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Pavlov, Michael
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Publications (10 of 20) Show all publications
Zhang, J., Pavlov, M. & Ehrenberg, M. (2018). Accuracy of genetic code translation and its orthogonal corruption by aminoglycosides and Mg2+ ions. Nucleic Acids Research, 46(3), 1362-1374
Open this publication in new window or tab >>Accuracy of genetic code translation and its orthogonal corruption by aminoglycosides and Mg2+ ions
2018 (English)In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 46, no 3, p. 1362-1374Article in journal (Refereed) Published
Abstract [en]

We studied the effects of aminoglycosides and changing Mg2+ ion concentration on the accuracy of initial codon selection by aminoacyl-tRNA in ternary complex with elongation factor Tu and GTP (T-3) on mRNA programmed ribosomes. Aminoglycosides decrease the accuracy by changing the equilibrium constants of 'monitoring bases' A1492, A1493 and G530 in 16S rRNA in favor of their 'activated' state by large, aminoglycoside-specific factors, which are the same for cognate and near-cognate codons. Increasing Mg2+ concentration decreases the accuracy by slowing dissociation of T-3 from its initial codon-and aminoglycoside-independent binding state on the ribosome. The distinct accuracy-corrupting mechanisms for aminoglycosides and Mg2+ ions prompted us to re-interpret previous biochemical experiments and functional implications of existing high resolution ribosome structures. We estimate the upper thermodynamic limit to the accuracy, the 'intrinsic selectivity' of the ribosome. We conclude that aminoglycosides do not alter the intrinsic selectivity but reduce the fraction of it that is expressed as the accuracy of initial selection. We suggest that induced fit increases the accuracy and speed of codon reading at unaltered intrinsic selectivity of the ribosome.

Place, publisher, year, edition, pages
OXFORD UNIV PRESS, 2018
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-349357 (URN)10.1093/nar/gkx1256 (DOI)000425294400033 ()29267976 (PubMedID)
Funder
Knut and Alice Wallenberg FoundationSwedish Research Council
Available from: 2018-05-02 Created: 2018-05-02 Last updated: 2018-05-02Bibliographically approved
Pavlov, M. & Ehrenberg, M. (2018). Substrate-Induced Formation of Ribosomal Decoding Center for Accurate and Rapid Genetic Code Translation. Annual Review of Biophysics, 525-548
Open this publication in new window or tab >>Substrate-Induced Formation of Ribosomal Decoding Center for Accurate and Rapid Genetic Code Translation
2018 (English)In: Annual Review of Biophysics, ISSN 1936-122X, E-ISSN 1936-1238, p. 525-548Article, review/survey (Refereed) Published
Abstract [en]

Accurate translation of genetic information is crucial for synthesis of functional proteins in all organisms. We use recent experimental data to discuss how induced fit affects accuracy of initial codon selection on the ribosome by aminoacyl transfer RNA in ternary complex (T-3) with elongation factor Tu (EF-Tu) and guanosine-5'-triphosphate (GTP). We define actual accuracy (A(I)(nc)) of a particular protein synthesis system as its current accuracy and the effective selectivity (d(eI)(nc)) as A(I)(nc) in the limit of zero ribosomal binding affinity for T-3. Intrinsic selectivity (D-I(nc)), defined as the upper thermodynamic limit of d(eI)(nc), is determined by the free energy difference between near-cognate and cognate T-3 in the pre-GTP hydrolysis state on the ribosome. D-I(nc) is much larger than d(eI)(nc), suggesting the possibility of a considerable increase in d(eI)(nc) and A(I)(nc) at negligible kinetic cost. Induced fit increases A(I)(nc) and d(eI)(nc) without affecting D-I(nc), and aminoglycoside antibiotics reduce A(I)(nc) and d(eI)(nc) at unaltered D-I(nc).

Keywords
induced fit, ribosome, translation accuracy, initial selection, tautomers
National Category
Biophysics
Identifiers
urn:nbn:se:uu:diva-361439 (URN)10.1146/annurev-biophys-060414-034148 (DOI)000433056800024 ()29792818 (PubMedID)978-0-8243-1847-5 (ISBN)
Available from: 2018-12-10 Created: 2018-12-10 Last updated: 2018-12-10Bibliographically approved
Caban, K., Pavlov, M., Kaledhonkar, S., Fu, Z., Frank, J., Ehrenberg, M. & Gonzalez, R. L. . (2018). The Structural Basis for Initiation Factor 2 Activation during Translation Initiation. Paper presented at 62nd Annual Meeting of the Biophysical-Society, FEB 17-21, 2018, San Francisco, CA, USA. Biophysical Journal, 114(3), 593A-593A
Open this publication in new window or tab >>The Structural Basis for Initiation Factor 2 Activation during Translation Initiation
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2018 (English)In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 114, no 3, p. 593A-593AArticle in journal, Meeting abstract (Other academic) Published
Place, publisher, year, edition, pages
CELL PRESS, 2018
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-357498 (URN)000430563200723 ()
Conference
62nd Annual Meeting of the Biophysical-Society, FEB 17-21, 2018, San Francisco, CA, USA
Available from: 2018-08-17 Created: 2018-08-17 Last updated: 2018-08-17Bibliographically approved
Caban, K., Pavlov, M., Ehrenberg, M. & Gonzalez, R. L. . (2017). A conformational switch in initiation factor 2 controls the fidelity of translation initiation in bacteria. Nature Communications, 8, Article ID 1475.
Open this publication in new window or tab >>A conformational switch in initiation factor 2 controls the fidelity of translation initiation in bacteria
2017 (English)In: Nature Communications, ISSN 2041-1723, E-ISSN 2041-1723, Vol. 8, article id 1475Article in journal (Refereed) Published
Abstract [en]

Initiation factor (IF) 2 controls the fidelity of translation initiation by selectively increasing the rate of 50S ribosomal subunit joining to 30S initiation complexes (ICs) that carry an N-formyl-methionyl-tRNA (fMet-tRNA(fMet)). Previous studies suggest that rapid 50S subunit joining involves a GTP- and fMet-tRNA(fMet)-dependent "activation" of IF2, but a lack of data on the structure and conformational dynamics of 30S IC-bound IF2 has precluded a mechanistic understanding of this process. Here, using an IF2-tRNA single-molecule fluorescence resonance energy transfer signal, we directly observe the conformational switch that is associated with IF2 activation within 30S ICs that lack IF3. Based on these results, we propose a model of IF2 activation that reveals how GTP, fMet-tRNA(fMet), and specific structural elements of IF2 drive and regulate this conformational switch. Notably, we find that domain III of IF2 plays a pivotal, allosteric, role in IF2 activation, suggesting that this domain can be targeted for the development of novel antibiotics.

National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
urn:nbn:se:uu:diva-342388 (URN)10.1038/s41467-017-01492-6 (DOI)000415124000002 ()29133802 (PubMedID)
Funder
NIH (National Institute of Health), R01 GM 084288
Available from: 2018-02-22 Created: 2018-02-22 Last updated: 2018-02-22Bibliographically approved
Pavlov, M., Liljas, A. & Ehrenberg, M. (2017). A recent intermezzo at the Ribosome Club. Philosophical Transactions of the Royal Society of London. Biological Sciences, 372(1716), Article ID 20160185.
Open this publication in new window or tab >>A recent intermezzo at the Ribosome Club
2017 (English)In: Philosophical Transactions of the Royal Society of London. Biological Sciences, ISSN 0962-8436, E-ISSN 1471-2970, Vol. 372, no 1716, article id 20160185Article, review/survey (Refereed) Published
Abstract [en]

Two sets of ribosome structures have recently led to two different interpretations of what limits the accuracy of codon translation by transfer RNAs. In this review, inspired by this intermezzo at the Ribosome Club, we briefly discuss accuracy amplification by energy driven proofreading and its implementation in genetic code translation. We further discuss general ways by which the monitoring bases of 16S rRNA may enhance the ultimate accuracy (d-values) and how the codon translation accuracy is reduced by the actions of Mg2+ ions and the presence of error inducing aminoglycoside antibiotics. We demonstrate that complete freezing-in of cognate-like tautomeric states of ribosome-bound nucleotide bases in transfer RNA or messenger RNA is not compatible with recent experiments on initial codon selection by transfer RNA in ternary complex with elongation factor Tu and GTP. From these considerations, we suggest that the sets of 30S subunit structures from the Ramakrishnan group and 70S structures from the Yusupov/Yusupova group may, after all, reflect two sides of the same coin and how the structurally based intermezzo at the Ribosome Club may be resolved simply by taking the dynamic aspects of ribosome function into account. This article is part of the themed issue 'Perspectives on the ribosome'.

Keywords
ribosome, translation accuracy, proofreading, initial transfer RNA selection, tautomers
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-317503 (URN)10.1098/rstb.2016.0185 (DOI)000393403600008 ()
Available from: 2017-04-13 Created: 2017-04-13 Last updated: 2018-01-13
Borg, A., Pavlov, M. & Ehrenberg, M. (2016). Complete kinetic mechanism for recycling of the bacterial ribosome. RNA: A publication of the RNA Society, 22(1), 10-21
Open this publication in new window or tab >>Complete kinetic mechanism for recycling of the bacterial ribosome
2016 (English)In: RNA: A publication of the RNA Society, ISSN 1355-8382, E-ISSN 1469-9001, Vol. 22, no 1, p. 10-21Article in journal (Refereed) Published
Abstract [en]

How EF-G and RRF act together to split a post-termination ribosomal complex into its subunits has remained obscure. Here, using stopped-flow experiments with Rayleigh light scattering detection and quench-flow experiments with radio-detection of GTP hydrolysis, we have clarified the kinetic mechanism of ribosome recycling and obtained precise estimates of its kinetic parameters. Ribosome splitting requires that EF-G binds to an already RRF-containing ribosome. EF-G binding to RRF-free ribosomes induces futile rounds of GTP hydrolysis and inhibits ribosome splitting, implying that while RRF is purely an activator of recycling, EF-G acts as both activator and competitive inhibitor of RRF in recycling of the post-termination ribosome. The ribosome splitting rate and the number of GTPs consumed per splitting event depend strongly on the free concentrations of EF-G and RRF. The maximal recycling rate, here estimated as 25 sec(-1), is approached at very high concentrations of EF-G and RRF with RRF in high excess over EF-G. The present in vitro results, suggesting an in vivo ribosome recycling rate of 5 sec(-1), are discussed in the perspective of rapidly growing bacterial cells.

Keywords
bacterial ribosome recycling; elongation factor G; ribosome recycling factor; translation rate optimization; protein synthesis
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-258988 (URN)10.1261/rna.053157.115 (DOI)000368967600002 ()26527791 (PubMedID)
Funder
Swedish Research CouncilKnut and Alice Wallenberg Foundation
Available from: 2015-08-04 Created: 2015-07-23 Last updated: 2017-12-04Bibliographically approved
Borg, A., Pavlov, M. & Ehrenberg, M. (2016). Mechanism of fusidic acid inhibition of RRF- and EF-G-dependent splitting of the bacterial post-termination ribosome. Nucleic Acids Research, 44(7), 3264-3275
Open this publication in new window or tab >>Mechanism of fusidic acid inhibition of RRF- and EF-G-dependent splitting of the bacterial post-termination ribosome
2016 (English)In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 44, no 7, p. 3264-3275Article in journal (Refereed) Published
Abstract [en]

The antibiotic drug fusidic acid (FA) is commonly used in the clinic against gram-positive bacterial infections. FA targets ribosome-bound elongation factor G (EF-G), a translational GTPase that accelerates both messenger RNA (mRNA) translocation and ribosome recycling. How FA inhibits translocation was recently clarified, but FA inhibition of ribosome recycling by EF-G and ribosome recycling factor (RRF) has remained obscure. Here we use fast kinetics techniques to estimate mean times of ribosome splitting and the stoichiometry of GTP hydrolysis by EF-G at varying concentrations of FA, EF-G and RRF. These mean times together with previous data on uninhibited ribosome recycling were used to clarify the mechanism of FA inhibition of ribosome splitting. The biochemical data on FA inhibition of translocation and recycling were used to model the growth inhibitory effect of FA on bacterial populations. We conclude that FA inhibition of translocation provides the dominant cause of bacterial growth reduction, but that FA inhibition of ribosome recycling may contribute significantly to FA-induced expression of short regulatory open reading frames, like those involved in FA resistance.

National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-297902 (URN)10.1093/nar/gkw178 (DOI)000375800200033 ()27001509 (PubMedID)
Funder
Swedish Research Council, 2015-04682Knut and Alice Wallenberg Foundation, KAW 2009.0251
Available from: 2016-06-29 Created: 2016-06-28 Last updated: 2018-01-10Bibliographically 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
Indrisiunaite, G., Pavlov, M. Y., Heurgue-Hamard, V. & Ehrenberg, M. (2015). On the pH Dependence of Class-1 RF-Dependent Termination of mRNA Translation. Journal of Molecular Biology, 427(9), 1848-1860
Open this publication in new window or tab >>On the pH Dependence of Class-1 RF-Dependent Termination of mRNA Translation
2015 (English)In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 427, no 9, p. 1848-1860Article in journal (Refereed) Published
Abstract [en]

We have studied the pH dependence of the rate of termination of bacterial protein synthesis catalyzed by a class-1 release factor (RF1 or RF2). We used a classical quench-flow technique and a newly developed stopped-flow technique that relies on the use of fluorescently labeled peptides. We found the termination rate to increase with increasing pH and, eventually, to saturate at about 70 s(-1) with an apparent pK(a) value of about 7.6. From our data, we suggest that class-1 RF termination is rate limited by the chemistry of ester bond hydrolysis at low pH and by a stop-codon-dependent and pH-independent conformational change of RFs at high pH. We propose that RF-dependent termination depends on the participation of a hydroxide ion rather than a water molecule in the hydrolysis of the ester bond between the P-site tRNA and its peptide chain. We provide a simple explanation for why the rate of termination saturated at high pH in our experiments but not in those of others.

National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-255073 (URN)10.1016/j.jmb.2015.01.007 (DOI)000353929400006 ()25619162 (PubMedID)
Funder
Knut and Alice Wallenberg FoundationSwedish Research Council
Available from: 2015-06-15 Created: 2015-06-12 Last updated: 2019-04-26Bibliographically approved
Ieong, K.-W., Pavlov, M. Y., Kwiatkowski, M., Ehrenberg, M. & Forster, A. C. (2014). A tRNA body with high affinity for EF-Tu hastens ribosomal incorporation of unnatural amino acids. RNA: A publication of the RNA Society, 20(5), 632-643
Open this publication in new window or tab >>A tRNA body with high affinity for EF-Tu hastens ribosomal incorporation of unnatural amino acids
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2014 (English)In: RNA: A publication of the RNA Society, ISSN 1355-8382, E-ISSN 1469-9001, Vol. 20, no 5, p. 632-643Article in journal (Refereed) Published
National Category
Natural Sciences
Identifiers
urn:nbn:se:uu:diva-218734 (URN)10.1261/rna.042234.113 (DOI)000334677800005 ()
Available from: 2014-02-16 Created: 2014-02-16 Last updated: 2017-12-06Bibliographically approved
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