Document Type

Article

Publication Date

2012

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.

Publication Title

Nucleic Acids Research

Volume

40

Issue

1

First Page

360

Last Page

370

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.

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 License
This work is licensed under a Creative Commons Attribution-Noncommercial 3.0 License

Language

English

Format

application/pdf

Included in

Chemistry Commons

Share

COinS