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  • 1. Allen, Gregory S
    et al.
    Zavialov, Andrey
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology. Molekylärbiologi.
    Gursky, Richard
    Ehrenberg, Måns
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology. Molekylärbiologi.
    Frank, Joachim
    The cryo-EM structure of a translation initiation complex from Escherichia coli.2005In: Cell, ISSN 0092-8674, Vol. 121, no 5, 703-12 p.Article in journal (Other scientific)
  • 2. Andreev, Dmitri
    et al.
    Hauryliuk, Vasili
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Terenin, Ilya
    Dmitriev, Sergey
    Ehrenberg, Måns
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Shatsky, Ivan
    The bacterial toxin ReIE induces specific mRNA cleavage in the A site of the eukaryote ribosome2008In: RNA: A publication of the RNA Society, ISSN 1355-8382, E-ISSN 1469-9001, Vol. 14, no 2, 233-239 p.Article in journal (Refereed)
    Abstract [en]

    ReIE/ReIB is a well-characterized toxin-anti-toxin pair involved in nutritional stress responses in Bacteria and Archae. ReIE lacks any eukaryote homolog, but we demonstrate here that it efficiently and specifically cleaves mRNA in the A site of the eukaryote ribosome. The cleavage mechanism is similar to that in bacteria, showing the feasibility of A-site cleavage of mRNA for regulatory purposes also in eukaryotes. ReIE cleavage in the A-site codon of a stalled eukaryote ribosome is precise and easily monitored, making "ReIE printing" a useful complement to toeprinting to determine the exact mRNA location on the eukaryote ribosome and to probe the occupancy of its A site.

  • 3.
    Antoun, Ayman
    et al.
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology. Molekylärbiologi.
    Pavlov, Michael
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology. Molekylärbiologi.
    Tenson, Tanel
    Ehrenberg, Måns
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology. Molekylärbiologi.
    Ribosome formation from subunits studied by stopped-flow and Rayleigh light scattering2004In: Biol Procedure Online, Vol. 6, 35–54- p.Article in journal (Refereed)
    Abstract [en]

    Light scattering and standard stopped-flow techniques were used to monitor rapid association of ribosomal subunits during initiation of eubacterial protein synthesis. The effects of the initiation factors IF1, IF2, IF3 and buffer conditions on subunit association were studied along with the role of GTP in this process. The part of light scattering theory that is essential for kinetic measurements is high-lighted in the main text and a more general treatment of Rayleigh scattering from macromolecules is given in an appendix.

  • 4.
    Antoun, Ayman
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Pavlov, Michael Y.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Lovmar, Martin
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Ehrenberg, Måns
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    How initiation factors maximize the accuracy of tRNA selection in initiation of bacterial protein synthesis2006In: Molecular Cell, ISSN 1097-2765, E-ISSN 1097-4164, Vol. 23, no 2, 183-193 p.Article in journal (Refereed)
    Abstract [en]

    During initiation of bacterial protein synthesis, messenger RNA and fMet-tRNA(fMet) bind to the 30S ribosomal subunit together with initiation factors IF1, IF2, and IF3. Docking of the 30S preinitiation complex to the 50S ribosomal subunit results in a peptidyl-transfer competent 70S ribosome. Initiation with an elongator tRNA may lead to frameshift and an aberrant N-terminal sequence in the nascent protein. We show how the occurrence of initiation errors is minimized by (1) recognition of the formyl group by the synergistic action of IF2 and IF1, (2) uniform destabilization of the binding of all tRNAs to the 30S subunit by IF3, and (3) an optimal distance between the Shine-Dalgarno sequence and the initiator codon. We suggest why IF1 is essential for E. coli, discuss the role of the G-C base pairs in the anticodon stem of some tRNAs, and clarify gene expression changes with varying IF3 concentration in the living cell.

  • 5.
    Antoun, Ayman
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Pavlov, Michael Y
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Lovmar, Martin
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Ehrenberg, Måns
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    How initiation factors tune the rate of initiation of protein synthesis in bacteria.2006In: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, Vol. 25, no 11, 2539-50 p.Article in journal (Refereed)
    Abstract [en]

    The kinetics of initiator transfer RNA ( tRNA) interaction with the messenger RNA ( mRNA)-programmed 30S subunit and the rate of 50S subunit docking to the 30S preinitiation complex were measured for different combinations of initiation factors in a cell-free Escherichia coli system for protein synthesis with components of high purity. The major results are summarized by a Michaelis-Menten scheme for initiation. All three initiation factors are required for maximal efficiency ( k(cat)/K-M) of initiation and for maximal in vivo rate of initiation at normal concentration of initiator tRNA. Spontaneous release of IF3 from the 30S preinitiation complex is required for subunit docking. The presence of initiator tRNA on the 30S subunit greatly increases the rate of 70S ribosome formation by increasing the rate of IF3 dissociation from the 30S subunit and the rate of 50S subunit docking to the IF3-free 30S preinitiation complex. The reasons why IF1 and IF3 are essential in E. coli are discussed in the light of the present observations.

  • 6.
    Borg, Anneli
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Ehrenberg, Måns
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Determinants of the Rate of mRNA Translocation in Bacterial Protein Synthesis2015In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 427, no 9, 1835-1847 p.Article in journal (Refereed)
    Abstract [en]

    Studying the kinetics of translocation of mRNA and tRNAs on the translating ribosome is technically difficult since the rate-limiting steps involve large conformational changes without covalent bond formation or disruption. Here, we have developed a unique assay system for precise estimation of the full translocation cycle time at any position in any type of open reading frame (ORF). Using a buffer system optimized for high accuracy of tRNA selection together with high concentration of elongation factor G, we obtained in vivo compatible translocation rates. We found that translocation was comparatively slow early in the ORF and faster further downstream of the initiation codon. The maximal translocation rate decreased from the in vivo compatible value of 30 s(-1) at 1 mM free Mg2+ concentration to the detrimentally low value of 1 s(-1) at 6 mM free Mg2+ concentration. Thus, high and in vivo compatible accuracy of codon translation, as well as high and in vivo compatible translocation rate, required a remarkably low Mg2+ concentration. Finally, we found that the rate of translocation deep inside an ORF was not significantly affected upon variation of the standard free energy of interaction between a 6-nt upstream Shine-Dalgarno (SD)-like sequence and the anti-SD sequence of 16S rRNA in a range of 0-6 kcal/mol. Based on these experiments, we discuss the optimal choice of Mg2+ concentration for maximal fitness of the living cell by taking its effects on the accuracy of translation, the peptide bond formation rate and the translocation rate into account. (C) 2014 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).

  • 7.
    Borg, Anneli
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Holm, Mikael
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Shiroyama, Ikue
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Hauryliuk, Vasili
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Pavlov, Michael
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Sanyal, Suparna
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Ehrenberg, Måns
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Fusidic Acid Targets Elongation Factor G in Several Stages of Translocation on the Bacterial Ribosome2015In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 290, no 6, 3440-3454 p.Article in journal (Refereed)
    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.

  • 8.
    Borg, Anneli
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Pavlov, Michael
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Ehrenberg, Måns
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Complete kinetic mechanism for recycling of the bacterial ribosome2016In: RNA: A publication of the RNA Society, ISSN 1355-8382, E-ISSN 1469-9001, Vol. 22, no 1, 10-21 p.Article in journal (Refereed)
    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.

  • 9.
    Borg, Anneli
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Pavlov, Michael
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Ehrenberg, Måns
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Mechanism of fusidic acid inhibition of RRF- and EF-G-dependent splitting of the bacterial post-termination ribosome2016In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 44, no 7, 3264-3275 p.Article in journal (Refereed)
    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.

  • 10.
    Choi, Junhong
    et al.
    Stanford Univ, Sch Med, Dept Biol Struct, Stanford, CA 94305 USA.;Stanford Univ, Dept Appl Phys, Stanford, CA 94305 USA..
    Ieong, Ka-Weng
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Demirci, Hasan
    SLAC Natl Accelerator Lab, Stanford PULSE Inst, Menlo Pk, CA USA.;SLAC Natl Accelerator Lab, Stanford Synchrotron Radiat Lightsource, Menlo Pk, CA USA..
    Chen, Jin
    Stanford Univ, Sch Med, Dept Biol Struct, Stanford, CA 94305 USA.;Stanford Univ, Dept Appl Phys, Stanford, CA 94305 USA..
    Petrov, Alexey
    Stanford Univ, Sch Med, Dept Biol Struct, Stanford, CA 94305 USA..
    Prabhakar, Arjun
    Stanford Univ, Sch Med, Dept Biol Struct, Stanford, CA 94305 USA.;Stanford Univ, Program Biophys, Stanford, CA 94305 USA..
    O'Leary, Sean E.
    Stanford Univ, Sch Med, Dept Biol Struct, Stanford, CA 94305 USA..
    Dominissini, Dan
    Chaim Sheba Med Ctr, Canc Res Ctr, IL-52621 Tel Hashomer, Israel.;Univ Chicago, Dept Chem, 5735 S Ellis Ave, Chicago, IL 60637 USA..
    Rechavi, Gideon
    Chaim Sheba Med Ctr, Canc Res Ctr, IL-52621 Tel Hashomer, Israel.;Tel Aviv Univ, Israel & Sackler Sch Med, IL-69978 Tel Aviv, Israel..
    Soltis, S. Michael
    SLAC Natl Accelerator Lab, Stanford Synchrotron Radiat Lightsource, Menlo Pk, CA USA..
    Ehrenberg, Måns
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Puglisi, Joseph D.
    Stanford Univ, Sch Med, Dept Biol Struct, Stanford, CA 94305 USA..
    N-6-methyladenosine in mRNA disrupts tRNA selection and translation-elongation dynamics2016In: Nature Structural & Molecular Biology, ISSN 1545-9993, E-ISSN 1545-9985, Vol. 23, no 2, 110-+ p.Article in journal (Refereed)
    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.

  • 11. Dennis, Patrick P
    et al.
    Ehrenberg, Måns
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology. Molekylärbiologi.
    Bremer, Hans
    Control of rRNA synthesis in Escherichia coli: a systems biology approach.2004In: Microbiol Mol Biol Rev, ISSN 1092-2172, Vol. 68, no 4, 639-68 p.Article, review/survey (Other (popular scientific, debate etc.))
  • 12. Dittmar, Kimberly A
    et al.
    Sörensen, Michael A
    Elf, Johan
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology. Molekylärbiologi.
    Ehrenberg, Måns
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology. Molekylärbiologi.
    Pan, Tao
    Selective charging of tRNA isoacceptors induced by amino-acid starvation.2005In: EMBO Rep, ISSN 1469-221X, Vol. 6, no 2, 151-7 p.Article in journal (Other scientific)
  • 13.
    Ehrenberg, M
    et al.
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology. MOLEKYLÄRBIOLOGI.
    Tenson, T
    A new beginning of the end of translation2002In: journal, Vol. 9, 85-87 p.Article in journal (Refereed)
  • 14.
    Ehrenberg, Måns
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Bremer, Hans
    Dennis, Patrick P.
    Medium-dependent control of the bacterial growth rate2013In: Biochimie, ISSN 0300-9084, E-ISSN 1638-6183, Vol. 95, no 4, 643-658 p.Article, review/survey (Refereed)
    Abstract [en]

    By combining results from previous studies of nutritional up-shifts we here re-investigate how bacteria adapt to different nutritional environments by adjusting their macromolecular composition for optimal growth. We demonstrate that, in contrast to a commonly held view the macromolecular composition of bacteria does not depend on the growth rate as an independent variable, but on three factors: (i) the genetic background (i.e. the strain used), (ii) the physiological history of the bacteria used for inoculation of a given growth medium, and (iii) the kind of nutrients in the growth medium. These factors determine the ribosome concentration and the average rate of protein synthesis per ribosome, and thus the growth rate. Immediately after a nutritional up-shift, the average number of ribosomes in the bacterial population increases exponentially with time at a rate which eventually is attained as the final post-shift growth rate of all cell components. After a nutritional up-shift from one minimal medium to another minimal medium of higher nutritional quality, ribosome and RNA polymerase syntheses are co-regulated and immediately increase by the same factor equal to the increase in the final growth rate. However, after an up-shift from a minimal medium to a medium containing all 20 amino acids, RNA polymerase and ribosome syntheses are no longer coregulated; a smaller rate of synthesis of RNA polymerase is compensated by a gradual increase in the fraction of free RNA polymerase, possibly due to a gradual saturation of mRNA promoters. We have also analyzed data from a recent publication, in which it was concluded that the macromolecular composition in terms of RNA/protein and RNA/DNA ratios is solely determined by the effector molecule ppGpp. Our analysis indicates that this is true only in special cases and that, in general, medium adaptation also depends on factors other than ppGpp.

  • 15.
    Ehrenberg, Måns
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Hauryliuk, Vasili
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Crist, Colin G
    Nakamura, Yoshikazu
    Translation Termination, the Prion [Psi+], and Ribosomal Recycling2007In: Translational Control in Biology and Medicine / [ed] Michael B. Mathews, Nahum Sonenberg & John W.B. Hershey, Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press , 2007, 173-196 p.Chapter in book (Other academic)
  • 16.
    Ehrenberg, Måns
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Pavlov, Michael Yu
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Optimal Strategy for Rapid Proteome Re-Arrangements in Bacterial Populations2013In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 104, no 2, 205A-206A p.Article in journal (Other academic)
  • 17.
    Elf, Johan
    et al.
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology. Molekylärbiologi.
    Ehrenberg, Måns
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology. Molekylärbiologi.
    Near-critical behavior of aminoacyl-tRNA pools in E. coli at rate-limiting supply of amino acids.2005In: Biophys J, ISSN 0006-3495, Vol. 88, no 1, 132-46 p.Article in journal (Other scientific)
  • 18.
    Elf, Johan
    et al.
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology. Molekylärbiologi.
    Ehrenberg, Måns
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology. Molekylärbiologi.
    Spontaneous separation of bi-stable biochemical systems into spatial domains of opposite phases2004In: Systems Biology, ISSN 1741-2471, Vol. 1, no 2, 230-236 p.Article in journal (Refereed)
  • 19.
    Elf, Johan
    et al.
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology. Molekylärbiologi.
    Ehrenberg, Måns
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology. Molekylärbiologi.
    What makes ribosome-mediated transcriptional attenuation sensitive to amino2005In: PLoS Comput Biol, ISSN 1553-734X, Vol. 1, no 1, e2- p.Article in journal (Refereed)
  • 20.
    Elf, Johan
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Paulsson, Johan
    Berg, Otto
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Evolution, Genomics and Systematics, Molecular Evolution.
    Ehrenberg, Måns
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Mesoscopic kinetics and its applications in protein synthesis2005In: Systems Biology: Definitions and Perspectives / [ed] Lila Alberghina and H.V. Westerhoff, Heidelberg: Springer-Verlag GmbH , 2005, 95-118 p.Chapter in book (Other academic)
    Abstract [en]

    Molecular biology emerged through unification of genetics and nucleic acid chemistry that took place with the discovery of the double helix (Watson and Crick 1953). Accordingly, molecular biology could be defined as the sum of all techniques used to perform genetic experiments by manipulating DNA. One consequence of the development of these techniques is large-scale sequencing of genomes from an ever increasing number of organisms. However, it became clear from this development that genetic information per se is not enough to grasp the most interesting functional and evolutionary aspects of cells and multi-cellular organisms. In fact, understanding how genotype leads to phenotype depends on concepts and techniques from areas that so far have been largely alien to molecular biological research, like physics, mathematics, and engineering. From the bits and pieces from these and other scientific fields new tools must be generated to make possible an understanding of the dynamic, adapting, and developing living systems that somehow take shape from the instructions given by their genomes. The growing total of these tools and their integration in experimental and theoretical approaches to understand complex biological processes in ways previously out of reach could be a way to define systems biology, in analogy with the above definition of molecular biology.

  • 21.
    Elf, Johan
    et al.
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology. Daniel.
    Tenson, Tanel
    Ehrenberg, Måns
    Selective loss of charging of tRNA isoacceptors during amino acid limitation.2003In: Science, Vol. 300, 1718-1722 p.Article in journal (Refereed)
  • 22.
    Fange, David
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Mellenius, Harriet
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Dennis, Patrick P.
    Ehrenberg, Måns
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Thermodynamic Modeling of Variations in the Rate of RNA Chain Elongation of E-coli rrn Operons2014In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 106, no 1, 55-64 p.Article in journal (Refereed)
    Abstract [en]

    Previous electron-microscopic imaging has shown high RNA polymerase occupation densities in the 16S and 23S encoding regions and low occupation densities in the noncoding leader, spacer, and trailer regions of the rRNA (rrn) operons in E. coli. This indicates slower transcript elongation within the coding regions and faster elongation within the noncoding regions of the operon. Inactivation of four of the seven rrn operons increases the transcript initiation frequency at the promoters of the three intact operons and reduces the time for RNA polymerase to traverse the operon. We have used the DNA sequence-dependent standard free energy variation of the transcription complex to model the experimentally observed changes in the elongation rate along the rrnB operon. We also model the stimulation of the average transcription rate over the whole operon by increasing rate of transcript initiation. Monte Carlo simulations, taking into account initiation of transcription, translocation, and backward and forward tracking of RNA polymerase, partially reproduce the observed transcript elongation rate variations along the rrn operon and fully account for the increased average rate in response to increased frequency of transcript initiation.

  • 23. Frank, Joachim
    et al.
    Sengupta, Jayati
    Gao, Haixiao
    Li, Wen
    Valle, Mikel
    Zavialov, Andrey
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology. Molekylärbiologi.
    Ehrenberg, Måns
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology. Molekylärbiologi.
    The role of tRNA as a molecular spring in decoding, accommodation, and peptidyl transfer.2005In: FEBS Lett, ISSN 0014-5793, Vol. 579, no 4, 959-62 p.Article, review/survey (Other (popular scientific, debate etc.))
  • 24.
    Fu, Ziao
    et al.
    Columbia Univ Coll Phys & Surg, Integrated Program Cellular Mol & Biomed Studies, 630 W 168th St, New York, NY 10032 USA..
    Kaledhonkar, Sandip
    Columbia Univ, Dept Biochem & Mol Biophys, 630 W 168th St, New York, NY 10027 USA..
    Borg, Anneli
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Sun, Ming
    Columbia Univ, Dept Biol Sci, New York, NY 10027 USA..
    Chen, Bo
    Columbia Univ, Dept Biol Sci, New York, NY 10027 USA..
    Grassucci, Robert A.
    Columbia Univ, Dept Biochem & Mol Biophys, 630 W 168th St, New York, NY 10027 USA.;Columbia Univ, Howard Hughes Med Inst, New York, NY 10032 USA..
    Ehrenberg, Måns
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Frank, Joachim
    Columbia Univ, Dept Biochem & Mol Biophys, 630 W 168th St, New York, NY 10027 USA.;Columbia Univ, Dept Biol Sci, New York, NY 10027 USA.;Columbia Univ, Howard Hughes Med Inst, New York, NY 10032 USA..
    Key Intermediates in Ribosome Recycling Visualized by Time-Resolved Cryoelectron Microscopy2016In: Structure, ISSN 0969-2126, E-ISSN 1878-4186, Vol. 24, no 12, 2092-2101 p.Article in journal (Refereed)
    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.

  • 25. Gao, Haixiao
    et al.
    Valle, Mikel
    Ehrenberg, Måns
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology. Molekylärbiologi.
    Frank, Joachim
    Dynamics of EF-G interaction with the ribosome explored by classification of a heterogeneous cryo-EM dataset.2004In: J Struct Biol, ISSN 1047-8477, Vol. 147, no 3, 283-90 p.Article in journal (Other scientific)
  • 26. Gao, Haixiao
    et al.
    Zhou, Zhihong
    Rawat, Urmila
    Huang, Chenhui
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Bouakaz, Lamine
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Wang, Chernhoe
    Cheng, Zhihong
    Liu, Yuying
    Zavialov, Andrey
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Gursky, Richard
    Sanyal, Suparna
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Ehrenberg, Måns
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Frank, Joachim
    Song, Haiwei
    RF3 induces ribosomal conformational changes responsible for dissociation of class I release factors2007In: Cell, ISSN 0092-8674, E-ISSN 1097-4172, Vol. 129, no 5, 929-941 p.Article in journal (Refereed)
    Abstract [en]

    During translation termination, class II release factor RF3 binds to the ribosome to promote rapid dissociation of a class I release factor (RF) in a GTP-dependent manner. We present the crystal structure of E. coli RF3•GDP, which has a three-domain architecture strikingly similar to the structure of EF-Tu•GTP. Biochemical data on RF3 mutants show that a surface region involving domains II and III is important for distinct steps in the action cycle of RF3. Furthermore, we present a cryo-electron microscopy (cryo-EM) structure of the posttermination ribosome bound with RF3 in the GTP form. Our data show that RF3•GTP binding induces large conformational changes in the ribosome, which break the interactions of the class I RF with both the decoding center and the GTPase-associated center of the ribosome, apparently leading to the release of the class I RF.

  • 27. Gao, Ning
    et al.
    Zavialov, Andrey V
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology. Molekylärbiologi.
    Li, Wen
    Sengupta, Jayati
    Valle, Mikel
    Gursky, Richard P
    Ehrenberg, Måns
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology. Molekylärbiologi.
    Frank, Joachim
    Mechanism for the disassembly of the posttermination complex inferred from cryo-EM studies.2005In: Mol Cell, ISSN 1097-2765, Vol. 18, no 6, 663-74 p.Article in journal (Refereed)
  • 28. Hallier, Marc
    et al.
    Ivanova, Natalia
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology. Molekylärbiologi.
    Rametti, Armelle
    Pavlov, Michael
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology. Molekylärbiologi.
    Ehrenberg, Måns
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology. Molekylärbiologi.
    Felden, Brice
    Pre-binding of small protein B to a stalled ribosome triggers trans-translation.2004In: J Biol Chem, ISSN 0021-9258, Vol. 279, no 25, 25978-85 p.Article in journal (Other scientific)
  • 29.
    Hauryliuk, Vasili
    et al.
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology. Molekylärbiologi.
    Ehrenberg, Måns
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology. Molekylärbiologi.
    Two-step selection of mRNAs in initiation of protein synthesis.2006In: Mol Cell, ISSN 1097-2765, Vol. 22, no 2, 155-6 p.Article in journal (Refereed)
  • 30.
    Hauryliuk, Vasili
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Zavialov, Andrey
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Kisselev, Lev
    Ehrenberg, Måns
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Class-1 release factor eRF1 promotes GTP binding by class-2 release factor eRF32006In: Biochimie, ISSN 0300-9084, E-ISSN 1638-6183, Vol. 88, no 7, 747-757 p.Article in journal (Refereed)
    Abstract [en]

    In eukaryotes, termination of mRNA translation is triggered by the essential polypeptide chain release factors eRF1, recognizing all three stop codons, and eRF3, a member of the GTPase superfamily with a role that has remained opaque. We have studied the kinetic and thermodynamic parameters of the interactions between eRF3 and GTP, GDP and the non-hydrolysable GTP analogue GDPNP in the presence (K-D(GDP) = 1.3 +/- 0.2 mu M, K-D(GTP) approximate to 200 mu M and K-D(GDPNP) > 160 mu M) as well as absence (K-D(GDP) = 1.9 +/- 0.3 mu M, K-D(GTP) 0.7 +/- 0.2 mu M and K-D(GDPNP) approximate to 200 mu M) of eRF1. From the present data we propose that (i) free eRF3 has a strong preference to bind GDP compared to GTP (ii) eRF3 in complex with eRF1 has much stronger affinity to GTP than free eRF3 (iii) eRF3 in complex with PABP has weak affinity to GTP (iv) eRF3 in complex with eRF1 does not have strong affinity to GDPNP, implying that GDPNP is a poor analogue of GTP for eRF3 binding.

  • 31. Hennelly, Scott
    et al.
    Antoun, Ayman
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology. Molekylärbiologi.
    Ehrenberg, Måns
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology. Molekylärbiologi.
    Gualerzi, Claudio
    Knight, William
    Lodmell, Stephen
    Hill, Walter
    A Time-resolved Investigation of Ribosomal Subunit Association2005In: Journal of Molecular Biology, Vol. 346, no 5, 1243-1258 p.Article in journal (Refereed)
    Abstract [en]

    The notion that the ribosome is dynamic has been supported by various biochemical techniques, as well as by differences observed in high-resolution structures of ribosomal complexes frozen in various functional states. Yet, the mechanisms and extent of rRNA dynamics are still largely unknown. We have used a novel, fast chemical-modification technique to provide time-resolved details of 16 S rRNA structural changes that occur as bridges are formed between the ribosomal subunits as they associate. Association of different 16 S rRNA regions was found to be a sequential, multi-step process involving conformational rearrangements within the 30 S subunit. Our results suggest that key regions of 16 S rRNA, necessary for decoding and tRNA A-site binding, are structurally altered in a time-dependent manner by association with the 50 S ribosomal subunits.

  • 32.
    Holm, Mikael
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Borg, Anneli
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Ehrenberg, Måns
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Sanyal, Suparna
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Molecular mechanism of viomycin inhibition of peptide elongation in bacteria2016In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 113, no 4, 978-983 p.Article in journal (Refereed)
    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.

  • 33.
    Ieong, Ka-Weng
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Pavlov, Michael Y.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Kwiatkowski, Marek
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Ehrenberg, Måns
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Forster, Anthony C.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    A tRNA body with high affinity for EF-Tu hastens ribosomal incorporation of unnatural amino acids2014In: RNA: A publication of the RNA Society, ISSN 1355-8382, E-ISSN 1469-9001, Vol. 20, no 5, 632-643 p.Article in journal (Refereed)
  • 34.
    Ieong, Ka-Weng
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Pavlov, Michael Y.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Kwiatkowski, Marek
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Forster, Anthony C.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Ehrenberg, Måns
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Inefficient delivery but fast peptide bond formation of unnatural l -aminoacyl-tRNAs in translation2012In: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 134, no 43, 17955-17962 p.Article in journal (Refereed)
    Abstract [en]

    Translations with unnatural amino acids (AAs) are generally inefficient, and kinetic studies of their incorporations from transfer ribonucleic acids (tRNAs) are few. Here, the incorporations of small and large, non-N-alkylated, unnatural l-AAs into dipeptides were compared with those of natural AAs using quench-flow techniques. Surprisingly, all incorporations occurred in two phases: fast then slow, and the incorporations of unnatural AA-tRNAs proceeded with rates of fast and slow phases similar to those for natural Phe-tRNA Phe. The slow phases were much more pronounced with unnatural AA-tRNAs, correlating with their known inefficient incorporations. Importantly, even for unnatural AA-tRNAs the fast phases could be made dominant by using high EF-Tu concentrations and/or lower reaction temperature, which may be generally useful for improving incorporations. Also, our observed effects of EF-Tu concentration on the fraction of the fast phase of incorporation enabled direct assay of the affinities of the AA-tRNAs for EF-Tu during translation. Our unmodified tRNA Phe derivative adaptor charged with a large unnatural AA, biotinyl-lysine, had a very low affinity for EF-Tu:GTP, while the small unnatural AAs on the same tRNA body had essentially the same affinities to EF-Tu:GTP as natural AAs on this tRNA, but still 2-fold less than natural Phe-tRNA Phe. We conclude that the inefficiencies of unnatural AA-tRNA incorporations were caused by inefficient delivery to the ribosome by EF-Tu, not slow peptide bond formation on the ribosome.

  • 35.
    Ieong, Ka-Weng
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Uzun, Ülkü
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Selmer, Maria
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Ehrenberg, Måns
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Two proofreading steps amplify the accuracy of genetic code translation2016In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 13, no 48, 13744-13749 p.Article in journal (Refereed)
    Abstract [en]

    Aminoacyl-tRNAs (aa-tRNAs) are selected by the messenger RNA programmed ribosome in ternary complex with elongation factor Tu (EF-Tu) and GTP and then, again, in a proofreading step after GTP hydrolysis on EF-Tu. We use tRNA mutants with different affinities for EF-Tu to demonstrate that proofreading of aatRNAs occurs in two consecutive steps. First, aa-tRNAs in ternary complex with EF-Tu·GDP are selected in a step where the accuracy increases linearly with increasing aa-tRNA affinity to EF-Tu. Then, following dissociation of EF-Tu·GDP from the ribosome, the accuracy is further increased in a second and apparently EFTu−independent step. Our findings identify the molecular basis of proofreading in bacteria, highlight the pivotal role of EF-Tu for fast and accurate protein synthesis, and illustrate the importance of multistep substrate selection in intracellular processing of genetic information.

  • 36.
    Indrisiunaite, Gabriele
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Pavlov, Michael Y.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Heurgue-Hamard, Valerie
    Ehrenberg, Måns
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    On the pH Dependence of Class-1 RF-Dependent Termination of mRNA Translation2015In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 427, no 9, 1848-1860 p.Article in journal (Refereed)
    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.

  • 37.
    Ivanova, Natalia
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Lindell, Magnus
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Pavlov, Michael
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Holmberg Schiavone, Lovisa
    Wagner, Gerhart E. H.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Ehrenberg, Måns
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Structure probing of tmRNA in distinct stages of trans-translation2007In: RNA: A publication of the RNA Society, ISSN 1355-8382, E-ISSN 1469-9001, Vol. 13, no 5, 713-722 p.Article in journal (Refereed)
    Abstract [en]

    Ribosomes stalled on problematic mRNAs in bacterial cells can be rescued by transfer-messenger RNA (tmRNA), its helperprotein (small protein B, SmpB), and elongation factor Tu (EF-Tu) through a mechanism called trans-translation. In this work weused lead(II) footprinting to probe the interactions of tmRNA with SmpB and other components of the translation machinery atdifferent steps of the trans-translation cycle. Ribosomes with a short nascent peptide stalled on a truncated mRNA were reactedwith Ala-tmRNA EF-Tu GTP, SmpB, and other translation components to initiate and execute trans-translation. Free tmRNA was                  d      dprobed with lead(II) acetate with and without SmpB, and ribosome bound tmRNA was probed in one of four different trans-translation states stabilized by antibiotic addition or selective exclusion of translation components. For comparison, we alsoanalyzed lead(II) cleavage patterns of tmRNA in vivo in a wild-type as well as in an SmpB-deficient Escherichia coli strain. Weobserved some specific cleavages/protections in tmRNA for the individual steps of trans-translation, but the overall tmRNAconformation appeared to be similar in the stages analyzed. Our findings suggest that, in vivo, a dominant fraction of tmRNA isin complex with SmpB and that, in vitro, SmpB remains tmRNA bound at the initial steps of trans-translation.

  • 38.
    Ivanova, Natalia
    et al.
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology. Molekylärbiologi.
    Pavlov, Michael Y
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology. Molekylärbiologi.
    Bouakaz, Elli
    Molekylärbiologi.
    Ehrenberg, Måns
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology. Molekylärbiologi.
    Schiavone, Lovisa Holmberg
    Mapping the interaction of SmpB with ribosomes by footprinting of ribosomal RNA.2005In: Nucleic Acids Res, ISSN 1362-4962, Vol. 33, no 11, 3529-39 p.Article in journal (Refereed)
  • 39.
    Ivanova, Natalia
    et al.
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology. Molekylärbiologi.
    Pavlov, Michael Y
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology. Molekylärbiologi.
    Ehrenberg, Måns
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology. Molekylärbiologi.
    tmRNA-induced release of messenger RNA from stalled ribosomes.2005In: J Mol Biol, ISSN 0022-2836, Vol. 350, no 5, 897-905 p.Article in journal (Refereed)
  • 40.
    Ivanova, Natalia
    et al.
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology. Molekylärbiologi.
    Pavlov, Michael Y
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology. Molekylärbiologi.
    Felden, Brice
    Ehrenberg, Måns
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology. Molekylärbiologi.
    Ribosome rescue by tmRNA requires truncated mRNAs.2004In: J Mol Biol, ISSN 0022-2836, Vol. 338, no 1, 33-41 p.Article in journal (Refereed)
  • 41.
    Johansson, Magnus
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Ieong, Ka-Weng
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Pavlov, Michael Y.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Ehrenberg, Måns
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Rate and accuracy of ribosomal peptidyl transfer2011In: Ribosomes: Structure, Function and Dynamics / [ed] Marina V. Rodnina, Rachel Green, Wolfgang Wintermeyer, Springer-Verlag New York, 2011, 225-235 p.Conference paper (Refereed)
  • 42.
    Johansson, Magnus
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Zhang, Jingji
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Ehrenberg, Måns
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Genetic code translation displays a linear trade-off between efficiency and accuracy of tRNA selection2012In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 109, no 1, 131-136 p.Article in journal (Refereed)
    Abstract [en]

    Rapid and accurate translation of the genetic code into protein is fundamental to life. Yet due to lack of a suitable assay, little is known about the accuracy-determining parameters and their correlation with translational speed. Here, we develop such an assay, based on Mg(2+) concentration changes, to determine maximal accuracy limits for a complete set of single-mismatch codon-anticodon interactions. We found a simple, linear trade-off between efficiency of cognate codon reading and accuracy of tRNA selection. The maximal accuracy was highest for the second codon position and lowest for the third. The results rationalize the existence of proofreading in code reading and have implications for the understanding of tRNA modifications, as well as of translation error-modulating ribosomal mutations and antibiotics. Finally, the results bridge the gap between in vivo and in vitro translation and allow us to calibrate our test tube conditions to represent the environment inside the living cell.

  • 43. Kisselev, L
    et al.
    Ehrenberg, M
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Frolova, L
    Termination of translation: interplay of mRNA, rRNAs and release factors?2003In: EMBO J., Vol. 22, 175-182 p.Article in journal (Refereed)
  • 44. Liljas, Anders
    et al.
    Ehrenberg, Måns
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Structural aspects of protein synthesis2013 (ed. 2nd)Book (Other academic)
  • 45. Lindsley, Dale
    et al.
    Bonthuis, Paul
    Gallant, Jonathan
    Tofoleanu, Teodora
    Elf, Johan
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology. Molekylärbiologi.
    Ehrenberg, Måns
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology. Molekylärbiologi.
    Ribosome bypassing at serine codons as a test of the model of selective transfer RNA charging.2005In: EMBO Rep, ISSN 1469-221X, Vol. 6, no 2, 147-50 p.Article in journal (Other scientific)
  • 46.
    Lovmar, Martin
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Ehrenberg, Måns
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Rate, accuracy and cost of ribosomes in bacterial cells2006In: Biochimie, ISSN 0300-9084, E-ISSN 1638-6183, Vol. 88, no 8, 951-961 p.Article in journal (Refereed)
    Abstract [en]

    Recent biochemical data on the rate of peptidyl-transfer and missense error levels associated with the E. coli ribosome in conjunction with direct measurements of diffusion constants for proteins in the E. coli cell have been used to discuss protein synthesis in the living E. coli cell in the perspective of a previously developed maximal fitness theory. With these improved experimental parameters, i.e. k(cat) similar to 50 s(-1) for protein elongation and k(cat)/K-M similar to 4 mu M-1 s(-1) for cognate ternary complex binding to the ribosomal A site, theory predicts the experimentally observed variations in protein elongation rate, ribosome and ternary complex concentrations with varying quality of the growth medium. The theoretically predicted average missense error level is close the error levels estimated in vitro for special isoacceptor combinations, i.e. error levels about I per million. The future prospect of extensive integration of biochemistry, cell physiology and population genetics is discussed in the light of the maximal fitness theory and other, similar, theoretical approaches.

  • 47.
    Lovmar, Martin
    et al.
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology. Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology. Molekylärbiologi.
    Nilsson, Karin
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology. Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology. Molekylärbiologi.
    Vimberg, Vladimir
    Tenson, Tanel
    Nervall, Martin
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology. Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology. Strukturbiologi.
    Ehrenberg, Måns
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology. Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology. Molekylärbiologi.
    The molecular mechanism of peptide-mediated erythromycin resistance.2006In: J Biol Chem, ISSN 0021-9258, Vol. 281, no 10, 6742-50 p.Article in journal (Refereed)
  • 48.
    Lovmar, Martin
    et al.
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology. Molekylärbiologi.
    Tenson, Tanel
    Ehrenberg, Måns
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology. Molekylärbiologi.
    Kinetics of macrolide action: the josamycin and erythromycin cases.2004In: J Biol Chem, ISSN 0021-9258, Vol. 279, no 51, 53506-15 p.Article in journal (Refereed)
  • 49.
    Mellenius, Harriet
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Ehrenberg, Måns
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    DNA Template Dependent Accuracy Variation of Nucleotide Selection in Transcription2015In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 10, no 3, e0119588Article in journal (Refereed)
    Abstract [en]

    It has been commonly assumed that the effect of erroneous transcription of DNA genes into messenger RNAs on peptide sequence errors are masked by much more frequent errors of mRNA translation to protein. We present a theoretical model of transcriptional accuracy. It uses experimentally estimated standard free energies of double-stranded DNA and RNA/DNA hybrids and predicts a DNA template dependent transcriptional accuracy variation spanning several orders of magnitude. The model also identifies high-error as well a high-accuracy transcription motifs. The source of the large accuracy span is the context dependent variation of the stacking free energy of pairs of correct and incorrect base pairs in the ever moving transcription bubble. Our model predictions have direct experimental support from recent single molecule based identifications of transcriptional errors in the C. elegans transcriptome. Our conclusions challenge the general view that amino acid substitution errors in proteins are mainly caused by translational errors. It suggests instead that transcriptional error hotspots are the dominating source of peptide sequence errors in some DNA template contexts, while mRNA translation is the major cause of protein errors in other contexts.

  • 50.
    Pallesen, Jesper
    et al.
    Columbia University.
    Hashem, Yaser
    Columbia University.
    Korkmaz, Gürkan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Koripella, Ravi Kiran
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Huang, Chenhui
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Ehrenberg, Måns
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Sanyal, Suparna Chandra
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Frank, Joachim
    Columbia University.
    Cryo-EM visualization of the ribosome in termination complex with apo-RF3 and RF12013In: eLife, ISSN 2050-084X, Vol. 2, e00411- p.Article in journal (Refereed)
    Abstract [en]

    Termination of messenger RNA translation in Bacteria and Archaea is initiated by release factors (RFs) 1 or 2 recognizing a stop codon in the ribosomal A site and releasing the peptide from the P-site transfer RNA. After release, RF-dissociation is facilitated by the G-protein RF3. Structures of ribosomal complexes with RF1 or RF2 alone or with RF3 alone-RF3 bound to a non-hydrolyzable GTP-analog-have been reported. Here, we present the cryo-EM structure of a post-termination ribosome containing both apo-RF3 and RF1. The conformation of RF3 is distinct from those of free RF3•GDP and ribosome-bound RF3•GDP(C/N)P. Furthermore, the conformation of RF1 differs from those observed in RF3-lacking ribosomal complexes. Our study provides structural keys to the mechanism of guanine nucleotide exchange on RF3 and to an L12-mediated ribosomal recruitment of RF3. In conjunction with previous observations, our data provide the foundation to structurally characterize the complete action cycle of the G-protein RF3.

12 1 - 50 of 71
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