(A) Emission spectra of IF2^Cy3-containing 30S initiation complex mixed with 50S subunits in the presence of ThS or NoS. 50S was pre-incubated with antibiotics for 5 min at 37 °C and then rapidly mixed with 30S initiation complex at 20 °C, followed by 5 min incubation before measurements. Black solid trace: no antibiotics; gray dashed trace: ThS; gray solid trace: NoS; black dashed trace: 30S initiation complex alone. The final concentrations are IF1, IF3, fMet-tRNA^fMet, 0.45 µM; IF2^Cy3, 0.15 µM; mRNA, 0.90 µM; 30S, 0.30 µM; 50S 0.30 µM; GTP, 100 µM and ThS, or NoS, 1.5 µM.
(B) Dose response curves for the inhibition of fluorescence change on mixing IF2^Cy3-containing 30S initiation complex with 50S subunits in the presence of thiopeptide compounds. The Y-axis indicates the % ΔF in the presence of added compound relative to the ΔF in the absence of added compound relative to the fluorescence of 70SIC by itself. Final concentrations are IF2^Cy3, 0.15 µM; IF1, IF3, fMet-tRNA^fMet, 0.45 µM; 022AUGmRNA, 0.9 µM; 30S, 0.30 µM; 50S, 0.30 µM; GTP, 100 µM.

In vitro transcription-translation of GFP in the absence or presence of ThS 25 µM GE2270T, 5 µM NoS and 5 µM ThS (black bars), or in the presence (50 µM) of precursor families PA2–5, PB1–3, PC1–3 and PD1–2 (−ThS, white bars) or with additional presence of 5 µM thiostrepton (+ThS, grey bars). GFP fluorescence from microtiter plate wells shown above each lane were quantified and represented as bars, with the fluorescence detected in the absence of antibiotic assigned as 100 %.

(A) Overview of thiopeptide binding site on the large ribosomal subunit. Interface view with helix 43 and 44 (H43/44, orange), L11 (yellow) and ThS (green) highlighted with surface representation (from PDB ID 3CF5) (Harms et al., 2008). (B) The thiazole rings of ThS (green) interact with the RNA bases at the tips of H43/44 as well as the prolines in the N-terminal domain of L11 (yellow). (C) Model for precursor PA2 bound to the ribosome, based on the position of ThS. PA1, but not PA3 (see text), bind similarly. (D–F) Possible modes of binding for precursors PB1 and PC1 based on ring stacking interactions with RNA and protein components of the ribosome, whereas PC2 lacks one phenyl ring compared to PC1. (G) NoS (pink) interacts with the RNA bases at the tips of H43/44 as well as the N-terminal domain of L11 (yellow), but in a distinct manner compared to ThS (using PDB ID 2ZJP; (Harms et al., 2008). (H–I) Model for precursor PD1 and PD2 bound to the ribosome, based on the position of NoS. PD2 lacks one ring moiety, suggesting binding would be destabilized.

Chemical structures of the thiopeptide antibiotics (A) thiostrepton (ThS), (B) nosiheptide (NoS), (C) micrococcin (MiC), and (D) GE2270A/T/C1, and precursor families (E) PA1–5, (F) PB1–3, (G) PC1–3, and (H) PD1–2.

(A) Inhibition of uncoupled ribosome-dependent EF-G GTPase by thiopeptides ThS (1 µM), GE2270T (25 µM) and precursors PA2, PB2, PC1 and PC2 (50 µM). Closed circles indicate GTPase activity of EF-G in the absence of antibiotic.
(B) Inhibition of uncoupled ribosome-dependent TetM GTPase by ThS (1 µM) and precursors PA2, PB1 and PC2 (100 µM). Closed circles indicate GTPase activity of TetM in the absence of antibiotic.
(C) The dose response curves of Pi release in the presence of ThS, NoS, MiC or precursor compounds. The Y axis indicates the % ΔF due to Pi release in the presence of added compound relative to the ΔF in the absence of added compound relative to the fluorescence from EF-G interaction with the ribosome in the absence of any compound. The final concentrations are 70S, 0.3 µM; EF-G, 0.75 µM; MDCC-PBP, 1.5 µM; GTP,100 µM;7-methylguanine, 200 µM; nucleotide phosphorylase, 0.3 unit/ml.
(D) Dose response curves for reversal of ThS inhibition of Pi release by NoS, MiC, or precursor compounds. The final concentrations for each component are 70S, 0.3 µM; EF-G, 0.75 µM; ThS, 1.2 µM; MDCC-PBP, 1.5 µM; GTP, 100 µM; 7-methylguanine, 200 µM;nucleotide phosphorylase, 0.3 unit/ml.

Protection profiles of representative precursors from all described groups against chloramphenicol (Cam, 10 µM), NoS (5 µM), GE2270T (25 µM) and ThS (5 µM). GFP fluorescence in the absence of antibiotic is assigned as 100 %, whereas the precursor results are presented as the % protection, given as the difference between the inhibition of translation by the active compound (Cam, NoS, GE2270T or ThS) in the presence and absence of the precursor compound (50 µM).

(A) Structure of the thiopeptide GE2270A (green) bound to EF-Tu (yellow), with domains I, II and III indicated (PDB ID 2C77) (Parmeggiani et al., 2006). (B) GE2270A overlaps the binding position on EF-Tu of the terminal A76 and aminoacyl moiety of tRNA. Inset shows overview of EF-Tu●tRNA ternary complex (PDB ID 1TTT) (Nissen et al., 1995) with superimposition of GE2270A. (C) Superimposition of EF-Tu●GE2270A (yellow) and EF-Tu ●tRNA (blue), aligned on basis of domain II. Note that GE2270A (green) clashes with domain I of EF-Tu from the ternary complex (blue). (D) Model for precursor PD1 bound to EF-Tu based on EF-Tu●GE2270A complex (PDB ID 2C77) (Parmeggiani et al., 2006). (E) PD1 (pink) overlaps the binding position on EF-Tu of the terminal A76 and aminoacyl moiety of tRNA (blue). (F) as (C) but with PD1 instead of GE2270A. Note that PD1 does not clash with domain I of EF-Tu from the ternary complex (blue)