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  • 1.
    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, p. 110-+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.

  • 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.
    Ieong, Ka-Weng
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Rate and Accuracy of Bacterial Protein Synthesis with Natural and Unnatural Amino Acids2014Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    This thesis addresses different questions regarding the rate, efficiency, and accuracy of peptide bond formation with natural as well as unnatural amino acids: Which step is rate-limiting during peptide bond formation? How does the accuracy vary with different transfer RNAs (tRNAs) and codons and how is it relevant to the living cells? Does proofreading selection of codon reading occur in a single- or multi-step manner as theoretically suggested? How does the E. coli translation system discriminate unnatural amino acids? Based on that, how to improve the incorporation efficiencies of unnatural amino acids?

    Based on the study on pH dependence of peptide bond formation, we show that the rate of the chemistry of peptidyl transfer to aminoacyl-tRNA (AA-tRNA) Gly-tRNAGly or Pro-tRNAPro limits the rate of peptide bond formation at physiological pH 7.5, and this could possibly be true for peptidyl transfer to all natural AA-tRNAs at physiological condition.

    By studying the efficiency-accuracy trade-off for codon reading by seven AA-tRNA containing ternary complexes, we observe a large variation on the accuracy of initial codon selection and identify several error hot-spots. The maximal accuracy varied 400-fold from 200 to 84000 depending on the tRNA identity, the type and position of the mismatches.

    We also propose a proofreading mechanism that contains two irreversible steps in sequence. This could be highly relevant to the living cells in relation to maintaining both high accuracy and high efficiency in protein synthesis.

    Finally, we show that peptide bond formation with small and large non-N-alkylated L- unnatural amino acids proceed at rates similar to those with natural amino acids Phe and Ala on the ribosome. Interestingly, the large side chain of the bulky unnatural amino acid only weakens its binding for elongation factor Tu (EF-Tu) but not slows down peptidyl transfer on the ribosome. Our results also suggest that the efficiency of unnatural amino acid incorporation could be improved in general by increasing EF-Tu concentration, lowering the reaction temperature and / or using tRNA bodies with optimal affinities for EF-Tu in the translation system.

    List of papers
    1. pH-sensitivity of the ribosomal peptidyl transfer reaction dependent on the identity of the A-site aminoacyl-tRNA
    Open this publication in new window or tab >>pH-sensitivity of the ribosomal peptidyl transfer reaction dependent on the identity of the A-site aminoacyl-tRNA
    Show others...
    2011 (English)In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 108, no 1, p. 79-84Article in journal (Refereed) Published
    Abstract [en]

    We studied the pH-dependence of ribosome catalyzed peptidyl transfer from fMet-tRNA(fMet) to the aa-tRNAs Phe-tRNA(Phe), Ala-tRNA(Ala), Gly-tRNA(Gly), Pro-tRNA(Pro), Asn-tRNA(Asn), and Ile-tRNA(Ile), selected to cover a large range of intrinsic pK(a)-values for the α-amino group of their amino acids. The peptidyl transfer rates were different at pH 7.5 and displayed different pH-dependence, quantified as the pH-value, pK(a)(obs), at which the rate was half maximal. The pK(a)(obs)-values were downshifted relative to the intrinsic pK(a)-value of aa-tRNAs in bulk solution. Gly-tRNA(Gly) had the smallest downshift, while Ile-tRNA(Ile) and Ala-tRNA(Ala) had the largest downshifts. These downshifts correlate strongly with molecular dynamics (MD) estimates of the downshifts in pK(a)-values of these aa-tRNAs upon A-site binding. Our data show the chemistry of peptide bond formation to be rate limiting for peptidyl transfer at pH 7.5 in the Gly and Pro cases and indicate rate limiting chemistry for all six aa-tRNAs.

    Keywords
    Chromogranins, Granin-derived peptides, Granins, Immunohistochemistry, Neuroendocrine differentiation, Neuroendocrine tumours, Prohormone convertases, Secretogranins
    National Category
    Biological Sciences
    Identifiers
    urn:nbn:se:uu:diva-145815 (URN)10.1073/pnas.1012612107 (DOI)000285915000019 ()21169502 (PubMedID)
    Available from: 2011-02-11 Created: 2011-02-11 Last updated: 2017-12-11Bibliographically approved
    2. Large accuracy variation in initial codon selection by aminoacyl-tRNAs on the bacterial ribosome
    Open this publication in new window or tab >>Large accuracy variation in initial codon selection by aminoacyl-tRNAs on the bacterial ribosome
    (English)Manuscript (preprint) (Other academic)
    National Category
    Biochemistry and Molecular Biology
    Identifiers
    urn:nbn:se:uu:diva-235533 (URN)
    Available from: 2014-11-05 Created: 2014-11-05 Last updated: 2015-02-03
    3. Two proofreading steps amplify the accuracy of genetic code translation
    Open this publication in new window or tab >>Two proofreading steps amplify the accuracy of genetic code translation
    2016 (English)In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 13, no 48, p. 13744-13749Article in journal (Refereed) Published
    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.

    National Category
    Biochemistry and Molecular Biology
    Identifiers
    urn:nbn:se:uu:diva-235532 (URN)10.1073/pnas.1610917113 (DOI)000388835700066 ()27837019 (PubMedID)
    Available from: 2014-11-05 Created: 2014-11-05 Last updated: 2017-12-05Bibliographically approved
    4. Inefficient delivery but fast peptide bond formation of unnatural l -aminoacyl-tRNAs in translation
    Open this publication in new window or tab >>Inefficient delivery but fast peptide bond formation of unnatural l -aminoacyl-tRNAs in translation
    Show others...
    2012 (English)In: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 134, no 43, p. 17955-17962Article in journal (Refereed) Published
    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.

    National Category
    Natural Sciences
    Identifiers
    urn:nbn:se:uu:diva-186030 (URN)10.1021/ja3063524 (DOI)000310483500024 ()
    Available from: 2012-11-28 Created: 2012-11-27 Last updated: 2017-12-07Bibliographically approved
    5. A tRNA body with high affinity for EF-Tu hastens ribosomal incorporation of unnatural amino acids
    Open this publication in new window or tab >>A tRNA body with high affinity for EF-Tu hastens ribosomal incorporation of unnatural amino acids
    Show others...
    2014 (English)In: RNA: A publication of the RNA Society, ISSN 1355-8382, E-ISSN 1469-9001, Vol. 20, no 5, p. 632-643Article in journal (Refereed) Published
    National Category
    Natural Sciences
    Identifiers
    urn:nbn:se:uu:diva-218734 (URN)10.1261/rna.042234.113 (DOI)000334677800005 ()
    Available from: 2014-02-16 Created: 2014-02-16 Last updated: 2017-12-06Bibliographically approved
  • 4.
    Ieong, Ka-Weng
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Ribosomal Peptidyl Transfer with Natural and Unnatural Amino Acids2012Licentiate thesis, comprehensive summary (Other academic)
  • 5.
    Ieong, Ka-Weng
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology. Institute for Medical Engineering & Science, Department of Biological Engineering, and Synthetic Biology Center, Massachusetts Institute of Technology, Cambridge, MA, USA. Infectious Disease and Microbiome Program, Broad Institute, Cambridge, MA, USA.
    Indrisiunaite, Gabriele
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Ehrenberg, Måns
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    N6-methyladenosines in mRNA have profound effects on the accuracy of codon reading by tRNAs and peptide release factorsManuscript (preprint) (Other academic)
  • 6.
    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, p. 632-643Article in journal (Refereed)
  • 7.
    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, p. 17955-17962Article 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.

  • 8.
    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, p. 13744-13749Article 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.

  • 9.
    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, p. 225-235Conference paper (Refereed)
  • 10.
    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.

  • 11.
    Zhang, Jingji
    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.
    Johansson, Magnus
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Ehrenberg, Måns
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Accuracy of initial codon selection by aminoacyl-tRNAs on the mRNA-programmed bacterial ribosome2015In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 112, no 31, p. 9602-9607Article in journal (Refereed)
    Abstract [en]

    We used a cell-free system with pure Escherichia coli components to study initial codon selection of aminoacyl-tRNAs in ternary complex with elongation factor Tu and GTP on messenger RNA-programmed ribosomes. We took advantage of the universal rate-accuracy trade-off for all enzymatic selections to determine how the efficiency of initial codon readings decreased linearly toward zero as the accuracy of discrimination against near-cognate and wobble codon readings increased toward the maximal asymptote, the d value. We report data on the rate-accuracy variation for 7 cognate, 7 wobble, and 56 near-cognate codon readings comprising about 15% of the genetic code. Their d values varied about 400-fold in the 200-80,000 range depending on type of mismatch, mismatch position in the codon, and tRNA isoacceptor type. We identified error hot spots (d = 200) for U:G misreading in second and U:U or G:A misreading in third codon position by His-tRNA(His) and, as also seen in vivo, Glu-tRNA(Glu). We suggest that the proofreading mechanism has evolved to attenuate error hot spots in initial selection such as those found here.

  • 12.
    Zhang, Jingji
    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.
    Johansson, Magnus
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Ehrenberg, Måns
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Large accuracy variation in initial codon selection by aminoacyl-tRNAs on the bacterial ribosomeManuscript (preprint) (Other academic)
  • 13.
    Zhang, Jingji
    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.
    Mellenius, Harriet
    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.
    Proofreading neutralizes potential error hotspots in genetic code translation by transfer RNAs2016In: RNA: A publication of the RNA Society, ISSN 1355-8382, E-ISSN 1469-9001, Vol. 22, no 6, p. 896-904Article in journal (Refereed)
    Abstract [en]

    The ribosome uses initial and proofreading selection of aminoacyl-tRNAs for accurate protein synthesis. Anomalously high initial misreading in vitro of near-cognate codons by tRNAHis and tRNAGlu suggested potential error hotspots in protein synthesis, but in vivo data suggested their partial neutralization. To clarify the role of proofreading in this error reduction, we varied the Mg2+ ion concentration to calibrate the total accuracy of our cell-free system to that in the living Escherichia coli cell. We found the total accuracy of tRNA selection in our system to vary by five orders of magnitude depending on tRNA identity, type of mismatch, and mismatched codon position. Proofreading and initial selection were positively correlated at high, but uncorrelated at low initial selection, suggesting hyperactivated proofreading as a means to neutralize potentially disastrous initial selection errors.

1 - 13 of 13
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