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
Required Publisher's Statement
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. Paper 4.
Creative Commons License
This work is licensed under a Creative Commons Attribution-Noncommercial 3.0 License