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Ornithine capture by a translating ribosome controls bacterial polyamine synthesis

A Publisher Correction to this article was published on 19 March 2020

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Abstract

Polyamines are essential metabolites that play an important role in cell growth, stress adaptation and microbial virulence1,2,3. To survive and multiply within a human host, pathogenic bacteria adjust the expression and activity of polyamine biosynthetic enzymes in response to different environmental stresses and metabolic cues2. Here, we show that ornithine capture by the ribosome and the nascent peptide SpeFL controls polyamine synthesis in γ-proteobacteria by inducing the expression of the ornithine decarboxylase SpeF4, via a mechanism involving ribosome stalling and transcription antitermination. In addition, we present the cryogenic electron microscopy structure of an Escherichia coli ribosome stalled during translation of speFL in the presence of ornithine. The structure shows how the ribosome and the SpeFL sensor domain form a highly selective binding pocket that accommodates a single ornithine molecule but excludes near-cognate ligands. Ornithine pre-associates with the ribosome and is then held in place by the sensor domain, leading to the compaction of the SpeFL effector domain and blocking the action of release factor 1. Thus, our study not only reveals basic strategies by which nascent peptides assist the ribosome in detecting a specific metabolite, but also provides a framework for assessing how ornithine promotes virulence in several human pathogens.

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Fig. 1: Mechanism of speF induction.
Fig. 2: Structural basis for the specific recognition of L-ornithine by the SpeFL–70S complex.
Fig. 3: Inhibition of peptide release by SpeFL.
Fig. 4: Mechanism of ornithine sensing and capture by the SpeFL–70S complex.

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Data availability

The SpeFL-ESRF and SpeFL-DLS structures have been deposited with the Research Collaboratory for Structural Bioinformatics Protein Data Bank with accession codes 6TC3 and 6TBV; the cryo-EM maps have been deposited with the Electron Microscopy Data Bank with accession codes EMD-10458 and EMD-10453. Source data for Figs. 1–3 and Extended Data Figs. 2 and 10 are provided with the paper.

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Acknowledgements

A.H.d.V. is funded by a doctoral grant from the French Ministère de l’Enseignement Supérieur et de la Recherche. C.A.I., B.S. and G.S. have received funding for this project from the European Research Council under the European Union’s Horizon 2020 research and innovation programme (grant no. 724040). C.A.I. is a European Molecular Biology Organization Young Investigator and has received funding from the Fondation Bettencourt-Schueller. I.C.-M. is funded by Inserm and A.C.S. is funded by a joint doctoral grant from Inserm and the Aquitaine Regional Council (grant no. 2014-1R30404). We acknowledge Diamond for access and support of the cryo-EM facilities at the UK’s national electron bio-imaging centre (proposal no. EM 19716-1), funded by the Wellcome Trust, Medical Research Council and Biotechnology and Biological Sciences Research Council, and S. Neumann, A. Howe and J. Gilchrist for assistance. We also thank the European Synchrotron Radiation Facility for providing microscope time on the CM01 (ref. 39) and M. Hons for his assistance. This work has been supported by iNEXT (grant no. 3901) funded by the Horizon 2020 programme of the European Union. We thank I. Iost for help with gradient fractionation, Y. Hashem, A. Bezault and M. Decossas for help with grid preparation and screening, R. Fronzes for access to the Graphics Processing Unit cluster, C. Rapisarda for help with cryo-EM data processing and N. Vazquez-Laslop for providing the E. coli TB1 strain and pErmZα plasmid for the in vivo lacZ assay.

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Contributions

C.A.I. and B.S. designed the study. I.C.-M. identified speFL. A.H.d.V., B.S., G.S. and A.C.S. performed the biochemical experiments. A.H.d.V. performed the bacterial assays. A.H.d.V. prepared the cryo-EM sample. A.H.d.V. and C.A.I. processed the cryo-EM data. A.H.d.V., B.S., G.S., A.C.S. and C.A.I. interpreted the results. A.H.d.V., B.S. and C.A.I. wrote the paper.

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Correspondence to C. Axel Innis.

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Extended data

Extended Data Fig. 1 Conservation of SpeFL across γ-proteobacteria.

a, Phylogenetic tree showing the distribution of representative speFL sequences from several orders of γ-proteobacteria. b, Multiple sequence alignment of SpeFL homologs from different species. The sequence shown for Salmonella Typhimurium corresponds to the previously reported orf347. SpeFL and its homologs belong to the group of proteins of unknown function DUF261854. The stop codon found after each sequence is indicated on the right of the alignment.

Extended Data Fig. 2 The amino acid sequence of SpeFL is important for ornithine-dependent translational arrest.

Toeprinting assay8,9 to monitor the translation of wild type (WT) and double frameshifted (speFLfs) speFL in the presence ( + ) or absence (–) of 10 mM ornithine, release factors (RF1,2,3) or 90 μM puromycin. Arrows indicate ribosomes stalled with the codon for the indicated amino acid in the P-site (Ala33 – open triangle; Arg34 – filled triangle). This experiment was repeated three times independently with similar results.

Source data

Extended Data Fig. 3 Mechanism of the SpeFL- and Rho-dependent regulation of the speF operon.

The mRNA sequence of speFL and part of the adjacent intergenic region is shown at various stages of the induction process, namely (a) when the RNA polymerase pauses on a hairpin encompassing the 3′ end of speFL, (b) when the leading ribosome translating speFL unwinds the pause hairpin, (c) when the leading ribosome terminates translation in the absence of ornithine to allow Rho to bind to the rut site and (d) when the leading ribosome stalls in the presence of ornithine and blocks Rho binding, allowing the operon to be transcribed. The footprints of the ribosomes are in gray, speFL is in turquoise, the rut site is in yellow, rare codons R12 and R13 are in red and the UAG stop codon is indicated with an asterisk. The predicted 3′ end of the premature transcript is at position –1 of the consensus pause-inducing sequence element G–11G–10(C/T)–1G+113.

Extended Data Fig. 4 Purification of a SpeFL-70S complex stalled in the presence of ornithine.

a, Overlaid absorbance profiles of sucrose gradients containing a translation mixture incubated without ornithine (black), in the presence of 10 mM L-ornithine (red) or in the presence of 10 mM L-ornithine followed by treatment with 100 µM puromycin (blue). This experiment was repeated three times independently with similar results. A schematic diagram depicting the expected ribosomal species in each fraction is shown on the right. b, Overlaid absorbance profiles of sucrose gradients loaded with polysomal fractions from a, with (blue) or without (black) RNase H treatment. This experiment was repeated three times independently with similar results. Expected ribosomal species for each fraction are shown on the right. c, Schematic representation of the purification strategy for SpeFL-70S. The collected fractions are indicated with gray boxes.

Extended Data Fig. 5 Flowchart of cryo-EM data processing for the SpeFL-ESRF and SpeFL-DLS datasets.

Steps where Relion 2.140 and Relion 3.041 were used are shown in purple and green, respectively. The step where Cryosparc 0.642 was used is indicated with an asterisk. Note the increase in resolution when using Relion 3.0 compared to Relion 2.1. This increase was also matched by the quality of the resulting cryo-EM density. Both structures could be refined to an overall resolution of 2.7 Å using a Fourier shell correlation (FSC) cutoff of 0.143.

Extended Data Fig. 6 Quality of the cryo-EM reconstructions.

a, Refined cryo-EM density map obtained in Relion 3.041 filtered and colored by local resolution estimation values in Chimera51. A cross-section of the same map is also shown. bd, Representative cryo-EM densities for (b) a hydrated magnesium ion bound to the 23 S rRNA, (c) the tunnel extension of ribosomal protein uL22 and (d) helix H64 of the 23 S rRNA. This experiment was repeated two times independently with similar results.

Extended Data Fig. 7 Interactions between SpeFL and the ribosome.

A cartoon representation of SpeFL (turquoise) is shown in the middle panel. a, Potential hydrogen bond between Ala33 of SpeFL and the base of 23 S rRNA residue G2061. b, Potential hydrogen bonds between Asn32 of SpeFL and the base of 23 S rRNA residue U2584. c, Hydrophobic core of the SpeFL effector domain formed by residues Phe20, Phe26, Phe28, Phe30 and Phe31. Phe28, Phe30 and Phe31 of SpeFL form π-stacking interactions with the bases of 23 S rRNA residues U2586, G2505 and A2062, respectively. d, Potential hydrogen bond between Asn24 of SpeFL and Lys90 of ribosomal protein uL22, and electrostatic interaction between Arg23 of SpeFL and the phosphate backbone of 23 S rRNA residue m5U747. e, The HIRRXXH ornithine-binding motif of SpeFL, showing potential hydrogen bonds between His10 and His16 of SpeFL, and Gly91 and Lys90 of ribosomal protein uL22, respectively. π-stacking interaction between 23 S rRNA residue A1614 and His10 of SpeFL. f, Electrostatic interaction between residue Glu2 of SpeFL and residue Arg67 of ribosomal protein uL4. Potential hydrogen bond between residue Asn3 of SpeFL and the phosphate backbone of 23 S rRNA residue C796.

Extended Data Fig. 8 Sharpened cryo-EM density for L-ornithine and neighboring solvent molecules.

A single L-ornithine molecule (orange) surrounded by 4 solvent molecules (red) is fitted into the cryo-EM density of the ligand binding pocket obtained for the (a) SpeFL-ESRF and (b) SpeFL-DLS datasets. This experiment was repeated two times independently with similar results. As a result, peaks for the solvent molecules that are visible in the two independently determined cryo-EM maps cannot be attributed to random noise.

Extended Data Fig. 9 Small ligand-binding pockets in the ribosomal exit tunnel.

Overview and close-up view of a cross-section of the E. coli 70S ribosomal exit tunnel showing the L-ornithine molecule observed in this work (orange) together with small molecules that are known to bind to the ribosomal exit tunnel: blasticidin S (PDB: 4v9q, dark blue)55, chloramphenicol (PDB: 4v7w, light green)56, clindamycin (PDB: 4v7v, magenta)57, dalfopristin (PDB: 4u24, light blue)45, erythromycin (PDB: 4v7u, purple)57, hygromycin (PDB: 5dox, red)58, linezolid (PDB: 3dll, pink)59, puromycin (PDB: 1q82, cyan)60, sparsomycin (PDB: 1njn, dark green)61 and tryptophan (PDB: 4uy8, yellow)17. The different regions of the tunnel are highlighted: the tRNA binding pocket (red), the peptidyl transferase center (PTC) (orange), the upper tunnel (yellow), the constriction formed by uL22 and uL4 (green) and the lower tunnel (blue).

Extended Data Fig. 10 Importance of residues 1–7, 10, 11, 12, 13 and 16 of SpeFL.

a, Toeprinting assay8,9 to monitor the translation of (ac) wild-type speFL and (a) speFLΔ1–7, (b) speFL-R12A-R13A (A12A13) and speFL-R12K-R13K (K12K13), and (c) speFL-H10A, speFL-I11A and speFL-H16A, in the absence (–) or presence ( + ) of 10 mM ornithine, release factors (RF1,2,3) or 90 μM puromycin. Arrows indicate ribosomes stalled with the indicated amino acid in the P-site (Ala33 – open triangle; Arg34 – filled triangle). This experiment was repeated three times independently with similar results.

Source data

Supplementary information

Supplementary Information

Supplementary Fig. 1 and Tables 1–3.

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Source data

Source Data Fig. 1

Toeprinting assay8,9 to monitor the translation of speFL in the absence (–) or presence (+) of 10 mM ornithine, 10 mM putrescine, RF1, 2 and 3 or 90 μM puromycin. Arrows indicate ribosomes stalled with the codon for the indicated amino acid in the P-site (Ala33, open triangle; Arg34, filled triangle). A schematic representation of the DNA template used for toeprinting is provided (RBS, ribosome binding site; NV1 (ref. 36), sequence used to anneal the Yakima Yellow-labelled probe for reverse transcription). This experiment was repeated three times independently with similar results.

Source Data Fig. 2

Toeprinting assay8,9 to monitor the translation of WT speFL in the absence (–) or presence of 10 mM (+) of various small molecules (see Fig. 2e for details). All translation reactions were treated with 90 µM puromycin and contained release factors. Arrows indicate ribosomes stalled with the indicated amino acid in the P-site (Ala33, open triangle; Arg34, filled triangle). A schematic representation of the DNA template used for toeprinting is provided (RBS, ribosome binding site; NV1 (ref. 36), sequence used to anneal the Yakima Yellow-labelled probe for reverse transcription). This experiment was repeated three times independently with similar results.

Source Data Fig. 3

Toeprinting assay8,9 to monitor the translation of WT and mutant speFL in the absence (–) or presence (+) of 10 mM ornithine, RF1, 2, 3 or 90 μM puromycin. Arrows indicate ribosomes stalled with the indicated amino acid in the P-site (Ala33, open triangle; Arg34, filled triangle). A schematic representation of the DNA templates used for toeprinting is provided (RBS, ribosome binding site; NV1 (ref. 36), sequence used to anneal the Yakima Yellow-labelled probe for reverse transcription). These experiments were repeated three times independently with similar results.

Source Data Extended Data Fig. 2

Toeprinting assay8,9 to monitor the translation of wild type (WT) and double frameshifted (speFLfs) speFL in the presence (+) or absence (–) of 10 mM ornithine, RF1, 2, 3 or 90 μM puromycin. Arrows indicate ribosomes stalled with the codon for the indicated amino acid in the P-site (Ala33, open triangle; Arg34, filled triangle). Schematic representations of the WT and speFLfs DNA templates used for toeprinting are provided (RBS, ribosome binding site; NV1 (ref. 36) sequence used to anneal the Yakima Yellow-labelled probe for reverse transcription). This experiment was repeated three times independently with similar results.

Source Data Extended Data Fig. 10

Toeprinting assays8,9 to monitor the translation of wild-type speFL, speFLΔ1–7, speFL-R12A-R13A (A12A13), speFL-R12K-R13K (K12K13), speFL-H10A, speFL-I11A and speFL-H16A in the absence (–) or presence (+) of 10 mM ornithine, RF1, 2, 3 or 90 μM puromycin. Arrows indicate ribosomes stalled with the indicated amino acid in the P-site (Ala33, open triangle; Arg34, filled triangle). A schematic representation of the DNA template used for toeprinting is provided (RBS, ribosome binding site; NV1 (ref. 36), sequence used to anneal the Yakima Yellow-labelled probe for reverse transcription). This experiment was repeated three times independently with similar results.

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Herrero del Valle, A., Seip, B., Cervera-Marzal, I. et al. Ornithine capture by a translating ribosome controls bacterial polyamine synthesis. Nat Microbiol 5, 554–561 (2020). https://doi.org/10.1038/s41564-020-0669-1

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