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Ribosome Processivity and Co-Translational Protein Folding Ribosome Processivity and Co-translational Protein Folding Dissertation for the award of the degree “Doctor rerum naturalium” (Dr. rer. nat.) of the Georg-August-Universität Göttingen within the doctoral program GGNB – Biomolecules of the Georg-August University School of Science (GAUSS) submitted by Michael Thommen from Vevey, Switzerland Göttingen, 2015 Members of Examination Board /Thesis Committee Prof. Dr. Marina Rodnina (1st Referee) Department of Physical Biochemistry Max Planck Institute for Biophysical Chemistry Göttingen Germany Prof. Dr. Heinz Neumann (2nd Referee) Department of Applied Synthetic Biology Institute for Molecular Structural Biology Georg-August University Göttingen Germany Prof. Dr. Marina Bennati Electron Spin Resonance Spectroscopy group Max Planck Institute for Biophysical Chemistry Göttingen Germany Additional Members of the Examination Board Prof. Dr. Claudia Höbartner Institute for Organic and Biomolecular Chemistry Georg-August University Göttingen Germany Prof. Dr. Helmut Grubmüller Department of Theoretical and Computational Biophysics Max Planck Institute for Biophysical Chemistry Göttingen Germany Prof. Dr. Holger Stark 3D Cryo-Electron Microscopy group Max Planck Institute for Biophysical Chemistry Göttingen Germany Date of oral examination: 3rd of November 2015 I Affidavit I hereby declare that my thesis entitled "Ribosome Processivity and Co-translational Protein Folding" has been written independently and with no other sources and aids than quoted. This thesis (wholly or in part) has not been submitted elsewhere for any academic award or qualification. Michael Thommen August, 2015 Göttingen, Germany II Related publications Rudorf, S., Thommen, M., Rodnina, M.V., and Lipowsky, R*. (2014). Deducing the kinetics of protein synthesis in vivo from the transition rates measured in vitro. PLoS Comput Biol 10, e1003909. Buhr, F.†, Jha, S.†, Thommen, M.†, Mittelstaet, J., Kutz, F., Schwalbe, H. *, Rodnina, M.V. *, and Komar, AA*. (2016). Synonymous codons direct co-translational folding towards different protein conformations. Mol Cell 61, 341-51. * corresponding author † equal contribution III Table of Contents Abstract .................................................................................................................. 1 1 Introduction ..................................................................................................... 2 1.1 The ribosomal elongation cycle ................................................................... 2 1.2 Substrate selection in the A site ................................................................. 5 1.3 Incorporation of non-canonical amino acids ............................................... 8 1.4 Codon usage bias ..................................................................................... 10 1.5 Co-translation protein folding .................................................................. 13 1.6 Aim of the thesis ...................................................................................... 16 2 Results .......................................................................................................... 17 2.1 Modeling of translation elongation rates ................................................... 17 2.1.1 Parameters for modeling of elongation rates in vitro ........................... 21 2.1.2 Modeling of elongation rates in vivo .................................................... 24 2.2 Pausing during translation of natural mRNA ............................................ 28 2.2.1 Translation intermediates .................................................................. 28 2.2.2 Quantitative description of pausing during translation of HemK ........ 31 2.2.3 Hybridization of peptidyl-tRNA on micro-array chips ......................... 41 2.2.4 Hybridization of 3’-labeled tRNA on micro-array ................................ 45 2.3 Co-translational incorporation of fluorescent probes into nascent peptides ............................................................................................................ 50 2.3.1 Fluorescent labels at the N-terminus of nascent chains ..................... 50 Cys Cys 2.3.2 Activity of tRNA GCA and suppressor tRNA from E. coli .................. 53 2.3.3 Fluorescent labeling of Cys-tRNA ....................................................... 56 2.3.4 Incorporation of fluorescent probes at internal positions ................... 57 2.3.5 Context dependence for incorporation of fluorescent probes .............. 65 2.4 Codon-specific elongation rates modulate co-translational folding ........... 73 2.4.1 Harmonization of synonymous codon usage of GBC .......................... 73 2.4.2 Sequences of U and H yield differential expression of GBC in E. coli .. 76 2.4.3 Dynamics of translation elongation of sequences coding for GBC ...... 77 IV 2.4.4 Co-translational folding of GBC is modulated by the codon usage ..... 80 2.4.5 Monitoring the folding of the N-terminal domain of GBC by FRET ..... 83 3 Discussion ..................................................................................................... 87 3.1 Markov model for translation elongation .................................................. 87 3.2 Sequence specific, transient pausing ........................................................ 89 3.3 Incorporation of fluorescent labels mediates by Cys-tRNA ........................ 95 3.4 Translation elongation and co-translational folding ................................ 101 4 Material and Methods .................................................................................. 107 4.1 Equipment ............................................................................................. 107 4.2 Software ................................................................................................. 107 4.3 Chemicals and Consumables ................................................................. 108 4.4 Strains and Plasmids ............................................................................. 109 4.5 Buffers and solutions ............................................................................. 110 4.6 Molecular biology protocols .................................................................... 111 4.6.1 Site-directed mutagenesis following the QuikChange protocol ......... 111 4.6.2 Mutagenesis by isothermal assembly (Gibson Assembly) ................. 111 4.6.3 Transformation ................................................................................ 111 4.7 DNA constructs ...................................................................................... 112 4.7.1 HemK constructs ............................................................................. 112 4.7.2 Subcloning of gene for tRNACys ......................................................... 112 4.7.3 Subcloning of gene for adenylate kinase .......................................... 112 4.7.4 Subcloning of gene for phosphoglycerate kinase .............................. 112 4.7.5 Subcloning of the gene for codon-optimized HemK ........................... 112 4.7.6 Site-directed mutagenesis for tRNACys constructs ............................. 113 4.7.7 Site-direct mutagenesis for HemK constructs .................................. 113 4.7.8 Site-direct mutagenesis for GBC constructs ..................................... 113 4.8 General RNA protocols ........................................................................... 116 4.8.1 Ethanol or isopropanol precipitation ................................................ 116 4.8.2 Phenol or phenol:chloroform extraction ........................................... 116 V 4.8.3 General protocol for in vitro transcription ......................................... 116 4.9 mRNA constructs ................................................................................... 117 4.9.1 Short synthetic mRNA ..................................................................... 117 4.9.2 mRNA for single-turnover translation .............................................. 117 4.10 Protein purification ............................................................................. 118 4.10.1 CysRS-His6 ................................................................................... 118 4.10.2 Initiation and elongation factors ................................................... 118 4.11 Protocols related to tRNA .................................................................... 119 4.11.1 TCA precipitation of aminoacyl-tRNA ............................................ 119 4.11.2 Formation of EF-Tu•GTP•aa-tRNA ternary complex ...................... 119 4.11.3 Native PAGE analysis of aminoacylation level ............................... 119 4.11.4 Analysis of aa-tRNA by Acid Urea PAGE ....................................... 119 4.11.5 Analysis of peptidyl-tRNA by Bis-Tris PAGE .................................. 120 4.11.6 Aminoacylation of total tRNA ........................................................ 120 4.11.7 Purification of tRNACys transcripts ................................................ 120 4.11.8 Purification of tRNACys from E. coli total tRNA ............................... 121 4.11.9 Aminoacylation of tRNACys variants ............................................... 121 4.11.10 Labeling of Cys-tRNA with thiol-reactive probes. ........................... 121 4.11.11 Labeling of the α-amino group of Met-tRNAfMet .............................. 122 4.11.12 Separation of fluorescence-labeled tRNA by HPLC .......................
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