Peer review of: Mechanism of ribosome rescue by alternative ribosome-rescue factor B (2020)

Reviewer #1 (Remarks to the Author): 

Chen et al. describes several biochemical and kinetic studies to decipher the mode of action of the 
alternative ribosome-rescue factor ArfB along with presenting several high resolution cryo-EM 
structures of ArfB bound to a model non-stop mRNA ribosome complex. The presented body of work is 
quite comprehensive and provides important data helping to rule out other proposed functional roles 
of ArfB in the cell, i.e. rescuing ribosomes stalled on rare codon clusters in an mRNA. Furthermore, the 
authors can put forward a mechanism for ArfB-mediated ribosome rescue considering several 
previously unknown kinetic steps found in their thorough kinetic analysis to provide the most 
comprehensive understanding of ArfB function to date. The cryo-EM models presented in this 
manuscript showcases the full ArfB protein interacting with the ribosome including improved resolution 
for the flexible linker between the N- and C-terminal domains that improve the biochemical 
interpretation based on the earlier crystal structure of this complex (DOI: DOI: 
10.1126/science.1217443 ). The resolution of the linker domain provided insight into how the dynamic 
nature of ArfB contributes to its functional mechanism in the absence of other termination factors like 
RF2 and that is required for the function of alternative ribosome rescue factor ArfA. Additionally, the 
resolution of the linker domain revealed why the mutation of residues in the linker resulted in 
abolished ArfB activity as described by Kogure et al. (DOI: 10.1093/nar/gkt1280). Overall, the cryo-
EM models and the impressive kinetic analysis of ArfB-mediated ribosome rescue presented here 
further our understanding of ArfB not only in bacteria, but as to how the eukaryotic mitochondrial 
targeted ortholog, ICT1, may function to rescue ribosomes stuck on a truncated, non-stop mRNA. As 
such this manuscript is of general interest, providing important structural and mechanic understanding 
beyond the bacterial ribosome and is well suited be published in nature communication. 

Major Issues 
No major issues. 

Minor Issues 
I found the paragraph at the end of the results (line 232 – 252) slightly confusing containing some 
hard to follow leaps in logic. The dissociation rate constant for the experiment described in this 
paragraph derives from the data (Fig 4f) fit with an exponential curve, or by some other means? (Line 
237 – in the range of <1 s-1 at 20°C). In the subsequent sentence, lines 239-240 specifically, it is 
stated that the data in Fig. 4b is fit with a two-exponential equation, and that two rate constants 
where derived for each complex. It is then concluded that the real difference between the complexes 
is in the “fraction of the slow vs. fast steps,” (Line 241-243), which I assume is the proportion of the 
amplitude change attributed to each rate constant from the fit of the curve? Is the overall conclusion 
from the fast vs slow steps that there are two steps in the dissociation of ArfB from the ribosome, or 
that there are two populations of ArfB-ribosome complexes with one rate of dissociation each? It 
might benefit the reader to see which step in the proposed mechanism is being referred to by the 
“preceding step” (line 245). 
Furthermore, given that the dynamic properties if the linker and the C-terminal domain are critical for 
the specificity and selection described in the model it would be important to discuss the 
thermodynamic details / implications that such a model would have (e.g. with respect to enthalpic vs 
entropic contributions). (maybe around line 310 or/and 351) 
The discussion might also provide some more detail about the mechanism of ArfB dissociation based 
on the kinetic data reported by the authors (maybe around line 340) 
Line 179 – the word binding is in a different font, could just be the PDF proof. 
Line 198 – “Supplementary fig. e,f).” – no figure number not listed, just panel e,f. I assume sup. Fig. 
4 
Line 210 – why using different precision indicators for koff e.g. ~110s-1 vs. 110-140s-1. 
Line 345 – How do the authors envision mechanistically such a “guiding” process? 
Line 393 – Missing a period at the end of the sentence “…at 37°C.” 
Line 458 – Should ArfB[flu]-96C be ArfB[Flu]-96C? 
Line 754 – Fig 3e some means of precision / significance indication (e.g. error bars) should be 
provided to allow assessment of the differences between the values? 
Line 770 – There is a period after the | divide between “Supplementary Fig. 3” and “Quality of the 
ArfB models.” 
Line 776 – Are the authors wanting to have alpha spelled out or with the Greek letter? 



Reviewer #2 (Remarks to the Author): 

Review -”Mechanism of Ribosome Rescue by Alternative Release Factor B” 

Kai-Hsin Chan and coauthors have studied by biochemical and structural methods the multistep 
mechanism of ArfB action of ribosome rescue. Authors significantly expand the X-ray studies of ArfB 
bound to ribosome (T. Steiz lab). Presented data shows a kinetic profile of ArfB action with structural 
snapshots of several steps. 
The paper is a good example of an integrative approach of biochemical and structural to study of the 
problem. 
The paper is well written and I would like to recommend for publication in Nature Communication. 

Some comments: 
Autors suggested: “Mechanism of ribosome rescue by ArfB based on rapid kinetics and cryo-EM data”. 
One of the most important model in the mechanism is intermediate state - initial binding of ArfB to the 
ribosome which is based on the NMR ensemble of free ArfB and cryo-EM structures of the P+9 
ribosome-ArfB complex. This is not an experimental intermediate state complex. I would like to ask 
authors to discuss about possibility to form complex containing simultaneously mRNA P+99 and ArfB 
or mRNA P+99 and ArfB mutant without CTD domain. 



Reviewer #3 (Remarks to the Author): 

The manuscript by Chan et al. “Mechanism of ribosome rescue by alternative release factor B” 
provides kinetics and structural insights into the mechanism of ribosome rescue by ArfB. The authors 
use rapid kinetics to clarify previous findings that suggested that ArfB can act on ribosome stalled on 
long mRNAs, including those containing rare codons. The authors show that ArfB-mediated ribosome 
rescue more likely occurs on mRNAs with no or short mRNA overhangs extending downstream of the P 
site. The efficiency of ribosome rescue by ArfB decreases rapidly on longer mRNAs. The data suggest 
that ArfB first binds to the stalled ribosome independently of the length of the mRNA, and that the 
following accommodation step is then dependent on the presence of mRNA in the mRNA entry 
channel. Using cryo-EM, the authors visualize ArfB bound to stalled ribosome complexes in two major 
states: one with no A site overhang, and one with a 9-nucleotide overhang. The 9-nucleotide 
overhang structure shows that ArfB displaces the 3’ end of the mRNA from the mRNA entry channel, 
which agrees with the slower accommodation step on ribosome complexes with longer mRNAs. In both 
structures, the C-terminal tail of ArfB is bound in the mRNA entry channel, making it incompatible with 
longer mRNAs. This observation is in agreement with a previous crystal structure of YaeJ (ArfB) bound 
to the Thermus thermophilus 70S ribosome. Interestingly, the high-resolution cryo-EM reported here 
(2.6 Å) allows the authors to build a more accurate model for ArfB which appears to better explain the 
available mutational data. The work is well executed and the manuscript is clearly written. This 
reviewer has only minor comments and suggestions to improve the manuscript: 

Lines 86-87: Change the order of the sentence to “When more than 9 nts of the mRNA extended past 
the P-site codon, the rate of hydrolysis reaction decreases sharply, by about…” 

Lines 152-155: Could be clearer to change to “In agreement with mutational data, the cryo-EM-based 
model shows that the essential residues Arg105, Arg118, Leu119, Lys122, Lys129, and Arg132 form 
intricate side chain contacts with the ribosomal RNA (rRNA), whereas most non-essential side chains 
that are not involved in any interaction are less well-resolved.” 

Line 252: “…(Fig. 4g).” – There is no panel g in figure 4. 

Line 325: Could be clearer to write “…when the mRNA exceeds the P-site codon by more than 6 nts.” 

Line 346: “…to adopt to different…” Remove extra word, or different meaning? 

Line 773: “…3.1 Å crystal structure…” This is a 3.2 Å structure. Same comment in panel a, label 
underneath the gray structure.