title: Thiostrepton inhibits stable 70S ribosome binding and ribosome-dependent GTPase activation of elongation factor G and elongation factor 4.
abstract: Thiostrepton, a macrocyclic thiopeptide antibiotic, inhibits prokaryotic translation by interfering with the function of elongation factor G (EF-G). Here, we have used 70S ribosome binding and GTP hydrolysis assays to study the effects of thiostrepton on EF-G and a newly described translation factor, elongation factor 4 (EF4). In the presence of thiostrepton, ribosome-dependent GTP hydrolysis is inhibited for both EF-G and EF4, with IC(50) values equivalent to the 70S ribosome concentration (0.15 µM). Further studies indicate the mode of thiostrepton inhibition is to abrogate the stable binding of EF-G and EF4 to the 70S ribosome. In support of this model, an EF-G truncation variant that does not possess domains IV and V was shown to possess ribosome-dependent GTP hydrolysis activity that was not affected by the presence of thiostrepton (>100 µM). Lastly, chemical footprinting was employed to examine the nature of ribosome interaction and tRNA movements associated with EF4. In the presence of non-hydrolyzable GTP, EF4 showed chemical protections similar to EF-G and stabilized a ratcheted state of the 70S ribosome. These data support the model that thiostrepton inhibits stable GTPase binding to 70S ribosomal complexes, and a model for the first step of EF4-catalyzed reverse-translocation is presented.
authors: Department of Chemistry, Western Washington University, 516 High Street, MS 9150, Bellingham, WA 98225-9150, USA.
authors: Justin D
authors: JD
authors: Walter
authors: Margaret
authors: M
authors: Hunter
authors: Melanie
authors: M
authors: Cobb
authors: Geoff
authors: G
authors: Traeger
authors: P Clint
authors: PC
authors: Spiegel
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D029968:Escherichia coli Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D009154:Mutation; D020653:Peptide Elongation Factor G / Q000037:antagonists & inhibitors* / Q000378:metabolism; D010448:Peptide Initiation Factors; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology*; D012338:RNA, Ribosomal, 23S / Q000737:chemistry / Q000378:metabolism; D012270:Ribosomes / Q000187:drug effects* / Q000378:metabolism; D013883:Thiostrepton / Q000494:pharmacology*; D035602:Transcriptional Elongation Factors / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism
chemical_list: D000900:Anti-Bacterial Agents; D004791:Enzyme Inhibitors; D029968:Escherichia coli Proteins; C503923:GDPNP compound; C515651:LepA protein, E coli; D020653:Peptide Elongation Factor G; D010448:Peptide Initiation Factors; D011500:Protein Synthesis Inhibitors; D012338:RNA, Ribosomal, 23S; D035602:Transcriptional Elongation Factors; D006160:Guanosine Triphosphate; D013883:Thiostrepton