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Insights into the fidelity mechanism of mRNA decoding from characterization of viomycin induced miscoding in translation
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology. (Sanyal)
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
(English)Article in journal (Refereed) Submitted
Abstract [en]

Using pre-steady state kinetics and an E. coli based in vitro translation system we have studied the effect of the antibiotic viomycin on mRNA decoding. We find that viomycin binds to the ribosome during initial selection of tRNA, after binding of ternary complex but before GTP hydrolysis by EF-Tu. Viomycin binding renders the ribosome completely incapable of rejecting incorrect A-site bound tRNAs in both initial selection and proofreading. Viomycin sensitivity correlates with the accuracy of initial selection for the four different codon·anticodon pairs tested here. Our results demonstrate that, in contrast to current ideas about ‘induced-fit’, accuracy in initial selection is achieved primarily by increased dissociation rates for near-cognate tRNAs rather than by decreased rates of GTP hydrolysis. Further, our results imply that the ‘monitoring’ bases A1492 and A1493 rapidly fluctuate between active and inactive conformations when a near-cognate tRNA is present in the A site.

Place, publisher, year, edition, pages
Uppsala.
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:uu:diva-300234OAI: oai:DiVA.org:uu-300234DiVA: diva2:951206
Available from: 2016-08-08 Created: 2016-08-08 Last updated: 2016-08-26
In thesis
1. 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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