Peer review of: Distinct pre-initiation steps in human mitochondrial translation (2020)



REVIEWERS' COMMENTS: 

Reviewer #1 (Remarks to the Author): 

In my original review, I had concerns that mtIF3 might move on the mtSSU to accommodate tRNA. 
The additional structural analysis provided in the revised manuscript has allayed my concerns. 

My other major concern was that insufficient evidence was provided in the original manuscript to 
support the model that mRNA binds to the monosome but not the SSU. Here, the authors provide 
further support for their “monosome only” model through the use of single-molecule imaging (optical 
tweezers combined with confocal fluorescence microscopy). These experiments are a great addition to 
the paper. 

In light of the improvements to the manuscript, I recommend that the paper is published. The findings 
are novel and the structural biology is done to a very high standard. 

Reviewer #2 (Remarks to the Author): 

The initiation of mitochondrial translation is a process of fundamental importance, which is still poorly 
characterized. Here Khawaja et al. have studied the initiation of translation in mitochondria. A 
biochemical reconstitution included a mitochondrial small subunit (mtSSU) bound to mtIF3 to which 
recombinant mtIF2-GMPPNP and initiator tRNA were added. Samples of this reaction mixture were 
subsequently analyzed by cryo-EM to reveal two distinct states. The first state contains mtSSU-mtIF3 
whereas the second contains mtSSU-mtIF2-mtIF3. The structural biology analysis that is presented in 
this manuscript is of exceptional quality and the interpretation of the structural data is outstanding. 

Subsequent biochemical studies (using fluorescence cross-correlation spectroscopy and single-
molecule imaging) were used to obtain further insights into the mechanism of mitochondrial 
translation initiation where the authors found that monosomes but not mtSSU complexes are 
associated with messenger RNAs. The authors conclude that there are key differences between 
bacterial and mitochondrial translation initiation so that during mitochondrial translation initiation 
mRNA only associates with monosomes and not mtSSU complexes. 

The authors have revised several key points after their initial submission, and I believe that this 
manuscript is of clear interest to the readership of Nature Communications and should therefore be 
published provided that the following point is addressed: 

The available biochemical data can be explained by two different models for mitochondrial translation 
initiation (1. mRNA associates late (with monosomes; c.f. Figure 5) or 2. mRNA associates very 
weakly with mtSSU complexes earlier) while the authors have only illustrated the first model in Figure 
5. 

The existing biochemical data shows that: 

1. Monosomes are the only species that is found to be stably associated with mRNAs when using FCCS 
(Fig.2). 
2. Monosomes are the only species that stably bind to mRNA in single-molecule experiments (Fig. 5). 

The existing biochemical data however does not rule out that: 

1. mtSSU-mRNA complexes are very transient and cannot be detected with the employed techniques. 

In the absence of Shine-Dalgarno and anti-Shine-Dalgarno sequences it is expected that mtSSU-
mRNA interactions are weaker than in bacteria. The addition of the mtLSU would be expected to 
enhance mRNA affinity so that monosomes should have a higher affinity for mRNAs than mtSSUs. This 
is consistent with the data shown in Figures 2 and 5. 

Based on the above points, it is equally possible (but only stated by the authors in the last two 
sentences of the manuscript) that mitochondrial translation initiation could involve mRNA joining at an 
earlier point via transient intermediates. 

To address this point clearly, the easiest way would be to change the model proposed in Figure 5 so 
that mRNA joins at the same time as mtLSU and initiator tRNA because the current temporal 
resolution does not allow us to distinguish between these options. Importantly, this model would: 

a. Capture all available data as this point is still not fully addressed. 
b. Include a model in which mRNA may bind after mtLSU or before mtLSU binding. 
c. Leave enough room for further in-depth mechanistic analyses, which are beyond the scope of this 
manuscript, to be performed in the future. 

Reviewer #3 (Remarks to the Author): 

I had the pleasure of reviewing this manuscript in its previous incarnation. I was thoroughly impressed 
and found it compelling, as it reports a translation initiation pathway that is unique to mitochondria, 
albeit that it may occasionally occur in bacteria. The authors have now performed some very delicate 
molecular tweezers expts to address comments from a reviewer who has suggested that weak 
interaction of various components in the pre-initiation pathway may have been lost during sucrose 
gradient centrifugation. This new data fully supports the authors pathway. I very much like this work 
and strongly support its publication. In the Life Sciences, nothing is ever fully black and whilte. Of 
course, there are some highly unlikely scenarios that may occur that could argue against the data 
presented to support this pathway, but I think the authors have done more than enough to lead to 
publication of what is quite a provocative and exciting novelty that once again supports the idea that 
mitochondria do things their own way!