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Holm, M., Mandava, C. S., Ehrenberg, M. & Sanyal, S. (2019). The mechanism of error induction by the antibiotic viomycin provides insight into the fidelity mechanism of translation. eLIFE, 8, Article ID e46124.
Open this publication in new window or tab >>The mechanism of error induction by the antibiotic viomycin provides insight into the fidelity mechanism of translation
2019 (English)In: eLIFE, E-ISSN 2050-084X, Vol. 8, article id e46124Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
ELIFE SCIENCES PUBLICATIONS LTD, 2019
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-390687 (URN)10.7554/eLife.46124 (DOI)000473013700001 ()31172942 (PubMedID)
Funder
Swedish Research Council, 2018-05498Swedish Research Council, 2016-06264Knut and Alice Wallenberg Foundation, KAW 2011.0081Knut and Alice Wallenberg Foundation, KAW 2017.0055Carl Tryggers foundation , CTS 18: 338Wenner-Gren Foundations, UPD2017-0238
Available from: 2019-08-14 Created: 2019-08-14 Last updated: 2019-08-14Bibliographically approved
Holm, M. (2016). A tale of two antibiotics: Fusidic acid and Viomycin. (Doctoral dissertation). Uppsala: Acta Universitatis Upsaliensis
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. p. 64
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1399
Keywords
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
Holm, M., Borg, A., Ehrenberg, M. & Sanyal, S. (2016). Molecular mechanism of viomycin inhibition of peptide elongation in bacteria. Proceedings of the National Academy of Sciences of the United States of America, 113(4), 978-983
Open this publication in new window or tab >>Molecular mechanism of viomycin inhibition of peptide elongation in bacteria
2016 (English)In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 113, no 4, p. 978-983Article in journal (Refereed) Published
Abstract [en]

Viomycin is a tuberactinomycin antibiotic essential for treating multi-drug-resistant tuberculosis. It inhibits bacterial protein synthesis by blocking elongation factor G (EF-G) catalyzed translocation of messenger RNA on the ribosome. Here we have clarified the molecular aspects of viomycin inhibition of the elongating ribosome using pre-steady-state kinetics. We found that the probability of ribosome inhibition by viomycin depends on competition between viomycin and EF-G for binding to the pretranslocation ribosome, and that stable viomycin binding requires an A-site bound tRNA. Once bound, viomycin stalls the ribosome in a pretranslocation state for a minimum of similar to 45 s. This stalling time increases linearly with viomycin concentration. Viomycin inhibition also promotes futile cycles of GTP hydrolysis by EF-G. Finally, we have constructed a kinetic model for viomycin inhibition of EF-G catalyzed translocation, allowing for testable predictions of tuberactinomycin action in vivo and facilitating in-depth understanding of resistance development against this important class of antibiotics.

Keywords
protein synthesis, viomycin, ribosome, antibiotics, tuberculosis
National Category
Microbiology in the medical area
Identifiers
urn:nbn:se:uu:diva-277786 (URN)10.1073/pnas.1517541113 (DOI)000368617900047 ()26755601 (PubMedID)
Available from: 2016-02-23 Created: 2016-02-23 Last updated: 2018-01-10Bibliographically approved
Koripella, R. K., Holm, M., Dourado, D., Mandava, C. S., Flores, S. & Sanyal, S. (2015). A conserved histidine in switch-II of EF-G moderates release of inorganic phosphate. Scientific Reports, 5, Article ID 12970.
Open this publication in new window or tab >>A conserved histidine in switch-II of EF-G moderates release of inorganic phosphate
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2015 (English)In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 5, article id 12970Article in journal (Refereed) Published
Abstract [en]

Elongation factor G (EF-G), a translational GTPase responsible for tRNA-mRNA translocation possesses a conserved histidine (H91 in Escherichia coli) at the apex of switch-II, which has been implicated in GTPase activation and GTP hydrolysis. While H91A, H91R and H91E mutants showed different degrees of defect in ribosome associated GTP hydrolysis, H91Q behaved like the WT. However, all these mutants, including H91Q, are much more defective in inorganic phosphate (Pi) release, thereby suggesting that H91 facilitates Pi release. In crystal structures of the ribosome bound EF-G center dot GTP a tight coupling between H91 and the gamma-phosphate of GTP can be seen. Following GTP hydrolysis, H91 flips similar to 140 degrees in the opposite direction, probably with Pi still coupled to it. This, we suggest, promotes Pi to detach from GDP and reach the inter-domain space of EF-G, which constitutes an exit path for the Pi. Molecular dynamics simulations are consistent with this hypothesis and demonstrate a vital role of an Mg2+ ion in the process.

National Category
Biological Sciences
Identifiers
urn:nbn:se:uu:diva-261233 (URN)10.1038/srep12970 (DOI)000359373400001 ()
Funder
Swedish Research Council, 2011-6088 2014-4423 2008-6593Knut and Alice Wallenberg Foundation, KAW 2011.0081
Available from: 2015-09-07 Created: 2015-08-31 Last updated: 2017-12-04Bibliographically approved
Borg, A., Holm, M., Shiroyama, I., Hauryliuk, V., Pavlov, M., Sanyal, S. & Ehrenberg, M. (2015). Fusidic Acid Targets Elongation Factor G in Several Stages of Translocation on the Bacterial Ribosome. Journal of Biological Chemistry, 290(6), 3440-3454
Open this publication in new window or tab >>Fusidic Acid Targets Elongation Factor G in Several Stages of Translocation on the Bacterial Ribosome
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2015 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 290, no 6, p. 3440-3454Article 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.

National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-247496 (URN)10.1074/jbc.M114.611608 (DOI)000349456000020 ()25451927 (PubMedID)
Available from: 2015-03-19 Created: 2015-03-19 Last updated: 2018-01-11Bibliographically approved
Korkmaz, G., Holm, M., Wiens, T. & Sanyal, S. (2014). Comprehensive Analysis of Stop Codon Usage in Bacteria and Its Correlation with Release Factor Abundance. Journal of Biological Chemistry, 289(44), 30334-30342
Open this publication in new window or tab >>Comprehensive Analysis of Stop Codon Usage in Bacteria and Its Correlation with Release Factor Abundance
2014 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 289, no 44, p. 30334-30342Article in journal (Refereed) Published
Abstract [en]

We present a comprehensive analysis of stop codon usage in bacteria by analyzing over eight million coding sequences of 4684 bacterial sequences. Using a newly developed program called "stop codon counter," the frequencies of the three classical stop codons TAA, TAG, and TGA were analyzed, and a publicly available stop codon database was built. Our analysis shows that with increasing genomic GC content the frequency of the TAA codon decreases and that of the TGA codon increases in a reciprocal manner. Interestingly, the release factor 1-specific codon TAG maintains a more or less uniform frequency (similar to 20%) irrespective of the GC content. The low abundance of TAG is also valid with respect to expression level of the genes ending with different stop codons. In contrast, the highly expressed genes predominantly end with TAA, ensuring termination with either of the two release factors. Using three model bacteria with different stop codon usage (Escherichia coli, Mycobacterium smegmatis, and Bacillus subtilis), we show that the frequency of TAG and TGA codons correlates well with the relative steady state amount of mRNA and protein for release factors RF1 and RF2 during exponential growth. Furthermore, using available microarray data for gene expression, we show that in both fast growing and contrasting biofilm formation conditions, the relative level of RF1 is nicely correlated with the expression level of the genes ending with TAG.

National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-239577 (URN)10.1074/jbc.M114.606632 (DOI)000344549700016 ()25217634 (PubMedID)
Available from: 2014-12-30 Created: 2014-12-29 Last updated: 2017-12-05Bibliographically approved
Holm, M., Ge, X. & Sanyal, S.Biochemical characterization of ΔTlyA mediated viomycin resistance.
Open this publication in new window or tab >>Biochemical characterization of ΔTlyA mediated viomycin resistance
(English)Manuscript (preprint) (Other academic)
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-300235 (URN)
Available from: 2016-08-08 Created: 2016-08-08 Last updated: 2016-08-26
Koripella, R. K., Holm, M. & Sanyal, S.Essential role of Histidine 92 in elongation factor-G in GTP hydrolysis and inorganic phosphate release during elongation of protein synthesis.
Open this publication in new window or tab >>Essential role of Histidine 92 in elongation factor-G in GTP hydrolysis and inorganic phosphate release during elongation of protein synthesis
(English)Manuscript (preprint) (Other academic)
Abstract [en]

The histidine (H) residue at the apex of switch II is conserved in all translational GTPases. Thishistidine (H92) in elongation factor G (EF-G) has been implicated in GTP hydrolysis andinorganic phosphate (pi) release similar to H85 in elongation factor-Tu (EF-Tu). Mutagenesis ofH92 to alanine (A) and glutamic acid (E) showed different degrees of defect in different steps ofelongation. While H92A was ~7 times slower than wild type EF-G in ribosome mediated GTPhydrolysis, it was 100 times slower in both pi release and tRNA translocation. The H92E mutant,on the other hand, was 100 times slower in all these steps. Both mutants were significantlydefective (~1000 times slower) in tripeptide formation that which requires dissociation of EF-Gfrom the post-translocation state. Thus, our results indicate that GTP hydrolysis takes place priorto tRNA translocation, whereas Pi release occurs probably after or independent of thetranslocation step. Since translocation involves back ratcheting of the ribosomal subunits ourresults suggest that there is a cross-talk between GTP hydrolysis by EF-G and ribosomal subunitrotation. We further confirm that Pi release is essential for the next round of elongation.

National Category
Biological Sciences
Research subject
Molecular Biology
Identifiers
urn:nbn:se:uu:diva-207882 (URN)
Available from: 2013-09-20 Created: 2013-09-20 Last updated: 2017-01-25
Holm, M. & Sanyal, S. Insights into the fidelity mechanism of mRNA decoding from characterization of viomycin induced miscoding in translation.
Open this publication in new window or tab >>Insights into the fidelity mechanism of mRNA decoding from characterization of viomycin induced miscoding in translation
(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:nbn:se:uu:diva-300234 (URN)
Available from: 2016-08-08 Created: 2016-08-08 Last updated: 2016-08-26
Organisations
Identifiers
ORCID iD: ORCID iD iconorcid.org/0000-0002-4361-9554

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