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Functional and Biophysical Characterization of Trypanosoma Brucei Pentatricopeptide Repeat Proteins

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Functional and Biophysical Characterization of Trypanosoma Brucei Pentatricopeptide Repeat Proteins FUNCTIONAL AND BIOPHYSICAL CHARACTERIZATION OF TRYPANOSOMA BRUCEI PENTATRICOPEPTIDE REPEAT PROTEINS By Pakoyo Fadhiru Kamba A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of Cell and Molecular Biology – Doctor of Philosophy 2013 ABSTRACT FUNCTIONAL AND BIOPHYSICAL CHARACTERIZATION OF TRYPANOSOMA BRUCEI PENTATRICOPEPTIDE REPEAT PROTEINS By Pakoyo Fadhiru Kamba Shuttling of Trypanosoma brucei between mammalian hosts and the arthropod vector is aided by a tunable mitochondrion which downregulates its activity in the blood stream and upregulates it in the arthropod gut via differential expression of its genes. Trypanosomes, however, are deficient in transcription factors and regulate gene expression posttranscriptionally via transcript stability, differential processing, and translation. It is hypothesized that a milieu of RNA binding proteins are involved, most of which are still putative. In this thesis, I investigated the function and mode of action of T. brucei pentatricopeptide repeat (PPR) proteins, a family of α-helical, organellar RNA binding proteins characterized by tandem repeats of 35 amino acids. I had three aims: (i) to elucidate the RNA binding specificity of two T. brucei PPR proteins, one with a molecular mass of 27 kDa (PPR27) and another with a molecular mass of 41 kDa (PPR41); (ii) to understand the contribution of each PPR motif to PPR27-RNA binding; (iii) to develop suitable methods for production of concentrated, monodisperse, PPR proteins for structural studies. For all aims, recombinant protein was produced in Escherichia coli and used for in vitro experiments. First, the RNA binding specificity of PPR27 was determined. We found that PPR27 selectively binds single-stranded polyguanosine RNA quadruplexes over other sequences and nucleic acid forms. PPR27 binding did not disrupt the quadruplex structure commonly adopted by poly-G sequences. There is selective enrichment of G-tracts in maxicircle genes for extensively edited mRNAs, suggesting recognition of G-rich RNA was biologically relevant. A pull-down assay identified uncleaved RNA precursors as biological ligands. An association between PPR27 and the small mitoribosomal subunit was recently reported. Hence, PPR27 may aid crosstalk between pre-mRNA processing and translation. Next, the RNA binding activity and solubility of PPR27 deprived of one or more PPR motifs was analyzed. Deletion of PPR motifs modestly reduced the affinity of PPR27 for RNA, and RNA binding still occurred with only two intact PPR motifs. Except for the construct with only two PPR motifs, which suffered a 7-fold drop in RNA binding affinity for G12 ssRNA, the RNA binding Kd of truncated variants ranged between 2.5- and 3.5-fold that of wild type. Thus, all the PPR modules in PPR27 are seemingly involved in RNA contacts. Finally, screening of truncated PPR27 variants identified one soluble enough to attain structural biology concentrations. Thirdly, the RNA binding specificity of PPR41 was studied. Under non-equilibrium conditions, stable binding was only observed with poly(G) ssRNA. Under equilibrium conditions PPR41 showed strong and similar affinities for G12, U12, and (GGU)4, modest affinity for A12, and reduced affinity for C12 ssRNAs. PPR41 pulled down precursor RNA transcripts from a mitochondrial RNA extract. RNA editing inserts U’s into G-tracts. A role for PPR41 in either edited RNA binding or precursor RNA processing is therefore likely. Lastly, I probed the application to PPR proteins of some production systems which have previously been successful with some difficult proteins. I found that wild type PPR27 can be purified and refolded from inclusion bodies of its thioredoxin fusion protein, and showed that chimeras of PPR proteins with large solubility tags have promise for NMR spectroscopic and crystallization studies. Copyright by PAKOYO FADHIRU KAMBA 2013 DEDICATION To those who brought me into this world (Mom Ruth, Dad Sowali), all those who gave me a chance and made me smile, particularly my grandparents (Jessica Baluka and Late Silvesti Lodoi), my late senior uncles (John Dumba and Abubaker Mwanga), my late aunties (Sumba Agnes, Hadija Twalo, and Kanifa Katooko), and all those who believe in me. v ACKNOWLEDGEMENTS A generation has passed since I started this journey as a naive 5 year old unaware of what was at stake, at Kanginima Primary School in January 1983. Such a long adventure cannot be without numerous bumps. Therefore, this dream could not have come true without the love of my family, relatives, friends, teachers, employers, and the Almighty who either cared or taught me or lifted me up or celebrated with me or stood with me or fought with me in both happy and sad times. The last 5 years have been very challenging. I therefore thank my Graduate Advisor, Prof. Charles Hoogstraten for pulling me through and for giving me a chance in his group even when I seemed so naive. You invested in a very unpredictable startup, and I at least count you as one person I can lean on for a boost to start a high risk enterprise! I am also very thankful to Prof. John Wang, Prof. Honggao Yan, Prof. Donna Koslowsky, Prof. James Geiger, and Prof. Jennifer Ekstrom for their service on my Graduate Guidance Committee, and for counseling me and showing me the right scientific path in order to overcome what was a very daunting task. Prof. Koslowsky’s advice, resources, and time were very critical in the success of my RNA work, and I am very thankful. Prof. Ekstrom was fundamental in training me on experimental protein molecular biology and I am also thankful. Because of Prof. Geiger’s time, resources, and courtesy, I gained essential experience with protein crystallization. Prof. Wang’s sharp thinking was essential in integrating the numerous ideas which evolved out of my Committee and I am thankful. From the outset, Prof. Yan knew how difficult the project I had embarked on was and his counsel shaped our thinking on how a PhD thesis could be produced from such a project. Whenever Science floors you, your closest ones take the flack. I am therefore not only proud of my wife, Sikola Kamba Nakolantya, my daughters Vanessa Twalo Sumba and Melissa Asio vi Ruth, and my son Jason Madingi Kamba for their perseverance, I also apologize to them for the numerous times I turned my back on them in order to bring PPR proteins to order. My mother (Ruth Mary Asio Lodoi), my grandmother (Jessica Baluka), my late grandfather (Silvesti Lodoi), my mother’s sisters and brothers, and their loved ones, my special teachers (Rose Apia, Mr. Talima, Mr.Lipoto, Mr. Muzeyi, Keith Obot, Salim Nabiso, Robert Biibi), Uncle Peter Ojakol, and Aunt Florence Kayendeke have been very fundamental to my life and I am thankful. Some special friends in East Lansing have been fundamental to my success on this challenge. The CMB Program Directors (Prof. Susan Conrad and Prof. Kathy Meek) and the Graduate Secretaries have been very nice people and have always made sure the Program supports me financially whenever in need. I am thankful to Prof. James Kelly of MSU’s Crop Science Department, whom I met on an airplane, for supporting me financially, socially, and logistically, including welcoming me and my family into his family. I am grateful to Prof. Michael Boivin of MSU’s CHM (& his wife, Grace), Dr. Andrew Muganga Kizito (& his wife, Julie), Dr. Joel Lwande, Patrick Ochieng, David Achilla, and Dennis Katuuramu for helping me navigate life in MI. And, I thank the U.S embassy in Kampala and the Fulbright Scholarship Commission for taking a chance on me, and Dorothy Ngalombi of the Public Affairs unit at the Kampala U.S embassy for being such a caring person. And, I thank the following for research support: Dr. Alice Barkan (University of Oregon) for the pMALTEV-E30 plasmid gift; Dr. Lisa Lapidus for the fluorimeter and CD facilities as well as for advice on FRET; Dr. Shelagh Ferguson-Miller for sonicator, thermocycler, and crystallization robot; the MSU DOE-PRL for the fluorescence imager; and Mr Kermit Johnson/Dr. Daniel Holmes for NMR support. Finally, former colleagues in the Hoogstraten Lab who taught me some research techniques (Dr. Minako Sumita, Kristine Julien, and Dr. James Johnson Jr.) or worked with me (David Dickson) are greatly thanked. vii TABLE OF CONTENTS LIST OF TABLES ......................................................................................................................... xi LIST OF FIGURES ..................................................................................................................... xiii KEY TO ABBREVIATIONS ..................................................................................................... xvii CHAPTER 1 ................................................................................................................................... 1 REVIEW OF TRYPANOSOME BIOLOGY AND PENTATRICOPEPTIDE REPEAT PROTEINS ..................................................................................................................................... 1 1.1 BACKGROUND ................................................................................................................... 2 1.1.1 Research goal and outline of thesis ................................................................................ 2 1.1.2 Overview of T. brucei and trypanosomes ....................................................................... 2 1.1.3 T. brucei causes severe morbidity and mortality ...........................................................
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