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Sequence Selectivity of Macrolide-Induced Translational Attenuation Sequence selectivity of macrolide-induced translational attenuation Amber R. Davis, David W. Gohara, and Mee-Ngan F. Yap1 Edward A. Doisy Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, St. Louis, MO 63104 Edited by Jennifer A. Doudna, University of California, Berkeley, CA, and approved September 18, 2014 (received for review June 3, 2014) The prevailing “plug-in-the-bottle” model suggests that macrolide presence of the macrolide azithromycin (AZ). In addition to antibiotics inhibit translation by binding inside the ribosome tun- identifying previously unidentified ORFs and noncoding RNAs, nel and indiscriminately arresting the elongation of every nascent we found incomplete down-regulation of the translation in cells polypeptide after the synthesis of six to eight amino acids. To test exposed to AZ. Of particular interest, we found that AZ-medi- this model, we performed a genome-wide analysis of translation ated translation arrest selectively occurs at stalling sites that are in azithromycin-treated Staphylococcus aureus. In contrast to ear- enriched in proline and charged residues, despite a lack of lier predictions, we found that the macrolide does not preferen- overall sequence conservation. Surprisingly, many ribosomes do tially induce ribosome stalling near the 5′ end of mRNAs, but not enrich the N-terminal region as predicted; instead, they rather acts at specific stalling sites that are scattered throughout undergo arrest at a much later stage of translational elongation the entire coding region. These sites are highly enriched in prolines depending on the locations of specific stalling sites. Even more and charged residues and are strikingly similar to other ligand- unexpectedly, the AZ-bound ribosomes have the ability to over- independent ribosome stalling motifs. Interestingly, the addition of come the initial AZ inhibition within a multistalling-site-containing structurally related macrolides had dramatically different effects sequence, possibly by rerouting the path of the nascent chain. on stalling efficiency. Our data suggest that ribosome stalling can occur at a surprisingly large number of low-complexity motifs in Results a fashion that depends only on a few arrest-inducing residues and Ribosome Profiling in Staphylococcus aureus. Ribosome profiling the presence of a small molecule inducer. provides a snapshot of ribosomal movement on its template mRNA BIOCHEMISTRY (15). A high number of ribosome-protected fragments (RPFs) Staphylococcus aureus antibiotic | ribosome stalling | mapping to the transcriptome (mRNA-seq) is indicative of pro- longed ribosome occupancy at a given position, also known as ri- uring translation, nascent polypeptides travel through a bosome stalling/pausing. AZ causes ribosome stalling and is one of D∼100-Å-long aqueous tunnel in the large ribosomal sub- the most commonly used macrolide antibiotics. The minimum in- unit before entering the cytoplasm. The ribosome tunnel is highly hibitory concentration (MIC) of AZ for S. aureus NCTC 8325 is irregular in shape and contains numerous cavities and grooves (1). ∼0.125 μg/mL (Fig. S1A). We performed growth inhibition and Despite its narrow diameter (∼15 Å on average), recent studies pulse-chase analyses and found that residual protein synthesis per- have indicated that at least some nascent chains adopt a more sisted for 45 min, even at a dose of AZ ∼200-fold higher than the compact conformation (2–4). Furthermore, the detection of spe- MIC, whereas bacterial growth was completely halted within 24 h cific peptide sequence motifs can arrest elongation or termination (Fig. S1 B and C). Cell lethality may occur owing to the imbalance in both eukaryotes and prokaryotes (5). The cis-acting translational of the cellular proteome caused by the partial inhibition of a large attenuator serves to regulate the expression of downstream genes proportion of proteins (12). Long-lived protein bands observed in in response to physiological signals (6, 7) and small molecules, such – as antibiotics or amino acids (8 10). In bacteria, ribosome stalling Significance at the upstream element often promotes the rearrangement of structured mRNA and activates downstream translation. Many By globally mapping the position of stalled ribosomes in macro- resistant pathogens exploit the translational stalling mechanism to Staphylococcus aureus up-regulate the synthesis of antibiotic resistance genes (10). Curi- lide-treated , we identified the proteins ously, the ribosome-stalling peptides identified to date vary in whose synthesis is affected by this antibiotic. Rather than acting length and share no sequence homology. as a nonselective inhibitor, the binding of macrolides to the ri- The ribosome tunnel is the target of many clinically important bosome tunnel only induces stalling at a few unique sites that are macrolide antibiotics. Macrolides bind to the tunnel close to the enriched in proline and charged residues. The sequences that peptidyltransferase center (PTC) and are thought to inhibit trans- flank the stalling sites are highly variable but are essential for lation by physically obstructing the progression of the nascent chain, efficient stalling. Although it has long been assumed that mac- thereby leading to the premature drop-off of peptides that are only rolides inhibit translation after the synthesis of a few residues, we found that stalling could occur at any point during trans- six to eight amino acids long (11). The prevailing view of macrolide lation. Our results reveal a striking conservation of stalling motifs action suggests that these antibiotics indiscriminately block the among all known arrest peptides that strongly suggests a uni- elongation of every protein during the early stage of translation. In versal ribosome stalling mechanism. contrast to this model, recent studies have shown that several pro- teins are able to bypass the inhibition, presumably by threading Author contributions: M.-N.F.Y. designed research; A.R.D. and M.-N.F.Y. performed re- through the drug-bound tunnel or by stimulating dissociation of search; D.W.G. contributed new reagents/analytic tools; A.R.D., D.W.G., and M.-N.F.Y. antibiotic from the ribosome (12–14). Although the precise mech- analyzed data; and A.R.D. and M.-N.F.Y. wrote the paper. anism is unclear, the identification of a short N-terminal motif that The authors declare no conflict of interest. circumvents erythromycin blockage (12) suggests that the extent of This article is a PNAS Direct Submission. macrolide inhibition is highly sequence-specific. The full scope of Data deposition: The data reported in this paper have been deposited in the Gene Ex- this selectivity remains unknown. pression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no. GSE57175). To identify the protein sequence motifs that are susceptible 1To whom correspondence should be addressed. Email: [email protected]. and resistant to macrolide action, we performed ribosome pro- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. filing to examine the Staphylococcus aureus translatome in the 1073/pnas.1410356111/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1410356111 PNAS Early Edition | 1of6 Downloaded by guest on September 28, 2021 the pulse-chase analysis could in part be due to the slow protein turnover, accumulation of truncated products, or bypassing of AZ- mediated translational inhibition. To investigate the effects of AZ on the global translatome and whether AZ preferentially stalls synthesis of particular protein sequences, we performed ribosome profiling on AZ-treated and untreated S. aureus cells, with the hypothesis that AZ-induced ri- bosome stalling (and hence translation arrest) would result in an unusually high ribosome density at unique stalling sites. Exponen- tially grown S. aureus were treated with and without AZ for 15 min because longer treatment severely impaired the assembly of ribo- somal subunits. Before cell harvest, 2-min pretreatment of chlor- amphenicol was conducted to stabilize the translating ribosomes. Cell lysate was prepared by cryomilling (16). Sucrose density frac- tionation was performed after micrococcal nuclease (MNase) treatment to isolate ribosome-protected mRNAs from the mono- somes. We were unable to fully convert the “disome” peak into the monosomes (Fig. 1A), despite using different MNase sources and digestion conditions, unlike Escherichia coli and Bacillus subtilis, which produce homogenous monosomes after MNase digestion (16, 17). Additional analyses confirmed that the “disome” peak in the MNase-negative sample was indeed a mixture of 100S ribosomes (>85%) and disomes (∼10–15%) (Fig. S1F). The binding of hi- bernation promoting factor (HPF) to the interface of the 30S and 50S subunits has been shown to stimulate the dimerization of the 70S to form 100S ribosomes (18, 19). In contrast to E. coli 100S ribosomes, which only exist during stationary phase, S. aureus 100S ribosomes persist throughout all growth phases (18). We performed Western blots using an anti-S. aureus HPF (SaHPF) antibody and confirmed that the protein was highly expressed in S. aureus and was ubiquitously present in all ribosomal fractions. SaHPF was also particularly enriched in the MNase-treated “disome” peak (Fig. 1A and Fig. S1 D and E). The “disome” peak diminished dramatically Fig. 1. Analysis of RPFs and identification of previously unidentified ORFs. A S. aureus upon the inactivation of the hpf gene in S. aureus (Fig.
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