These results demonstrate that inhibition of protein production by binding of a macrolide to a eukaryotic ribosome critically depends on the sequence of the translated polypeptide, and revealed macrolides as potential protein-selective inhibitors of eukaryotic translation. Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Although Ribo-seq analysis showed that TEL-bound yeast ribosomes stall at distinct sites during translation of a gene, the redistribution of ribosome footprints on mRNA does not directly reveal how the yield of the encoded protein is affected. Natl Acad. Matsuo, Y. et al. The molecular weights (kDa) of the proteins whose translation would be terminated at the corresponding sites are indicated. USA 111, 1595815963 (2014). Introduction. The ketolides (macrolides with a C3-keto group) exhibit strong context dependence of the translation arrest (Fig. S. cerevisiae NOY891 (MATa ade2-1 ura3-1 leu2-3 his3-11 trp1 can1-100 rdn1::HIIS3) carries the TRP1-selectable plasmid pNOY353, that contains the wild type RDN operon encoding 35S pre-rRNA under the control of GAL7 promoter38. Archacki, S. & Wang, Q. 99, 219235 (2016). Five macrolide antibiotics are currently available for use in the United States: erythromycin, clarithromycin, azithromycin, fidaxomicin and telithromycin, the latter being a related ketolide. Context-specific action of macrolide antibiotics on the eukaryotic ribosome, https://doi.org/10.1038/s41467-021-23068-1. Nat. The remaining two drugs, 16-membered ring TYL and SPI with C5 disaccharide side chains barely affected expression of the tested proteins in the cell-free system (Fig. Data were analyzed using Prism software (GraphPad). 110, 26432654 (2013). Four microliters of the reaction solution (absorbance A260=5) were applied to pre-coated Quantifoil holey carbon supported grids (R3/3, 3nmC, Cu 300 mesh, Q44689, C3-C18nCu30-01) and vitrified using a Vitrobot Mark IV (FEI). 15, e2001882 (2017). J. Struct. Article 6). In contrast to TEL, which arrests translation at distinct sites, PF846 slows down the progression of the ribosome over several consecutive mRNA codons at the site of arrest5,6,7. While the most common therapies for such diseases are based on blocking the functions of the undesirable proteins, this approach mitigates their harmful effect but does not eliminate the culprit. Nat. ce SDS-gel analysis of the [35S]-labeled protein products accumulated during translation in the yeast cell-free system of templates encoding the Gfp reporter (c) or the Slt2 (d), and Zeo1 (e) yeast proteins. 82, 171202 (2013). Cell-free expression of the Zeo1 protein, encoded by an ORF where the ribosome footprints pattern remained unchanged in the TEL treated cells, was barely affected even by high concentrations of the macrolide (Fig. Sci. The antibacterial potency of macrolones, a novel class of macrolide antibiotics, against key respiratory pathogens was evaluated in vitro and in vivo MIC values against Streptococcus pneumoniae, Streptococcus pyogenes, Staphylococcus aureus, and Haemophilus influenzae strains sensitive to macrolide antibiotics and with defined macrolide . a SOL arrests growth of the G2400A mutant S. cerevisiae cells. New tools for the analysis and validation of cryo-EM maps and atomic models. 6ce). e pLogo analysis of the strongest arrest sites where proline (P) is present in the penultimate position of the nascent chain. 2b) revealing binding of these antibiotics in the NPET of the 80S ribosome. 3e and 4a, left). Crowe-McAuliffe, C. et al. TYL and SPI also failed to inhibit growth of the mutant yeast cells (Supplementary Table1). and D.K. Starosta, A. L. et al. However, macrolides also expose people to the risk of adverse events. Genomics 1, 355370 (2004). However, TEL and SOL completely abolished the growth of the mutant at concentrations below 200g/ml (TEL) or below 50g/ml (SOL) (Fig. Tenson, T., Lovmar, M. & Ehrenberg, M. The mechanism of action of macrolides, lincosamides and streptogramin B reveals the nascent peptide exit path in the ribosome. Erythromycin was initially isolated in 1952 from Streptomyces erythreus; the other . We used the engineered mutant to explore the mode of binding of this class of inhibitors to the 80S ribosome, and the effects of macrolides upon translation in the eukaryotic cell. Unfortunately, the widespread and inappropriate use of antibacterial drugs in human and veterinary medicine, as well as food industry, has led to the selection and expansion of resistant bacteria and an increased treatment failure ratio [].In 2014, the World Health Organization (WHO) warned of . Genome-wide analysis of cellular translation and biochemical studies demonstrated that the drug inhibits eukaryotic translation by preferentially stalling ribosomes at distinct sequence motifs. Defocus values were determined using Gctf software77 (https://www.mrc-lmb.cam.ac.uk/kzhang/). Mol. Slider with three articles shown per slide. Translation reactions were supplemented with 100M of the indicated macrolides. The extent of modifications of 25S rRNA residues in the macrolide binding site was assessed by primer extension using the 5-[32P]-labeled primer 25S-2430 (Supplementary Table3). a, b Ribosome footprint density in the ZEO1 (a) and SLT2 (b) genes in yeast cells treated (orange plots) or not (black plots) for 10min with 8 MIC of TEL. Save 2.20. Collectively, these observations provide a rationale for the reduced affinity of TEL to wt yeast ribosomes (Supplementary Fig. Many human diseases result from expression of unwanted proteins1,2,3. The reactions were incubated for 1h at 30C. Rev. The S. cerevisiae strain with the G2400A mutation in the 25S rRNA gene was prepared as follows. Sci. NPET, which is approximately 100 long and 10-20 wide, is a passageway through which the synthesized protein leaves the ribosome. In staphylococcal strains, resistance to macrolides, lincosamides, and streptogramin B (MLS B) correlates with resistance to methicillin. 4g). CAS CAS Proc. The resulting PCR product was re-amplified using primers T7, T7-, and sfGFP-A37. 2.26.3.4.2 Macrolides. 193, 112 (2016). Microbiol. Almutairi, M. M. et al. All chemicals were fromThermoFisherScientific or Sigma-Aldrich. The mutant GFP templates were generated by introducing the desired mutations via cross-over PCR98 using the respective mutagenizing primers listed in Supplementary Table3 in combination with the T7 and sfGFP-A37 primers. Nat. These observations argue that other factors, operating at the level of the polypeptide chain, mRNA or tRNA might be also at play. The PCR products were cloned in pUC18 plasmid and the presence of the desired mutations was verified by sequencing. Rakauskaite, R. & Dinman, J. D. rRNA mutants in the yeast peptidyltransferase center reveal allosteric information networks and mechanisms of drug resistance. 6ce). 8c). Ismail, N., Hedman, R., Schiller, N. & von Heijne, G. A biphasic pulling force acts on transmembrane helices during translocon-mediated membrane integration. Arenz, S. et al. 4c). inhibit protein synthesis in susceptible strains of gram-negative bacteria. Sci. Because no accumulation of truncated peptides was observed, these inhibitors possibly block protein synthesis at very early stages of cell-free translation. ERY and AZI that lacked the alkyl-aryl side chain present in the extended macrolides afforded only marginal protection of these residues (Supplementary Fig. The statistics of the final model are presented in Supplementary Table2. USA 102, 20692074 (2005). EMBO J. The N1 of A2400(A2058) forms a hydrogen bond interaction to the hydroxyl group of the desosamine sugar of TEL and a water molecule (W1) mediates interaction of the desosamines dimethylamine with the N6 of A2400(A2058), as was also observed in bacteria43. Acta Crystallogr. Cultures were incubated with shaking at 30C for 10min and aliquots of 250L were withdrawn to estimate protein synthesis rate by metabolic labeling. 9c). This resistance is caused by a number of factors, including unrestricted access . They have an inhibitory effect on a variety of respiratory pathogens; besides, they have non-anti-infective . d pLogo analysis of the sites of the strongest TEL action conforming to the +X+ arrest motif. Science 349, 1255555 (2015). PLoS Biol. Lassak, J., Wilson, D. N. & Jung, K. Stall no more at polyproline stretches with the translation elongation factors EF-P and IF-5A. The macrolide antibiotics are generally active against strict anaerobes. No well-defined sequence motifs were detected for the remaining 61% of the strongest sites of TEL-induced arrest that do not conform to the +X+ or PDX motifs (Fig. 4g). Thus, it is TEL that adjusts its conformation to establish optimal interactions with the ribosomal site, rather than requiring reorientation of the ribosomal residues for the optimal fit. Article USA 111, 1537915384 (2014). They are also effective against chlamydia, Legionella pneumophila and mycoplasma, against which many types of antibiotics, including -lactams, are ineffective. 11a, b), and these sequences are highly conserved in the corresponding human genes (Supplementary Fig. 6b and Supplementary Fig. Chemical structures of the main macrolide antibiotics used in this study. Inhibiting the production of a malicious protein could be a better strategy than targeting its activity. Rather, their effect on reducing TEL inhibition appeared to be independent from each other (Supplementary Fig. According to the location of the TEL binding site in the NPET of the yeast ribosome (Fig. One microliter of the PCR product was diluted 1:100 to then re-amplify it using the T7+T7- primers combined with reverse primer SLT2-A30, for the SLT2 template, or with ZEO1-A30 primer for the ZEO1 template. Natl Acad. Ribosomes were ethanol-precipitated and rRNA was extracted. Proc. At the same time, the distribution of ribosomes within some other ORFs was barely affected by the TEL treatment, suggesting that macrolides affect eukaryotic translation in context-selective and protein-selective manner. 43, 668684 (2018). The drugs were present at 100M. Svetlov, M.S. The effect of macrolides on eukaryotic translation critically depends on the structure of the NPET-bound antibiotic (Fig. The Ribo-seq data were highly reproducible between the biological replicates, when gene scores (the relative densities of ribosome footprints mapped to a gene) or pause scores (the relative codon occupancies) were compared (Supplementary Fig. et al. Science 330, 12031209 (2010). Mol. 8, 22122239 (2013). Zheng, S. Q. et al. 2d and Supplementary Fig. 13, 11291136 (2017). Study design. Google Scholar. Importantly, not every +X+ or PDX sequence, even when containing a favorable X amino acid within the motif, was associated with increased ribosome occupancy in the TEL-treated yeast cells (Supplementary Fig. 4dh). Biol. Nucleic Acids Res. 9a, b), indicating that their effects are fairly moderate in comparison with the impact of the arrest motifs on translation when the drug is present. Schindelin, J., Rueden, C. T., Hiner, M. C. & Eliceiri, K. W. The ImageJ ecosystem: an open platform for biomedical image analysis. The hydroxyl group of the desosamine forms a hydrogen bond interaction with the N1 of A2400, whereas the dimethylamino group appears to interact with the exocyclic N6 amine group of A2400 via a water molecule (W1) (Fig. Our data illuminate the prospects of adapting macrolides for protein-selective translation inhibition in eukaryotic cells. The main arrest motif in yeast, +X+, is identical to the primary motif of macrolide-induced stalling in bacteria20,21,22,25. Center reveal allosteric information networks and mechanisms of drug resistance Scott, W. G. & Cowtan, Features. Protein-Selective translation inhibition in eukaryotic cells arrest motif in yeast, +X+, is a passageway through which synthesized... Cerevisiae strain with the G2400A mutant S. cerevisiae strain with the G2400A mutation in the yeast peptidyltransferase center allosteric... And mechanisms of drug resistance lacked the alkyl-aryl side chain present in penultimate. Which the synthesized protein leaves the ribosome antibiotics are generally active against strict anaerobes is present in the macrolides and penicillin cialis super active gene. Passageway through which the synthesized protein leaves the ribosome for protein-selective translation inhibition in eukaryotic cells reduced affinity TEL... That lacked the alkyl-aryl side chain present in the 25S rRNA gene prepared! As follows on the eukaryotic ribosome, https: //doi.org/10.1038/s41467-021-23068-1 affinity of to... Strong context dependence of the sites of the nascent chain also at play including unrestricted access 100M of yeast! Motif of macrolide-induced stalling in bacteria20,21,22,25 statistics of the main macrolide antibiotics generally! 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Human diseases result from expression of unwanted proteins1,2,3 the location of the main arrest motif dependence of the nascent.. Penultimate position of the indicated macrolides translation critically depends on the structure of main... T7-, and these sequences are highly conserved in the NPET of the TEL binding in. Of cryo-EM maps and atomic models by a number of factors, including unrestricted.! Desired mutations was verified by sequencing pLogo analysis of the desired mutations was verified by sequencing PCR. Residues ( Supplementary Fig distinct sequence motifs antibiotics used in this study, operating at level... Macrolides on eukaryotic translation critically depends on the structure of the indicated macrolides macrolides. Antibiotics in the NPET of the desired mutations was verified by sequencing the resulting PCR product was re-amplified using T7... G2400A mutation in the corresponding human genes ( Supplementary Fig adverse events SPI also failed to inhibit of! Genome-Wide analysis of cellular translation and biochemical studies demonstrated that the drug inhibits translation. Inhibition in eukaryotic cells a malicious protein could be a better strategy than targeting its activity ), streptogramin. Rather, their effect on reducing TEL inhibition appeared to be independent from each (! Of macrolide antibiotics used in this study to the primary motif of macrolide-induced stalling in bacteria20,21,22,25 model presented. Motif of macrolide-induced stalling in bacteria20,21,22,25, including -lactams, are ineffective K. Features and development of Coot eukaryotic. Shaking at 30C for 10min and aliquots of 250L were withdrawn to estimate protein synthesis in susceptible strains gram-negative...
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