MOLECULAR RECOGNITION OF INHIBITORS, METAL IONS AND SUBSTRATES BY RIBONUCLEASE P by Xin Liu A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy (Chemistry) in the University of Michigan 2013 Doctoral Committee: Professor Carol A. Fierke, Chair Professor David R. Engelke Professor George A. Garcia Professor Anna K. Mapp Professor Nils G. Walter © Xin Liu 2013 DEDICATION To Lei Shi (石磊) Lei, you believe in me and listen to my wildest dreams. You bring out the best in me. Thank you for your endless love, patience and support! 愛你的馨! ii ACKNOWLEDGEMENTS This work would not be possible without support from many mentors, friends and family members. First of all, I want to thank my advisor Professor Carol Fierke for her great guidance and enormous patience for the past few years. Without her encouragement and critical suggestions, I would have not been able to learn such a great deal of experimental and analytical methods and to finish this dissertation. Carol has set a role model for me as an extraordinary scientist and inspiring mentor that I hope to learn from in my future careers. I would also like to thank my committee members who have been patient and helped me in many different ways. Prof. David Engelke helped me to think and communicate my research more clearly during the monthly RNase P meetings. Prof. Ann Mapp allowed me to use the TECAN instruments in her laboratory and wrote letters for supporting my various fellowship applications. I have learned about many different research projects when attending the Midwest enzyme mechanism conference organized by Prof. George Garcia. I have also used the HPLC instrument and received help from many members in Prof. Nils Walter’s laboratory (Matt Marek, Mario Blanco, Visha Krishnan and Sethu Pitchaiya). I want to acknowledge all past and current members of the Fierke lab for being such wonderful and supportive colleagues. Dr. Daina Zeng showed me the basic experimental procedures for handling RNA samples and measuring RNase P iii activity. Dr. John Hsieh has been answering all my questions about RNase P over the years. Dr. Wan Lim is a great friend and provided many insightful discussions. Dr. Elaina Zverina has been a great friend since my first day of class in graduate school at Michigan and offered me numerous help in adjusting to a foreign country and gave me many excellent suggestions for my experiments. Yu Chen is a colleague, friend but also a mentor who inspired me in improving efficiency and communications in my research. I want to thank Andrea Stoddard for providing her expertise in molecular biology, gracious support and encouragement to me over the years. I also thank Elia Wright for offering me experimental help as well as work-life balance strategies. Thanks also go to Michael Howard who taught me valuable tricks in protein purification and has been a great source of inspiration. I would like to thank Noah Wolfson for his support and discussions about research, science and life. I want to acknowledge all members of the RNase P subgroup for insightful discussions during the monthly meetings (Prof. Carol Fierke, Prof. David Engelke, Dr. John Hsieh, Dr. Nathan Zahler, Dr. Kristin Koutmos, Dr. Scott Walker, Dr. Michael Marvin, Dr. Wan Lim, Yu Chen, Micheal Howard, and Bradley Klemm). I also thank Dr. Manoj Cheriyan, Dr. James Hougland and Dr. Terry Watt for their help in my early years in the lab. Special thanks go to Paul Lin and Justin Liedel whom are the first two undergraduate students I have worked with. They have done great experiments and inspired me in many ways. The work presented in each of my dissertation chapters was conducted not only by me, but also by many collaborators. I want to also list my acknowledgement to my collaborators and source of funding. For Chapter 2, I thank Dr. Lyra Chang, iv Professor Jason Gestwicki, Professor Anna Mapp, Dr. Elaina Zverina, Dr. John Hsieh, Dr. Daina Zeng and Yu Chen for help with the development of this assay. I want to thank Dr. Jonas Hojfeldt, Dr. Chinmay Majmudar and Dr. Ningkun Wang (Mapp lab) for sharing their experience with fluorescence polarization and high- throughput screening techniques. I also acknowledge the help of Martha Larsen, Steven Swaney and Paul Kirchhoff at the Center for Chemical Genetics at University of Michigan with the compound library screen and data mining. I want to thank two internal grants that partially made the high-throughput screening project possible: the Rackham Graduate Student Research Grant and a Pilot Screening Grant from the Life Science Institute at University of Michigan. For Chapter 3, I want to thank Yu Chen, Dr. Katja Petzold and Prof. Hashim Al-Hashimi for their insightful discussions on the dynamics and structure studies of P4 helix. For Chapter 4, I want to specially thank Paul Lin for performing molecular cloning experiments for subcloning human mt-pre-tRNA genes and for his general help in preparing cell cultures, growth media and plates in the laboratory. I would like to thank Andrea Stoddard and Elia Wright for their help in growing HEK293 cells. The initial subcloning, purification and activity measurements of each human mtRNase P proteins were accomplished by Dr. Wan Lim and I also thank her for sharing regents and useful ideas during discussions. I would like to thank Michael Howard for help with protein purifications. Dr. Elaina Zverina provided valuable suggestions for purification of MRPP3 and experimental assistance in purification of MRPP1∙MRPP2 complex. Thanks also go to Dr. Lubomir Dostal who performed ICP-MS experiments. I want to thank Yueyang Zhong (Ruotolo lab) for her passion and expertise in the IM-MS experiments. I would also v like to thank Department of Chemistry for a Research Excellence Award, which funded me for one winter semester when a large part of this work was undertaken. Special thanks go to who have critically read my thesis chapters (Prof. Carol Fierke, Yu Chen, Carol Ann Pitcairn, Elia Wright, Nancy Wu, Andrea Stoddard, Byung Chul Kim and Michael Howard). Last but not least, millions of thanks go to my dear friends and families whom I have not yet mentioned above. I must thank Dr. Tracy Xiao Liu for bringing happiness in my life and for being understanding and caring to me all these years. Dr. Lyra Chang is my role model as a scientist and a dear friend whom I can trust even without much talking between us. Dr. Wenjing Chen taught me optimism and resilience. Dr. Jing Chen, Dr. Chenxi Shen, Dr. Penny Bo Peng and Dr. Liuling Gong also took care of me during hard times and taught me accountability. Dr. Yipei Wang showed me importance of efficiency in work and in life. I also want to thank Dr. Boyangzi Li for her encouragement and inspiration for pursuing life’s dream courageously. I also thank Guangming Lang for inspiring me with wisdom in Buddhism. I want to express my utmost gratitude to my parents, who raised me up with unconditional love and support. Thanks go to my parents-in-laws who also gave me endless love. Finally, I thank my husband Lei Shi. I could not get to this point without his love, support and help. Thank you all! vi TABLE OF CONTENTS DEDICATION............................................................................................................. ii ACKNOWLEDGEMENTS ........................................................................................ iii LIST OF FIGURES ................................................................................................. xiv LIST OF TABLES ................................................................................................... xix ABSTRACT ............................................................................................................. xx CHAPTER 1 INTRODUCTION: MOLECULAR RECOGNITION BY RNASE P ........ 1 Introduction ...................................................................................................... 1 RNase P Enzymes: Essential yet Diverse ....................................................... 6 1.2.1 Ribonucleoprotein (RNP) RNase P .......................................................... 6 1.2.2 RNase P without RNA ............................................................................ 15 1.2.3 RNase P as a Drug Target ..................................................................... 19 Catalytic Mechanism ...................................................................................... 23 1.3.1 Catalysis and Role of Divalent Metal Ions .............................................. 23 1.3.2 Identification of Metal Binding Sites in RNP RNase P ............................ 25 Substrate Recognition ................................................................................... 30 1.4.1 Structural Features of tRNA ................................................................... 30 1.4.2 Sequence-specific Interactions by Bacterial RNase P ............................ 31 vii 1.4.3 Substrate Specificity ............................................................................... 34 Conclusion ..................................................................................................... 39 Objectives of this Research ........................................................................... 39 References .................................................................................................... 41 CHAPTER 2 DEVELOPMENT OF A FLUORESCENCE POLARIZATION ASSAY TO MEASURE ACTIVITY AND SCREEN FOR INHIBITORS OF B. SUBTILIS RNASE P ................................................................................................................