Volume 56, Issue 4, Pages 531-540 (November 2014) Amicoumacin A Inhibits Translation by Stabilizing mRNA Interaction with the Ribosome  Yury S. Polikanov, Ilya A. Osterman, Teresa Szal, Vadim N. Tashlitsky, Marina V. Serebryakova, Pavel Kusochek, David Bulkley, Irina A. Malanicheva, Tatyana A. Efimenko, Olga V. Efremenkova, Andrey L. Konevega, Karen J. Shaw, Alexey A. Bogdanov, Marina V. Rodnina, Olga A. Dontsova, Alexander S. Mankin, Thomas A. Steitz, Petr V. Sergiev  Molecular Cell  Volume 56, Issue 4, Pages 531-540 (November 2014) DOI: 10.1016/j.molcel.2014.09.020 Copyright © 2014 Elsevier Inc. Terms and Conditions

Molecular Cell 2014 56, 531-540DOI: (10.1016/j.molcel.2014.09.020) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 1 Inhibition of Protein Synthesis In Vitro by AMI and Its Electron Density Map (A) Chemical structure of AMI. (B) Difference Fourier map of AMI in complex with the T. thermophilus 70S ribosome. The refined model of AMI (yellow) is displayed in its respective electron density before refinement. The unbiased (Fobs − Fcalc) difference electron density map is contoured at 2σ. Nitrogens are colored blue; oxygens are colored red. (C) Relative inhibition of RNA, DNA, or protein synthesis by AMI determined by macromolecular synthesis inhibition. The effects of AMI on in vitro protein synthesis carried out in cell lysate. Data are represented as mean ± SEM. (D and E) The effects of AMI on in vitro protein synthesis carried out in cell lysate (D) or in the reaction assembled from purified components (E). Firefly luciferase was translated in the cell extract and green fluorescence protein was translated in the PURE system. Molecular Cell 2014 56, 531-540DOI: (10.1016/j.molcel.2014.09.020) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 2 AMI Binding Site on the 70S Ribosome (A–C) Overview of the AMI binding site (yellow) on the T. thermophilus 70S ribosome viewed from three different perspectives. 30S subunit is shown in light yellow; 50S subunit is in light blue. mRNA is shown in magenta, and tRNAs are displayed in green for the A site, in dark blue for the P site, and in orange for the E site. In (A), only the anticodon stems of tRNAs are shown, and the 30S subunit is viewed from the intersubunit interface (50S subunit is removed for clarity), as indicated by the inset. The view in (B) is from the cytoplasm onto the E site. 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 the inset. The anticodon stem loop (ASL) of the E-site tRNA is omitted for clarity in (C). (D–F) Close-up views of the AMI binding site shown in (A), (B), and (C), respectively. Nitrogens are colored blue, oxygens and magnesium-coordinated waters are colored red, and magnesium ions are colored green. E. coli numbering of the nucleotides in the 16S rRNA is used. See also Figure S1 and Movie S1. Molecular Cell 2014 56, 531-540DOI: (10.1016/j.molcel.2014.09.020) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 3 AMI Is a Likely Inhibitor of Ribosome Translocation (A) Induction of a pRFPCER-TrpL2A reporter, sensitive to inhibitors of the ribosome progression. Spots of PCT 1.2 μg, erythromycin 1.5 μg (ERY), and AMI 0.5 μg were placed on the surface of an agar plate containing E. coli cells transformed with the reporter plasmid. Green circles surrounding inhibition zones correspond to induction of cer gene expression. ERY (a positive control) interferes with the ribosome progression along mRNA; PCT (a negative control) inhibits translation initiation (Wilson, 2014). (B) Toe-printing analysis of inhibition of protein synthesis in the PURE system by antibiotics binding in the vicinity of h24 in 16S rRNA, AMI, PCT, and EDE. The control lanes contain no antibiotic (“no drug”) or thiostrepton (THS), which arrests ribosome at the start codon (Orelle et al., 2013a; Vázquez-Laslop et al., 2011). Antibiotics THS, PCT, and EDE were present in the reaction at 100 μM and AMI at 500 μM. The sequence of mRNA is shown on the left. The band corresponding to the ribosome occupying the initiator codon is indicated by the black triangle. (C) The inhibitory action of AMI in a model translocation assay in which the movement of the ribosome from the initiator codon to the second codon of the phage T4 gene 32 is monitored by toe-printing (Shoji et al., 2006). The translocation reaction, initiated by addition of EF-G, was terminated after 30 s while translocation in the absence of the inhibitor is still incomplete. The extent of the inhibition of translocation by AMI was calculated relative to the “no AMI” control, which was taken as 100%. Data are represented as mean ± SEM. Triangles indicate the toe-printing bands corresponding to the position of the ribosome at the initiator codon (white triangle, empty A site; gray triangle, Phe-tRNAPhe-occupied A site) or the ribosome translocated to the second codon (black triangle). The intensities of the bands indicated by gray, and black triangles were used for calculating the extent of translocation. (D) Inhibition of in vitro synthesis of firefly luciferase (white bars) by AMI (black bars) without or with addition of extra 1 μM of EF-Tu, wild-type EF-G, or the G542V mutant EF-G. Data are represented as mean ± SEM. See also Figures S2 and S3. Molecular Cell 2014 56, 531-540DOI: (10.1016/j.molcel.2014.09.020) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 4 Antibiotics in the E Site on the Small Ribosomal Subunit (A) Overview of the superimposed binding sites of AMI (yellow), PCT (blue), KSG (red), and EDE (green) on the 30S subunit. The view and the coloring of 16S rRNA, mRNA, and tRNAs are the same as in Figure 2A. AMI and PCT structures are from the current work, KSG is from PDB entry 1VS5 (Schuwirth et al., 2006), and EDE is from PDB entry 1I95 (Pioletti et al., 2001). All four structures were aligned based on h24 of the 16S rRNA (nucleotides 769–810). (B–E) Close-up views of the binding sites shown in (A) for AMI, PCT, KSG, and EDE, respectively. Steric clashes between antibiotics and parts of the ribosome are indicated by red arrows. Note that AMI tethers mRNA to the 16S rRNA and does not clash with any parts of the ribosome, while PCT, KSG, and EDE clash with mRNA. See also Figure S4 and Movie S2. Molecular Cell 2014 56, 531-540DOI: (10.1016/j.molcel.2014.09.020) Copyright © 2014 Elsevier Inc. Terms and Conditions