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Structural Characterization of Proteinaceous Rnase P from Arabidopsis Thaliana Franziska Pinker

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Structural Characterization of Proteinaceous Rnase P from Arabidopsis Thaliana Franziska Pinker Structural characterization of proteinaceous RNase P from Arabidopsis thaliana Franziska Pinker To cite this version: Franziska Pinker. Structural characterization of proteinaceous RNase P from Arabidopsis thaliana. Biologie végétale. Université de Strasbourg, 2014. Français. NNT : 2014STRAJ093. tel-01236246 HAL Id: tel-01236246 https://tel.archives-ouvertes.fr/tel-01236246 Submitted on 1 Dec 2015 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. UNIVERSITÉ DE STRASBOURG ÉCOLE DOCTORALE des Sciences de la Vie et de la Santé Institut de Biologie Moléculaire des Plantes - IBMP THÈSE présentée par : Franziska PINKER soutenue le : 15 septembre 2014 pour obtenir le grade de : !"#$%&'($')R%+ iversité de Strasbourg Discipline/ Spécialité : Sciences du Vivant /Biochimie, Biologie Moléculaire et Structurale Structural characterization of proteinaceous RNase P from Arabidopsis thaliana THÈSE dirigée par : M. Claude SAUTER Dr, Université de Strasbourg M. Philippe GIEGE Dr, Université de Strasbourg RAPPORTEURS : M. Walter ROSSMANITH Dr, Université de Vienne Mme Emmanuelle SCHMITT Dr, Ecole Polytechnique AUTRES MEMBRES DU JURY : Mme Anita MARCHFELDER Professeur, !"#$%&"' )*R ,- Mme Pascale ROMBY Dr, Université de Strasbourg Fur¨ meine Familie Acknowledgements First of all I would like to thank the members of my jury, Dr. Emmanuelle Schmitt, Dr. Pascale Romby, Dr. Anita Marchfelder and Dr. Walter Rossmanith, for having agreed to judge my work. I would like to offer my special thanks to my Ph.D. advisors Dr. Claude Sauter and Dr. Philippe Gieg e´ for having accepted me in their laboratories and for their infinite patience and support during the last three years. I would like to express my very great appreciation to Agn es` and Anthony who gave me so much advice, good discussions and encouragement at the IBMC and IBMP. Furthermore, I thank all the labmembers: Catherine, Marie, Bernard, J orn,¨ Loukmane, Ayoub, Thalia and G eraldine.´ I would like to offer my thanks to all the members of the IBMC and IBMP for answering all kind of questions and making working so much fun, especially Jo elle,¨ Kamel, Anne-So, M elodie,´ Hagen, Anja and Bernadette. Finally, I wish to thank my family and my friends that supported me during the years, who cheered me up and who most importantly encouraged me to go on. Heiko und Dieter: danke, dass Ihr mich mit auf die tollsten Berge und die sch onsten¨ Trails der Region genommen habt. Ohne euch w are¨ Badisch noch ein Buch mit sieben Siegeln f ur¨ mich. i Abbreviations aaRS . Aminoacyl-tRNA-synthetase CBD . Chitin binding domain cv . Column volume Da . Dalton DLS . Dynamic light scattering EMSA . Electromobility shift assay HPLC . High performance liquid chromatography ICP-MS . Inductively coupled plasma mass spectrometry LB . Lysogeny broth MALS . Multi-angle light scattering MN . Micrococcal nuclease mRNA . Messenger RNA MRPP . Mitochondrial ribonuclease P protein MST . Microscale thermophoresis MTS . Mitochondrial targeting signal MWCO . Molecular weight cut-off NEP . Nuclear-encoded phage-type RNA polymerases NiNTA . Nickel nitrilotriacetic acid nts . Nucleotides PAA . Polyacrylamide PNPase . Polynucleotide phosphorylase pre-tRNA . Precursor tRNA PRORP . Proteinaceous RNase P RNase P . Ribonuclease P rRNA . Ribosomal RNA SAXS . Small angle X-ray scattering SEC . Size exclusion chromatography snRNA . Small nuclear RNA snRNP . Small nuclear ribonucleoprotein particles SRCD . Synchrotron radiation circular dichroism SV . Sedimentation velocity TCEP . Tris(2-carboxyethyl)phosphine TFIII . Transcription factor III ii Contents Acknowledgements . i Abbreviations . ii Contents . vi List of Figures . viii List of Tables . ix Introduction ........................................... 1 1.1 Transfer RNAs . 1 1.1.1 Transfer RNAs - structure and function . 1 1.1.2 Prokaryotic tRNA transcription . 2 1.1.3 tRNA transcription in eukaryotic nuclei . 2 1.1.4 tRNAs in the model plant Arabidopsis thaliana ................ 3 1.2 tRNA maturation steps . 5 1.2.1 Prokaryotic tRNA maturation steps . 5 1.2.2 Eukaryotic tRNA maturation steps . 7 1.2.2.1 5’ end maturation . 7 1.2.2.2 3’ end maturation . 7 1.2.2.3 CCA addition . 7 1.2.2.4 Nucleotide modifications . 7 1.2.2.5 Splicing and other factors . 8 1.2.2.6 The La protein . 8 1.3 5’ end maturation of tRNAs in different domains of life . 9 1.3.1 Ribonucleoproteic RNase P . 10 1.3.1.1 Bacterial RNase P . 10 1.3.1.2 Archaeal RNase P . 12 1.3.1.3 Eukaryotic RNase P . 13 1.3.2 Proteinaceous RNase P . 14 1.3.2.1 Animals: First proof of protein-only RNase P in human mitochondria 15 1.3.2.2 Plants: Early work on spinach chloroplasts . 16 1.4 Life without RNase P . 16 1.5 What about RNase MRP . 17 1.6 Publication 1: PPR proteins shed a new light on RNase P biology . 18 1.7 PRORP: A Pentatricopeptide repeat protein . 32 1.7.1 PPR distribution in all domains of life . 32 iii 1.7.2 PPR classifications . 32 1.7.3 Functions of PPR proteins . 32 1.8 The RNA recognition code . 33 1.9 Structural information on PPR/RNA interactions . 33 1.10 Objectives of my thesis . 34 Material and methods ..................................... 36 2.1 Materials . 36 2.1.1 Bacterial strains . 36 2.1.2 Plasmids . 36 2.1.2.1 Protein expression vectors . 36 2.1.2.2 RNA transcription vectors . 37 2.1.3 Protein constructs . 37 2.1.4 RNA substrates . 37 2.1.5 Primers . 38 2.1.6 Devices . 39 2.2 Methods . 40 2.2.1 Protein production . 40 2.2.1.1 PRORP overexpression in E. coli Rosetta 2 cells . 40 2.2.1.2 Affinity chromatography of His-tagged proteins on a nickel column . 41 2.2.1.3 Affinity chromatography of the intein-tagged proteins on a chitin matrix 42 2.2.1.4 Size exclusion chromatography . 43 2.2.2 RNA production . 44 2.2.2.1 Template preparation . 44 2.2.2.2 In vitro transcription . 44 2.2.2.3 Polyacrylamide gel electrophoresis . 45 2.2.2.4 Chromatography . 46 2.2.3 Protein quality control . 47 2.2.3.1 Dynamic light scattering . 47 2.2.3.2 Activity assay of PRORP proteins . 49 2.2.4 Inductively coupled plasma mass spectrometry . 49 2.2.5 Methods to determine affinity parameters of the PRORP/tRNA interaction . 50 2.2.5.1 Isothermal titration calorimetry . 50 2.2.5.2 Microscale thermophoresis . 51 2.2.5.3 Analytical ultracentrifugation . 54 2.2.6 Analyzing PRORP/pre-tRNA interactions . 56 2.2.6.1 Size exclusion chromatography as a tool for studying PRORP-tRNA interactions . 56 2.2.6.2 Electromobility shift assay . 56 2.2.6.3 Complex modeling . 56 2.2.6.4 Crosslinking . 56 iv 2.2.7 Synchrotron radiation circular dichroism . 57 2.2.8 Small angle x-ray scattering . 58 2.2.9 Macromolecule crystallization . 61 2.2.9.1 Initial screening . 63 2.2.9.2 Crystal optimization . 63 2.2.10 X-ray diffraction data collection . 64 3 Protein and RNA production ............................... 66 3.1 PRORP1-2-3 constructs . 66 3.2 Recloning of PRORP2 in pTYB-vectors . 68 3.3 Optimizing PRORP purification containing a His-tag . 70 3.4 tRNA purification . ..
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