Inhibition by Siomycin and Thiostrepton of Both Aminoacyl-tRNA and Factor G Binding to Ribosomes

Abstract

Siomycin, a peptide antibiotic that interacts with the 50S ribosomal subunit and inhibits binding of factor G, is shown also to inhibit binding of aminoacyltRNA; however, it does not impair binding of fMet-tRNA and completion of the initiation complex. Moreover, unlike other inhibitors of aminoacyl-tRNA binding (tetracycline, sparsomycin, and streptogramin A), siomycin completely abolishes the GTPase activity associated with the binding of aminoacyl-tRNA catalyzed by factor Tu. A single-site interaction of siomycin appears to be responsible for its effect on both the binding of the aminoacyl-tRNA-Tu-GTP complex and that of factor G.Factor G, one of the supernatant proteins necessary for ribosomal activity in Escherichia coli, is involved in the coupling of GTP hydrolysis to polypeptide elongation. This hydrolysis is thought to provide the energy necessary for the rearrangements of tRNA, mRNA, and ribosomal subunits, in each round of peptide bond formation, that result in translocation of mRNA and peptidyl-tRNA (reviewed in ref. 1). GTP hydrolysis can also occur in the absence of mRNA and peptidyl-tRNA, the reaction requiring only factor G and ribosomes (2, 3). In recent work this reaction has been used to show that there is a binding site for factor G (the G-site) on the 50S subunit (4, 5). Our studies have further shown that a peptide antibiotic, siomycin, inactivates this site, preventing the interaction of this site with factor G. Moreover, the G-site is evidently distinct from the peptidyl transferase center on the 50S subunit (5).We have now shown that siomycin-treated ribosomes can carry out the initiation step in protein synthesis, with either viral messenger or triplet AUG, and the bound fMet-tRNA can reach the puromycin-reactive position. However, no peptide bond can be formed because the ribosomes do not bind aminoacyl-tRNA in the recognition site (the A-site). Moreover, the siomycin-treated ribosomes can complement neither the GTPase activity associated with factor T nor that associated with factor G. These effects appear to be the consequence of a single action of siomycin on the 50S subunit, thus suggesting a relationship between the binding site of factor G and that of the aminoacyl-tRNA-T.-GTP complex. MATERIALS AND METHODSRibosomes washed with ammonium chloride (1 M) and crude initiation factors from E. coli MRE600 were prepared as described (6, 7). Homogeneous initiation factor F1 and partially purified factor F2 were a gift of Dr. S. Ochoa. Elongation factors G and T (T. plus Tu) and ['y-82P]GTP were prepared as described (2, 8 Binding of aminoacyl-tRNA to ribosomes was assayed by filtration (9). Reaction mixtures for the binding of fMettRNA contained: 80 mM NH4Cl, 50 mM Tris HOl (pH 7.7), 5 mM Mg(acetate)2, 6 mM mercaptoethanol, 0.4 mM GTP, 0.3 mg/ml of crude initiation factors, 4.7 pmol f-[3H]MettRNA per A260 unit ribosomes, 1.2 mg/ml of R17-RNA, 35 A260 units/ml of ribosomes, and other additions as specified. Unless otherwise indicated, incubation was at 34°C for 10 min and was follo...

Ribosomally synthesized and post-translationally modified peptide natural products: overview and recommendations for a universal nomenclature

“…231–233 These thiopeptides bind near the GTPase-associated center that engages a number of translation factors during initiation and elongation. 234–236 Structural studies revealed that thiopeptides bind to a cleft between the ribosomal protein L11 and the 23S rRNA, and the precise intermolecular interactions differ according to the exact structural features of the thiopeptide. 231,237 Complexation of the ribosome and a thiopeptide disrupts the conformational changes ordinarily communicated from the translation factors to the ribosome, ultimately halting translocation along the mRNA template.…”

“…231,237 Complexation of the ribosome and a thiopeptide disrupts the conformational changes ordinarily communicated from the translation factors to the ribosome, ultimately halting translocation along the mRNA template. 229,233,235,236 A subset of thiopeptides, including the thiomuracins and GE2270A, exert a separate mode of antibacterial action by the inhibition of elongation factor Tu (EF-Tu). EF-Tu ordinarily delivers aminoacylated tRNAs to the ribosome following translocation.…”

Subinhibitory Concentrations of Bacteriostatic Antibiotics Induce relA -Dependent and relA -Independent Tolerance to β-Lactams

“…The cyclic peptide thiostrepton is an efficient inhibitor of both translational GTPases, targeting initiation factor IF2 and elongation factors EF-Tu and EF-G on the ribosome (26–28), and E. coli RelA (at least in the test tube [29, 30]).…”

Differential Effects of Thiopeptide and Orthosomycin Antibiotics on Translational GTPases

“…This region is part of the GTPase-associated center (GAC), so named because it is involved in binding of translation factors and stimulation of their GTPase activities: Consistently, thiopeptide antibiotics have been shown to inhibit IF2-dependent 70S initiation complex (70SIC) formation (Brandi et al, 2004; Grigoriadou et al, 2007), EF-Tu-dependent delivery of aminoacyl-tRNAs to the ribosome (Brandi et al, 2004; Gonzalez et al, 2007; Modelell et al, 1971; Otaka and Kaji, 1974), translocation of the tRNA 2 -mRNA complex through the ribosome (Munro et al, 2010; Pan et al, 2007; Pestka, 1970; Pestka and Brot, 1971; Rodnina et al, 1997), and stringent factor RelA-dependent synthesis of ppGpp (Cundliffe and Thompson, 1981; Jenvert and Schiavone, 2005). Surprisingly, however, ThS and MiC exhibit differential effects on the uncoupled ribosome-dependent EF-G GTPase activities: ThS strongly inhibits multiple-turnover GTP hydrolysis of EF-G (Pestka, 1970; Weisblum and Demohn, 1970) by preventing Pi release and thus trapping EF-G on the ribosome (Rodnina et al, 1999; Seo et al, 2006).…”