View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by OPUS: Open Uleth Scholarship - University of Lethbridge Research Repository University of Lethbridge Research Repository OPUS http://opus.uleth.ca Theses Arts and Science, Faculty of 2013 Kinetic analysis of pseudouridine formation in tRNA by archaeal Cbf5 and Pus10 Kamalampeta, Rajashekhar Lethbridge, Alta. : University of Lethbridge, Dept. of Chemistry and Biochemistry http://hdl.handle.net/10133/3464 Downloaded from University of Lethbridge Research Repository, OPUS KINETIC ANALYSIS OF PSEUDOURIDINE FORMATION IN TRNA BY ARCHAEAL CBF5 AND PUS10 RAJASHEKHAR KAMALAMPETA M.Sc. University of Skövde, 2008 A Thesis Submitted to the School of Graduate Studies of the University of Lethbridge in Partial Fulfillment of the Requirements for the Degree DOCTOR OF PHILOSOPHY IN BIOMOLECULAR SCIENCE Department of Chemistry and Biochemistry University of Lethbridge LETHBRIDGE, ALBERTA, CANADA © Rajashekhar Kamalampeta, 2013 Dedicated to My parents for their unconditional love and support & Those passionate teachers for instilling my interest in science iii Abstract Pseudouridines (), the most common modifications in RNA, are formed by stand-alone synthases in all organisms. In addition, archaea and eukaryotes use H/ACA small ribonucleoproteins for pseudouridylation. Cbf5, the catalytic component of these complexes, can also introduce 55 in archaeal tRNAs in a guide RNA-independent manner. Here, kinetic and thermodynamic analyses revealed that both Pyrococcus furiosus Nop10 and Gar1 proteins enhance the catalytic ability of Cbf5 and increase its affinity for tRNA. Pus10, representing a novel synthase family, is the in vivo archaeal tRNA 55 synthase. Characterization of several Pus10 variants demonstrated the importance of the thumb loop for catalysis, a potential role of the THUMP domain in tRNA binding and a new catalytic arginine which may flip the target uridine into Pus10’s active site. The quantitative characterization of the archaeal pseudouridine synthases Cbf5 and Pus10 reported here sheds light on their cellular roles in RNA modification. iv Acknowledgements First and foremost, I would like to express my sincere gratitude to Dr. Ute Kothe, my supervisor, for her invaluable guidance, mentorship, encouragement, and constructive suggestions throughout my Ph.D. I wish to thank Dr. Marc Roussel, my co-supervisor, for his helpful advice and constant support. I would also like to sincerely thank my supervisory committee members Drs. Steven Mosimann and Elizabeth Schultz for their valuable suggestions and critical feedback. I would like to extend special thanks to Dr. Hans-Joachim Wieden for his supervision in the absence of my supervisor and for his critical advice on my projects. I would also like to extend my thanks to Dr. George Owttrim for accepting to be the external examiner, and to Dr. Paul Hayes for chairing the examination committee. I am grateful to all Kothe and Wieden lab members for making this journey a great learning experience. Special appreciation goes to Laura, Jenna, Evan Caton, Jessica, Jaden, Roxanne, Lisza, Alvin, Evan, Evelina, Jeff, Marion, Tobias, Fan, Kirsten, Mike, Lindsay and many other wonderful people for their critical discussions and continuous help during my thesis. I must also thank Andy for all his helpful conversations. I would also like to acknowledge all those brilliant students, Aruna, Chaitra, Christian, Dominik, Justin, Jenna, and Vanessa for assisting me in the preparatory work and for giving me a great opportunity to supervise them. Also thanks are due to the previous Kothe lab members who contributed to this project. v Special thanks goes to Susan and many people from the School of Graduate Studies for their help. Finally, I wish to thank my family and friends for their support and encouragement. I must not forget to thank my wife Malini for being there during all ups and downs and for contributing immensely to my success. vi Table of Contents Abstract ........................................................................................................................ iv Acknowledgements ........................................................................................................v Table of Contents ........................................................................................................ vii List of Tables .................................................................................................................x List of Figures .............................................................................................................. xi List of Abbreviations .................................................................................................. xii Chapter 1: Introduction ................................................................................................1 1.1 Ribosome biogenesis ..............................................................................................1 1.1.1 Organization of genes encoding ribosomal RNA and proteins ..........................2 1.1.2. Eukaryotic rRNA processing and ribosome assembly......................................3 1.2 RNA modifications ................................................................................................6 1.2.1 Brief introduction to RNA ...............................................................................6 1.2.2 Modifications in RNA and their significance....................................................7 1.2.3 Pseudouridine ................................................................................................ 11 1.2.3.1 Structure and important properties of pseudouridine ................................ 11 1.2.3.2 Distribution of in RNA and its biological significance ......................... 13 1.3 synthases .......................................................................................................... 17 1.3.1 Stand-alone synthases................................................................................. 17 1.3.1.1 synthase families and their structural organization ............................... 18 1.3.1.2 Substrate recognition and catalysis .......................................................... 22 1.3.2 H/ACA small ribonucleoprotein complex ...................................................... 26 1.3.2.1 H/ACA snoRNAs .................................................................................... 27 1.3.2.2 Structure and function of H/ACA sRNP complex .................................... 28 1.3.2.3 Eukaryotic H/ACA snoRNP biogenesis ................................................... 31 1.4 Objectives ............................................................................................................ 33 Chapter 2: tRNA pseudouridylation by archaeal Cbf5 and the contribution of Nop10 and Gar1........................................................................................................... 35 2.1 Introduction .......................................................................................................... 35 2.2 Methods ............................................................................................................... 38 vii 2.2.1 Buffers and reagents ...................................................................................... 38 2.2.2 Molecular cloning and mutagenesis ............................................................... 38 2.2.3 Protein expression and purification ................................................................ 40 2.2.4 In vitro transcription and purification of tRNA and H/ACA guide RNA ......... 42 2.2.5 Nitrocellulose Filtration ................................................................................. 43 2.2.6 Tritium Release Assay ................................................................................... 43 2.3 Results ................................................................................................................. 45 2.3.1 Optimal conditions required for pseudouridylation reactions .......................... 45 2.3.2 Multiple-turnover catalysis of tRNA modification by Cbf5 in absence and presence of Nop10 and Gar1 ................................................................................... 48 2.3.3 Steady-state kinetic analysis of tRNA modification by Cbf5 .......................... 50 2.3.4 Substrate binding by Cbf5 alone and in complex with Nop10 and/or Gar1 ..... 52 2.3.5 Single-turnover tRNA modification by Cbf5 and Cbf5 complexes ................. 56 2.3.6 Interaction of Cbf5 complexes with modified product tRNA and H/ACA guide RNA ....................................................................................................................... 58 2.4 Discussion ............................................................................................................ 60 Chapter 3: Structure-guided functional analysis of archaeal Pus10 in pseudouridine 55 formation in tRNA .................................................................................................. 67 3.1 Introduction .......................................................................................................... 67 3.2 Materials and Methods ......................................................................................... 72 3.2.1 Buffers and reagents ...................................................................................... 72 3.2.2 Molecular cloning and mutagenesis ..............................................................