Trypanosoma Brucei Trna Editing Deaminase: Conserved Deaminase Core, Unique Deaminase Features
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Trypanosoma brucei tRNA Editing Deaminase: Conserved Deaminase Core, Unique Deaminase Features DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Jessica Lynn Spears Graduate Program in Microbiology The Ohio State University 2011 Dissertation Committee: Dr. Juan Alfonzo, Advisor Dr. Michael Ibba Dr. Chad Rappleye Dr. Venkat Gopalan Copyright by Jessica Lynn Spears 2011 Abstract Inosine, a guanosine analog, has been known to function in transfer RNAs (tRNAs) for decades. When inosine occurs at the wobble position of the tRNA, it functionally expands the decoding capability of a single tRNA because inosine can base pair with cytosine, adenosine, and uridine. Because inosine is not genomically encoded, essential enzyme(s) are responsible for deaminating adenosine to inosine by a conserved zinc-mediated hydrolytic deamination mechanism. Collectively called ADATs (Adenosine deaminase acting on tRNA), these enzymes are heterodimeric in eukaryotes and are comprised of subunits called ADAT2 and ADAT3. ADAT2 is presumed to be the catalytic subunit while ADAT3 is thought to be just a structural component. Although these enzymes are essential for cell viability and their products (inosine-containing tRNAs) have a direct effect on translation, little is known about ADAT2/3. Questions such as what is ADAT3’s role in enzyme activity, how many zinc ions are coordinated, how many tRNAs are bound per heterodimer per catalytic cycle and what is the nature of the tRNA binding domain were all open questions until this work. The focus of chapter two is on the specific contributions of each subunit to catalysis. Steady state kinetic measurements with a series of ADAT2/3 mutants show that ADAT3 contributes directly to catalysis via participation in inter-subunit zinc coordination. A molecular model is presented that is corroborated by ICP (inductively coupled plasma) studies which further show that unlike other multimeric deaminase, one ii zinc ion is necessary and sufficient for deaminase activity. This effectively means that ADAT2/3 has one complete active site and a pseudo-active site (which is not complete because of a naturally missing catalytic glutamate). Furthermore, electrophoresis mobility shift assays (EMSAs) show a predicted 1:1 stoichiometry of tRNA: ADAT2/3 heterodimer. In chapter three, the focus shifts from the catalytic site and moves to the tRNA binding domain(s). In silico work predicted an RNA binding domain away from the active site and at the C-terminus of ADAT2 (KR-domain). A combination of enzyme kinetics and EMSAs show that not only are the positively charged arginines and lysines critical for substrate binding but also the pseudo-active site is critical for binding. Moreover, these two binding sites work cooperatively to bind and position a single tRNA for catalysis. The final study presented in chapter four examines inosine formation away from 1 the anticodon. In archaeal tRNAs, A57 in the TΨC loop is first methylated (m A57) by the 1 SAM-dependent TrmI and then deaminated (m I57) by an unknown enzyme(s). Using a combination of bioinformatics and protein purification via column chromatography, progress was made towards the final goal of identifying the enzyme responsible for m1A to m1I activity. In summary this dissertation presents interesting findings with respect to tRNA editing deaminases and fills important data gaps including new idea about the active site works and how protein binds its tRNA substrate. iii Acknowledgments I would like to acknowledge and thank first and foremost my advisor, Juan “Sir” Alfonzo, without whom most of the work presented herein would not have been possible. His constant guidance, life lessons, and invaluable discussions are appreciated and will be greatly missed. He demands nothing less than excellence from himself and those around him and I am a stronger person and scientist because of it. And of course there are no words to express the deepest of gratitude to the wonderful Mary Anne Rubio, to whom I will be forever indebted for becoming my Columbus mom, assisting in technical challenges and keeping the lab a functional and (mostly) sane place to be. I am also very thankful to have had a very thoughtful dissertation committee. Their suggestions, advice, and willingness to help me succeed will not soon be forgotten. To all of my labmates (especially Zdenek Paris, Kirk Gaston, Ashlie Tseng, and Jessica Wohlgamuth-Benedum) who made working long hours in a windowless building enjoyable, I thank you. To all of my labmates (namely Zdenek, Paul Sample, and Ian Fleming) who have and will continue to take the lab to higher heights, it has been a pleasure working with all of you. I also owe a huge thank you to one of my dearest friends, Michael Carter. He has been a very valuable sounding board for many ideas, scientific and otherwise. I would also like to acknowledge my parents Donna and Steve Spears (Dee Dee and Sparky) and my sister, Nicole Brei, who have been supportive not just during my graduate studies but in all my life endeavors. They perhaps still don’t quite understand iv what a Ph.D. is or why tRNA editing is important but they never stopped believing in me or reminding me that I could do it when I wanted to just give up. And last but certainly not least I would like to acknowledge my very loving and supportive partner, Adriane Brown. She not only had to bear the brunt of my moodiness and stress during my dissertation process but also had to do it while teaching and writing her own dissertation. She has been a constant source of love and support through it all and to her I will be forever grateful. v Vita 2005 .................................................... B.S. Biology, University of Wisconsin-Eau Claire 2005 to present .................................. Graduate Teaching and Research Associate, Department of Microbiology, The Ohio State University Publications Peer-reviewed journal articles Spears, JL, Rubio, MA, Gaston, KW, Wywial, E, Strikoudis, A, Papavasiliou, FN, Bujnicki, JM, and Alfonzo, JD. (2011) One Zinc ion is sufficient to create an active Trypanosoma brucei heterodimeric tRNA editing deaminase. JBC (Apr 20 Epub ahead of print). Ragone, F*, Spears, JL*, Wohlgamuth, JM, Kreel, N, Rubio, MA, Papavasiliou, FN and Alfonzo, JD. (2011) The C-terminal end of T. brucei adenosine deaminase acting on tRNA plays a critical role in tRNA binding and editing. RNA (In Press). *These authors contributed equally to the work. Gaston, KW, Rubio, MA, Spears, JL, Pastar, I, Papavasiliou, FN, and Alfonzo, JD. (2007) C to U editing at position 32 of the anticodon loop precedes tRNA 5’ leader removal in trypanosomatids. Nucleic Acid Res. 35(20): 6740-6749. Book Chapters Spears, JL, Rubio, MA, Sample, PJ, and Alfonzo JD. (2011) Working title: tRNA biogenesis and processing in Trypanosome review (In press). Spears, JL, Gaston, KW, and Alfonzo JD. (2011) Analysis of tRNA Editing in Native and Synthetic Substrates. Methods Mol Biol. 2011; 718: 209-26. vi Fields of Study Major Field: Microbiology vii Table of Contents Page Abstract ......................................................................................................................... ii Acknowledgements ...................................................................................................... iv Vita ............................................................................................................................... vi Publications .................................................................................................................. vi Field of Study .............................................................................................................. vii List of Tables ................................................................................................................ xi List of Figures .............................................................................................................. xii Chapters: 1. Introduction 1.1 Brief introduction to tyrpanosomes .............................................................. 1 1.2 Medical significance of trypanosomes ......................................................... 2 1.3 tRNA processing in trypansomes ................................................................ 3 1.3.1 Trimming of 5’ and 3’ ends to generate full length tRNA ............... 5 1.3.2 Intron removal .............................................................................. 6 1.3.3 tRNA export to the cytoplasm and mitochondrial import ................ 6 1.4 RNA editing ................................................................................................. 7 1.4.1 Insertion/deletion editing ............................................................... 8 1.4.2 Nucleotide substitution editing by deamination ............................. 9 1.4.3 mRNA editing: APOBEC-1 ......................................................... 11 1.4.4 mRNA editing: ADAR1 and ADAR2 ............................................ 13 1.4.5 tRNA editing ............................................................................... 13 1.4.5.1 C to U tRNA editing ...................................................... 14 1.4.5.2 A to I tRNA editing........................................................ 17 2. A Single Zinc Ion is Sufficient for an Active Trypanosoma brucei tRNA Editing Enzyme: Insights into the Function of Multimeric Deaminases 2.1 Introduction