Comparative studies on elongation and termination of the three RNA polymerases from S. cerevisiae in vitro DISSERTATION ZUR ERLANGUNG DES DOKTORGRADES DER NATURWISSENSCHAFTEN (DR. RER. NAT.) DER FAKULTÄT FÜR BIOLOGIE UND VORKLINISCHE MEDIZIN DER UNIVERSITÄT REGENSBURG vorgelegt von Philipp Merkl aus Regensburg im Jahr 2013 Das Promotionsgesuch wurde eingereicht am: 30. September 2013 Die Arbeit wurde angeleitet von: Prof. Dr. Herbert Tschochner Prüfungsausschuss: Vorsitzender: PD Dr. Joachim Griesenbeck 1. Prüfer: Prof. Dr. Herbert Tschochner 2. Prüfer: Prof. Dr. Thomas Moss (Université Laval, Québec, Kanada) 3. Prüfer: Prof. Dr. Klaus Grasser Die vorliegende Arbeit wurde im Zeitraum von Oktober 2009 bis September 2013 am Lehrstuhl für Biochemie III des Instituts für Biochemie, Genetik und Mikrobiologie der Universität Regensburg unter Anleitung von Prof. Dr. Herbert Tschochner angefertigt. Ich erkläre hiermit, dass ich diese Arbeit selbst verfasst und keine anderen als die angegebenen Quellen und Hilfsmittel verwendet habe. Diese Arbeit war bisher noch nicht Bestandteil eines Prüfungsverfahrens. Andere Promotionsversuche wurden nicht unternommen. Regensburg, den Philipp Merkl 5 Table of contents CHAPTER I – SUMMARY 13 CHAPTER II – INTRODUCTION 15 2.1 RNA polymerases – ubiquitous conserved enzymes ................................... 15 2.1.1 Outline ......................................................................................................................... 15 2.1.2 T7 RNA polymerase ..................................................................................................... 16 2.1.3 Bacterial RNA polymerase ........................................................................................... 17 2.1.4 Archaeal RNA polymerase ........................................................................................... 18 2.2 Eukaryotic RNA polymerases ...................................................................... 20 2.2.1 From one to five – an overview ................................................................................... 20 2.2.2 Structure and subunit composition of yeast RNA Pol I, II and III ................................ 20 2.2.3 Cellular localization and spatial organization of the rRNA genes ............................... 23 2.2.4 The only yeast Pol I target in vivo: The rDNA locus ..................................................... 24 2.3 The transcriptional cycles of yeast RNA Pol I, II and III ................................ 26 2.3.1 Transcription initiation ................................................................................................ 26 A) Pol I............................................................................................................................................ 26 B) Pol II ........................................................................................................................................... 28 C) Pol III .......................................................................................................................................... 29 2.3.2 Transcription elongation ............................................................................................. 29 A) Pol I............................................................................................................................................ 29 B) Pol II ........................................................................................................................................... 31 C) Pol III .......................................................................................................................................... 32 6 TABLE OF CONTENTS __________________________________________________________________________________ 2.3.3 Transcription termination ........................................................................................... 33 A) Pol I (reviewed in Nemeth et al. 2013) ...................................................................................... 33 B) Pol II ........................................................................................................................................... 37 C) Pol III .......................................................................................................................................... 38 2.4 Transcription of nucleosomal templates by Pol I, II and III ......................... 39 A) Pol I............................................................................................................................................ 40 B) Pol II ........................................................................................................................................... 42 C) Pol III .......................................................................................................................................... 44 2.5 Objectives .................................................................................................. 45 CHAPTER III – MATERIAL & METHODS 47 3.1 Material ...................................................................................................... 47 3.1.1 Chemicals ..................................................................................................................... 47 3.1.2 Media ........................................................................................................................... 47 3.1.3 Buffers.......................................................................................................................... 49 A) General buffers ......................................................................................................................... 49 B) Solutions for Coomassie and silver staining .............................................................................. 52 C) Buffers for RNA polymerase/transcription factor IIF (TFIIF) purification .................................. 52 D) Buffers for purification of recombinant proteins via the FLAG tag ........................................... 53 E) Buffers for purification of recombinant Reb1-His6 and Hmo1-His6 .......................................... 54 F) Buffers for purification of the recombinant A49-His6/A34.5 dimer .......................................... 55 G) Buffers for chromatin assembly ................................................................................................ 56 H) Buffers for in vitro transcription ............................................................................................... 56 3.1.4 Nucleic acids ................................................................................................................ 57 A) Nucleotides ............................................................................................................................ 57 B) Oligonucleotides .................................................................................................................... 57 C) Plasmids ................................................................................................................................. 67 D) DNA and RNA size markers .................................................................................................... 77 3.1.5 Enzymes and polypeptides .......................................................................................... 77 TABLE OF CONTENTS 7 __________________________________________________________________________________ 3.1.6 Antibodies .................................................................................................................... 78 3.1.7 Organisms .................................................................................................................... 79 A) Escherichia coli strains ........................................................................................................... 79 B) Saccharomyces cerevisiae strains .......................................................................................... 80 C) SF21 insect cells ..................................................................................................................... 82 3.1.8 Baculoviruses ............................................................................................................... 82 3.1.9 Kits ............................................................................................................................... 82 3.1.10 Equipment ................................................................................................................. 83 3.1.11 Consumables ............................................................................................................. 84 3.1.12 Software .................................................................................................................... 85 3.2 Methods ................................................................................................. 86 3.2.1 Enzymatic DNA manipulation ...................................................................................... 86 A) Polymerase chain reaction (PCR) .............................................................................................. 86 B) Sequence specific restriction digest .......................................................................................... 87 C) Introduction of single strand breaks ......................................................................................... 87 D) Blunting of single strand DNA overhangs ................................................................................