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Removal and Replacement of Ribosomal Proteins

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The Road Not Taken - Robert Frost Two roads diverged in a yellow wood, And sorry I could not travel both And be one traveler, long I stood And looked down one as far as I could To where it bent in the undergrowth; Then took the other, as just as fair, And having perhaps the better claim, Because it was grassy and wanted wear; Though as for that the passing there Had worn them really about the same, And both that morning equally lay In leaves no step had trodden black. Oh, I kept the first for another day! Yet knowing how way leads on to way, I doubted if I should ever come back. I shall be telling this with a sigh Somewhere ages and ages hence: Two roads diverged in a wood, and I— I took the one less traveled by, And that has made all the difference. List of Papers This thesis is based on the following papers, which are referred to in the text by their Roman numerals. I Tobin, C., Mandava, CS., Ehrenberg, M., Andersson, DI., San- yal, S. (2010) Ribosomes lacking protein S20 are defective in mRNA binding and subunit association. Journal of Molecular Biology, 397(3):767–76 II Tobin, C., Nord, S., Wikström, PM., Näsvall, J., Sanyal, S., Andersson, DI. (2011) Ribosomal protein L1 is required for balanced subunit formation. (Manuscript) III Lind, PA., Tobin, C., Berg, OG., Kurland, CG., Andersson, DI. (2010) Compensatory gene amplification restores fitness after inter-species gene replacements. Molecular Microbiology, 75(5):1078-89 Reprints were made with permission from the respective publishers. Cover illustration made in PyMol (www.pymol.org) using the coordinates of the X- ray crystal structure of the T. thermophilus 70S ribosome (PDB I.D. 2J00 & 2J01) (Selmer et al. 2006). Contents Preface .......................................................................................................... 11 The Magnificence of the Machine ........................................................... 11 Background................................................................................................... 13 The ribosome is a ribozyme but what about the proteins?....................... 13 The necessary tools for understanding the ribosome and translation ...... 14 The nuts and bolts of the translation machinery ...................................... 14 Messenger RNA (mRNA) ................................................................... 16 Transfer RNA (tRNA)......................................................................... 17 Translation factors............................................................................... 19 The ribosome at work .............................................................................. 19 Initiation .............................................................................................. 20 Elongation ........................................................................................... 22 Termination ......................................................................................... 26 Ribosome recycling............................................................................. 28 Ribosome biogenesis and assembly......................................................... 29 rRNA synthesis.................................................................................... 29 R-protein synthesis .............................................................................. 30 Processing and modification of rRNA and r-proteins ......................... 31 Subunit assembly................................................................................. 33 Degradation and turnover of ribosomal components .......................... 37 Ribosomal Proteins .................................................................................. 38 General features................................................................................... 38 Roles in and outside the ribosome....................................................... 39 Conservation........................................................................................ 41 Non-essential r-proteins ...................................................................... 43 R-proteins of particular interest........................................................... 44 Ribosomal complexity – an evolutionary riddle ...................................... 47 Concept of fitness..................................................................................... 49 Adaptive and non-adaptive forces drive evolution .................................. 49 Compensatory evolution and adaptation.................................................. 50 Ribosomal complexity – a consequence of compensation? ................ 51 Horizontal Gene Transfer (HGT)............................................................. 52 Constraints on HGT............................................................................. 52 Present Investigations ................................................................................... 54 The model organism................................................................................. 55 Construction and characterization of r-protein mutants........................... 55 Recombineering................................................................................... 55 In vitro system ..................................................................................... 57 In vivo system...................................................................................... 57 Fitness costs of r-protein removal and replacement................................. 59 Removal of r-proteins confers substantial fitness costs ...................... 59 Fitness costs of replacement are generally weaker than removal ....... 60 Small fitness costs restrict HGT of r-proteins ..................................... 60 Phenotypes of S20 and L1 mutants..................................................... 61 Removal of S20 perturbs mRNA binding and subunit association..... 61 S20 is required for correct structural positioning of h44..................... 62 Removal of L1 impairs ribosome biogenesis ...................................... 63 Mutations in 50S r-proteins mitigate fitness costs of L1 removal ...... 64 High functional conservation of r-proteins .............................................. 66 Increased dosage rescues sub-optimal r-proteins..................................... 67 Future Perspectives .................................................................................. 68 Paper I.................................................................................................. 68 Paper II ................................................................................................ 68 Paper III............................................................................................... 70 Concluding remarks...................................................................................... 72 Acknowledgements....................................................................................... 74 References..................................................................................................... 77 Abbreviations DNA Deoxyribonucleic acid RNA Ribonucleic acid rRNA ribosomal RNA mRNA messenger RNA tRNA transfer RNA aa-tRNA amino-acyl tRNA r-protein ribosomal protein MDa Mega Dalton Å Ångström bps base pairs nt nucleotide ss single-stranded ds double-stranded CP Central Protuberance PTC Peptidyl-Transferase Centre SD Shine-Dalgarno sequence aSD anti-Shine-Dalgarno sequence TIR Translation Initiation Region IC Initiation Complex ASL Anticodon stem loop AARSs Amino-acyl tRNA synthetases A-site Amino-acyl site P-site Peptidyl site E-site Exit site IF Initiation Factor EF Elongation Factor RF Release Factor RRF Ribosome recycling factor GTP Guanosine triphosphate GDP Guanosine diphosphate ATP Adenosine triphosphate ppGpp Guanosine 5’-diphosphate, 3’- diphosphate pppGpp Guanosine 5’-triphosphate, 3’- diphosphate RBS Ribosome Binding Site ITS Internal Transcribed Spacer IVS Intervening sequence Cryo-EM Cryogenic Electron Microscopy FRET Fluorescence Resonance Energy Transfer Met Methionine fMet formylated Methionine GEF Guanine nucleotide Exchange Factor ram ribosomal ambiguity E. coli Escherichia coli S. typhimurium Salmonella enterica var Typhi- murium LT2 T. thermophilus Thermus thermophilus H. marismortui Haloarcula marismortui HIV Human Immunodeficiency Virus HGT Horizontal Gene Transfer H-NS Histone-like Nucleoid Structuring Protein SRP Signal Recognition Particle mitoribosome Mitochondrial ribosome Fe Iron ORF Open reading frame NG N-methyl-N'-nitro-N- nitrosoguanidine IPTG Isopropyl -D-1- thiogalactopyranoside CAI Codon Adaptation Index Preface The Magnificence of the Machine Of all the molecules present in the living organism, proteins are the most abundant and arguably the most fundamental. Often referred to as the bio- logical workhorses of the cell, proteins form the cornerstone of the most elementary cellular processes such as enzyme catalysis, structural support and immune defence, to name but a few. As such, efficient and orderly pro- duction of proteins is vital for cell fitness and survival. This fact is reflected in the recognition that an expanding catalogue of human diseases is caused by modifications of various components that function in protein synthesis. For example, mutated variants of mammalian ribosomal protein S19 are the cause of approximately 25% of cases of the rare erythropoietic disorder Diamond-Blackfan anaemia (DBA) (Scheper et al. 2007). In every living
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