Orthosomycin antibiotics inhibit protein synthesis by binding to the large ribosomal subunit in the tRNA accommodation corridor, which is traversed by incoming aminoacyl-tRNAs. Structural and biochemical studies suggested that orthosomycins block accommodation of any aminoacyl-tRNAs in the ribosomal A-site. However, the mode of action of orthosomycins in vivo remained unknown. Here, by carrying out genome-wide analysis of antibiotic action in bacterial cells, we discovered that orthosomycins primarily inhibit the ribosomes engaged in translation of specific amino acid sequences. Our results reveal that the predominant sites of orthosomycin-induced translation arrest are defined by the nature of the incoming aminoacyl-tRNA and likely by the identity of the two C-terminal amino acid residues of the nascent protein. We show that nature exploits this antibiotic-sensing mechanism for directing programmed ribosome stalling within the regulatory open reading frame, which may control expression of an orthosomycin-resistance gene in a variety of bacterial species.
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The scripts for Ribo-seq analysis can be found at the following link: https://github.com/mmaiensc/RiboSeq. The metagene analysis custom scripts are available at https://github.com/adamhockenberry/ribo-t-sequencing. The eUIgene.py Python script is available at https://github.com/GCA-VH-lab/eUIgene.
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We thank A. Innis and E. Leroy (University of Bordeaux) for structural insights, S. Meydan (University of Illinois at Chicago) for advice with some experiments, M. Svetlov for help with the figures and M. Ibba (Ohio State University) for sharing the B. subtilis strains. We are grateful to Y.-M. Hou (Thomas Jefferson University) for insights into the role of tRNA modification in decoding and to P. Mann and T. Black (Merck) for the advice on antibiotic-sensitive bacterial strains and information about preclinical development of evernimicin (Ziracin). This work was supported by the NIH grant R35-GM127134 to A.S.M. K.M. was supported by a NIH Training Grant (5T32AT007533). G.C.A. was supported by grants from the Knut and Alice Wallenberg Foundation (KAW 2020.0037), Umeå University Medical Faculty (Biotechnology grant to G.C.A), Kempestiftelserna (SMK-1858.3 to G.C.A), Carl Tryggers Stiftelse för Vetenskaplig Forskning (CTS19:24), and the Swedish Research Council (2019-01085). C.K.S. acknowledges support from Stiftelsen J.C. Kempes Stipendiefond.
The authors declare no competing interests.
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Extended Data Fig. 1 Action of EVN is gene specific and does not involve an obligatory translation initiation arrest.
a, In vitro toeprinting experiments to analyze EVN-dependent translation arrest. Selection of the model genes was based on the results of Ribo-seq analysis (hupA) or those tested in previous reports (hns and ermBL)7,13. Open arrows indicate toeprint bands representing ribosomes stalled at the start codons by the action of initiation inhibitor oncocin (Onc)21. Black arrows point to the bands representing EVN-stalled ribosomes. Shown is a representative gel from at least two independent experiments. b, The nucleotide sequences of the relevant portions of the mRNA templates used in the toeprinting experiments shown in (a) and amino acid sequences of the encoded proteins. The open and filled arrows are as in (a). c, Ribosome profiling does not show any pronounced EVN-induced ribosome stalling at the hns start codon in E. coli cells. The first strong drug-induced translation arrest (green arrow) occurs at the codon 5 of hns.
Sucrose gradient analysis of polysomes prepared from untreated E. coli cells (no drug) or treated with 0.5 µg/mL (1x MIC) or with 50 µg/mL (100x MIC) EVN during 30 min or 4 min, respectively.
Extended Data Fig. 3 Conditions and reproducibility of the Ribo-seq experiments to elucidate the mode of action of EVN.
a, Residual global protein synthesis (evaluated as incorporation of [35S]-Met into the TCA-insoluble protein fraction) of E. coli cells exposed to 50 µg/mL EVN for 4 min. Incorporation of [35S]-Met in a sample of the bacterial culture taken right before the addition of EVN was set as 100%. Open and closed circles represent the results of two independent experiments. b-d, Reproducibility of the two independent Ribo-seq experiments as judged by: (b) the EVN stall scores of all the considered codon positions (n = 118,222, Pearson’s r = 0.72); (c) of the mean stall score for codons at the ribosomal A site (n = 61, Pearson’s r = 0.96); (d) of the mean score for considered tripeptide motifs (n = 4039, Pearson’s r = 0.83) described in Figs. 2 and 3.
Comparison of ribosome occupancy profiles of individual genes in untreated cells (no drug) with those treated with EVN.
a,b, Ribosome occupancy profiles of individual genes where, in the presence of EVN, ribosomes translate successfully or not CGA codons (a) or PEP sequences (belonging to the identified PXP EVN arrest motif, see Fig. 3a, b) (b).
a, Modifications of the gltX template to facilitate toeprinting analysis of EVN-mediated stalling at the PSP arrest sequence. The thin arrows indicate the mutations introduced in the wild-type sequence of the first 14 codons of the gltX gene encoding the EVN arrest motif Pro9-Ser10-Pro11. Gly13 codon was mutagenized to Trp13 in order to trap translating ribosomes at the preceding Thr12 (open arrow) when Trp-tRNA is depleted by the addition of indolmycin, an inhibitor of tryptophanyl-tRNA synthetase. In addition, the Lys4 codon was mutagenized to Asn4 codon to disrupt the +X + motif Lys2-Ile3-Lys4 of GltX, where mild EVN-mediated stalling occurs during in vitro translation (grey arrow). The red and open arrows indicate toeprint band representing EVN-bound ribosomes stalled at Ser10 codon and the ribosomes trapped at the Thr12 codon due to the depletion of Trp-tRNA, respectively. b, Toeprinting analysis of EVN-mediated ribosome stalling at the original PXP motif Pro9-Ser10-Pro11 (X = Ser) of gltX or mutant templates where the Ser10 codon was mutated to encode different amino acids (Met, Leu, Ile, or Ala). c, Synonymous mutations of the P-site Ser10 codon have little effect on stalling, whereas replacement of the Ser10codon with the Ala codon has a strong effect. Representative gels from two independent experiments are shown in panels a, b and c.
Alignment of representative EmtA sequences identified in sequenced bacterial genomes (see Extended Data Fig. 8 for the complete phylogenetic tree).
Branch lengths are proportional to the number of substitutions. The tree is annotated with circles showing the presence (red) or absence (blue) of the emtAL uORF in 5’UTR of the emtA genes, where the 5’UTR is confidently alignable. The sequences of the encoded leader peptides are indicated. The asterisk indicates peptides from two annotated neighboring ORFs in Paenactinomyces guangxiensis, corresponding to the N and C terminal regions of EmtA. The interruption of the emtA ORF suggests pseudogenation caused by a frameshift.
Extended Data Fig. 9 Distal segments of the nascent peptide in the ribosomal exit tunnel contribute to EVN-dependent translation arrest within the emtAL ORF.
a, The VFL motif within the EmtAL peptide affords only partial translation arrest. Top: gel electrophoretic analysis of accumulation of MMMAVF-tRNA and MMMAVFL-tRNA during in vitro translation of the truncated emtAL template at increasing concentrations of EVN. Bottom: quantification of the gel (from two independent experiments) showing the fraction of incomplete translation product (MMMAVF-tRNA). b, Toeprinting analysis of EVN-mediated ribosome stalling in templates encoding the E. faecium emtAL ORF where the identities of codons 2–4 were simultaneously modified by introducing single-nucleotide compensatory frame-shifting mutations (indicated by black arrows and red letters). Representative gels from two independent experiments are shown in panels a and b.
Possible secondary structure of the 5’UTR of the E. faecium emtA gene. The emtAL ORF and its Shine-Dalgarno sequence are highlighted in green. The emtA ORF and its Shine-Dalgarno sequence are shown in red.
Uncropped toeprinting gels.
Uncropped toeprinting gel.
Uncropped toeprinting gels.
Uncropped toeprinting and protein gels.
Uncropped toeprinting gel.
Uncropped toeprinting gels.
Uncropped protein and toeprinting gels.
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Mangano, K., Marks, J., Klepacki, D. et al. Context-based sensing of orthosomycin antibiotics by the translating ribosome. Nat Chem Biol 18, 1277–1286 (2022). https://doi.org/10.1038/s41589-022-01138-9