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Mechanistic and Bioinformatic Studies of Mitochondrial Ribosomes and Auxiliary Translational Factors Domenick Gabriel Grasso

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Mechanistic and Bioinformatic Studies of Mitochondrial Ribosomes and Auxiliary Translational Factors Domenick Gabriel Grasso Mechanistic and Bioinformatic Studies of Mitochondrial Ribosomes and Auxiliary Translational Factors Domenick Gabriel Grasso A dissertation submitted to the faculty of the University of North Carolina at Chapel Hill in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Chemistry. Chapel Hill 2007 Approved by: Dr. Linda Spremulli Dr. Gary Pielak Dr. Dorothy Erie Dr. Kevin Weeks Dr. Nancy Thompson © 2007 Domenick Gabriel Grasso ALL RIGHTS RESERVED ii ABSTRACT DOMENICK GABRIEL GRASSO: Mechanistic and Bioinformatic Studies of Mitochondrial Ribosomes and Auxiliary Translational Factors (Under the direction of Dr. Linda L. Spremulli) This body of work is focused on mitochondrial translational systems. This apparatus synthesizes vital components of the electron transport chain facilitating the production of energy in eukaryotic cells. Of particular interest is the role of mammalian mitochondrial initiation factor 2 (IF2mt) in this process. This factor recruits the initiatior fMet-tRNA to begin protein synthesis within this organelle. IF2mt is unique in that it possesses an insertion. The work described here demonstrates that the insertion acts as the factor equivalent of initiation factor 1 (IF1). IF1 is an essential gene in prokaryotes and eukaryotes (eIF1A) but has yet to be discovered in mitochondria. IF2mt is a GTPase and the guanine ring binding region in this factor was probed to determine the effect of mutations on the X residue of the guanine ring binding motif NKXD. Quite surprisingly despite the connotation of X being random, this residue is very sensitive to perturbation. Other work included in this dissertation includes a bioinformatics analysis of the proteins found in a Leishmania tarentolae mitochondrial ribosomal small subunit particle. L. tarentolae is a member of the trypanosomitids family, whose members are known to cause African sleeping sickness and South American Chagas disease. Examination of this organism offers a deeper understanding to the mechanism of action of these disease causing organisms and offers the potential development of therapies for these illnesses. This study explored the iii limitations of “hits” produced by search algorithms as well as insight into mitochondrial ribosomal protein evolution. This last chapter of this dissertation attempts to further the understanding of the mechanisms of translation in the mammalian mitochondria by probing for previously unknown translational factors. Multiple approaches are employed to find these factors including alignments to factors known to be involved in the prokaryotic translational apparatus, such as LepA, as well as promoter and import sequence analysis. ACKNOWLEDGEMENTS I cannot faithfully say that I have done this on my own. There is one person who, without their guidance, this work would have never been possible: my advisor, mentor and friend Linda Spremulli. She is a beacon of fortitude and kindness that I will always admire and respect. Words cannot express my thanks to her for guiding me these past five years. I hope to live up to the potential she sees in me and make her proud through my accomplishments in science and life. Of course one cannot escape their genes so my parents deserve a lot of the credit for turning me into the curious creature that I am today. I love them and my family very much. And last I have been blessed to have been surrounded by amazing and beautiful people. I’d like to thank those who have stood by me, believed in me and loved me. Time and distance can only separate me physically from the people I care for because I will always hold those joyful memories shared between us close to my heart. TABLE OF CONTENTS LIST OF TABLES .................................................................................................................. vii LIST OF FIGURES ............................................................................................................... viii ABBREVIATIONS ...................................................................................................................x Chapter I. MECHANISM OF PROTEIN SYNTHESIS ..........................................................1 INTRODUCTION ...................................................................................................2 PROKARYOTIC PROTEIN SYNTHESIS .............................................................4 EUKARYOTIC PROTEIN SYNTHESIS .............................................................10 MITOCHONDRIAL PROTEIN SYNTHESIS .....................................................14 REFERENCES ......................................................................................................20 II. STRUCTURAL AND BIOINFORMATIC STUDIES OF MITOCHONDRIAL RIBOSOMES ........................................................................................................23 INTRODUCTION .................................................................................................24 MATERIALS AND METHODS ...........................................................................35 RESULTS ..............................................................................................................39 DISCUSSION ........................................................................................................46 REFERENCES ......................................................................................................48 III. ANALYSIS OF THE ROLES OF THE NKXD MOTIF AND THE INSERTION DOMAIN IN MAMMALIAN MITOCHONDRIAL INITIATION FACTOR 2..50 INTRODUCTION .................................................................................................51 vi MATERIALS AND METHODS ...........................................................................60 RESULTS ..............................................................................................................69 DISCUSSION ........................................................................................................79 REFERENCES ......................................................................................................83 IV. AUXILIARY TRANSLATIONAL FACTORS OF MAMMALIAN MITOCHONDRIAL RIBOSOMES ......................................................................87 INTRODUCTION .................................................................................................88 MATERIALS AND METHODS ...........................................................................92 RESULTS ..............................................................................................................96 DISCUSSION ......................................................................................................109 REFERENCES ....................................................................................................111 vii LIST OF TABLES Table 1-1. Comparison of the E. coli and Mammalian Mitochondrial Translational Machinery ..............................................................................................................16 1-2. Further Comparison of the E. coli and Mammalian Mitochondrial Translational Machinery .............................................................................................................19 2-1. The Primers Used to Created MRPL13, MPRL15, MRPS2 and MRPS11 ...........36 2-2. Preliminary Analysis of the 45S SSU* Particle LC/MS/MS Peptide Hits ............43 2-3. Comparison of Leishmania, Yeast and Human Mitochondrial and Cytoplasmic Ribosomal Proteins to that of Eubacteria...............................................................45 3-1. Primers Used to Created the NKXD Variants, IF2mtΔ37 and EcoIF2::37 ...........62 4-1. Conservation of Bacterial Translational Factors....................................................90 4-2. Mitochondrial Import and Promoter Analysis of Various Factors ......................103 viii LIST OF FIGURES Figure 1-1. Placement of tRNAs on the SSU and the Peptide Exit Channel ..............................3 1-2. Overview of the Steps of Initiation in Prokaryotes ..................................................6 1-3. Overview of the Elongation Cycle in Prokaryotes ..................................................8 1-4. Overview of Termination and Recycling in Prokaryotes ........................................9 1-5. Overview of Eukaryotic Initiation .........................................................................12 1-6. Overview of Elongation and Termination in Eukaryotes ......................................13 1-7. The Mammalian Mitochondrial Genome...............................................................15 2-1. The Prokaryotic LSU and SSU ..............................................................................26 2-2. The Secondary and Tertiary Structure of Prokaryotic rRNA ................................27 2-3. The Mammalian Mitochondrial Ribosome ............................................................29 2-4. The Mammalian Mitochondrial rRNA ..................................................................30 2-5. Structure and Isolation of Leishmania tarentolae Mitochondrial Ribosomes .......33 2-6. The Secondary Structure of Leishmania tarentolae SSU 9S rRNA ......................34 2-7. Modeling of the Mammalian Mitochondrial Proteins for Accessibility ................40 2-8. Expression and Western Analysis of MRP Proteins and Antibodies ....................41 3-1. Domain Organization and Structure of IF2 ...........................................................52 3-2. The
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