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- Holm, Mikael, 1984-
(författare)
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A tale of two antibiotics : Fusidic acid and Viomycin
- 2016
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- 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.
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- Holm, Mikael, 1984-, et al.
(författare)
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Insights into the fidelity mechanism of mRNA decoding from characterization of viomycin induced miscoding in translation
- 2024
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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- Holm, Mikael, 1984-, et al.
(författare)
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The mechanism of error induction by the antibiotic viomycin provides insight into the fidelity mechanism of translation
- 2019
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Ingår i: eLIFE. - : ELIFE SCIENCES PUBLICATIONS LTD. - 2050-084X. ; 8
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Tidskriftsartikel (refereegranskat)abstract
- Applying pre-steady state kinetics to an Escherichia-coli-based reconstituted translation system, we have studied how the antibiotic viomycin affects the accuracy of genetic code reading. We find that viomycin binds to translating ribosomes associated with a ternary complex (TC) consisting of elongation factor Tu (EF-Tu), aminoacyl tRNA and GTP, and locks the otherwise dynamically flipping monitoring bases A1492 and A1493 into their active conformation. This effectively prevents dissociation of near- and non-cognate TCs from the ribosome, thereby enhancing errors in initial selection. Moreover, viomycin shuts down proofreading-based error correction. Our results imply a mechanism in which the accuracy of initial selection is achieved by larger backward rate constants toward TC dissociation rather than by a smaller rate constant for GTP hydrolysis for near- and non-cognate TCs. Additionally, our results demonstrate that translocation inhibition, rather than error induction, is the major cause of cell growth inhibition by viomycin.
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- Kim, Changil, et al.
(författare)
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Optimization of a fluorescent-mRNA based real-time assay for precise kinetic measurements of ribosomal translocation
- 2021
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Ingår i: RNA Biology. - : Informa UK Limited. - 1547-6286 .- 1555-8584. ; 18:12, s. 2363-2375
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Tidskriftsartikel (refereegranskat)abstract
- Kinetic characterization of ribosomal translocation is important for understanding the mechanism of elongation in protein synthesis. Here we have optimized a popular fluorescent-mRNA based translocation assay conducted in stopped-flow, by calibrating it with the functional tripeptide formation assay in quench-flow. We found that a fluorescently labelled mRNA, ten bases long from position +1 (mRNA+10), is best suited for both assays as it forms tripeptide at a fast rate equivalent to the longer mRNAs, and yet produces a large fluorescence change upon mRNA movement. Next, we compared the commonly used peptidyl tRNA analog, N-acetyl-Phe-tRNAPhe, with the natural dipeptidyl fMet-Phe-tRNAPhe in the stopped-flow assay. This analog translocates about two times slower than the natural dipeptidyl tRNA and produces biphasic kinetics. The rates reduce further at lower temperatures and with higher Mg2+ concentration, but improve with higher elongation factor G (EF-G) concentration, which increase both rate and amplitude of the fast phase significantly. In summary, we present here an improved real time assay for monitoring mRNA-translocation with the natural- and an N-Ac-analog of dipeptidyl tRNA.
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