Dissertation zur Erlangung des Doktorgrades der Fakultät für Chemie und Pharmazie der Ludwig-Maximilians-Universität München Auxiliary factors regulating ribosome function: Roles in biogenesis, antibiotic resistance and ribosome recycling Alexandra Dönhöfer aus München 2012 1 Erklärung Diese Dissertation wurde im Sinne von § 7 der Promotionsordnung vom 28. November 2011 von Herrn Prof. Dr. Roland Beckmann betreut. Eidesstattliche Versicherung Diese Dissertation wurde eigenständig und ohne unerlaubte Hilfe erarbeitet. München,................................ ....................................................... Alexandra Dönhöfer Dissertation eingereicht am: 09.02.2012 1. Gutachter: Herr Prof. Dr. Roland Beckmann 2. Gutachter: Herr Dr. Daniel N. Wilson Mündliche Prüfung am: 29.03.2012 2 Table of Contents LIST OF PUBLICATIONS ........................................................................................................7 CONTRIBUTION REPORT ......................................................................................................8 ABBREVIATIONS ..................................................................................................................10 1 INTRODUCTION .................................................................................................................13 1.1 The Flow of Genetic Information ................................................................................... 13 1.2 The Ribosome ................................................................................................................. 13 1.3 Ribosome Biogenesis in Eubacteria .............................................................................14 1.3.1 Processing of ribosomal RNA ....................................................................................15 1.3.2 Modification of ribosomal RNA ...................................................................................16 1.3.3 Modification of ribosomal Proteins ............................................................................. 19 1.3.4 Non-ribosomal Factors involved in Ribosome Assembly ............................................ 20 1.4 Ribosomal Proteins ........................................................................................................ 23 1.4.1 Ribosomal Proteins in Eubacteria ..............................................................................23 1.4.2 Ribosomal Proteins in Eukaryotes and Archaea ........................................................24 1.4.3 Specific Ribosomal Proteins in Organelles .................................................................25 1.4.3.1 Chloroplasts .........................................................................................................25 1.4.3.2 Mitochondria ........................................................................................................26 1.5 Translation Mechanism in Eubacteria...........................................................................27 1.5.1 Initiation Phase ........................................................................................................... 27 1.5.2 Elongation Phase: Decoding, Peptide-Bond Formation and Translocation ................ 29 1.5.3 Termination & Recycling ............................................................................................31 1.6 Inhibition of Translation: Antibiotics ............................................................................33 1.6.1 Stages of Inhibition during Translation .......................................................................35 1.6.2 Mechanisms of Antibiotic Resistance and Strategies to overcome this Problem ....... 37 3 2 OBJECTIVES AND AIMS OF THE THESIS .......................................................................40 3 CUMULATIVE THESIS: SUMMARY OF PUBLISHED RESULTS ..................................... 42 3.1 Paper I .............................................................................................................................. 42 3.2 Paper II ............................................................................................................................. 43 3.3 Paper III ............................................................................................................................ 44 3.4 Paper IV ........................................................................................................................... 45 3.5 Paper V ............................................................................................................................ 46 4 EXTRA-RIBOSOMAL FACTORS IN RIBOSOME BIOGENESIS ....................................... 48 4.1 Project Aims .................................................................................................................... 48 4.2 Introduction ..................................................................................................................... 48 4.2.1 Post-transcriptional Modification by Methyltransferases............................................. 49 4.2.2 Guanine 966 specific Methyltransferase YhhF (RsmD)..............................................50 4.2.3 Ψ1915 specific Methyltransferase RlmH (YbeA) ........................................................51 4.2.4 Ribosome maturation factor M (RimM) .......................................................................52 4.3 Results ............................................................................................................................. 54 4.3.1 Purification of E. coli YhhF protein .............................................................................54 4.3.2 In vitro binding assay: Eco30SyhhF + YhhF + sinefungin ........................................ 55 4.3.3 3D-reconstruction of the [Eco30SyhhF·YhhF] complex ........................................... 56 4.3.4 In vitro binding assay: Eco70SrluD/rlmH + RlmH + sinefungin ................................ 57 4.3.5 3D-reconstruction of the [Eco70SrluD/rlmH·RlmH] complex .................................... 59 4.3.6 Ribosome profiles and purification of E. coli RimM .................................................... 61 4.3.7 In vitro binding assay: Eco30S/Eco30SrimM + RimM .............................................. 62 4.3.8 3D-reconstruction of the [Eco30SrimM·RimM] complex ........................................... 63 4.4 Conclusions and Outlook ..............................................................................................65 5 ORGANELLAR-SPECIFIC RIBOSOMAL PROTEINS ........................................................67 4 5.1 Project Aims .................................................................................................................... 67 5.2 Introduction ..................................................................................................................... 67 5.2.1 Mitochondrion-specific ribosomal proteins (MSRPs) ..................................................67 5.3 Results ............................................................................................................................. 68 5.3.1 Expression and purification of yeast MSRPs ..............................................................68 5.3.2 In vivo association of individual MSRPs with E. coli ribosomes ................................. 70 5.3.3 Purification of [Eco70S·MRPL3] complexes ...............................................................72 5.3.4 3D-reconstruction of [Eco70S·MRPL3] complexes ....................................................73 5.4 Conclusions and Outlook ..............................................................................................74 6 ANTIBIOTIC RESISTANCE PROTEINS .............................................................................76 6.1 Project Aims .................................................................................................................... 76 6.2 Introduction ..................................................................................................................... 76 6.2.1 Ribosome protection protein Tet(M) ...........................................................................76 6.3 Results ............................................................................................................................. 78 6.3.1 Purification of E. faecalis Tet(M) ................................................................................78 6.3.2 In vitro binding assay: Eco70S + Tet(M) + GDPNP +/- Tgc ....................................... 79 6.3.3 3D-reconstruction of [Eco70S·Tet(M)·GDPNP·Tgc] complex .................................... 81 6.4 Conclusions and Outlook ............................................................................................101 7 MATERIAL AND METHODS .............................................................................................106 7.1 Methods in Molecular Biology .....................................................................................106 7.1.1 Media and Supplements ...........................................................................................106 7.1.2 Strains ...................................................................................................................... 106 7.1.3 Polymerase Chain Reaction (PCR) ..........................................................................106 7.1.4 Agarose Gel Electrophoresis ....................................................................................108 7.1.5 Cloning