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Uncovering the Identity and Metabolism of Bacterial Coa-RNA The University of Southern Mississippi The Aquila Digital Community Dissertations Spring 5-2017 Uncovering the Identity and Metabolism of Bacterial CoA-RNA Joseph R. Spangler University of Southern Mississippi Follow this and additional works at: https://aquila.usm.edu/dissertations Part of the Biochemistry Commons, Cell Biology Commons, and the Molecular Biology Commons Recommended Citation Spangler, Joseph R., "Uncovering the Identity and Metabolism of Bacterial CoA-RNA" (2017). Dissertations. 890. https://aquila.usm.edu/dissertations/890 This Dissertation is brought to you for free and open access by The Aquila Digital Community. It has been accepted for inclusion in Dissertations by an authorized administrator of The Aquila Digital Community. For more information, please contact [email protected]. UNCOVERING THE IDENTITY AND METABOLISM OF BACTERIA COA-RNA by Joseph Rankin Spangler A Dissertation Submitted to the Graduate School and the Department of Chemistry and Biochemistry at The University of Southern Mississippi in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy Approved: _________________________________________ Dr. Faqing Huang, Committee Chair Professor, Chemistry and Biochemistry _________________________________________ Dr. Sabine Heinhorst, Committee Member Professor, Chemistry and Biochemistry _________________________________________ Dr. Vijayaraghavan Rangachari, Committee Member Associate Professor, Chemistry and Biochemistry _________________________________________ Dr. Douglas Masterson, Committee Member Associate Professor, Chemistry and Biochemistry _________________________________________ Dr. YanLin Guo, Committee Member Professor, Biological Sciences _________________________________________ Dr. Karen S. Coats Dean of the Graduate School May 2017 COPYRIGHT BY Joseph Rankin Spangler 2017 Published by the Graduate School ABSTRACT UNCOVERING THE IDENTITY AND METABOLISM OF BACTERIA COA-RNA by Joseph Rankin Spangler May 2017 Coenzyme A is an indispensable molecule in all known life with roles in metabolism, gene regulation, and macromolecule synthesis. As CoA is derived from RNA itself, it’s incorporation into RNA by in vitro methods has proven useful in research probing the origin of life based on the RNA World theory. The discovery in contemporary bacteria of RNA modified with CoA, however, provided an unexpected twist to previously well-characterized bacterial systems. The identity of sequences associated with CoA-RNA has been elusive since their discovery in 2009 based on the difficulties in isolation while maintaining RNA quality. The aim of this study is to develop a specific CoA-RNA capture protocol to uncover the sequence identities for the further understanding of CoA-RNA function. While the free thiol of CoA can be used to purify the CoA-RNA from total bacterial RNA, the success of process depends on the specific release from the purification matrix. This work details the characterization of recombinant Nudix CoA pyrophosphohydrolases from various organisms for their use in CoA hydrolysis and CoA-RNA metabolism, as well as for the development and successful implementation of a specific CoA-RNA capture protocol. The use of this protocol for the construction of high quality NGS libraries is vital to the elucidation of CoA-RNA function via bioinformatics analysis, for its potential ii ranges from a simple stabilization adduct to contemporary examples of RNA World era coenzyme-conjugated ribozymes. iii ACKNOWLEDGMENTS This work was made possible by the ongoing communication and openness of the Department of Chemistry and Biochemistry, especially the Rangachari, Heinhorst, and Guo labs. I would like to convey special thanks to symbolize my deep gratitude to my advisor, Dr. Faqing Huang, whose patience was as unending as his inspirational drive to obtain the best results from any situation. iv DEDICATION This work is dedicated to my wife and family, whose patience and support were unquestioning and absolute. v TABLE OF CONTENTS ABSTRACT ...........................................................................................................ii ACKNOWLEDGMENTS .......................................................................................iv DEDICATION ....................................................................................................... v LIST OF TABLES ................................................................................................. x LIST OF ILLUSTRATIONS ...................................................................................xi LIST OF SCHEMES ........................................................................................... xiii LIST OF ABBREVIATIONS ................................................................................ xiv CHAPTER I – INTRODUCTION ........................................................................... 1 1.1 RNA ............................................................................................................ 1 1.1.1 Origin of Life ......................................................................................... 1 1.1.2 Contemporary RNA Functions .............................................................. 4 1.2 Coenzymes ............................................................................................... 13 1.2.1 Origin and Functions .......................................................................... 13 1.2.2 CoA .................................................................................................... 16 1.3 The Nudix Superfamily .............................................................................. 22 1.3.1 Structure and Functions ..................................................................... 22 1.3.2 Coenzyme Pyrophosphohydrolases ................................................... 24 1.4 Coenzyme-RNA Conjugates ..................................................................... 34 1.4.1 In Vitro Conjugates ............................................................................. 34 vi 1.4.2 In Vivo Conjugates ............................................................................. 41 CHAPTER II - OBJECTIVES OF RESEARCH ................................................... 54 CHAPTER III – MATERIALS AND METHODS ................................................... 60 3.1 Instrumentation and Equipment ................................................................ 60 3.2 Chemicals, Reagents, and Kits ................................................................. 61 3.3 Buffers and Commonly Used Reagents .................................................... 65 3.4 Experimental Procedures .......................................................................... 76 3.4.1 Polymerase Chain Reaction ............................................................... 76 3.4.2 Preparation of Cloning Fragments ...................................................... 76 3.4.3 Transformation ................................................................................... 79 3.4.4 Colony PCR, Plasmid Analysis, and Culture Handling ....................... 79 3.4.5 Small Scale Protein Expression and Solubility Assay......................... 81 3.4.6 Large Scale Protein Expression and Cell Lysate Preparation ............ 82 3.4.7 Protein Purification ............................................................................. 83 3.4.8 Enzyme Activity Assays ...................................................................... 84 3.4.9 RNA and CoA-RNA Preparation ......................................................... 85 3.4.10 CoA Conjugation to Resin ................................................................ 87 3.4.11 CoA Release Assays ........................................................................ 87 3.4.12 RNA Ligation and Reverse Transcription ......................................... 88 3.4.13 CoA-RNA Capture Protocol .............................................................. 89 vii 3.5 DNA and RNA Oligos ................................................................................ 91 3.6 Cell Lines and Genotypes ......................................................................... 94 CHAPTER IV – THE CLONING AND ACTIVITY OF NUDIX COA PYROPHOSPHOHYDROLASES ....................................................................... 96 4.1 Introduction ............................................................................................... 96 4.2 Results and Discussion ............................................................................. 98 4.2.1 C. Elegans NDX8 ............................................................................... 98 4.2.1.1 Cloning of NDX8 .......................................................................... 98 4.2.1.2 Cloning and Expression of SUMO-NDX8 ................................... 104 4.2.1.3 Cloning and Expression of TrxA-NDX8 ...................................... 109 4.2.2 M. musculus Nudt7 ........................................................................... 113 4.2.2.1 Cloning and Expression of SUMO-Nudt7 ................................... 113 4.2.2.2 Nudt7 Activity ............................................................................. 115 4.2.3 E. coli NudC ..................................................................................... 120 4.2.3.1 Cloning and Expression of MBP-NudC ...................................... 120 4.2.3.2 Cloning and Expression of SUMO-NudC ..................................
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