A bacterial regulatory uORF senses multiple classes of ribosome-targeting antibiotics

Reviewer #1 (Public Review):

Summary:

The manuscript reports that expression of the E. coli operon topAI/yjhQ/yjhP is controlled by the translation status of a small open reading frame, that authors have discovered and named toiL, located in the leader region of the operon. The authors propose the following model for topAI activation: Under normal conditions, toiL is translated but topAI is not expressed because of Rho-dependent transcription termination within the topAI ORF and because its ribosome binding site and start codon are trapped in an mRNA hairpin. Ribosome stalling at various codons of the toiL ORF, caused by the presence of some ribosome-targeting antibiotics, triggers an mRNA conformational switch which allows translation of topAI and, in addition, activation of the operon's transcription because the presence of translating ribosomes at the topAI ORF blocks Rho from terminating transcription. Even though the model is appealing and several of the experimental data support some aspects of it, several inconsistencies remain to be solved. In addition, even though TopAI was shown to be an inhibitor of topoisomerase I (Yamaguchi & Inouye, 2015, NAR 43:10387), the authors suggest, without offering any experimental support, that, because ribosome-targeting antibiotics act as inducers, expression of the topAI/yjhQ/yjhP operon may confer resistance to these drugs.

Strengths:

- There is good experimental support of the transcriptional repression/activation switch aspect of the model, derived from well-designed transcriptional reporters and ChIP-qPCR approaches.

- There is a clever use of the topAI-lacZ reporter to find the 23S rRNA mutants where expression topAI was upregulated. This eventually led the authors to identify that translation events occurring at toiL are important to regulate the topAI/yjhQ/yjhP operon. This section can be strengthened if the authors suggest an explanation for how mutant ribosomes translating toiL increased topAI expression. Is there any published evidence that ribosomes with the identified mutations translate slowly (decreased fidelity does not necessarily mean slow translation, does it?)?

- Authors incorporate relevant links to the antibiotic-mediated expression regulation of bacterial resistance genes. Authors can also mention the tryptophan-mediated ribosome stalling at the tnaC leader ORF that activates the expression of tryptophan metabolism genes through blockage of Rho-mediated transcriptional attenuation.

Weaknesses:

The main weaknesses of the work are related to several experimental results that are not consistent with the model, or related to a lack of data that needs to be included to support the model.

The following are a few examples:

- It is surprising that authors do not mention that several published Ribo-seq data from E. coli cells show active translation of toiL (for example Li et al., 2014, Cell 157: 624). Therefore, it is hard to reconcile with the model that starts codon/Shine-Dalgarno mutations in the toiL-lux reporter have no effect on luciferase expression (Figure 2C, bar graphs of the no antibiotic control samples).

- The SHAPE reactivity data shown in Figure 5A are not consistent with the toiL ORF being translated. In addition, it is difficult to visualize the effect of tetracycline on mRNA conformation with the representation used in Figure 5B. It would be better to show SHAPE reactivity without/with Tet (as shown in panel A of the figure).

- The "increased coverage" of topAI/yjhP/yjhQ in the presence of tetracycline from the Ribo-seq data shown in Figure 6A can be due to activation of translation, transcription, or both. For readers to know which of these possibilities apply, authors need to provide RNA-seq data and show the profiles of the topAI/yjhQ/yjhP genes in control/Tet-treated cells.

- Similarly, to support the data of increased ribosomal footprints at the toiL start codon in the presence of Tet (Figure 6B), authors should show the profile of the toiL gene from control and Tet-treated cells.

- Representation of the mRNA structures in the model shown in Figure 5, does not help with visualizing 1) how ribosomes translate toiL since the ORF is trapped in double-stranded mRNA, and 2) how ribosome stalling on toiL would lead to the release of the initiation region of topAI to achieve expression activation.

- The authors speculate that, because ribosome-targeting antibiotics act as expression inducers [by the way, authors should mention and comment that, more than a decade ago, it had been reported that kanamycin (PMID: 12736533) and gentamycin (PMID: 19013277) are inducers of topAI and yjhQ], the genes of the topAI/yjhQ/yjhP operon may confer resistance to these antibiotics. Such a suggestion can be experimentally checked by simply testing whether strains lacking these genes have increased sensitivity to the antibiotic inducers.

Reviewer #2 (Public Review):

Summary:

In this important study, Baniulyte and Wade describe how the translation of an 8-codon uORF denoted toiL upstream of the topAI-yjhQP operon is responsive to different ribosome-targeting antibiotics, consequently controlling translation of the TopAI toxin as well as Rho-dependent termination with the gene.

Strengths:

I appreciate that the authors used multiple different approaches such as a genetic screen to identify factors such as 23S rRNA mutations that affect topA1 expression and ribosome profiling to examine the consequences of various antibiotics on toiL-mediated regulation. The results are convincing and clearly described.

Weaknesses:

I have relatively minor suggestions for improving the manuscript. These mainly relate to the figures.

Reviewer #3 (Public Review):

Summary:

The authors nicely show that the translation and ribosome stalling within the ToiL uORF upstream of the co-transcribed topAI-yjhQ toxin-antitoxin genes unmask the topAI translational initiation site, thereby allowing ribosome loading and preventing premature Rho-dependent transcription termination in the topAI region. Although similar translational/transcriptional attenuation has been reported in other systems, the base pairing between the leader sequence and the repressed region by the long RNA looping is somehow unique in toiL-topAI-yjhQP. The experiments are solidly executed, and the manuscript is clear in most parts with areas that could be improved or better explained. The real impact of such a study is not easy to appreciate due to a lack of investigation on the physiological consequences of topAI-yjhQP activation upon antibiotic exposure (see details below).

Strengths:

>Conclusion/model is supported by the integrated approaches consisting of genetics, in vivo SHAPE-seq and Ribo-Seq.

>Provide an elegant example of cis-acting regulatory peptides to a growing list of functional small proteins in bacterial proteomes.

Author Response:

The reviewers suggested that we determine whether the functions of TopAI, YjhQ, and/or YjhP are connected to antibiotic susceptibility.

We fully agree with the reviewers that the function of TopAI/YjhQ/YjhP is an important topic. Our preliminary studies (not included in the paper) failed to identify a function connected to antibiotic susceptibility, although these studies were far from exhaustive. There are many environmental stressors that can stall ribosomes, making it challenging to find the functionally relevant stressor(s). We feel that further work on this topic is outside the scope of this manuscript.

The reviewers suggested that the SHAPE data are inconsistent with our conclusions about translation of toiL.

We believe the SHAPE data are consistent with our model, although we acknowledge that interpretation of base reactivity is somewhat subjective. We will address the reviewers’ comments on this topic in more detail in our full response.

The reviewers suggested that published Ribo-Seq data are inconsistent with our data showing that toiL start codon/Shine-Dalgarno mutations have no effect on expression of luciferase reporters in the absence of antibiotics.

Our assays with these mutations looked at expression of topAI, not toiL. Our model predicts that mutations that prevent toiL translation will not induce expression of the downstream genes. We did not look at the effect of these mutations on expression of toiL itself.

The reviewers suggested we use RNA-seq to complement the Ribo-seq data for cells grown +/- tetracycline (Figure 5).

In principle, RNA-seq data would allow us to determine whether tetracycline specifically induces translation of topAI, as opposed to only increasing the RNA level. We did not generate RNA-seq data because prior work from other groups suggests that topAI is too weakly expressed to accurately measure translation efficiency in non-inducing conditions. However, the major conclusion from Figure 5 is that tetracycline stalls ribosomes at start codons, including the start codon of toiL.