications for 
proofreading. 

The authors have maximally exploited the versatility of their system to capture new translocation 
states and add insightful details to their previous observations. Moreover, the mechanisms proposed 
probably apply generally to other viral RdRPs and may aid antiviral drug development. Thus the 
manuscript is worthy of publication. While the analysis is generally well argued, some aspects require 
clarification. 

1. The authors describe several structures with alternative conformations of the nucleic acid strands 
visible. However, they do not comment whether strand movement is accompanied also by protein 
movement. Are alternative positions of protein residues seen and modelled? Are the residues 
interacting with the product static, or do they follow the product strand, in other words, is the product 
floating in a static environment or does the protein ‘paddle’ the product strand? 

2. The authors should discuss whether any other parts of the active site might facilitate the product 
strand movement. For example, in influenza polymerase the conserved Met rich motif B loop, has 
been proposed to push on the product strand, in a ‘stroke’ like fashion (Kouba et a NSMB, 2019). Is it 
possible, that the equivalent of motif B in enterovirus 71 could play a similar role? Do authors observe 
any significant and perhaps coordinated movement of motif F (in particular Ile176) together with motif 
B in the translocation structures? How does the Ile176 move out of the way to allow the +2 base to 
move into the +1 position and then back to support the +1 base? A more detailed figure of the active 
site motifs movement should be included to accompany Figure 4A and 4C and perhaps the discussion 
on lines 310-313 extended. 

3. The authors describe motif G Thr114 and Ser115 as the ‘hurdle’ for the template translocation, but 
how is the hurdle overcome? A relatively conserved positively charge residue just preceding these two 
residues (His113 in EV71, positively charged residue -ve strand RNA viruses) directly interacts with 
product strand at position -6 Is it possible that His113 senses product translocation and induces some 
movement of the G motif Thr114 and Ser115 that releases of the ‘hurdle’ and trigger template 
translocation? If so, might it be interesting to enzymology characterize His113 mutants? 

4. Lys127/Arg188, contacting the template strand in the pre-translocation state, are packed over the 
G-motif to reach the template. That means that when motif G moves, Lys127/Arg188 might be shifted 
away, loosening the contact with template, thus releasing it to translocate. Could authors comment on 

such a scenario? 

5. After the ‘chemistry’ step, the presented results show the product strand translocases first, thus 
leaving the +1 incoming NTP position empty. Is it possible that the incoming NTP insertion towards 
the partially moved -1 template position, and subsequent base-paring in +1 position might play a role 
in completing template translocation? 

Minor comments: 
Line 56-57. Could authors compare the ‘push’ mechanism with recent findings about motif B in 
transcriptionally active influenza B strain RdRP (Kouba et a NSMB, 2019). 
Line 14-16: Not very clear. 
Line 57: Change to: An analogous 
Line 90. Explain results from the ‘tested’ C291M mutation or reword. 
Lines 123-125: Relative high level of freedom of the template contradicts line 107-108 from previous 
paragraph. 
Line 391: Explain the principle of the assay in one sentence. 
Figure S4. The difference between the experiments to obtain C0S6M and C2S6M is not very clear. The 
template RNAs should be listed in some table and incorporated nucleotides in the reaction should be 
marked. 

Reviewer #2 (Remarks to the Author): 

Summary 
The manuscript by Wang, et al. characterizes translocation intermediates for viral RNA-dependent RNA 
polymerase (RdRP) elongation complexes (EC) during the nucleotide addition cycle (NAC). This study 
provides insight into the forward and reverse translocation events, the role of RdRP motif G, and a 
transition state during translocation. The study uses X-ray crystallography to solve structures of 
enterovirus 71 (EV71) RdRP ECs and stopped-flow fluorescence-based single nucleotide incorporation 
assay to explore the role of RdRP motif G in controlling template RNA strand movement. The 
combination of structures with enzymology of mutant RdRP provides a convincing model of 
translocation and identify the residues that are important in this step. The data are compelling and 
appear rigorous. The findings are also significant as the molecular details of translocation by RNA and 
DNA dependent RNAPs are still fairly uncharacterized. I do have some critiques, and they are listed 
below. I recommend publishing upon revision. 

The aims of this study are listed below and were demonstrated convincingly: 
1. Structural elucidation of EV71 RdRP ECs translocation states 
2. Examination of the role of the RdRP motif G in translocation 

The approaches used to address these aims include: 

1. X-ray crystallography to determine the structures of EV71 RdRP ECs using NTP incorporation and 
pyrophosphate (PPi) to capture forward and reverse translocation states 
2. Characterization of the motif G hurdle residues using mutational analysis and stopped-flow 
fluorescence-based single nucleotide incorporation assays 
3. X-ray crystallography to determine the structural consequences of a mutation in the motif G for 
template strand restriction during translocation 

Major critiques 

1. In the introduction, the authors claim there is no structural evidence of translocation intermediates 
with a kinked BH. That is not true. Brueckner & Cramer (NSMB, 2008) published a paper describing a 
structure of a possible pol II translocation intermediate. Weixlbaumer (Cell 2013) also determined a 
structure with relevant similarities to the Brueckner/Cramer structure. This does not take away from 
the significance of this work but should be mentioned and this work should be discussed in the context 
of those studies in the intro and discussion. 

2. I think the NAC needs to be the first figure. 

3. Please include a pipeline of the initial template, initial RNA and which NTPs were added to 
accompany the structures. 

4. Because the crystals are a combination of nucleic acid translocation intermediates, the rest of the 
protein (not interacting with the nucleic acid) should have uniform high resolution around it, can the 
authors supply some supplemental figures illustrating the rest of the protein represents the high-
resolution data. 

5. Fig 1B has letters A,C, B and F but are not described in the legend. 

6. Authors need to give more background on why they focused T114 and S115 in the results as the 
hurdle residues. They just delve into “these two positions” (line 174) without much context. 

7. Fig. 3 needs to be explained better. The authors assume everyone is familiar with their system. For 
example, is it just CTP being added? It isn’t but is presented such. Does F indicate the fluorophore? 
Please add a pipeline or include more details in Fig. or legend. 

8. Authors do not provide illustrations of the electron density for the motif G hurdle residues (T114-
S115) and the basic residues K127/R188 which make key interactions with the nucleic acids. The 
hurdle resides (T114-S115) are only shown as atomic spheres in already built models (e.g. Figures 4A, 
4D, S1B, S4B). The density of the electrostatic interactions between the K127/R188 side chains and 
the -1/-2 template phosphates is not shown for any of the intermediates. However, authors show 
electron density for other residues such as I176, D238, and D329 but do not mention them in the 
main text. The authors should provide electron density figures for residues T114-S115 and K127/R188 
to show how they are modeled and how they are interacting with the RNA. 

9. The authors outlined a procedure for their RdRP EC crystallization, which involves prolonged 
incubation of the RNA scaffold in the presence of EV71 RdRP (Main Text, Page 17, Line 368-375). 
RNAs are susceptible to degradation especially if the protein component of the complex is 
contaminated with RNases that are carried over during purification. Degradation of the RNA scaffold 
would have effects on the observed structural intermediates. What measures did the authors use to 
minimize RNA degradation (e.g RNase-free purification of EV71 RdRP)? Did should run a gel on mature 
EC crystals to see if the RNA scaffold and product RNA are still intact after crystallization. 

10. The main structural focus of the paper is the nucleic acids near the site of catalysis. RNA-RNA 
(template-product) and nucleic acid/protein interactions with neighboring RdRP motifs are compared 
and highlighted between the translocation intermediates. However, the authors later state that 'the 
goal of translocation is to “move” the entire polymerase' (Main Text, Page 11, Line 241) and that the 
'conformational changes in translocation are not expected to be localized around the active site' (Main 
Text, Page 12, Line 243-244). Could the authors elaborate on any conformational changes that they 
observe in the overall structure of the EV71 RdRP translocation intermediates? Perhaps, the authors 
can align each structure and provide RMSD values for the comparisons (e.g. overall RP comparisons 
and nucleic acid/motifs comparisons)? 

11. In the S6M structure, the authors show a loss of interactions between K127/R188 and the -1/-2 

template phosphates that are usually maintained in the forward (S6A and S6B) and reverse (S6RA 
and S6RB) translocation intermediates. Authors should expand on the conservation and the roles of 
the K127/R188 residues. Have the authors performed mutational studies on these residues to assess 
their roles in catalysis or translocation? 

12. The authors capture an intermediate EC state (S6M) by using the T114S-S115T mutant. They 
proposed the stability of this state is comparable to pre- and post-translocated states (Main Text, Page 
11, Line 221-223). To test this, they soaked CTP into the crystal to induce forward translocation (C2) 
and found that they arrived at the same intermediate state as C0 (absence of CTP) (Main Text, Page 
11, Line 223-229). The authors should look at reverse translocation with the S6M intermediate by 
using PPi and divalent metal ions (similar to what they did to obtain S6RA and S6RB). This would 
provide evidence that the observed intermediate seen with mutant is between the pathway from pre- 
to post-translocation (Figure 5C). This is not necessary for this paper, just a friendly suggestion. 

Minor comments 
1. Main Text, Page 2, Line 14. Change 'that similar strategy' to 'that a similar strategy'. 

2. Main Text, Page 2, Line 9. Change ‘regular' to forward' 

3. Main Text, Page 3, Line 32. Change 'the understands of the' to 'insights into'. 

4. Main Text, Page 5, Line 91. Change 'since equivalent' to 'since the equivalent'. 

5. Main Text, Page 6, Line 117. Change 'this type of intermediates' to 'these types of intermediates'. 

6. Main Text, Page 10-11, Line 216-217. The authors should define what the 'regular conformation' is 
and how the -1/-2 template phosphates in the intermediate 'switch' to this conformation. In Figure 
4D, the -1/-2 phosphate positions are very different between S5 and S6M. 

7. Main Text, Page 11, Line 234. Change 'the 10s-100s-ncleotide per second rapid synthesis in a 
processive manner' to 'rapid synthesis in a processive manner (tens to hundreds of nucleotides per 
second)' 

8. Main Text, Page 11, Line 240. Remove 'as an entity'. 

9. Main Text, Page 12, Line 261. Change 'intermediates for easily' to 'intermediates from easily'. 

10. Main Text, Page 13, Line 279-280. Change 'rate-limiting step with high energy barrier comparable 
to that in the regular translocation may not exist' to 'the rate-limiting step present in regular 
translocation may not exist'. 

11. Main Text, Page 25, Figure 3A. Could the authors comment on the conservation of G117 in the 
motif G? Did they also mutate this residue in their enzymological studies? 

12. Supplementary Information, Page 3, Figure S2 (bottom-right). C2S4/5 should be labeled with 
'(Mn)' since it was generated using PPi and MnCl2. 

13. Supplementary Information, Page 4, Figure S3, Class V. 'amplitue' should be corrected to 
'amplitude'. 

14. Supplementary Information, Page 5, Figure S4. Show electron density for the C2S6M map similar 
to Figures 1B, 2B, 4A, S2. 

15. Supplementary Information, Page 7, Supplementary Video Legends. Provide timestamps for each 

structure shown in the video. For clarity, authors should label the states and key residues and bases in 
Video S1 and Video S2.