Sarecycline interferes with tRNA accommodation and tethers mRNA to the 70S ribosome

Figures

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  Fig. 1.. Chemical structures and electron density maps of ribosome-bound SAR. (A–C) Chemical structures of tetracycline-class antibiotics: TET (A), TIG (B), and SAR (C). The standard numbering of carbon atoms is indicated for each drug. Key chemical moieties are highlighted for TIG and SAR. (D) Unbiased Fo-Fc (green mesh) and 2Fo-Fc (blue mesh) electron difference Fourier maps of SAR in complex with the T. thermophilus 70S ribosome programmed with the UUC-mRNA. The refined model of SAR is displayed in the electron density maps contoured at 2.5 σ and 1.0 σ, respectively. Carbon atoms are colored yellow, nitrogens are blue, and oxygens are red.

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  Fig. 2.. Structure of SAR in complex with the 70S ribosome, UUC-mRNA, and P-site tRNA at 2.8-Å resolution. (A–C) Overview of the SAR binding site (yellow) on the T. thermophilus 70S ribosome viewed from three different perspectives. The 30S subunit is shown in light gray, the 50S subunit is dark gray, the mRNA is magenta, and the P-site tRNA is colored dark blue. In A, the 30S subunit is viewed from the intersubunit interface, as indicated by Inset (the 50S subunit and parts of the P-site tRNA are removed for clarity). The view in B is a transverse section of the 70S ribosome. The view in C is from the top after removing the head of the 30S subunit and protuberances of the 50S subunit, as indicated by Inset. (D–F) Close-up views of the SAR interactions with the decoding center on the 30S ribosomal subunit. The E. coli numbering of the nucleotides in the 16S rRNA is used. Potential H bond interactions are indicated with dashed lines. Nucleotides of the mRNA are numbered relative to the first adenine in the P-site codon. Note that the C7 extension of SAR appears in close proximity to the third nucleotide of the A-site codon.

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  Fig. 3.. Antibiotics that bind in the decoding center on the small ribosomal subunit. (A and B) The location of the SAR binding site relative to the binding sites of other antibiotics known to target the decoding center of the small ribosomal subunit: TET (blue), negamycin (NEG, red), odilorhabdin NOSO-641 (ODL, green), streptomycin (STR, cyan), paromomycin (PAR, salmon). In B, the 16S rRNA nucleotides critical for decoding are shown as sticks.

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  Fig. 4.. Structure of SAR in complex with the 70S ribosome, UAA-mRNA, and P-site tRNA at 3.0-Å resolution. (A) 2Fo-Fc electron density map (blue mesh) of SAR (yellow) in complex with the T. thermophilus 70S ribosome programmed with UAA-mRNA (teal). The refined model of SAR and mRNA with UAA codon in the A site are displayed in the electron density map contoured at 1.0 σ. (B) Close-up view of the SAR interactions with the mRNA and the nucleotides of the decoding center of the 30S ribosomal subunit. The E. coli numbering of the nucleotides in the 16S rRNA is used. Note that the exocyclic amino group (N6 atom) of the third adenine residue in the codon is within H bond distance (3.2 Å) from the oxygen of the C7 moiety of SAR (dotted lines).

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  Fig. 5.. TET resistance involving ribosome protection protein TetM. (A) Superposition of the structure of ribosome-bound TetM protein (green, PDB ID code 3J9Y; ref. 21) with the structures of ribosome-bound TET (blue, PDB ID code 4V9A; ref. 13), TIG (teal, PDB ID code 4V9B; ref. 13), or SAR (yellow). All structures were aligned based on the 16S rRNA. mRNA is colored magenta; P-site tRNA is dark blue. (B–D) Close-up views of the sterical clashes between the amino acid residue(s) at the tip of the loop 3 of the domain IV of TetM and the ribosome-bound TET (B), TIG (C), or SAR (D). Note that TET exhibits clash only with the Pro509 residue, while the extended C9 and C7 moieties of TIG and SAR, respectively, exhibit more severe clashes with Ser508, Pro509, and Val510 residues of TetM.

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  Fig. 6.. SAR inhibits the initiation step during protein synthesis. Ribosome stalling by SAR on csrA mRNA in comparison with other translation inhibitors (edeine, EDE; retapamulin, RET; and TET), as revealed by reverse-transcription primer-extension inhibition (toe-printing) assay in a cell-free translation system. csrA mRNA nucleotide sequence and the corresponding amino acid sequence are shown at Left. Colored triangles show ribosome stalling at various codons. Note that owing to the large size of the ribosome, the reverse transcriptase stops at the nucleotide +16 relative to the codon located in the P site.