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 tothe 70S ribosome concentration (0.15 μM). Further studies indicate the mode of thiostrepton inhibition is to abrogate the stable bind- ing 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.
Nucleic Acids Research
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The Author(s) 2011. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/3.0), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Spiegel, P. Clint; Walter, Justin D.; Hunter, Margaret; Cobb, Melanie; and Traeger, Geoff, "Thiostrepton Inhibits Stable 70S Ribosome Binding and Ribosome-Dependent GTPase Activation of Elongation Factor G and Elongation Factor 4" (2012). Chemistry Faculty and Staff Publications. 4.
Subjects - Topical (LCSH)
GTPase-activating protein--Inhibitors; Guanosine triphosphatase; Oligopeptides
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This work is licensed under a Creative Commons Attribution-Noncommercial 3.0 License