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Structural Framework for the Mechanism of Archaeal Exosomes in RNA Processing

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Structural Framework for the Mechanism of Archaeal Exosomes in RNA Processing Dissertation zur Erlangung des Doktorgrades der Fakultät für Chemie und Pharmazie der Ludwig-Maximilians-Universität München Structural Framework for the Mechanism of Archaeal Exosomes in RNA processing Structural Insights into DNA Duplex Separation by the Archaeal Superfamily 2 Helicase Hel308 Katharina Büttner aus Wolfratshausen 2007 Erklärung Diese Dissertation wurde im Sinne von § 13 Abs. 3 bzw. 4 der Promotionsordnung vom 29. Januar 1998 von Herrn Prof. Dr. Karl-Peter Hopfner betreut. Ehrenwörtliche Versicherung Diese Dissertation wurde selbständig, ohne unerlaubte Hilfe erarbeitet. München, am 24. April 2007 .................................................... (Katharina Büttner) Dissertation eingereicht am 24. April 2007 1. Gutachter Herr Prof. Dr. Karl-Peter Hopfner 2. Gutachter Herr Prof. Dr. Patrick Cramer Mündliche Prüfung am 03. Juli 2007 The presented thesis was prepared in the time from December 2003 to March 2007 in the laboratory of Professor Dr. Karl-Peter Hopfner at the Gene Center of the Ludwig-Maximilians-University of Munich (LMU). Parts of this PhD thesis have been published: Büttner K., Nehring S., and Hopfner K.P., (2007). Structural basis for DNA duplex separation by a Superfamily 2 Helicase. NSMB 2007, Jul;14(7):647-52. Büttner K. and Hopfner K.P., (2006). Das Exosom. Bioforum 2006; 29:19-21. Büttner K., Wenig K., and Hopfner K.P., (2006). The exosome: a macromolecular cage for controlled RNA degradation. Mol. Microbiology 2006;61:1372-1379. Büttner K., Wenig K.. and Hopfner K.P., (2005). Structural Framework for the Mechanism of Archaeal Exosomes in RNA Processing. Mol Cell. 2005 Nov 11;20(3):461-71. Other publications: Karcher A., Büttner K., Märtens B., Jansen R.P. and Hopfner K.P., (2005). X-ray structure of RLI, an essential twin cassette ABC ATPase involved in ribosome biogenesis and HIV capsid assembly. Structure. 2005 Apr. 13(4):649-59. Presentations at international conferences: Büttner K., Wenig K., and Hopfner KP. Structural Framework for the Mechanism of Archaeal Exosomes in RNA Processing Hybrid Methods; March 15-19, 2006, Lake Tahoe, CA, USA (Poster) Our Father who art in heaven, Hallowed be Thy name. Thy kingdom come. Thy will be done on Earth, as it is in heaven. Give us this day our daily bread. And forgive us our debts, as we forgive our debtors. And lead us not into temptation. But deliver us from evil. For Thine is the kingdom, and the power, and the glory. Forever, Amen Matthew, 6:8-13 PART I Structural Framework for the Mechanism of Archaeal Exosomes in RNA processing Table of contents Table of contents 1 INTRODUCTION....................................................................................................................................1 1.1 THE EXOSOME IS CONSERVED IN EUKARYOTES AND ARCHAEA ..........................................................1 1.2 FUNCTIONS OF THE EXOSOME AND COACTIVATOR COMPLEXES .........................................................4 1.2.1 Cytosolic RNA decay and quality control .............................................................................4 1.2.2 Nuclear RNA processing and degradation.............................................................................6 1.3 STRUCTURE DETERMINATION BY X-RAY CRYSTALLOGRAPHY ..........................................................8 1.3.1 Crystallization........................................................................................................................8 1.3.2 Theory of X-ray diffraction ...................................................................................................9 1.3.3 The Phase problem ................................................................................................................9 1.3.4 Solving the phase problem...................................................................................................11 1.3.4.1 Patterson Function......................................................................................................................... 11 1.3.4.2 Single- and multiple anomalous dispersion (SAD/MAD) ............................................................. 11 1.3.4.3 Molecular replacement.................................................................................................................. 13 1.4 OBJECTIVES .....................................................................................................................................14 2 MATERIALS AND METHODS...........................................................................................................15 2.1 MATERIALS......................................................................................................................................15 2.1.1 Chemicals ............................................................................................................................15 2.1.2 Bacterial strains ...................................................................................................................15 2.1.3 Media and supplements .......................................................................................................16 2.1.4 Buffers and solutions ...........................................................................................................17 2.2 METHODS ........................................................................................................................................18 2.2.1 Bioinformatic Methods........................................................................................................18 2.2.1.1 Homology searches and alignments .............................................................................................. 18 2.2.1.2 Structural homology searches........................................................................................................ 18 2.2.1.3 Calculation of protein parameters.................................................................................................. 18 2.2.1.4 Structure visualization and analyzation.........................................................................................18 2.2.2 Molecular methods ..............................................................................................................19 2.2.2.1 Oligonucleotide design and Polymerase Chain Reaction (PCR) ................................................... 19 2.2.2.2 Site-directed mutagenesis.............................................................................................................. 19 2.2.2.3 Restriction cleavage and ligation................................................................................................... 19 2.2.2.4 Transformation of E. coli and isolation of plasmid DNA.............................................................. 20 2.2.2.5 Preparation of competent cells ...................................................................................................... 20 2.2.3 Electrophoretic methods ......................................................................................................20 2.2.3.1 Electrophoretic separation of DNA ...............................................................................................20 2.2.3.2 Protein separation by SDS-PAGE ................................................................................................. 21 2.2.4 Proteinchemical methods.....................................................................................................21 2.2.4.1 Protein expression ......................................................................................................................... 21 2.2.4.2 Selenomethionine labelling ........................................................................................................... 21 Table of contents 2.2.4.3 Gelfiltration experiments and determination of molecular weight ................................................ 22 2.2.5 Structure determination of the archaeal exosome................................................................22 2.2.5.1 Cloning and expression of the exosome complexes from Archaeoglobus fulgidus....................... 22 2.2.5.2 Purification of the two exosome isoforms ..................................................................................... 22 2.2.5.3 Crystallization and structure determination of the exosome complexes........................................ 25 2.2.5.4 Tungstate soaking experiments ..................................................................................................... 26 2.2.6 Biochemical Methods ..........................................................................................................27 2.2.6.1 Radioactive Labelling of RNA...................................................................................................... 27 2.2.6.2 RNA degradation and polyadenylation assays .............................................................................. 27 2.2.6.3 RNA binding assays...................................................................................................................... 28 3 RESULTS................................................................................................................................................29 3.1 PURIFICATION, CRYSTALLIZATION AND STRUCTURE DETERMINATION OF THE ARCHAEAL EXOSOMES 29 3.1.1 Purification of the Rrp4-exosome........................................................................................30 3.1.2 Purification of the Csl4-exosome ........................................................................................31
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