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The Small Subunit of the Mitoribosome from Andalucia Godoyi. Isolation and Study of Its Protein Composition”

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The Small Subunit of the Mitoribosome from Andalucia Godoyi. Isolation and Study of Its Protein Composition” UNIVERSITÉ DE MONTRÉAL DÉPARTEMENT DE BIOCHIMIE ET MÉDECINE MOLÉCULAIRE FACULTÉ DE MÉDECINE “The small subunit of the mitoribosome from Andalucia godoyi. Isolation and study of its protein composition” Présenté par José Angel Gonzalez Alcazar Mémoire présenté à la Faculté de médecine en vue de l’obtention du grade de maîtrise en biochimie 20 mars 2018 © José Angel Gonzalez Alcazar, 2018 UNIVERSITÉ DE MONTRÉAL DÉPARTEMENT DE BIOCHIMIE ET MÉDECINE MOLÉCULAIRE FACULTÉ DE MÉDECINE Ce mémoire intitulé: “The small subunit of the mitoribosome from Andalucia godoyi. Isolation and study of its protein composition” Présenté par : José Angel Gonzalez Alcazar a été évalué par un jury composé des personnes suivantes : Dr. Sebastian Pechmann Dre. Gertraud Burger Dr. Martin Schmeing 1 TABLE OF CONTENTS 1. INTRODUCTION ................................................................................................... 10 1.1 Mitochondrial genomes.................................................................................... 10 1.2 The most bacteria-like mtDNAs...................................................................... 11 1.3 Prokaryotic translation .................................................................................... 12 1.4 Translation in mammalian mitochondria ...................................................... 13 1.4 Differences of translation in bacteria, and mammalian and yeast mitochondria ................................................................................................................ 15 1.5 Ribosome composition ..................................................................................... 17 1.6 The mammalian mitoribosome ....................................................................... 18 1.7 The yeast mitoribosome ................................................................................... 21 1.8 Mitoribosome composition in kinetoplastids ................................................. 22 1.9 Mitoribosome evolution ................................................................................... 23 1.9.1 Evolution of the mitoribosome structure.................................................... 23 1.9.2 Evolution of mitoribosome protein composition........................................ 24 1.10 Andalucia godoyi ............................................................................................... 27 2. HYPOTHESIS ......................................................................................................... 28 3. MATHERIALS AND METHODS ......................................................................... 28 3.1 A. godoyi cell culture ........................................................................................ 28 3.2 Sucrose gradient purification of the small subunit of the mitoribosome (mt- SSU ribosome) ............................................................................................................. 29 3.3 Validation of the enrichment of the mt-SSU-rRNA ...................................... 32 3.3.1 RNA quantification......................................................................................... 32 3.3.2 Electrophoretic separation of rRNAs ........................................................... 32 3.3.3 Radiolabelling of probes ................................................................................ 33 3.3.4 Northern blot hybridization .......................................................................... 33 3.3.5 SDS-PAGE of mitoribosome proteins (mito-r-proteins) ............................. 35 3.4 Mass spectrometry analysis ............................................................................. 35 3.5 Sequence analysis ............................................................................................. 37 4. RESULTS ................................................................................................................. 37 4.1 Sucrose gradient purification of the A. godoyi small subunit mitoribosome 37 2 4.2 Validating the enrichment of A. godoyi mt-SSU-rRNA ................................ 39 4.3 Mass spectrometry analysis and comparison with predicted SSU mito-r- proteins ......................................................................................................................... 42 4.4 Additional proteins detected in the mt-SSU ribosomal fraction of Andalucia godoyi ........................................................................................................................... 47 4.5 Tracing the evolutionary history of SSU mitoribosomal proteins in Discoba 48 5. DISCUSSION ........................................................................................................... 49 5.1 Technical challenges in the purification of A. godoyi mt-SSU ribosome ..... 49 5.2 Identification and analysis of SSU mitoribosomal proteins from A. godoyi 49 5.3 The composition of A. godoyi mt-SSU ribosome............................................ 50 5.4 Evolution of protein composition in the mt-SSU ribosome of Discoba ....... 51 6. OUTLOOK AND FUTURE WORK ..................................................................... 52 7. REFERENCES ........................................................................................................ 54 8. APPENDICES .......................................................................................................... 60 8.1 Materials ........................................................................................................... 60 8.2 Solutions and buffers ....................................................................................... 63 8.3 Primers used in RT-PCR ................................................................................. 74 8.4 Probes used in Northern blot .......................................................................... 74 LIST OF FIGURES AND TABLES Figure 1. Translation on mammalian mitochondria proceeds in four consecutive steps: Initiation, elongation, termination, and recycling ............................................. 14 Figure 2. Structural view of the bacterial ribosome .................................................... 17 Figure 3. Differences of mRNA entrance site and polypeptide exit site in bacterial ribosomes and human mitoribosomes ........................................................................... 20 Figure 4. Representation of the constructive evolution of mitoribosomes ................ 24 Figure 5. Phylogenetic tree of eukaryotes used to predict the evolutionary history of mitoribosome proteins .................................................................................................... 25 Figure 6. Prediction of mito-r-proteins possessed by the LECA and its following evolution in Bikonts and Unikonts ................................................................................ 26 Figure 7. Light micrographs of A. godoyi ..................................................................... 27 Figure 8. Linear regression analysis performed for protein quantification .............. 36 Figure 9. RNA extracted from fractions of a 5 ml 15-40% sucrose gradient ............ 38 Figure 10. Amplification of SSU-rRNAs in single gradient fractions ........................ 39 3 Figure 11. The enrichment of the 16S bt-rRNA and the 23S bt-rRNA was analyzed by Northern blot .............................................................................................................. 40 Figure 12. The enrichment of the mt-SSU-rRNA was quantified by Northern blot 41 Figure 13. The enrichment of the mt-LSU-rRNA was analyzed by Northern blot .. 41 Figure 14. Protein electrophoresis ................................................................................. 42 Figure 15. Total number of proteins in the SSU of E. coli and the mt-SSU ribosome of select eukaryotes ......................................................................................................... 46 Figure 16. Number of bacterial homolog proteins in the mt-SSU ribosome of different organisms ......................................................................................................... 46 Figure 17. Number of eukaryotic-specific proteins in the mt-SSU ribosome of different organisms ......................................................................................................... 47 Table 1. Summary of the composition in several characterized ribosomes ............... 19 Table 2. Primers used for RT of rRNAs ....................................................................... 31 Table 3. Primers pairs and number of cycles used to amplify rRNAs ....................... 31 Table 4. RNA quantification of samples used in Northern blot ................................. 32 Table 5. Probes used for Northern blot of rRNAs ....................................................... 34 Table 6. BSA standards used in the linear regression analysis for protein quantification................................................................................................................... 36 Table 7. Mito-r-proteins inferred from the A. godoyi nuclear and mitochondrial genome sequences............................................................................................................ 43 Table 8. Proteins in the small subunit of the mitoribosome ........................................ 44 Table 9. Inference of the set of mito-r-proteins present in the mt-SSU ribosome of the last common ancestor of Discobaa ..........................................................................
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