Insights into the molecular mechanism of translation inhibition by the ribosome-targeting antibiotic thermorubin

Figures

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  Figure 1.. Thermorubin inhibits protein synthesis both in vivo and in vitro. (A) Chemical structure of thermorubin (THR). (B) Induction of a two-color dual reporter system sensitive to inhibitors of the ribosome progression or inhibitors of DNA replication. Spots of erythromycin (ERY), levofloxacin (LEV) and thermorubin (THR) were placed on the surface of an agar plate containing E. coli ΔtolC cells transformed with the pDualrep2 plasmid (14). Induction of expression of Katushka2S (red) is triggered by translation inhibitors, while RFP (green) is induced upon DNA damage. (C) Time-courses of inhibition of sfGFP synthesis by THR (blue and red curves) and sarecycline (SAR, magenta curve) in the in vitro cell-free translation system. AU, arbitrary units. (D, E) Ribosome stalling by THR and other protein synthesis inhibitors on ermBL(D) and yrbA(E) mRNA templates as revealed by reverse transcription inhibition (toe-printing) in a recombinant cell-free translation system. Nucleotide sequences of wild-type ermBL and yrbA genes, and the corresponding amino acid sequence, are shown on the left in each panel. Sequencing lanes (C, U, A, G) are on the left of each gel. Due to the large size of the…

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  Figure 2.. X-ray and cryo-EM maps of ribosome-bound thermorubin. (A) 2Fo-Fc Fourier electron density map of THR in complex with the T. thermophilus 70S ribosome (blue mesh) carrying only the P-site tRNA viewed from two different angles. The refined model of THR is displayed within its respective electron density map contoured at 1.0σ. (B) Cryo-EM map of the ribosome-bound THR in complex with the E. coli 70S ribosome (blue mesh) carrying both A- and P-site tRNAs. Carbon and oxygen atoms are colored red and salmon in (A), and yellow and red in (B), respectively. Magnesium ion is colored in teal. Note that the locations of all chemical moieties of THR can be unambiguously determined from the electron density map.

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  Figure 3.. X-ray crystal structure of 70S-THR complex with P-site tRNA. (A) Overview of the drug-binding site (red) in the T. thermophilus 70S ribosome carrying mRNA (magenta) and deacylated initiator tRNA in the P site (navy) viewed as a cross-cut section through the nascent peptide exit tunnel. The 30S subunit is shown in light yellow; the 50S subunit is light blue. Helices 69 and 44 of the 23S and 16S rRNAs are highlighted in blue and pale yellow, respectively. (B) Superposition of the ribosome-bound THR in the presence of P-site tRNA (red) with the previous structure of THR bound to a vacant ribosome (blue, PDB entry 4V8A (8)). The structures were aligned based on helix 44 of the 16S rRNA. (C, D) Close-up views of the THR bound in the decoding center of the 70S ribosome (E. coli numbering of the 23S and 16S rRNA nucleotides is used). Potential H-bond interactions are indicated with dashed lines. Note that binding of THR to the 16S–23S bridge B2a causes nucleotide C1914 to flip out of its usual location in the absence of A-site tRNA. Also note that THR binding, even with the empty A site, induces nucleotides A1492 and A1493 of the 16S rRNA to flip out of helix 44.

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  Figure 4.. Cryo-EM structure of 70S-THR complex with both A- and P-site tRNAs. (A) Overview of the THR binding site (yellow) in the E. coli 70S ribosome containing mRNA (magenta) and full-length Phe-tRNAPhe (green) and tRNAifMet (navy) in the A and P sites, respectively, viewed as a cross-cut section through the ribosome. (B) Close-up view of THR bound in the decoding center in the presence of fully accommodated A-site tRNA. Note that nucleotide C1914 adopts a previously unseen conformation, in which it does not interfere with tRNA binding. (C) Structural re-arrangements in the decoding center (red dashed arrows) upon THR binding and tRNA accommodation. Superpositioning of the previous structure of drug-free ribosome carrying both A- and P-site tRNAs (PDB entry 6XHW (25)) with the two new 70S-THR structures with or without A-site tRNA. The structures were aligned based on helix 44 of the 16S rRNA. Note that in the presence of THR and A-site tRNA, nucleotide C1914 flips halfway out from its normal position in Helix 69 of the 23S rRNA, allowing for tRNA accommodation (light blue to blue transition). However, in the absence of A-site tRNA, nucleotide C1914 is unrestricted and fully flips out…

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  Figure 5.. Ribosome-bound THR impairs the elongation cycle. (A) Pre-steady state kinetics of A-site tRNA binding upon interaction of the ternary complex EF-Tu•GTP•Phe-tRNAPhe(Prf16/17) (0.1 μM) with the 70S initiated ribosomes (0.4 μM) containing fMet-tRNAifMet in the P site. Shown are time courses in the absence (blue) and presence (red) of THR. (B) Pre-steady state kinetics of translocation upon interaction of the 70S pre-translocation ribosome complexes (60 nM) containing deacylated-tRNAifMet in the P site and fMet-Phe-tRNAPhe(Prf16/17) in the A site with EF-G (2 μM). Color scheme is the same as in panel A. Each time course represents the average of five to seven experimental replicates. Standard deviations associated with the kinetics were calculated using GraphPad Prism software. (C) Time courses of dissociation of peptidyl-tRNA from the A site of the 70S pre-translocation ribosome complexes in the absence (blue) or presence (red) of THR.

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  Figure 6.. Ribosome-bound THR likely prevents binding of release factor to the A site. (A) Superimposition of the ribosome-bound RF1 (PDB entry 3D5A (57)) with the new structure of 70S-THR complex carrying both A- and P-site tRNAs. The structures were aligned based on helix 44 of the 16S rRNA. Note that the THR-induced flipped-out conformation of C1914 would cause steric clashes with RF1 bound in the A site. (B) Structural re-arrangements in the decoding center that are required for RF1 to bind to the 70S ribosome (dashed red arrows). Note that binding of RF1/2 to the ribosome requires nucleotides A1492, A1493 of the 16S rRNA and A1913 of the 23S rRNA to adopt positions that are incompatible with ribosome-bound THR.