Purdue University Purdue e-Pubs Open Access Dissertations Theses and Dissertations January 2016 Ribozymes of the RNA World: Investigating Ancient Enzymes through Modern Self-Cleaving Ribozymes. Aamir Mir Purdue University Follow this and additional works at: https://docs.lib.purdue.edu/open_access_dissertations Recommended Citation Mir, Aamir, "Ribozymes of the RNA World: Investigating Ancient Enzymes through Modern Self-Cleaving Ribozymes." (2016). Open Access Dissertations. 1465. https://docs.lib.purdue.edu/open_access_dissertations/1465 This document has been made available through Purdue e-Pubs, a service of the Purdue University Libraries. Please contact [email protected] for additional information. Graduate School Form 30 Updated PURDUE UNIVERSITY GRADUATE SCHOOL Thesis/Dissertation Acceptance This is to certify that the thesis/dissertation prepared By Aamir Mir Entitled Ribozymes of the RNA World: Investigating Ancient Enzymes through Modern Self-Cleaving Ribozymes. For the degree of Doctor of Philosophy Is approved by the final examining committee: Barbara L. Golden Chair Daisuke Kihara Frederick S. Gimble Elizabeth J. Tran To the best of my knowledge and as understood by the student in the Thesis/Dissertation Agreement, Publication Delay, and Certification Disclaimer (Graduate School Form 32), this thesis/dissertation adheres to the provisions of Purdue University’s “Policy of Integrity in Research” and the use of copyright material. Approved by Major Professor(s): Barbara L. Golden Approved by: Andrew D. Mesecar 11/29/2016 Head of the Departmental Graduate Program Date RIBOZYMES OF THE RNA WORLD: INVESTIGATING ANCIENT ENZYMES THROUGH MODERN SELF-CLEAVING RIBOZYMES A Dissertation Submitted to the Faculty of Purdue University by Aamir Mir In Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy December 2016 Purdue University West Lafayette, Indiana ii To my mother iii ACKNOWLEDGEMENTS There are many people who I would like to acknowledge who helped me get here. First, I am very grateful to my advisor, Dr. Barbara Golden for patiently guiding me through the ups and downs of my graduate school. Without her support and guidance, none of this would have been possible. I am thankful to her for helping me with research, publications, writing of this thesis, and for other opportunities she provided me with to further my career as a scientist. I would also like to thank the rest of my thesis committee members, Dr. Elizabeth Tran, Dr. Frederick Gimble, and Dr. Daisuke Kihara, for their critical feedback and support during my graduate career. I am also grateful to the Tran lab members (past and present) for their helpful comments, and help with research. I am especially grateful to Dr. Ji Chen for his help with lab work, and for insightful discussions. Other graduate students of the Golden lab have also been instrumental in my success including Samantha Lee and Rui Gan. I am also thankful to my undergraduate mentees Jaclyn Goodman, Emma Lendy, Kyle Robinson, and others for their contributions to my thesis project. Lastly, I would like to acknowledge the support of my friends and family during my Ph.D. iv TABLE OF CONTENTS Page LIST OF TABLES .................................................................................................................. viii LIST OF FIGURES .................................................................................................................. ix LIST OF ABBREVIATIONS .................................................................................................... xii ABSTRACT .......................................................................................................................... xiii CHAPTER 1. THE RNA WORLD HYPOTHESIS .................................................................. 1 1.1 Perspective ................................................................................................................ 1 1.2 Making a case for the existence of the RNA world based on observations from the modern world ..................................................................................................................... 2 1.3 Emergence of the early RNA – from prebiotic soup to an RNA polymer .................. 4 1.4 Beginnings of Natural Selection - non-enzymatic RNA replication systems ............. 7 1.5 The ribozymes of the RNA world – lessons from in-vitro evolution studies ............. 9 1.5.1 RNA Polymerases ........................................................................................ 10 1.5.2 RNA processing ribozymes (ligation and cleavage) .................................... 11 1.5.3 Other ribozymes ......................................................................................... 13 1.6 Role of compartmentalization in the RNA world .................................................... 16 1.7 Objectives of this work ............................................................................................ 18 CHAPTER 2. CATALYTIC STRATEGIES USED BY RIBOZYMES IN THE MODERN WORLD 19 2.1 Perspective .............................................................................................................. 19 2.2 Splicing ribozymes – group I introns, group II introns, and the spliceosome ......... 20 2.3 Peptidyl transferase ribozyme – the ribosome ....................................................... 28 2.4 Branching ribozyme (lariat capping) – the GIR1 or LC ribozyme ............................ 32 2.5 Hydrolase ribozyme (trans-cleaving) – Ribonuclease P .......................................... 35 v 2.6 Self-cleaving ribozymes ........................................................................................... 39 2.6.1 Self-cleaving ribozymes derived from viral RNAs ....................................... 42 2.6.1.1 The hairpin ribozyme .............................................................................. 42 2.6.1.2 The HDV ribozyme ................................................................................... 45 2.6.1.3 The VS ribozyme ...................................................................................... 49 2.6.1.4 Twister and twister sister ribozymes ...................................................... 52 2.6.1.5 The pistol ribozyme ................................................................................. 56 2.6.1.6 The hatchet ribozyme ............................................................................. 58 2.6.2 Self-cleaving ribozymes found in pre-mRNAs ............................................. 59 2.6.2.1 The glmS ribozyme .................................................................................. 62 2.6.2.2 Other self-cleaving ribozymes: CPEB3, Vg1, CoTc, and CLEC2 ribozymes 65 2.7 Lessons from existing natural ribozymes, and the need to explore more ............. 68 CHAPTER 3. TWO DIVALENT METALS IONS AND CONFORMATIONAL CHANGES PLAY ROLES IN THE HAMMERHEAD RIBOZYME CLEAVAGE REACTION ..................................... 73 3.1 Declaration of collaborative work ........................................................................... 73 3.2 Introduction ............................................................................................................. 74 3.3 Materials and Methods ........................................................................................... 80 3.3.1 RNA Synthesis ............................................................................................. 80 3.3.2 RNA Crystallization ...................................................................................... 80 3.3.3 Structure determination and refinement ................................................... 82 3.3.4 Kinetic Assays .............................................................................................. 82 3.4 Results ..................................................................................................................... 84 3.4.1 Crystal structure of RzB hammerhead ribozyme ........................................ 84 3.4.2 The G12A mutation is disruptive to catalysis. ............................................ 92 3.4.3 A pH-dependent conformational switch in the hammerhead ribozyme active site ................................................................................................................... 96 3.4.4 The G12A mutation exhibits a metal ion specificity switch. ....................... 99 3.4.5 A Zn2+ ion binding pocket exists near A12 ................................................ 102 vi 3.4.6 There is a divalent metal ion binding pocket at G10.1 ............................. 103 3.5 Discussion .............................................................................................................. 104 3.5.1 The curious case of G12 ............................................................................ 105 3.5.2 G12 positions an active site metal ion ...................................................... 107 3.5.3 Zn2+ could induce tautomerization of A12................................................ 110 3.5.4 Active site rearrangements during the hammerhead ribozyme cleavage reaction. 113 3.6 Conclusions: a new strategy in RNA catalysis ....................................................... 115 CHAPTER 4. TWO ACTIVE SITE DIVALENT IONS IN THE CRYSTAL STRUCTURE OF THE HAMMERHEAD RIBOZYME BOUND TO A TRANSITION STATE ANALOGUE .................... 117 4.1 Declaration of collaborative work ........................................................................