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Ehrenberg, Måns
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Publications (10 of 74) Show all publications
Choi, J., Indrisiunaite, G., DeMirci, H., Leong, K.-W., Wang, J., Petrov, A., . . . Puglisi, J. D. (2018). 2 '-O-methylation in mRNA disrupts tRNA decoding during translation elongation. Nature Structural & Molecular Biology, 25(3), 208-216
Open this publication in new window or tab >>2 '-O-methylation in mRNA disrupts tRNA decoding during translation elongation
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2018 (English)In: Nature Structural & Molecular Biology, ISSN 1545-9993, E-ISSN 1545-9985, Vol. 25, no 3, p. 208-216Article in journal (Refereed) Published
Abstract [en]

Chemical modifications of mRNA may regulate many aspects of mRNA processing and protein synthesis. Recently, 2 '-O-methylation of nucleotides was identified as a frequent modification in translated regions of human mRNA, showing enrichment in codons for certain amino acids. Here, using single-molecule, bulk kinetics and structural methods, we show that 2 '-O-methylation within coding regions of mRNA disrupts key steps in codon reading during cognate tRNA selection. Our results suggest that 2 '-O-methylation sterically perturbs interactions of ribosomal-monitoring bases (G530, A1492 and A1493) with cognate codon-anticodon helices, thereby inhibiting downstream GTP hydrolysis by elongation factor Tu (EF-Tu) and A-site tRNA accommodation, leading to excessive rejection of cognate aminoacylated tRNAs in initial selection and proofreading. Our current and prior findings highlight how chemical modifications of mRNA tune the dynamics of protein synthesis at different steps of translation elongation.

Place, publisher, year, edition, pages
NATURE PUBLISHING GROUP, 2018
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-350295 (URN)10.1038/s41594-018-0030-z (DOI)000426704000006 ()29459784 (PubMedID)
Funder
Knut and Alice Wallenberg FoundationSwedish Research Council
Available from: 2018-05-09 Created: 2018-05-09 Last updated: 2018-05-09Bibliographically approved
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
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
Reuveni, S., Ehrenberg, M. & Paulsson, J. (2017). Ribosomes are optimized for autocatalytic production. Nature, 547(7663), 293-297
Open this publication in new window or tab >>Ribosomes are optimized for autocatalytic production
2017 (English)In: Nature, ISSN 0028-0836, E-ISSN 1476-4687, Vol. 547, no 7663, p. 293-297Article in journal (Refereed) Published
Abstract [en]

Many fine-scale features of ribosomes have been explained in terms of function, revealing a molecular machine that is optimized for error-correction, speed and control. Here we demonstrate mathematically that many less well understood, larger-scale features of ribosomes-such as why a few ribosomal RNA molecules dominate the mass and why the ribosomal protein content is divided into 55-80 small, similarly sized segments-speed up their autocatalytic production.

Place, publisher, year, edition, pages
NATURE PUBLISHING GROUP, 2017
National Category
Biological Sciences
Identifiers
urn:nbn:se:uu:diva-332850 (URN)10.1038/nature22998 (DOI)000405844900023 ()28726822 (PubMedID)
Funder
Swedish Research Council
Available from: 2017-11-08 Created: 2017-11-08 Last updated: 2017-11-08Bibliographically approved
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
Fu, Z., Kaledhonkar, S., Borg, A., Sun, M., Chen, B., Grassucci, R. A., . . . Frank, J. (2016). Key Intermediates in Ribosome Recycling Visualized by Time-Resolved Cryoelectron Microscopy. Structure, 24(12), 2092-2101
Open this publication in new window or tab >>Key Intermediates in Ribosome Recycling Visualized by Time-Resolved Cryoelectron Microscopy
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2016 (English)In: Structure, ISSN 0969-2126, E-ISSN 1878-4186, Vol. 24, no 12, p. 2092-2101Article in journal (Refereed) Published
Abstract [en]

Upon encountering a stop codon on mRNA, polypeptide synthesis on the ribosome is terminated by release factors, and the ribosome complex, still bound with mRNA and P-site-bound tRNA (post-termination complex, PostTC), is split into ribosomal subunits, ready for a new round of translational initiation. Separation of post-termination ribosomes into subunits, or "ribosome recycling,'' is promoted by the joint action of ribosome-recycling factor (RRF) and elongation factor G (EF-G) in a guanosine triphosphate (GTP) hydrolysis-dependent manner. Here we used a mixing-spraying-based method of time-resolved cryo-electron microscopy (cryo-EM) to visualize the short-lived intermediates of the recycling process. The two complexes that contain (1) both RRF and EF-G bound to the PostTC or (2) deacylated tRNA bound to the 30S subunit are of particular interest. Our observations of the native form of these complexes demonstrate the strong potential of time-resolved cryo-EM for visualizing previously unobservable transient structures.

National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-316969 (URN)10.1016/j.str.2016.09.014 (DOI)000393211300009 ()27818103 (PubMedID)
Funder
NIH (National Institute of Health), R01 GM55440 GM29169Swedish Research Council, 2015-04682Knut and Alice Wallenberg Foundation
Available from: 2017-03-08 Created: 2017-03-08 Last updated: 2018-01-13Bibliographically 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
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
Choi, J., Ieong, K.-W., Demirci, H., Chen, J., Petrov, A., Prabhakar, A., . . . Puglisi, J. D. (2016). N-6-methyladenosine in mRNA disrupts tRNA selection and translation-elongation dynamics. Nature Structural & Molecular Biology, 23(2), 110-+
Open this publication in new window or tab >>N-6-methyladenosine in mRNA disrupts tRNA selection and translation-elongation dynamics
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2016 (English)In: Nature Structural & Molecular Biology, ISSN 1545-9993, E-ISSN 1545-9985, Vol. 23, no 2, p. 110-+Article in journal (Refereed) Published
Abstract [en]

N-6-methylation of adenosine (forming m(6)A) is the most abundant post-transcriptional modification within the coding region of mRNA, but its role during translation remains unknown. Here, we used bulk kinetic and single-molecule methods to probe the effect of m(6)A in mRNA decoding. Although m(6)A base-pairs with uridine during decoding, as shown by X-ray crystallographic analyses of Thermus thermophilus ribosomal complexes, our measurements in an Escherichia coli translation system revealed that m(6)A modification of mRNA acts as a barrier to tRNA accommodation and translation elongation. The interaction between an m(6)A-modified codon and cognate tRNA echoes the interaction between a near-cognate codon and tRNA, because delay in tRNA accommodation depends on the position and context of m(6)A within codons and on the accuracy level of translation. Overall, our results demonstrate that chemical modification of mRNA can change translational dynamics.

National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-277998 (URN)10.1038/nsmb.3148 (DOI)000369220800006 ()26751643 (PubMedID)
Funder
NIH (National Institute of Health), GM51266NIH (National Institute of Health), GM099687Knut and Alice Wallenberg FoundationSwedish Research CouncilNIH (National Institute of Health), GM111858
Available from: 2016-02-23 Created: 2016-02-23 Last updated: 2018-01-10Bibliographically approved
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