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Article
Thiostrepton Inhibits Stable 70S Ribosome Binding and Ribosome-Dependent GTPase Activation of Elongation Factor G and Elongation Factor 4
Nucleic Acids Research
  • P. Clint Spiegel, Western Washington University
  • Justin D. Walter, Western Washington University
  • Margaret Hunter
  • Melanie Cobb
  • Geoff Traeger
Document Type
Article
Publication Date
1-1-2012
Disciplines
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 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.

Required Publisher's Statement

Published online 9 September 2011

Comments

Published online 9 September 2011

Subjects - Topical (LCSH)
GTPase-activating protein--Inhibitors; Guanosine triphosphatase; Oligopeptides
Genre/Form
articles
Type
Text
Creative Commons License
Creative Commons Attribution-Noncommercial 3.0
Language
English
Format
application/pdf
Citation Information
P. Clint Spiegel, Justin D. Walter, Margaret Hunter, Melanie Cobb, et al.. "Thiostrepton Inhibits Stable 70S Ribosome Binding and Ribosome-Dependent GTPase Activation of Elongation Factor G and Elongation Factor 4" Nucleic Acids Research Vol. 40 Iss. 1 (2012) p. 360 - 370
Available at: http://works.bepress.com/pc_spiegel/1/