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  • 1.
    Caban, Kelvin
    et al.
    Columbia Univ, Dept Chem, New York, NY 10027 USA..
    Pavlov, Michael
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Kaledhonkar, Sandip
    Columbia Univ, Dept Biochem & Mol Biophys, New York, NY USA..
    Fu, Ziao
    Columbia Univ, Dept Biochem & Mol Biophys, New York, NY USA..
    Frank, Joachim
    Columbia Univ, Dept Biochem & Mol Biophys, New York, NY USA.;Columbia Univ, Dept Biol Sci, New York, NY 10027 USA..
    Ehrenberg, Måns
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Gonzalez, Ruben L., Jr.
    Columbia Univ, Dept Chem, New York, NY 10027 USA..
    The Structural Basis for Initiation Factor 2 Activation during Translation Initiation2018In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 114, no 3, p. 593A-593AArticle in journal (Other academic)
  • 2.
    Choi, Junhong
    et al.
    Stanford Univ, Appl Phys, Stanford, CA 94305 USA..
    Indrisiunaite, Gabriele
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    DeMirci, Hasan
    SLAC Natl Accelerator Lab, Menlo Pk, CA USA..
    Ieong, Ka-Weng
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Wang, Jinfan
    Stanford Univ, Stanford, CA 94305 USA..
    Petrov, Alexey
    Stanford Univ, Stanford, CA 94305 USA..
    Prabhakar, Arjun
    Stanford Univ, Stanford, CA 94305 USA..
    Rechavi, Gideon
    Chaim Sheba Med Ctr, Canc Res Ctr, Tel Hashomer, Israel.;Tel Aviv Univ, Tel Aviv, Israel..
    Dominissini, Dan
    Tel Aviv Univ, Tel Aviv, Israel.;Chaim Sheba Med Ctr, Tel Hashomer, Israel..
    He, Chuan
    Univ Chicago, Chicago, IL 60637 USA..
    Ehrenberg, Måns
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Puglisi, Joseph D.
    Stanford Univ, Stanford, CA 94305 USA..
    How 2 '-O-Methylation in mRNA Disrupts tRNA Decoding during Translation Elongation2018In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 114, no 3, p. 592A-592AArticle in journal (Other academic)
  • 3.
    Choi, Junhong
    et al.
    Stanford Univ, Sch Med, Dept Biol Struct, Stanford, CA 94305 USA.;Stanford Univ, Dept Appl Phys, Stanford, CA 94305 USA..
    Indrisiunaite, Gabriele
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    DeMirci, Hasan
    SLAC Natl Accelerator Lab, Stanford PULSE Inst, Menlo Pk, CA USA.;SLAC Natl Accelerator Lab, Biosci Div, Menlo Pk, CA USA..
    Leong, Ka-Weng
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Wang, Jinfan
    Stanford Univ, Sch Med, Dept Biol Struct, Stanford, CA 94305 USA..
    Petrov, Alexey
    Stanford Univ, Sch Med, Dept Biol Struct, Stanford, CA 94305 USA.;Auburn Univ, Dept Biol Sci, Auburn, AL 36849 USA..
    Prabhakarl, Arjun
    Stanford Univ, Sch Med, Dept Biol Struct, Stanford, CA 94305 USA.;Stanford Univ, Program Biophys, Stanford, CA 94305 USA..
    Rechavi, Gideon
    Chaim Sheba Med Ctr, Canc Res Ctr, Tel Hashomer, Israel.;Chaim Sheba Med Ctr, Wohl Ctr Translat Med, Tel Hashomer, Israel.;Tel Aviv Univ, Sackler Sch Med, Tel Aviv, Israel..
    Dominissini, Dan
    Chaim Sheba Med Ctr, Canc Res Ctr, Tel Hashomer, Israel.;Chaim Sheba Med Ctr, Wohl Ctr Translat Med, Tel Hashomer, Israel.;Tel Aviv Univ, Sackler Sch Med, Tel Aviv, Israel..
    He, Chuan
    Univ Chicago, Dept Biochem & Mol Biol, Dept Chem, 920 E 58Th St, Chicago, IL 60637 USA.;Univ Chicago, Inst Biophys Dynam, Chicago, IL 60637 USA.;Univ Chicago, Howard Hughes Med Inst, 5841 S Maryland Ave, Chicago, IL 60637 USA..
    Ehrenberg, Måns
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Puglisi, Joseph D.
    Stanford Univ, Sch Med, Dept Biol Struct, Stanford, CA 94305 USA..
    2 '-O-methylation in mRNA disrupts tRNA decoding during translation elongation2018In: Nature Structural & Molecular Biology, ISSN 1545-9993, E-ISSN 1545-9985, Vol. 25, no 3, p. 208-216Article in journal (Refereed)
    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.

  • 4.
    Fislage, Marcus
    et al.
    VIB VUB Ctr Struct Biol, Brussels, Belgium;Columbia Univ, Dept Biochem & Mol Biophys, New York, NY 10027 USA;Vrije Univ Brussel, Struct Biol Brussels, Brussels, Belgium.
    Zhang, Jingji
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology. Columbia Univ, Dept Biochem & Mol Biophys, New York, NY 10027 USA.
    Brown, Zuben Patrick
    Osaka Univ, Inst Prot Res, Lab Prot Synth & Express, Osaka, Japan;Columbia Univ, Dept Biochem & Mol Biophys, New York, NY 10027 USA.
    Mandava, Chandra Sekhar
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    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
    Columbia Univ, Dept Biol Sci, New York, NY 10027 USA;Columbia Univ, Dept Biochem & Mol Biophys, New York, NY 10027 USA.
    Cryo-EM shows stages of initial codon selection on the ribosome by aa-tRNA in ternary complex with GTP and the GTPase-deficient EF-Tu(H84A)2018In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 46, no 11, p. 5861-5874Article in journal (Refereed)
    Abstract [en]

    The GTPase EF-Tu in ternary complex with GTP and aminoacyl-tRNA (aa-tRNA) promotes rapid and accurate delivery of cognate aa-tRNAs to the ribosomal A site. Here we used cryo-EM to study the molecular origins of the accuracy of ribosome-aided recognition of a cognate ternary complex and the accuracy-amplifying role of themonitoring bases A1492, A1493 and G530 of the 16S rRNA. We used the GTPase-deficient EF-Tu variant H84A with native GTP, rather than non-cleavable GTP analogues, to trap a near-cognate ternary complex in high-resolution ribosomal complexes of varying codon-recognition accuracy. We found that ribosome complexes trapped by GTPase-deficicent ternary complex due to the presence of EF-TuH84A or non-cleavable GTP analogues have very similar structures. We further discuss speed and accuracy of initial aa-tRNA selection in terms of conformational changes of aa-tRNA and stepwise activation of the monitoring bases at the decoding center of the ribosome.

  • 5.
    Ge, Xueliang
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Mandava, Chandra Sekhar
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Lind, Christoffer
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational Biology and Bioinformatics.
    Åqvist, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational Biology and Bioinformatics.
    Sanyal, Suparna
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Complementary charge-based interaction between the ribosomal-stalk protein L7/12 and IF2 is the key to rapid subunit association2018In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 115, no 18, p. 4649-4654Article in journal (Refereed)
    Abstract [en]

    The interaction between the ribosomal-stalk protein L7/12 (L12) and initiation factor 2 (IF2) is essential for rapid subunit association, but the underlying mechanism is unknown. Here, we have characterized the L12–IF2 interaction on Escherichia coli ribosomes using site-directed mutagenesis, fast kinetics, and molecular dynamics (MD) simulations. Fifteen individual point mutations were introduced into the C-terminal domain of L12 (L12-CTD) at helices 4 and 5, which constitute the common interaction site for translational GTPases. In parallel, 15 point mutations were also introduced into IF2 between the G4 and G5 motifs, which we hypothesized as the potential L12 interaction sites. The L12 and IF2 mutants were tested in ribosomal subunit association assay in a stopped-flow instrument. Those amino acids that caused defective subunit association upon substitution were identified as the molecular determinants of L12–IF2 interaction. Further, MD simulations of IF2 docked onto the L12-CTD pinpointed the exact interacting partners—all of which were positively charged on L12 and negatively charged on IF2, connected by salt bridges. Lastly, we tested two pairs of charge-reversed mutants of L12 and IF2, which significantly restored the yield and the rate of formation of the 70S initiation complex. We conclude that complementary charge-based interaction between L12-CTD and IF2 is the key for fast subunit association. Considering the homology of the G domain, similar mechanisms may apply for L12 interactions with other translational GTPases.

  • 6.
    Goronzy, I. N.
    et al.
    Stanford Univ, Dept Chem, Stanford, CA 94305 USA..
    Rawle, R. J.
    Univ Virginia, Dept Mol Physiol & Biomed Engn, Box 800886, Charlottesville, VA 22908 USA..
    Boxer, S. G.
    Stanford Univ, Dept Chem, Stanford, CA 94305 USA..
    Kasson, Peter M.
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology. Univ Virginia, Dept Mol Physiol & Biomed Engn, Box 800886, Charlottesville, VA 22908 USA..
    Cholesterol enhances influenza binding avidity by controlling nanoscale receptor clustering2018In: Chemical Science, ISSN 2041-6520, E-ISSN 2041-6539, Vol. 9, no 8, p. 2340-2347Article in journal (Refereed)
    Abstract [en]

    Influenza virus infects cells by binding to sialylated glycans on the cell surface. While the chemical structure of these glycans determines hemagglutinin-glycan binding affinity, bimolecular affinities are weak, so binding is avidity-dominated and driven by multivalent interactions. Here, we show that membrane spatial organization can control viral binding. Using single-virus fluorescence microscopy, we demonstrate that the sterol composition of the target membrane enhances viral binding avidity in a dose-dependent manner. Binding shows a cooperative dependence on concentration of receptors for influenza virus, as would be expected for a multivalent interaction. Surprisingly, the ability of sterols to promote viral binding is independent of their ability to support liquid-liquid phase separation in model systems. We develop a molecular explanation for this observation via molecular dynamics simulations, where we find that cholesterol promotes small-scale clusters of glycosphingolipid receptors. We propose a model whereby cholesterol orders the monomeric state of glycosphingolipid receptors, reducing the entropic penalty of receptor association and thus favoring multimeric complexes without phase separation. This model explains how cholesterol and other sterols control the spatial organization of membrane receptors for influenza and increase viral binding avidity. A natural consequence of this finding is that local cholesterol concentration in the plasma membrane of cells may alter the binding avidity of influenza virions. Furthermore, our results demonstrate a form of cholesterol-dependent membrane organization that does not involve lipid rafts, suggesting that cholesterol's effect on cell membrane heterogeneity is likely the interplay of several different factors.

  • 7.
    Kipper, Kalle
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Eremina, Nadja
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Marklund, Emil
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Tubasum, Sumera
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Mao, Guanzhong
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Lehmann, Laura Christina
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology.
    Elf, Johan
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology.
    Deindl, Sebastian
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Structure-guided approach to site-specific fluorophore labeling of the lac repressor LacI2018In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 13, no 6, article id e0198416Article in journal (Refereed)
    Abstract [en]

    The lactose operon repressor protein LacI has long served as a paradigm of the bacterial transcription factors. However, the mechanisms whereby LacI rapidly locates its cognate binding site on the bacterial chromosome are still elusive. Single-molecule fluorescence imaging approaches are well suited for the study of these mechanisms but rely on a functionally compatible fluorescence labeling of LacI. Particularly attractive for protein fluorescence labeling are synthetic fluorophores due to their small size and favorable photophysical characteristics. Synthetic fluorophores are often conjugated to natively occurring cysteine residues using maleimide chemistry. For a site-specific and functionally compatible labeling with maleimide fluorophores, the target protein often needs to be redesigned to remove unwanted native cysteines and to introduce cysteines at locations better suited for fluorophore attachment. Biochemical screens can then be employed to probe for the functional activity of the redesigned protein both before and after dye labeling. Here, we report a mutagenesis- based redesign of LacI to enable a functionally compatible labeling with maleimide fluorophores. To provide an easily accessible labeling site in LacI, we introduced a single cysteine residue at position 28 in the DNA-binding headpiece of LacI and replaced two native cysteines with alanines where derivatization with bulky substituents is known to compromise the protein's activity. We find that the redesigned LacI retains a robust activity in vitro and in vivo, provided that the third native cysteine at position 281 is retained in LacI. In a total internal reflection microscopy assay, we observed individual Cy3-labeled LacI molecules bound to immobilized DNA harboring the cognate O-1 operator sequence, indicating that the dye-labeled LacI is functionally active. We have thus been able to generate a functional fluorescently labeled LacI that can be used to unravel mechanistic details of LacI target search at the single molecule level.

  • 8.
    Liljeruhm, Josefine
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Funk, Saskia K.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Tietscher, Sandra
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Edlund, Anders D.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology. Uppsala Univ, iGEM Uppsala, Uppsala, Sweden.
    Jamal, Sabri
    Uppsala Univ, iGEM Uppsala, Uppsala, Sweden.
    Yuen, Pikkei
    Uppsala Univ, iGEM Uppsala, Uppsala, Sweden.
    Dyrhage, Karl
    Uppsala Univ, iGEM Uppsala, Uppsala, Sweden.
    Gynnå, Arvid H.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology. Uppsala Univ, iGEM Uppsala, Uppsala, Sweden.
    Ivermark, Katarina
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Biology Education Centre.
    Lövgren, Jessica
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Biology Education Centre.
    Törnblom, Viktor
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Biology Education Centre.
    Virtanen, Anders
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Lundin, Erik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala Univ, iGEM Uppsala, Uppsala, Sweden.
    Wistand-Yuen, Erik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Forster, Anthony C.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Engineering a palette of eukaryotic chromoproteins for bacterial synthetic biology2018In: Journal of Biological Engineering, ISSN 1754-1611, E-ISSN 1754-1611, Vol. 12, article id 8Article in journal (Refereed)
    Abstract [en]

    Background: Coral reefs are colored by eukaryotic chromoproteins (CPs) that are homologous to green fluorescent protein. CPs differ from fluorescent proteins (FPs) by intensely absorbing visible light to give strong colors in ambient light. This endows CPs with certain advantages over FPs, such as instrument-free detection uncomplicated by ultra-violet light damage or background fluorescence, efficient Forster resonance energy transfer (FRET) quenching, and photoacoustic imaging. Thus, CPs have found utility as genetic markers and in teaching, and are attractive for potential cell biosensor applications in the field. Most near-term applications of CPs require expression in a different domain of life: bacteria. However, it is unclear which of the eukaryotic CP genes might be suitable and how best to assay them.

    Results: Here, taking advantage of codon optimization programs in 12 cases, we engineered 14 CP sequences (meffRed, eforRed, asPink, spisPink, scOrange, fwYellow, amilGFP, amajLime, cjBlue, mefiBlue, aeBlue, amilCP, tsPurple and gfasPurple) into a palette of Escherichia coil BioBrick plasmids. BioBricks comply with synthetic biology's most widely used, simplified, cloning standard. Differences in color intensities, maturation times and fitness costs of expression were compared under the same conditions, and visible readout of gene expression was quantitated. A surprisingly large variation in cellular fitness costs was found, resulting in loss of color in some overnight liquid cultures of certain high-copy-plasmid-borne CPs, and cautioning the use of multiple CPs as markers in competition assays. We solved these two problems by integrating pairs of these genes into the chromosome and by engineering versions of the same CP with very different colors.

    Conclusion: Availability of 14 engineered CP genes compared in E coil, together with chromosomal mutants suitable for competition assays, should simplify and expand CP study and applications. There was no single plasmid-borne CP that combined all of the most desirable features of intense color, fast maturation and low fitness cost, so this study should help direct future engineering efforts.

  • 9.
    Mao, Guanzhong
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Srivastava, Abhishek S.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology. AstraZeneca R&D, Discovery Sci, Cambridge Sci Pk, Cambridge, England..
    Wu, Shiying
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Kosek, David
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Lindell, Magnus
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Kirsebom, Leif
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Critical domain interactions for type A RNase P RNA catalysis with and without the specificity domain2018In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 13, no 3, article id e0192873Article in journal (Refereed)
    Abstract [en]

    The natural trans-acting ribozyme RNase P RNA (RPR) is composed of two domains in which the catalytic (C-) domain mediates cleavage of various substrates. The C-domain alone, after removal of the second specificity (S-) domain, catalyzes this reaction as well, albeit with reduced efficiency. Here we provide experimental evidence indicating that efficient cleavage mediated by the Escherichia coli C-domain (Eco CP RPR) with and without the C5 protein likely depends on an interaction referred to as the "P6-mimic". Moreover, the P18 helix connects the C-and S-domains between its loop and the P8 helix in the S-domain (the P8/P18-interaction). In contrast to the "P6-mimic", the presence of P18 does not contribute to the catalytic performance by the C-domain lacking the S-domain in cleavage of an all ribo model hairpin loop substrate while deletion or disruption of the P8/P18-interaction in full-size RPR lowers the catalytic efficiency in cleavage of the same model hairpin loop substrate in keeping with previously reported data using precursor tRNAs. Consistent with that P18 is not required for cleavage mediated by the C-domain we show that the archaeal Pyrococcus furiosus RPR C-domain, which lacks the P18 helix, is catalytically active in trans without the S-domain and any protein. Our data also suggest that the S-domain has a larger impact on catalysis for E. coli RPR compared to P. furiosus RPR. Finally, we provide data indicating that the absence of the S-domain and P18, or the P8/P18-interaction in full-length RPR influences the charge distribution near the cleavage site in the RPR-substrate complex to a small but reproducible extent.

  • 10.
    Mellenius, Harriet
    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, Molecular Biology.
    Transcriptional accuracy modeling suggests two-step proofreading by RNA polymerase2017In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 45, no 20, p. 11582-11593Article in journal (Refereed)
    Abstract [en]

    We suggest a novel two-step proofreading mechanism with two sequential rounds of proofreading selection in mRNA transcription. It is based on the previous experimental observations that the proofreading RNA polymerase cleaves off transcript fragments of at least 2 nt and that transcript elongation after a nucleotide misincorporation is anomalously slow. Taking these results into account, we extend the description of the accuracy of template guided nucleotide selection beyond previous models of RNA polymerase-dependent DNA transcription. The model derives the accuracy of initial and proofreading base selection from experimentally estimated nearest-neighbor parameters. It is also used to estimate the small accuracy enhancement of polymerase revisiting of previous positions following transcript cleavage.

  • 11.
    Moriou, Céline
    et al.
    CNRS, Inst Chim Subst Nat, Gif Sur Yvette, France.
    Da Silva, Adilson D.
    Univ Fed Juiz de Fora, Dept Quim, ICE, BR-Juiz De Fora, MG, Brazil.
    Vianelli Prado, Marcos Joel
    Univ Fed Juiz de Fora, Dept Quim, ICE, BR-Juiz De Fora, MG, Brazil.
    Denhez, Clément
    Univ Reims, Inst Chim Mol Reims, CNRS, UMR 7312,UFR Pharm, 51 Rue Cognacq Jay, Reims, France; Univ Reims, Multiscale Mol Modelling Platform, UFR Sci Exactes & Nat, Reims, France.
    Plashkevych, Oleksandr
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Chattopadhyaya, Jyoti
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Guillaume, Dominique
    Univ Reims, Inst Chim Mol Reims, CNRS, UMR 7312,UFR Pharm, 51 Rue Cognacq Jay, Reims, France.
    Clivio, Pascale
    Univ Reims, Inst Chim Mol Reims, CNRS, UMR 7312, UFR Pharm, 51 Rue Cognacq Jay, Reims, France.
    C2 '-F Stereoconfiguration As a Puckering Switch for Base Stacking at the Dinucleotide Level2018In: Journal of Organic Chemistry, ISSN 0022-3263, E-ISSN 1520-6904, Vol. 83, no 4, p. 2473-2478Article in journal (Refereed)
    Abstract [en]

    Fluorine configuration at C2′ of the bis(2′-fluorothymidine) dinucleotide is demonstrated to drive intramolecular base stacking. 2′-β F-Configuration drastically reduces stacking compared to the 2′-α series. Hence, base stacking emerges as being tunable by the C2′-F stereoconfiguration through dramatic puckering variations scrutinized by NMR and natural bond orbital analysis. Accordingly, 2′-β F-isomer photoreactivity is significantly reduced compared to that of the 2′-α F-isomer.

  • 12.
    Pavlov, Michael
    et al.
    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.
    Substrate-Induced Formation of Ribosomal Decoding Center for Accurate and Rapid Genetic Code Translation2018In: Annual Review of Biophysics, ISSN 1936-122X, E-ISSN 1936-1238, p. 525-548Article, review/survey (Refereed)
    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).

  • 13.
    Togtema, Melissa
    et al.
    Thunder Bay Reg Hlth Res Inst, Probe Dev & Biomarker Explorat, Thunder Bay, ON, Canada; Lakehead Univ, Biotechnol Program, Thunder Bay, ON, Canada.
    Jackson, Robert
    Thunder Bay Reg Hlth Res Inst, Probe Dev & Biomarker Explorat, Thunder Bay, ON, Canada; Lakehead Univ, Biotechnol Program, Thunder Bay, ON, Canada.
    Grochowski, Jessica
    Thunder Bay Reg Hlth Res Inst, Probe Dev & Biomarker Explorat, Thunder Bay, ON, Canada; UHN, Michener Inst Educ, Genet Technol Program, Toronto, ON, Canada.
    Villa, Peter L.
    Thunder Bay Reg Hlth Res Inst, Probe Dev & Biomarker Explorat, Thunder Bay, ON, Canada; Lakehead Univ, Dept Biol, Thunder Bay, ON, Canada.
    Mellerup, Miranda
    Thunder Bay Reg Hlth Res Inst, Probe Dev & Biomarker Explorat, Thunder Bay, ON, Canada; Univ Ottawa, Dept Biol, Ottawa, ON, Canada.
    Chattopadhyaya, Jyoti
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Zehbe, Ingeborg
    Thunder Bay Reg Hlth Res Inst, Probe Dev & Biomarker Explorat, Thunder Bay, ON, Canada; Lakehead Univ, Dept Biol, Thunder Bay, ON, Canada.
    Synthetic siRNA targeting human papillomavirus 16 E6: a perspective on in vitro nanotherapeutic approaches2018In: Nanomedicine, ISSN 1743-5889, E-ISSN 1748-6963, Vol. 13, no 4, p. 455-474Article in journal (Other academic)
    Abstract [en]

    High-risk human papillomaviruses infect skin and mucosa, causing approximately 5% of cancers worldwide. In the search for targeted nanotherapeutic approaches, siRNAs against the viral E6 transcript have been molecules of interest but have not yet seen successful translation into the clinic. By reviewing the past approximately 15 years of in vitro literature, we identify the need for siRNA validation protocols which concurrently evaluate ranges of key treatment parameters as well as characterize downstream process restoration in a methodical, quantitative manner and demonstrate their implementation using our own data. We also reflect on the future need for more appropriate cell culture models to represent patient lesions as well as the application of personalized approaches to identify optimal treatment strategies.

  • 14.
    Volkov, Ivan
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology.
    Lindén, Martin
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology.
    Aguirre, Javier
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology.
    Ieong, Ka-Weng
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Metelev, Mikhail
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology.
    Elf, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology.
    Johansson, Magnus
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology.
    tRNA tracking for direct measurements of protein synthesis kinetics in live cells2018In: Nature Chemical Biology, ISSN 1552-4450, E-ISSN 1552-4469, Vol. 14, no 6, p. 618-626Article in journal (Refereed)
    Abstract [en]

    Our ability to directly relate results from test-tube biochemical experiments to the kinetics in living cells is very limited. Here we present experimental and analytical tools to directly study the kinetics of fast biochemical reactions in live cells. Dye-labeled molecules are electroporated into bacterial cells and tracked using super-resolved single-molecule microscopy.Trajectories are analyzed by machine-learning algorithms to directly monitor transitions between bound and free states. In particular, we measure the dwell time of tRNAs on ribosomes, and hence achieve direct measurements of translation rates inside living cells at codon resolution. We find elongation rates with tRNA(Phe) that are in perfect agreement with previous indirect estimates, and once fMet-tRNA(fMet) has bound to the 30S ribosomal subunit, initiation of translation is surprisingly fast and does not limit the overall rate of protein synthesis. The experimental and analytical tools for direct kinetics measurements in live cells have applications far beyond bacterial protein synthesis.

  • 15.
    Zhang, Jingji
    et al.
    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, Molecular Biology.
    Ehrenberg, Måns
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Accuracy of genetic code translation and its orthogonal corruption by aminoglycosides and Mg2+ ions2018In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 46, no 3, p. 1362-1374Article in journal (Refereed)
    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.

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