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Function of Elongation Factor P in Translation

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Function of Elongation Factor P in Translation Function of Elongation Factor P in Translation Dissertation for the award of the degree ”Doctor rerum naturalium“ of the Georg-August-Universität Göttingen within the doctoral program Biomolecules: Structure–Function–Dynamics of the Georg-August University School of Science (GAUSS) submitted by Lili Klara Dörfel from Berlin Göttingen, 2015 Members of the Examination board / Thesis Committee Prof. Dr. Marina Rodnina, Department of Physical Biochemistry, Max Planck Institute for Biophysical Chemistry, Göttingen (1st Reviewer) Prof. Dr. Heinz Neumann, Research Group of Applied Synthetic Biology, Institute for Microbiology and Genetics, Georg August University, Göttingen (2nd Reviewer) Prof. Dr. Holger Stark, Research Group of 3D Electron Cryo-Microscopy, Max Planck Institute for Biophysical Chemistry, Göttingen Further members of the Examination board Prof. Dr. Ralf Ficner, Department of Molecular Structural Biology, Institute for Microbiology and Genetics, Georg August University, Göttingen Dr. Manfred Konrad, Research Group Enzyme Biochemistry, Max Planck Institute for Biophysical Chemistry, Göttingen Prof. Dr. Markus T. Bohnsack, Department of Molecular Biology, Institute for Molecular Biology, University Medical Center, Göttingen Date of the oral examination: 16.11.2015 I Affidavit The thesis has been written independently and with no other sources and aids than quoted. Sections 2.1.2, 2.2.1.1 and parts of section 2.2.1.4 are published in (Doerfel et al, 2013); the translation gel of EspfU is published in (Doerfel & Rodnina, 2013) and section 2.2.3 is published in (Doerfel et al, 2015) (see list of publications). Lili Klara Dörfel November 2015 II List of publications EF-P is Essential for Rapid Synthesis of Proteins Containing Consecutive Proline Residues Doerfel LK†, Wohlgemuth I†, Kothe C, Peske F, Urlaub H, Rodnina MV*. Science (2013); 339(6115):85- 8. Elongation Factor P: Function and Effects on Bacterial Fitness Doerfel LK, Rodnina MV*. Biopolymers (2013); 99(11):837-45. Entropic contribution of elongation factor P to proline positioning at the catalytic center of the ribosome Doerfel LK†, Wohlgemuth I†, Kubyshkin V, Starosta AL, Wilson DN*, Budisa N*, Rodnina MV*. Journal of the American Chemical Society (2015); 137(40): 12997-13006 * Corresponding author † Equal contribution III Content ABSTRACT ................................................................................................................................................ 1 1 INTRODUCTION .................................................................................................................................. 2 1.1 Translation .................................................................................................................................. 2 1.1.1 General overview ................................................................................................................ 2 1.1.2 Peptide bond formation ..................................................................................................... 4 1.2 Elongation factor P ..................................................................................................................... 6 1.3 Proline ....................................................................................................................................... 10 1.4 Aims of the thesis ..................................................................................................................... 12 2 RESULTS ............................................................................................................................................ 13 2.1 The catalytic function of EF-P ................................................................................................... 13 2.1.1 Initiation ............................................................................................................................ 13 2.1.2 Elongation ......................................................................................................................... 14 2.1.2.1 The P-site substrate ................................................................................................................ 14 2.1.2.2 The A-site substrate ................................................................................................................ 15 2.1.2.3 Sequence context .................................................................................................................... 16 2.1.2.4 Proline induced stalling leads to peptidyl tRNA dissociation .................................................. 17 2.1.2.5 EF-P alleviates Pro-induced ribosome stalling in longer model peptides ................................ 18 2.1.2.6 Ribosomes stall at PP/G and can be rescued by EF-P .............................................................. 19 2.1.2.7 EF-P enhances synthesis of E. coli proteins containing polyproline motifs ............................. 21 2.1.2.8 Stalling is mainly caused by the Pro moiety of Pro-tRNAPro .................................................... 23 2.1.3 Release .............................................................................................................................. 25 2.2 Investigation of the catalytic mechanism of EF-P ..................................................................... 26 2.2.1 The function of the EF-P body and the modification ........................................................ 27 2.2.1.1 EF-P modification increases its catalytic proficiency ............................................................... 27 2.2.1.2 Slow peptide bond formation competes with the translocation process ................................ 30 2.2.1.3 Increasing MgCl2 concentrations reduce peptidyl-tRNA dissociation ...................................... 32 2.2.1.4 EF-P selectively accelerates peptide bond formation with poor substrates. ........................... 33 2.2.1.5 Impact of the EF-P modification on peptidyl transfer ............................................................. 34 2.2.2 Variation of physico-chemical parameters ....................................................................... 35 2.2.2.1 pH-dependence of peptide bond formation with a native A-site substrate ............................ 35 2.2.2.2 Activation parameters ............................................................................................................ 38 2.2.3 Variation of the substrate ................................................................................................. 41 2.2.3.1 Substitution on the prolyl ring modulates steric and electronic properties of proline ............ 41 2.2.3.2 Pro derivatives were accepted by the translation machinery ................................................. 42 2.2.3.3 Substitutions in the prolyl ring strongly modulate rate of peptide bond formation ............... 42 IV 2.2.3.4 The ribosome-catalysed reaction is more sensitive to substituent effects than the in-solution reaction 45 2.2.3.5 The reactivity does not correlate with the electrophilicity of the P-site substrate .................. 47 2.2.3.6 Isomery .................................................................................................................................... 48 2.2.3.7 Impact of Pro analogs on activation parameters .................................................................... 50 3 DISCUSSION ...................................................................................................................................... 52 3.1 The biological role of EF-P ........................................................................................................ 52 3.1.1 EF-P inactivation causes pleiotropic in-vivo phenotypes ................................................. 52 3.1.1.1 Susceptibility to external stimuli ............................................................................................. 53 3.1.1.2 Motility .................................................................................................................................... 53 3.1.1.3 Virulence.................................................................................................................................. 54 3.1.2 Potential regulation by the EF-P modification state ......................................................... 54 3.2 What makes proline slow in peptide bond formation? ........................................................... 55 3.2.1 Poor reactivity of proline .................................................................................................. 55 3.2.2 Proline in proteins and in the cell ..................................................................................... 56 3.2.3 Sequence context effects of Pro-induced stalling ............................................................ 56 3.3 EF-P binds to the ribosome in a tRNAPro-specific fashion ......................................................... 57 3.4 How does EF-P catalyze peptide bond formation? .................................................................. 58 3.4.1 EF-P body stabilizes and positions the peptidyl-tRNA ...................................................... 58 3.4.2 The modification of EF-P contributes to catalysis ............................................................ 59 3.4.3 EF-P function does not involve general acid or base catalysis ......................................... 60 3.4.4 A possible catalytic mechanism of EF-P ...........................................................................
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