BIOSYNTHESIS AND PHYSIOLOGICAL ROLE OF ARCHAEOSINE IN THE EXTREME HALOPHILIC ARCHAEON Haloferax volcanii By GABRIELA PHILLIPS A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2011 1 © 2011 Gabriela Phillips 2 To my husband for his love, understanding, patience 3 ACKNOWLEDGMENTS Abundant gratitude belongs to Dr. Valerie de Crécy-Lagard for supervision, support, encouragement throughout all the years we worked together in the field. Her knowledgeable and valuable input stimulated this dissertation from preliminary levels to actualization. I would sincerely like to thank my committee members, James Preston, Nemat Keyhani, Claudio Gonzalez, Nigel Richards for their support, time, and helpful insights that helped me become a better prepared scholar in the field. I would like to express my deep and sincere gratitude to Basma el Yacoubi for her helpful teachings, discussions, understanding; her precious support helped me enormously to cope with the difficulties of my doctoral studies. I am grateful to Marc Bailly for insightful discussions and for developing a better procedure for bulk tRNA extraction and purification as well as setting up the protocol for extraction and purification of E. coli tRNAAsp. I am especially indebted to Sophie Alvarez (Danforth Plant Science Center, Proteomics and Mass Spectrometry Facility, St. Louis, MO.) for her LC-MS/MS analysis on bulk tRNA. I also want to thank to Kirk Gaston (Pat A. Limbach Research Group University of Cincinnati) for his prompt E. coli tRNAAsp sequencing and analysis. I am grateful to Dr. Julie Maupin-Furlow (MCB, UF) for the H. volcanii H26 and H. salinarum NRC-1 strains. I also thank her for H. volcanii expression plasmid pJAM202; without it, I would not have been able to perform all the H. volcanii phenotype complementation tests. I will miss my coworkers Crysten Haas, Ian Blaby, and Patrick Thiaville for helpful discussions. My undergraduate studies where directed by the advice of Dr. Madeline Rasche who introduced me to my first serious scientific experiments and believed in my 4 scholastic abilities. Finally, I need to thank my family for unceasing support and patience. I would not have completed this task without their love and understanding. 5 TABLE OF CONTENTS page ACKNOWLEDGMENTS .................................................................................................. 4 LIST OF TABLES ............................................................................................................ 9 LIST OF FIGURES ........................................................................................................ 10 LIST OF ABBREVIATIONS ........................................................................................... 13 ABSTRACT ................................................................................................................... 17 CHAPTER 1 tRNA BIOGENESIS IN ARCHAEA ......................................................................... 19 tRNA Role in Translation ........................................................................................ 19 tRNA Structure ........................................................................................................ 21 tRNA Processing .................................................................................................... 23 tRNA Processing in Archaea .................................................................................. 23 Overview of Archaea Domain ........................................................................... 23 Maturation of tRNA 5‟-end ................................................................................ 25 Maturation of tRNA 3‟-end ................................................................................ 28 Introns in Archaeal tRNA Transcripts ............................................................... 30 M. kandleri C-to-U tRNA Editing ....................................................................... 34 Posttranscriptional Modifications of tRNA Nucleosides .................................... 35 Agmatidine, a recently discovered tRNA modification essential for decoding ................................................................................................. 37 Wyosine derivatives biosynthesis pathways in Archaea ............................ 38 Modification of Adenosine to N1-methyladenosine to N1-methylinosine, an archaeal site specific modification ..................................................... 40 Guide RNA dependent modifications of tRNAs .......................................... 41 Archaeosine, an archaeal tRNA specific modification ................................ 43 2 MATERIAL AND METHODS .................................................................................. 54 Materials ................................................................................................................. 54 Bioinformatics Tools................................................................................................ 54 Three Dimensional (3D) Structure Superimposition and Visualization ............. 55 Strains, Media, Growth and Transformation ..................................................... 55 H. volcanii Competent Cells And Transformation Protocols ............................. 57 Competent cells ......................................................................................... 57 Transformation ........................................................................................... 57 Polymerase Chain Reaction ............................................................................. 58 DNA Electrophoresis ........................................................................................ 59 Plasmid Isolation and Transformation .............................................................. 59 6 Site-Directed Mutagenesis ............................................................................... 59 General Cloning ............................................................................................... 59 Plasmids and Strains Construction ................................................................... 60 Plasmids construction for bacterial complementation assays .................... 60 Plasmids construction for archaeal complementation assays .................... 61 Chromosomal gene deletions .................................................................... 61 Southern Blot .................................................................................................... 63 Functional Complementation Assays ............................................................... 64 Thymidine auxotrophy phenotype complementation .................................. 64 Queuosine deficient phenotype complementation ..................................... 64 Archaeosine deficient phenotype complementation ................................... 64 tRNA Work ....................................................................................................... 65 Bulk tRNA extraction .................................................................................. 65 tRNAAsp purification .................................................................................... 66 Bulk tRNA digestion for LC-MS/MS analysis .............................................. 66 tRNAAsp digestion ....................................................................................... 67 3 ARCHAEOSINE BIOSYNTHESIS IN H. volcanii .................................................... 68 Background ............................................................................................................. 68 Results .................................................................................................................... 70 In the Extreme Halophilic Archaeon H. volcanii, Archaeosine Is Not Essential for growth ....................................................................................... 70 HVO_2348, Encoding FolE2 Homolog, Is Involved in Both Folate and Archaeosine Biosynthesis ............................................................................. 72 HVO_1718, Encoding QueD Homolog, Is Involved in Archaeosine Biosynthesis. ................................................................................................. 75 HVO_1717, Encoding QueE Homolog, and HVO_1716, Encoding QueC Homolog, Are Involved in Archaeosine Biosynthesis .................................... 76 ArcS Is the Last Step in Archaeosine Biosynthesis in H. volcanii ..................... 77 Discussion .............................................................................................................. 79 4 ALTERNATIVE ARCHAEOSINE BIOSYNTHESIS ROUTES ................................. 96 Background ............................................................................................................. 96 Results .................................................................................................................... 96 In Some Crenarchaea, QueF-like Protein Catalyzes the Last Step in Archaeosine Biosynthesis. ............................................................................ 98 In Other Crenarchaea, GATII-QueC Protein Catalyzes the Last Step in Archaeosine Biosynthesis ............................................................................. 99 Bacterial Tgt Charges Archaeosine at Position 34 of tRNAAsp .......................