Characterization of r-protein variants in Saccharomyces cerevisiae DISSERTATION ZUR ERLANGUNG DES DOKTORGRADES DER NATURWISSENSCHAFTEN (DR. RER. NAT.) DER NATURWISSENSCHAFTLICHEN FAKULTÄT III BIOLOGIE UND VORKLINISCHE MEDIZIN DER UNIVERSITÄT REGENSBURG vorgelegt von Andreas Neueder aus Bogen Regensburg, 2010 Promotionsgesuch eingereicht am: 13.04.2010 Prüfungausschuss: Vorsitzender: Prof. Dr. A. Kurtz 1. Gutachter: Prof. Dr. H. Tschochner 2. Gutachter: Prof. Dr. R. Sterner 3. Prüfer: Prof. Dr. W. Seufert Ersatzprüfer: Prof. Dr. T. Dresselhaus Diese Arbeit wurde unter der Anleitung von Prof. Dr. Herbert Tschochner und Dr. Philipp Milkereit am Lehrstuhl für Biochemie III des Instituts für Mikrobiologie, Genetik und Biochemie der Universität Regensburg erstellt. Ich erkläre hiermit, dass ich die vorliegende Arbeit ohne unzulässige Hilfe Dritter und ohne Benutzung anderer als der angegebenen Hilfsmittel angefertigt habe. Die aus anderen Quellen direkt oder indirekt übernommenen Daten und Konzepte sind unter Angabe der Quelle gekennzeichnet. Insbesondere habe ich nicht die entgeltliche Hilfe von Vermittlungs- bzw. Beratungsdiensten (Promotionsberater oder andere Personen) in Anspruch genommen. Niemand hat von mir unmittelbar oder mittelbar geldwerte Leistungen für Arbeit erhalten, die im Zusammenhang mit dem Inhalt der vorgelegten Dissertation stehen. Die Arbeit wurde bisher weder im In- noch im Ausland in gleicher oder ähnlicher Form einer anderen Prüfungsbehörde vorgelegt. Regensburg, den Table of contents Table of contents 1 Introduction .............................................................................................................1 1.1 The Ribosome ............................................................................................................1 1.2 Ribosome structure ...................................................................................................1 1.2.1 Composition ......................................................................................................................1 1.2.2 Structures of prokaryotic ribosomes ..................................................................................4 1.2.3 Structures of eukaryotic ribosomes ...................................................................................8 1.2.4 Comparison and assessment of pro- and eukaryotic ribosome structures .......................11 1.3 Ribosome function ..................................................................................................12 1.3.1 Translation initiation .........................................................................................................12 1.3.2 Translation elongation .....................................................................................................14 1.3.3 Translation termination and recycling ..............................................................................15 1.4 Ribosome biogenesis ..............................................................................................16 1.4.1 Production of ribosomal constituents ...............................................................................16 1.4.2 Ribosome biogenesis factors and snoRNPs ....................................................................17 1.4.3 Maturation of ribosomal RNAs .........................................................................................20 1.4.4 Folding of precursor subunits and assembly of r-proteins ...............................................23 1.4.5 Transport of precursor subunits .......................................................................................27 1.4.6 Regulation, quality control and homeostasis of ribosome production ..............................29 1.5 Ribosomal proteins ..................................................................................................32 1.5.1 The roles of r-proteins in mature ribosomes ....................................................................32 1.5.2 The roles of r-proteins in ribosome biogenesis ................................................................34 1.5.3 Extra-ribosomal functions ................................................................................................35 1.6 Objectives .................................................................................................................37 2 Results ...................................................................................................................39 2.1 General strategies to design variant r-protein alleles for functional studies in S. cerevisiae .........................................................................................................39 2.1.1 General considerations ...................................................................................................39 2.1.2 Expression of archaeal homologues of r-proteins in S. cerevisiae ..................................41 2.1.3 Truncation and fusion mutants of yeast r-proteins ...........................................................42 2.1.4 Amino acid substitution mutants of yeast r-proteins .........................................................42 2.1.5 Table of described variant r-proteins in this work .............................................................43 2.2 Characterization of r-protein variants in S. cerevisiae .........................................44 2.2.1 rpS15 and its variants ......................................................................................................44 2.2.2 rpS17 and its variants ......................................................................................................50 2.2.3 rpS2 and its variants ........................................................................................................56 2.2.4 rpS5 and its variants ........................................................................................................61 2.2.5 rpS14 and its variants ......................................................................................................74 2.2.6 rpS20 and its variants ......................................................................................................78 2.3 Nob1p interaction with precursor subunits ...........................................................89 2.3.1 Nob1p co-purified pre-SSU rRNA independent of the in vivo assembly of some platform or head domain r-proteins .................................................................................89 2.3.2 20S pre-rRNA might be stabilized through TAP-tag fusion of Nob1p ...............................91 I Table of contents 3 Discussion and perspective ................................................................................93 3.1 Conservation of r-protein – rRNA interactions between Eukarya and Archaea .....................................................................................................................93 3.2 The influence of ribosomal proteins on final pre-18S rRNA maturation .............95 3.3 The role of ribosomal proteins in nuclear export and surveillance of nascent eukaryotic ribosomes ...............................................................................99 4 Summary – Zusammenfassung .........................................................................103 5 Materials & Methods ...........................................................................................106 5.1 Materials .................................................................................................................106 5.1.1 Escherichia coli strains ..................................................................................................106 5.1.2 Saccharomyces cerevisiae strains ................................................................................106 5.1.3 Oligonucleotides ............................................................................................................109 5.1.4 Plasmids ........................................................................................................................114 5.1.5 Enzymes .......................................................................................................................120 5.1.6 Antibodies ......................................................................................................................120 5.1.7 Chemicals .....................................................................................................................121 5.1.8 Other Materials ..............................................................................................................121 5.1.9 Media ............................................................................................................................122 5.1.10 Equipment ...................................................................................................................123 5.1.11 Software ......................................................................................................................124 5.2 Methods ..................................................................................................................124 5.2.1 Work with Escherichia coli .............................................................................................124 5.2.2 Work with Saccharomyces cerevisiae ...........................................................................125 5.2.3 Protein analysis .............................................................................................................125