Nevertheless, recent studies have identified AGO-mediated translation repression as an additional RNA silencing mechanism against plant viruses Bhattacharjee et al. J Exp Bot Translational repression of viral mRNAs was first observed in association with the defense response activated by the interaction between a dominant resistance gene and a viral elicitor Bhattacharjee et al. Co-expression of a resistance protein with nucleotide-binding NB and leucine-rich repeat LRR domain NB—LRR and its cognate viral effector results in an antiviral response that inhibits the translation of virus-encoded proteins in Nicotiana benthamiana Bhattacharjee et al.
Both the translational repression of viral transcripts and NB-LRR-mediated virus resistance were impaired by the downregulation of Argonaute 4-like genes. These results suggest that AGO proteins are involved in the specific translational control of viral transcripts in virus resistance mediated by NB—LRR proteins Bhattacharjee et al.
Another example of AGO-dependent translational repression mechanism was observed in N. In this interaction, symptom recovery follows an initial symptomatic systemic infection. These authors also showed that the recovery of ToRSV-infected plants is associated with a reduction in the steady-state levels of viral proteins and decreased translation of the corresponding viral RNA.
In vivo labeling experiments revealed efficient synthesis of the RNA2-encoded coat protein CP early in infection, but reduced RNA2 translation later in infection. Among the well-characterized VSRs, some of them e. PLoS Pathog e P0 proteins destabilize AGO1 through an F-box-like domain and induce subsequent degradation through the autophagy pathway Baumberger et al.
Likewise, the silencing suppressor P25 of Potato virus X interacts with AGO1 and mediates its degradation through the proteasome pathway Chiu et al. PLoS Pathog 6:e J Virol 86 The immune receptor NIK1 [nuclear shuttle protein NSP -interacting kinase 1] has a remarkable role in the defense response against begomoviruses. It belongs to the receptor-like kinase RLK family of plant receptors, and it was first identified as a virulence target of the begomovirus nuclear shuttle protein NSP Fontes et al.
Begomoviruses replicate their genome in the nuclei of infected plants via rolling circle replication. In the case of begomoviruses, it has been shown that in addition to encoding suppressors for siRNA-mediated defenses, these viruses enhance their pathogenicity in susceptible hosts by suppressing the antiviral activity of the transmembrane receptor NIK1 by the viral NSP Fontes et al. PLoS One 4:e Plant Biotechnol J Development BMC Plant Biol Several lines of evidence further support a NIK role in antiviral defense.
PLoS Pathog 4:e Finally, mutations in the activation loop A-loop of NIK1 that block its autophosphorylation activity also impair the capacity of NIK1 to elicit a response against begomoviruses Santos et al. EMBO J Structure NIK1 kinase activity has been shown to be dependent on the phosphorylation status of the A-loop Fontes et al. NIK1 is phosphorylated in vitro at the conserved positions Thr and Thr, and mutations within the A-loop interfere in the NIK1 capacity of autophosphorylation Santos et al.
Replacement of Thr with alanine TA strongly inhibits the autophosphorylation activity. In contrast, replacement of Thr with a phosphomimetic aspartate residue increases autophosphorylation activity and results in constitutive activation of a NIK1 mutant receptor that it is no longer inhibited by the begomovirus NSP Santos et al. The biological relevance of these findings has been certified by in vivo complementation assays. In contrast, ectopic expression of the Arabidopsis phosphomimetic TD mutant in tomato transgenic lines confers higher level of tolerance to tomato-infecting begomoviruses than expression of an intact NIK1 receptor Brustolini et al.
Collectively, these results implicate the phosphorylation at the essential Thr residue within the A-loop as a key regulatory mechanism for NIK activation. Consistent with an RPL10 role in antiviral defense, loss of RPL10 function recapitulated the nik1 enhanced susceptibility phenotype to begomovirus infection, as the rpl10 knockout lines developed similar severe symptoms and displayed similar infection rate as nik1 Carvalho et al.
In fact, RPL10 is localized in the cytoplasm, but is phosphorylated and redirected to the nucleus by co-expression with NIK1 Carvalho et al. Nevertheless, several lines of evidence indicate that the nucleocytoplasmic shuttling of RPL10 is dependent on the phosphorylation status and kinase activity of NIK1. Finally, mutations in the A-loop similarly affect the NIK1 capacity to mediate a phosphorylation-dependent nuclear relocalization of the RPL10 downstream component and to trigger an antiviral response Carvalho et al.
To gain further mechanistic insights into the role of NIK1 in antiviral immunity, the induced and repressed transcriptome by expressing the NIK1 phosphomimetic gain-of-function mutant TD was assessed in Arabidopsis Zorzatto et al. NIK1 constitutive activation does not induce the expression of typical defense marker genes associated to gene silencing, salicylic acid, or PAMP-triggered immunity PTI pathways but rather it down-regulates translation-related genes, causing suppression of global in vivo translation and decreased loading of host mRNA in actively translating polysomes PS fractions.
Likewise, induction of TD expression through a dexamethasone-inducible promoter also impairs global translation, which was correlated with a reduction of both PS and monosome NPS fractions, as well as of the RNA content associated with these fractions in the TD lines. Ectopic expression of TD controls begomovirus infection, causing symptomless infection, delayed course of infection and reduced accumulation of viral DNA in systemically infected leaves.
Additionally, in infected TD lines, the loading of coat protein viral mRNA in actively translating polysomes is reduced as compared to that of wild type infected lines, suggesting that the translation of viral transcripts is strongly impaired by NIK1-mediated signaling.
Thus, begomovirus cannot sustain high levels of viral mRNA translation in the TD-expressing lines, indicating that suppression of global protein synthesis may effectively protect plant cells against DNA viruses Zorzatto et al. Arch Virol In addition, the gain-of-function mutant TD from Arabidopsis functions similarly in tomato plants, as it causes a general down-regulation of translation machinery-related genes, affects translation in transgenic tomato lines and decreases viral mRNA association with the polysome fractions Brustolini et al.
Therefore, the enhanced tolerance to tomato-infecting begomovirus displayed by the TD-expressing lines is associated with the translational control branch of the NIK-mediated antiviral responses. These observations demonstrate the potential of a sustained NIK1-mediated defense pathway to confer broad-spectrum tolerance to begomoviruses in distinct plant species.
Nevertheless, in the Arabidopsis homologous system, the level of translational inhibition by the constitutive activation of NIK1 causes stunted growth in transgenic lines grown under short-day conditions, whereas, in tomato, ectopic expression of the TD mutant does not impact development under greenhouse conditions Brustolini et al.
As a possible explanation for this phenotype, tomato plants may not need maximal translational capacity for optimal growth under greenhouse conditions; thereby, the level of translational inhibition mediated by NIK1 activation does not reach a threshold that would impact growth. Additionally or alternatively, the TD-mediated translational suppression provokes a constant perception of stress in the transgenic lines, which, in turn, promotes acclimation to maintain normal growth under greenhouse conditions.
Therefore, the intrinsic capacity of agronomically relevant crops to withstand the deleterious effect from the suppression of global translation is a relevant agronomic trait to be considered for engineering the NIK1-mediated resistance against begomoviruses in crops.
Collectively, these results provide both genetic and biochemical evidence that the LIMYB gene functions as a downstream component of the NIK1-mediated signaling pathway linking NIK1 activation to global translation suppression and tolerance to begomiviruses.
Despite the advances in the elucidation of NIK-mediated antiviral signaling pathway, there is a complete lack of information on the critical early event that triggers the NIK1 signaling and transduction, which culminates with the suppression of host global translation as an antiviral response.
Recently, a comparison between the transcriptomes induced by begomovirus infection and by expression of the gain-of-function TD mutant revealed that begomovirus infection is the activating stimulus of NIK1-mediated defense, although the molecular basis for this elicitation is still unknown Machado et al. BioEssays A mechanistic model for a NIK1-mediated defense signaling pathway and its interaction with the begomovirus NSP is illustrated in Figure 3. Upon begomovirus infection, the extracellular domain of NIK undergoes oligomerization, allowing the intracellular kinase domains to transphosphorylate on a key threonine residue at position T and to activate one another Santos et al.
Alternatively or additionally, NIK1 may serve as a co-receptor for a defense-signaling cascade and interacts with an unidentified ligand-dependent LRR-RLK receptor in response to virus infection. The phosphorylation-dependent activation of NIK leads to the phosphorylation of RPL10 and the phosphorylated RPL10 is translocated to the nucleus, where it interacts with LIMYB to fully down-regulate translation machinery-related genes, leading to host global translation suppression that affects the translation of the begomovirus mRNAs Carvalho et al.
Thus, this down-regulation of cytosolic translation underlies at least partially the molecular mechanisms involved in the NIK1-mediated antiviral defense, which can be suppressed by binding of NSP to the NIK1 kinase domain. Due to the agronomic importance of plant virus as pathogens, the development of antiviral strategies aiming crop protection has been continually on focus. In this context, the identification and characterization of host factors targeted during infection constitute one of the most important goals of the virology research.
In vitro translation extracts lacking endogenous mRNAs were programmed with in vitro-synthesized capped and polyadenylated mRNAs. Non-programmed extracts served as controls. Total protein is a control from non-programmed T cell in vitro translation extracts.
Silencing eF4GI had no effect on c-Jun protein levels. Results are representative of three independent experiments. We also suspect that the use of different pathways of translation initiation, including the canonical eIF4E-eIF4GI cap-dependent translation, as well as IRES-dependent translation, are likely dependent on cell type and tissue specificity, which deserves further investigation.
Translation initiation is a well-established and critical regulatory point in gene expression. It has also emerged more recently as a remarkably plastic response to a variety of physiological changes by altering the type of translation initiation mechanism used, thereby dynamically reprogramming the types of mRNAs that are translated in response, for example, to stress, drug resistance, and transformation 3 , 24 , Understanding these different mechanisms is paramount, as they fundamentally underlie the manifestation of diseases as widely represented as cancer and autoimmunity.
See Supplementary Fig. Cells were transfected with plasmids using Lipofectamine Invitrogen as described by the manufacturer. The design primers containing the T7 promoter followed by sequences of interest are:. The shRNA cassette sequences were as follows:. Target sequences were:. Cells were lysed and subjected to immunoblot analysis. Puromycin incorporation in neo-synthesized proteins a measure of the rate of mRNA translation in vitro was assessed with an anti-puromycin antibody 12D10 monoclonal antibody.
The integrated density of positive bands was quantified using ImageJ software. Proteins were eluted from beads with HA-peptide Sigma. Samples from immunoprecipitates were reduced, alkylated, and loaded onto SDS-polyacrylamide gel electrophoresis gels to remove LC-MS incompatible reagents. Gel plugs were excised, destained, and subjected to proteolytic digestion with trypsin and resulting peptides extracted and desalted, as previously described The results were filtered to only include proteins identified by at least two peptides.
In addition, the data were analyzed using the SAINT algorithm 27 , including experiments 52—54 from the crapome. Polysome isolation was performed by separation of ribosome-bound mRNAs via sucrose gradient.
Briefly, Beckman Ultra-Clear centrifuge tubes were loaded with 5. To quantify translational efficiency, the difference in log 2 intensity between matched polysomal mRNA and total mRNA was determined. To examine differences in transcription and translation, total mRNA and polysome mRNAs quantile normalized independently. Statistical analysis was performed using the limma R package Cells were harvested, washed with PBS, and incubated with 0.
RNAs were purified by phenol—chloroform extraction and ethanol precipitation. In vitro translation extracts were made from human T cells as described Fractions were collected from the gradient and RNA purified by phenol—chloroform extraction and ethanol precipitation, and protein precipitated with trichloroacetic acid.
Data supporting the findings of this manuscript are available from the corresponding author upon request. Sonenberg, N. Regulation of translation initiation in eukaryotes: mechanisms and biological targets. Cell , — Silvera, D. Translational control in cancer.
Cancer 10 , — Ramirez-Valle, F. Cell Biol. Sharma, D. Role of eukaryotic initiation factors during cellular stress and cancer progression. Nucleic Acids , Translation initiation factors and their relevance in cancer. Svitkin, Y. Eukaryotic translation initiation factor 4E availability controls the switch between cap-dependent and internal ribosomal entry site-mediated translation.
Shatsky, I. Cells 30 , — Holcik, M. Internal ribosome initiation of translation and the control of cell death.
Trends Genet. Hellen, C. Internal ribosome entry sites in eukaryotic mRNA molecules. Genes Dev. Liberman, N. Nucleic Acids Res. Virgili, G. Structure 21 , — Marash, L. Cell 30 , — Moreover, several transcriptome-wide analyses of mRNA structure in E. We asked how mRNA structure differs between annotated start sites and internal AUG codons that are not annotated as start sites. From transcripts with sufficient coverage, we calculated the median SHAPE reactivity over a nt window surrounding annotated start sites and compared it to non-annotated AUGs Figure 5E.
For annotated initiation sites, the level of mRNA structure is significantly lower for a region 30 nt in length on both sides of the AUG codon shown in red as previously reported Del Campo et al. In contrast, except for a sharp dip in reactivity at the aligned AUG codon due to sequence bias, we see that mRNA structure is consistently high across this window for the set of non-annotated AUGs shown in blue.
These differences may be due in part to the ability of ribosomes to melt RNA structure during translation; indeed, initiation leads to the unfolding of RNA, which facilitates initiation by another 30S subunit Espah Borujeni and Salis, ; Andreeva et al. Originally developed for in vitro studies of ribosomes containing lethal rRNA mutations Youngman et al. Of particular interest are rRNA variants in bacterial genomes that are expressed differentially in response to changes in the environment and are proposed to have different specificities or functions Kurylo et al.
Variant rRNA alleles have also been reported for eukaryotic cells Parks et al. In addition, the functions of the highly variable rRNA expansion segments in eukaryotes are poorly understood Spahn et al. Previous genome-wide studies in bacteria have shown little or no correlation between SD strength and ribosome occupancy Li et al.
Using MS2RP, we are able for the first time to reveal the role of SD motifs in promoting initiation across the transcriptome. In other words, the mutant ribosomes translate genes with strong SD motifs worse than those with weak SD motifs Figure 2B.
There are two possible explanations for this negative correlation. It may be that the binding of wild-type ribosomes to mRNAs with strong SD motifs occludes their ribosome-binding sites, preventing mutant ribosomes from initiating and efficiently translating these genes. Alternatively, mRNA structure and other features may outweigh the impact of SD motifs, masking their effects, explaining why conventional ribosome profiling studies failed to observe correlations between SD strength and ribosome occupancy.
Regardless of which of these explanations is correct, the MS2RP strategy allows us to subtract the cumulative contribution to ribosome occupancy of all of such other mRNA features, and thus to focus exclusively on the contribution to ribosome occupancy of the SD:ASD interaction genome-wide.
Given that the SD motif functions through a well-defined mechanism and is widely conserved throughout bacteria, it has been thought to provide an important mechanism for start codon selection and translational output. Consistent with such a view, SD motifs are underrepresented within ORFs in order to avoid spurious initiation at internal start codons Hockenberry et al. Strikingly, however, we find that ribosomes with altered ASDs still find the correct start codons about as efficiently as wild-type ribosomes Figure 3.
Start peaks for all four ribosome types are observed at annotated start sites regardless of the affinity of the ribosome binding site for the ASD. These observations also hold true at the occasional non-annotated AUG codons where some initiation occurs Figure 4. These data are consistent with the conclusion that SD motifs are not essential for determining where translation starts on mRNAs genome-wide. What, then, are other mechanisms that could be used for start codon selection?
Local mRNA structure and RNA folding kinetics clearly must play a critical role in allowing ribosomes to find the start codon. A number of mechanistic studies have demonstrated that RNA structure around the start codon lowers translation levels Hall et al. Recent transcriptome-wide analyses of mRNA structure in E. Comparison of annotated start sites and non-annotated AUGs across several bacterial genomes shows that this mechanism is widespread Figure 5—figure supplement 1.
Indeed, in a recent study, Fredrick and co-workers used ribosome profiling in F. We envision that this sequence, like the Shine-Dalgarno motif, acts as a translational enhancer, fine-tuning the efficiency of initiation.
The mechanism by which A-rich sequences enhance initiation is not clear. A-rich sequences have been reported to enhance translation in a variety of eukaryotic contexts including Drosophila and wheat germ and reticulocyte lysates Ranjan and Hasnain, ; Sano et al. It may be that A-rich sequences interact with conserved elements of the ribosome across the domains of life.
Our findings have broad implications for the evolution of translational mechanisms in bacteria. Not all bacteria utilize SD motifs to promote translational initiation—SD motifs are notably lacking in Bacteroidetes and Cyanobacteria. Because the prevalence of SD motifs is a feature of the genome in general and not of a single gene, it makes sense that evolutionary selective pressure for or against SD usage would act at the level of the transcriptome. The nature of these selective pressures remains unclear, although Hockenberry recently argued that bacteria with high levels of SD usage tend to have higher maximal growth rates Hockenberry et al.
Future studies will clarify the evolutionary relationship between the growth environment, levels of SD usage among bacterial species, and their transcriptome-wide effects. IPTG was added 0. Cells were harvested by filtration using a Kontes 99 mm filtration apparatus and 0.
Cells were lysed in lysis buffer 20 mM Tris pH 8. For MS2RP, 1. Another 2 mL of lysis buffer was passed through the resin and collected. The flow-through fractions were then combined. Fractionation was performed on a Piston Gradient Fractionator Biocomp. RNA was purified by hot-phenol extraction. Cells were diluted fold in TBS. Reads were aligned using bowtie version 1. Reads that failed to align to those sequences were aligned to E.
To calculate average ribosome density, for each AUG we took the rpm at each position across this window, divided it by the total rpm in the window, and then computed the mean of these values for all AUGs included in the calculation. Probability logos were generated by kpLogo Wu and Bartel, using its default settings. For Figure 5A , input and background sequences are described in the figure legend. In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.
This paper uses an innovative twist on ribosome profiling to investigate the importance of Shine-Dalgarno sequences in bacterial translation initiation.
Surprisingly, the data show that strong base-pairing between the 16S ribosomal RNA and the mRNA Shine-Dalgarno sequence is neither necessary nor sufficient for translation initiation.
This suggests that start-codons are "hard-wired" into the genome, largely independent of Shine-Dalgarno sequence. What follows is the decision letter after the first round of review. Thank you for submitting your work entitled "Shine-Dalgarno sequences fine-tune translation genome-wide but are not the primary determinants of start-site selection" for consideration by eLife. Your article has been reviewed by three peer reviewers, including Joe Wade as the Reviewing Editor and Reviewer 1, and the evaluation has been overseen by Jim Manley as the Senior Editor.
The following individual involved in review of your submission has agreed to reveal their identity: Shura Mankin Reviewer 2. Our decision has been reached after an extensive discussion involving the three reviewers. The reviewers were enthusiastic about parts of the manuscript, in particular the method itself; however, there was some disagreement as to the significance of the work.
Much of the discussion focused on the data in Figure 6, which the reviewers considered to present potentially the most important result. We felt that further analysis is needed to fully address the key questions of i whether annotated start codons are inherently good at binding ribosomes, independent of the SD, and ii whether an SD alone e. Put more simply, we felt that further analysis is required to show that start-codons are "hard-wired" into the genome, largely independent of SD sequence.
We are therefore rejecting the paper because the outcome of the new analysis is unclear. Nonetheless, we would be willing to consider a revised version if the analyses suggested below, or something equivalent, provide stronger support for the idea that start-codons are hard-wired, independent of the SD sequence.
We suggest that a more appropriate control set of ATGs would be those with a good predicted match to the altered ASD sequence. We also suggest limiting the analysis in Figure 6C to start codons that have a poor match to the modified ASD. Another way to look at this would be to compare which annotated start codons are recognized by the different modified ribosomes; if all three types of ribosome recognize the same subset of start codons, it's safe to conclude that this is occurring independent of the SD.
If these or other analyses can provide stronger support for the "hard-wired" model, that would likely be sufficient for publication. In addition to the re-analysis of data from Figure 6, it's important to improve the clarity of the paper, which was at times confusing see the detailed reviews for more information on this.
Additionally, reviewer 3 makes some important points about the calculation of hybridization energies, such as considering a full, 9 nt SD sequence with variable spacing. Lastly, the manuscript would benefit from a clearer description of what is already known about features other than SD sequence that contribute to translation initiation see comments from reviewer 3. This paper describes an innovative approach to probe the importance of Shine-Dalgarno S-D sequences in translation initiation in Escherichia coli.
By performing ribosome profiling on modified ribosomes, the authors are able to observe translation by ribosomes with altered anti-S-D sequences. This method reveals that despite no correlation between S-D strength and translation levels, there is a contribution of S-D strength that is apparent when all confounding factors have been controlled for.
Interestingly, this effect of S-Ds is lost during other growth conditions, although for cold shock that is largely consistent with previous work, and it is unclear what the mechanism is in stationary phase.
While I think the topic is interesting and the primary method is ingenious, I'm not convinced that the authors have learned much about the relative importance of S-Ds in translation initiation. As they acknowledge, previous studies have failed to see a correlation between S-D strength and translation initiation levels, and the importance of secondary structure and of A-rich sequences has been described previously.
The fact that predictions of S-D strength correlate with translation initiation levels once factors other than S-D have been accounted for indicates that these predictions are fairly accurate. This is important, since it accounts for the possibility that the lack of correlation between predicted S-D strength and translation initiation is because of our inability to predict S-D strength. However, the impact of this advance is small.
I also have concerns about the interpretation of Figures 6 and 7 that impact the overall conclusions. The overall conclusion is that S-D-dependence is lost at almost all genes during cold shock. However, a small subset of genes appears to depend strongly on the S-D.
The distinction between the effect on the majority of genes and the effect on a small subset should be explained more clearly. The simplest interpretation of these data is that most start codons are highly structured during cold shock, but those that are do not rely on their S-Ds. This model is largely consistent with previous work. The data show that for the altered ribosomes, annotated start codons are used far more efficiently than the collection of all other ATG sequences within ORFs.
However, there are many more ATGs within ORFs than annotated start codons, and even if translation relies heavily on S-D sequences, you would expect that most ATGs within ORFs would not be selected by alternative ribosomes because only a small subset will have appropriate S-D sequences, and many may be weakly expressed.
My interpretation of these data is that alternative ribosomes do use annotated start codons, but there is no way to tell how selectively they do this. A more appropriate comparison would be of i annotated start codons to ii ATGs within ORFs where the ATG is associated with a sequence that is predicted to function as a good S-D for the alternative ribosome.
Figure 2C suggests that the number with good matches will be fairly high. Is the ribosome density at annotated start codons simply due to the subset of start codons that have reasonable SD matches to the altered ASD? Another way to think about this is to ask whether the start codons contributing to the signal in Figure 6C are the same start codons that contribute to the signal in Supplementary Figure 5A-B.
Similar to Figure 6, these data highlight the contribution of non-SD sequences to translation initiation, but they do not provide any information about the relative importance of the different sequence elements. The paper of Saito and al. Using a clever approach, the authors use ribosome profiling to compare mRNA occupancy by wt ribosomes and ribosomes with the altered anti-SD sequence ASD. In confirmation of previous findings from the Weissman lab, they find that the general translation efficiency does not correlate with the predicted strength of SD-ASD interactions.
However, when all the other factors are masked, they observe a strong dependence of the initiation rate on the strength of SD-ASD pairing. They also noted that a subset of genes expressed in the stressed cells depend heavily on recognition of the SD sequence by the ribosomes. One of the unexpected, but highly important findings is the observation that the ribosomes with the altered ASD can nevertheless correctly and selectively initiate translation at the known start sites underscoring the importance of factors other than SD-ASD interactions in the start codon selection.
Importantly, the reported work reveals the prevalence of A-rich motifs in the ribosome binding sites of the genes with weak SD sequences in E. This trend becomes especially prominent in the bacterial species that do not rely on SD-ASD interactions for translation initiation.
This is an interesting, intriguing and important study. The results are nice and clean and the implications are important for unraveling the fundamental mechanism of translation initiation in bacteria.
Although the paper is generally well written, it was hard at times to follow the authors logic and I strongly encourage the authors to try to clarify the message, which often was hard to extract. This is hard to digest.
However, they sound nearly identical and thus, do not accurately communicate the point the authors apparently are trying to make. Although the presence of unprocessed sequences at the 5' and 3' end of the ASD-mutant 16S rRNA would not likely change the general conclusions of the paper, hypothetically it could affect the functionality and elongation rate of the mutant ribosomes.
I am wondering whether authors have checked how well their mutant 16S rRNAs are processed. Irrespectively, I believe a more detailed discussion of the general functionality of the ribosomes with altered ASD, especially in relation to the elongation rate, would be beneficial. To do this, they carry out ribosome profiling experiments to measure genome-wide ribosome densities on aSD-modified strains, including during exponential, stationary, and cold shock growth phases.
Overall, they find that changing the last 9 nucleotides of the 16S rRNA has a significant effect on the transcriptomes' translation rates.
Overall, the collected measurements are interesting and potentially useful. However, the analysis suffers from a terribly incomplete knowledge of what controls a mRNA's translation rate. The statistics applied are tailored for a 1-factor problem, when in fact, there are many factors that control translation rate.
There are also inconsistencies and errors in the authors' calculations that should be corrected. The authors' conclusions are not well supported by their analysis. The manuscript requires significant work for it to productively add to our knowledge of what controls translation rate in bacteria.
The authors write that "Initiation rates vary depending on how well an mRNA recruits 30S subunits to the start codon, and in bacteria, the working model is that this is accomplished primarily by Shine-Dalgarno SD motifs.
The current working model is that a mRNA's translation initiation rate is controlled by at least five important molecular interactions, only one is the hybridization between the last 9 nucleotides of the 16S rRNA and the mRNA.
They include:.
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