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Fusidic Acid Targets Elongation Factor G in Several Stages of Translocation on the Bacterial Ribosome
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
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2015 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 290, no 6, 3440-3454 p.Article in journal (Refereed) Published
Abstract [en]

The antibiotic fusidic acid (FA) targets elongation factor G (EF-G) and inhibits ribosomal peptide elongation and ribosome recycling, but deeper mechanistic aspects of FA action have remained unknown. Using quench flow and stopped flow experiments in a biochemical system for protein synthesis and taking advantage of separate time scales for inhibited (10 s) and uninhibited (100 ms) elongation cycles, a detailed kinetic model of FA action was obtained. FA targets EF-G at an early stage in the translocation process (I), which proceeds unhindered by the presence of the drug to a later stage (II), where the ribosome stalls. Stalling may also occur at a third stage of translocation(III), just before release of EF-G from the post-translocation ribosome. We show that FA is a strong elongation inhibitor (K-50% approximate to 1 mu M), discuss the identity of the FA targeted states, and place existing cryo-EM and crystal structures in their functional context.

Place, publisher, year, edition, pages
2015. Vol. 290, no 6, 3440-3454 p.
National Category
Cell and Molecular Biology
Identifiers
URN: urn:nbn:se:uu:diva-247496DOI: 10.1074/jbc.M114.611608ISI: 000349456000020PubMedID: 25451927OAI: oai:DiVA.org:uu-247496DiVA: diva2:796429
Available from: 2015-03-19 Created: 2015-03-19 Last updated: 2017-12-04Bibliographically approved
In thesis
1. Mechanisms and Inhibition of EF-G-dependent Translocation and Recycling of the Bacterial Ribosome
Open this publication in new window or tab >>Mechanisms and Inhibition of EF-G-dependent Translocation and Recycling of the Bacterial Ribosome
2015 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The GTPase elongation factor G (EF-G) is an important player in the complex process of protein synthesis by bacterial ribosomes. Although extensively studied much remains to be learned about this fascinating protein. In the elongation phase, after incorporation of each amino acid into the growing peptide chain, EF-G translocates the ribosome along the mRNA template. In the recycling phase, when the synthesis of a protein has been completed, EF-G, together with ribosome recycling factor (RRF), splits the ribosome into its subunits. We developed the first in vitro assay for measuring the average time of a complete translocation step at any position along the mRNA. Inside the open reading frame, at saturating EF-G concentration and low magnesium ion concentration, translocation rates were fast and compatible with elongation rates observed in vivo. We also determined the complete kinetic mechanism for EF-G- and RRF-dependent splitting of the post-termination ribosome. We showed that splitting occurs only when RRF binds before EF-G and that the rate and GTP consumption of the reaction varies greatly with the factor concentrations.

The antibiotic fusidic acid (FA) inhibits bacterial protein synthesis by binding to EF-G when the factor is ribosome bound, during translocation and ribosome recycling. We developed experimental methods and a theoretical framework for analyzing the effect of tight-binding inhibitors like FA on protein synthesis. We found that FA targets three different states during each elongation cycle and that it binds to EF-G on the post-termination ribosome both in the presence and absence of RRF. The stalling time of an FA-inhibited ribosome is about hundred-fold longer than the time of an uninhibited elongation cycle and therefore each binding event has a large impact on the protein synthesis rate and may induce queuing of ribosomes on the mRNA. Although ribosomes in the elongation and the recycling phases are targeted with similar efficiency, we showed that the main effect of FA in vivo is on elongation. Our results may serve as a basis for modelling of EF-G function and FA inhibition inside the living cell and for structure determination of mechanistically important intermediate states in translocation and ribosome recycling.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2015. 60 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1268
Keyword
Protein synthesis, Elongation factor G, Translocation, Ribosome recycling, Fusidic acid
National Category
Biochemistry and Molecular Biology
Research subject
Biology with specialization in Molecular Biotechnology
Identifiers
urn:nbn:se:uu:diva-258990 (URN)978-91-554-9289-2 (ISBN)
Public defence
2015-09-25, B22, BMC, Husargatan 3, Uppsala, 10:00 (English)
Opponent
Supervisors
Available from: 2015-09-04 Created: 2015-07-23 Last updated: 2015-10-01
2. A tale of two antibiotics: Fusidic acid and Viomycin
Open this publication in new window or tab >>A tale of two antibiotics: Fusidic acid and Viomycin
2016 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Antibiotics that target the bacterial ribosome make up about half of all clinically used antibiotics. We have studied two ribosome targeting drugs: Fusidic acid and Viomycin. Fusidic acid inhibits bacterial protein synthesis by binding to elongation factor G (EF-G) on the ribosome, thereby inhibiting translocation of the bacterial ribosome. Viomycin binds directly to the ribosome and inhibits both the fidelity of mRNA decoding and translocation. We found that the mechanisms of inhibition of these two antibiotics were unexpectedly complex. Fusidic acid can bind to EF-G on the ribosome during three separate stages of translocation. Binding of the drug to the first and most sensitive state does not lead to stalling of the ribosome. Rather the ribosome continues unhindered to a downstream state where it stalls for around 8 seconds. Dissociation of fusidic acid from this state allows the ribosome to continue translocating but it soon reaches yet another fusidic acid sensitive state where it can be stalled again, this time for 6 seconds. Viomycin inhibits translocation by binding to the pre-translocation ribosome in competition with EF-G. If viomycin binds before EF-G it stalls the ribosome for 44 seconds, much longer than a normal elongation cycle. Both viomycin and fusidic acid probably cause long queues of ribosomes to build up on the mRNA when they bind. Viomycin inhibits translational fidelity by binding to the ribosome during initial selection. We found that the concentration of viomycin required to bind to the ribosome with a given probability during decoding is proportional to the accuracy of the codon∙anticodon pair being decoded. This demonstrated that long standing models about ribosomal accuracy cannot be correct. Finally, we demonstrated that a common viomycin resistance mutation increases the drug binding rate and decreases its dissociation rate. Our results demonstrate that ribosome targeting drugs have unexpectedly complex mechanisms of action. Both fusidic acid and viomycin preferentially bind to conformations of the ribosome other than those that they stabilize. This suggests that determining the structures of stable drug-bound states may not give sufficient information for drug design.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2016. 64 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1399
Keyword
Protein Synthesis, Antibiotics, Fusidic acid, Viomycin, Translocation, Accuracy
National Category
Biochemistry and Molecular Biology
Research subject
Molecular Biotechnology
Identifiers
urn:nbn:se:uu:diva-300479 (URN)978-91-554-9644-9 (ISBN)
External cooperation:
Public defence
2016-09-23, B41 BMC, Husargatan 3, Uppsala, 13:15 (English)
Opponent
Supervisors
Available from: 2016-09-01 Created: 2016-08-09 Last updated: 2016-09-05

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Borg, AnneliHolm, MikaelPavlov, MichaelSanyal, SuparnaEhrenberg, Måns

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