Yale University EliScholar – A Digital Platform for Scholarly Publishing at Yale Yale Medicine Thesis Digital Library School of Medicine Spring 5-2010 RNA-dependent selenocysteine biosynthesis in eukaryotes and Archaea Sotiria Palioura Yale University. Follow this and additional works at: http://elischolar.library.yale.edu/ymtdl Part of the Medicine and Health Sciences Commons Recommended Citation Palioura, Sotiria, "RNA-dependent selenocysteine biosynthesis in eukaryotes and Archaea" (2010). Yale Medicine Thesis Digital Library. 2253. http://elischolar.library.yale.edu/ymtdl/2253 This Open Access Dissertation is brought to you for free and open access by the School of Medicine at EliScholar – A Digital Platform for Scholarly Publishing at Yale. It has been accepted for inclusion in Yale Medicine Thesis Digital Library by an authorized administrator of EliScholar – A Digital Platform for Scholarly Publishing at Yale. For more information, please contact [email protected]. RNA-dependent Selenocysteine Biosynthesis in Eukaryotes and Archaea A Dissertation Presented to the Faculty of the Graduate School of Yale University in Candidacy for the Degree of Doctor of Philosophy by Sotiria Palioura Dissertation Director: Dieter G. Soil May 2010 Abstract RNA-dependent Selenocysteine Biosynthesis in Eukaryotes and Archaea Sotiria Palioura Yale University 2010 Selenocysteine (Sec), the 21st genetically encoded amino acid, is the major metabolite of the micronutrient selenium. Sec is inserted into nascent proteins in response to a UGA codon. The substrate for ribosomal protein synthesis is selenocysteinyl-tRNASec. While the formation of Sec-tRNASec from seryl-tRNA5^ by a single bacterial enzyme selenocysteine synthase (SelA) has been well described, the mechanism of Sec-tRNASec formation in archaea and eukaryotes remained poorly understood. Herein, biochemical and genetic data provide evidence that, in contrast to bacteria, eukaryotes and archaea utilize a different route to Sec-tRNASec that requires the tRNASec-dependent conversion of O-phosphoserine (Sep) to Sec. In this two-step pathway, O-phosphoseryl-tRNA kinase (PSTK) first converts Ser-tRNASec to Sep-tRNASec. This misacylated tRNA is the obligatory precursor for a Sep-tRNA: Sec-tRNA synthase (SepSecS); this protein was previously annotated as Soluble Liver Antigen/Liver Pancreas (SLA/LP). SepSecS genes from Homo sapiens, the lower eukaryote Trypanosoma brucei and the archaea Methanocaldococcus jannaschii and Methanococcus maripaludis complement an Escherichia coli AselA deletion strain in vivo. Furthermore, genetic analysis of selenoprotein biosynthesis in T. brucei in vivo demonstrated that eukaryotes have a single pathway to Sec-tRNASec that requires Sep-tRNASec as an intermediate. Finally, purified recombinant SepSecS converts Sep-tRNASec into Sec-tRNASec in vitro in the presence of sodium selenite and purified E. coli selenophosphate synthetase. The final step in Sec biosynthesis was further investigated by a structure-based mutational analysis of the M. maripaludis SepSecS and by determining the crystal structure of human SepSecS complexed with tRNAScc, phosphoserine and thiophosphate at 2.8 A resolution. In vivo and in vitro enzyme assays support a mechanism of Sec- tRNASec formation based on pyridoxal phosphate, while the lack of active site cysteines demonstrates that a perselenide intermediate is not involved in SepSecS-catalyzed Sec formation. Two tRNA5" molecules, with a fold distinct from other canonical tRNAs, bind to each human SepSecS tetramer through their unique 13 base-pair acceptor-T^C arm. The tRNA binding induces a conformational change in the enzyme's active site that allows a Sep covalently attached to tRNA5", but not free Sep, to be oriented properly for the reaction to occur. ACKNOWLEDGEMENTS This dissertation completes an 11-year cycle at Yale University since I first joined Yale College in August 1999. A completed dissertation, though, cannot be and is not the work of one person alone. It is rather, a collective effort of a group of individuals that includes advisors, co-authors, family, friends, relatives, and colleagues amongst others. To all the people that supported me at various points during this process, I am and will always remain grateful. First and foremost, I would like to thank my PhD advisor, Dieter Soil, for his never-ending encouragement and support since my first experience in his laboratory as an undergraduate in 2001.1 feel very privileged to have trained in science under Dieter's guidance and inspiration. Dieter has always been extremely generous in sharing his insight and knowledge with me not only about science but also about life. Being always humble, he has taught me the elegance of research, the joy of discovering a new insight, and the true ethos of a scholar. Although my road to this first «Ithaca » has not been very long, it was certainly « full of adventure, full of knowledge ». I primarily owe this to Dieter and I aspire to be an equally good mentor in the future as the example he has set for me. I would like to thank my research committee, Susan Baserga and Gary Brudvig, for their patience, support, insightful critiques and thoughtful guidance. I would also like to thank the director of the MD/PhD program at Yale University School of Medicine, James Jamieson, for his time, advice and shrewd guidance. The first part of this work was done in collaboration with Jing Yuan, whom I thank for her patience and help all along. The trypanosomal work was done in collaboration with Eric Aeby in the laboratory of Andre Schneider (University of Bern, Bern, Switzerland) and Elisabetta Ullu (Yale University, New Haven, CT), whom I thank for introducing me to trypanosomal biochemistry. The archaeal SepSecS structure was determined by Yuhei Araiso in the laboratory of Osamu Nureki (University of Tokyo, Tokyo, Japan). I thank Miljan Simonovic for introducing me to X-ray crystallography and for his guidance and support during the last part of my thesis work. I would also like to thank Tom Steitz for his insight into the human SepSecS-tRNA^0 complex structure. I am indebted to my fellow colleagues in the Soil lab, Anselm Sauerwald, Juan v Salazar, Alex Ambrogelly, Lynn Sherrer, Markus Englert, Michael Hohn, Patrick O'Donoghue, Theodoros Rampias, Dan Su and Kelly Sheppard, and to Gregor Blaha, Axel Innis and Bill Eliason in the Steitz lab for making this process an educational and enjoyable one. Outside the lab, I especially would like to thank the circle of my closest friends - Ioannis Ioannou, Roula Tavoulari, Amanda Psyrri, Nasos Roussias, Zoe Cournia, Antonios Finitsis, Tasos Gogakos, Dimitrios Zattas, Haralampos Stratigopoulos and Michael Gousgounis - who have been by my side every single time I needed them without thinking twice. They have been my closest "family" in the United States. I also sincerely thank all the great friends that I made here, in New Haven, for their help and support: Dario Englot, Eric Huebner, Mike Martinez, Yanni Hatzaras, George Galanis, Spyros Alogoskoufis, Olia Galenianou, Marios Panayides, Daniela Donno-Panayides, Andira Hernandez, Eleni Gousgounis, Irene Hatzidimitriou, Nikos Angelopoulos, Nicola Santoro, Ebe D'Adamo, Flavius Schackert, Mihalis Maniatakos and Antonis Stamboulis. Lastly and most importantly, I would like to thank my family, and in particular, my father Dimitris, my mother Elisavet, and my sister Vassiliki, as well as my uncle Manthos and my aunt Zoe, for their patience throughout the years, for believing in me since day one, and without whom I could have never made it this far. I am thankful and humbled to be surrounded by all these amazing people and I can only hope that each and every one of them will continue to support and challenge me in the years ahead. VI To my beloved sister, Vassiliki Vll TABLE OF CONTENTS Page ACKNOWLEDGEMENTS v PUBLICATIONS x LIST OF FIGURES xi LIST OF ABBREVIATIONS xiii INTRODUCTION 1 A. Selenocysteine, a versatile amino acid 1 B. Selenocysteine, the 21st genetically encoded amino acid 4 C. Selenocysteine is the universal exception to Crick's paradigm 6 D. Biosynthesis of selenocysteine and UGA recoding in bacteria 9 E. Selenocysteine biosynthesis in eukaryotes and archaea: the last riddle in aminoacyl- tRNA synthesis 10 F. Scope of Dissertation 15 1. Solving the riddle of selenocysteine formation in eukaryotes and archaea 15 2. The PSTK/SepSecS pathway is the only route to Sec in Trypanosomes, and likely in all eukaryotes 16 3. Insights into archaeal Sec synthesis from the structure of M. maripaludis SepSecS....17 4. The mechanism of tRNASec recognition and Sec formation by the human SepSecS..19 MATERIALS AND METHODS 21 A. General 21 B. Bacterial strains and plasmids 21 C. In vivo SepSecS assay 23 D. Metabolic labeling with [75Se]selenite 24 E. Protein expression and purification 24 F. Preparation and purification of tRNA gene transcripts 25 G. Purification and crystallization of the SepSecS-tRNA^ complex 27 H. Ligand soaks and data collection 27 I. Preparation of 32P-labeled Sep-tRNASec 28 J. In vitro conversion of Sep-tRNASec to Cys-tRNA^ 29 RESULTS 32 A. The missing step of selenocysteine biosynthesis in archaea and eukaryotes 32 vm 1. SepSecS rescues selenoprotein biosynthesis in an E. coli AselA deletion strain 32 2. SepSecS converts Sep-tRNASec to Sec-tRNASec in vitro 34 B. In vivo evidence for a single pathway of Sec-tRNA^ formation in T. brucei 36 C. Structural insights into archaeal RNA-dependent selenocysteine biosynthesis 40 1. Experimental approach 40 2. Overall structure of SepSecS from Methanococcus maripaludis 40 3. Archaeal SepSecS is active only as a tetramer 41 4. Active site mutants define PLP recognition 45 5. The active site 52 D. The human SepSecS-tRNA^ complex 55 1. SepSecS can bind tRNASec only as a tetramer 55 2. The antigenic region 61 3. The unique 9/4 fold of tRNASec is recognized by the Sec-specific proteins 63 4. Sep binds to the active site only in the presence of tRNAScc 67 5.