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Riboswitch-Targeted Drug Discovery: Investigation of Factors That Affect the T Box

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Riboswitch-Targeted Drug Discovery: Investigation of Factors That Affect the T Box Riboswitch-targeted Drug Discovery: Investigation of Factors that Affect the T Box Transcription Antitermination Mechanism A dissertation presented to the faculty of the College of Arts and Sciences of Ohio University In partial fulfillment of the requirements for the degree Doctor of Philosophy Chunxi Zeng April 2016 © 2016 Chunxi Zeng. All Rights Reserved. 2 This dissertation titled Riboswitch-targeted Drug Discovery: Investigation of Factors that Affect the T Box Transcription Antitermination Mechanism by CHUNXI ZENG has been approved for the Department of Chemistry and Biochemistry and the College of Arts and Sciences by Jennifer V. Hines Professor of Chemistry and Biochemistry Robert Frank Dean, College of Arts and Sciences 3 ABSTRACT ZENG, CHUNXI, Ph.D., April 2016, Molecular and Cellular Biology Riboswitch-targeted Drug Discovery: Investigation of Factors that Affect the T Box Transcription Antitermination Mechanism Director of Dissertation: Jennifer V. Hines The T box riboswitch is a regulation mechanism at the level of transcription or translation which controls expression of amino acids related genes, including a lot of essential genes, in many bacteria. The T box riboswitch interacts with cognate tRNAs and senses their aminoacylation status. A charged cognate tRNA allows formation of the thermodynamically more stable terminator structure which induces transcription termination. An uncharged tRNA stabilizes the alternative antiterminator structure and prevents formation of the terminator. Transcription proceeds and leads to expression of the downstream gene(s). The T box riboswitch is a novel and promising drug target since multiple genes essential to bacterial survival are regulated by this mechanism in many pathogenic bacteria. In order to further study the T box riboswitch mechanism and screen a synthetic ligands library, a fluorescently monitored multi-round in vitro antitermination assay with an enhanced throughput was successfully developed and comprehensively evaluated. Using this assay, the effects of molecular crowding, spermidine and DMSO on the T box riboswitch function were studied and 304 ligands were screened. A total of nine ligands showed specific inhibition to the tRNA-induce antitermination. Combining melting 4 temperature analysis and structural probing, the binding of spermidine to the antiterminator was also characterized. 5 DEDICATION To my beautiful wife, Weijun Meng and my little angles, Molly and Max. 6 ACKNOWLEDGMENTS I would like to express my great gratitude to my advisor, Dr. Jennifer V. Hines. The enclosed research and this dissertation would not be possible without her essential guidance and support. I would like to thank Dr. Stephen C. Bergmeier and his lab members, Dr. George Acquaah-Harrison, Dr. Crina M. Orac and Dr. Weihe Zhang, Rumita Laha, Ian Armstrong, for providing all the synthetic ligands for this research. I would like to give special thanks to Dr. Xiaozhuo Chen for his support in the Molecular and Cellular Biology Program and his guidance when I encountered difficulties during coursework. I would like to thank Dr. Shiyong Wu for being my committee member and providing valuable insights for my research. I would like to thank Dr. Shu Zhou for teaching me many important lab skills when I first joined the lab. Her preliminary work also paved the way for this research. I would like to thank Vivian Hogan for her assistance to improve the tRNA purification method. I thank the Molecular and Cellular Biology Program and the Department of Chemistry and Biochemistry for providing me financial support. And I want to thank all the lab colleagues, Dr. Shu Zhou, Jia Liu, Ali Al-Dhumani, Vivian Hogan, Jacob Sieg for helpful research discussions. My great appreciation goes to my wife, Weijun Meng, who spent tremendous amount of time to take good care of our children and me. I also want to thank my parents and my parents-in-law for giving financial support to my family. 7 TABLE OF CONTENTS Page Abstract ............................................................................................................................... 3 Dedication ........................................................................................................................... 5 Acknowledgments............................................................................................................... 6 List of Tables .................................................................................................................... 11 List of Figures ................................................................................................................... 12 List of Abbreviations ........................................................................................................ 15 Chapter 1. Introduction ..................................................................................................... 18 1.1 RNA Structure......................................................................................................... 18 1.2 Folding of RNA ...................................................................................................... 18 1.2.1 Folding Kinetics ............................................................................................... 18 1.2.2 Folding Thermodynamics ................................................................................ 21 1.2.3 Factors Affecting RNA Folding ....................................................................... 22 1.3 Bacteria and Antibiotics .......................................................................................... 25 1.3.1 Pathogenic Bacteria.......................................................................................... 26 1.3.2 Current Antibiotics ........................................................................................... 26 1.4 Riboswitch .............................................................................................................. 31 1.4.1 Riboswitches in General .................................................................................. 31 1.4.2 Classification of Riboswitch ............................................................................ 32 1.4.3 Factors Affecting Riboswitch Function ........................................................... 33 1.4.4 Riboswitches as Drug Targets ......................................................................... 34 1.5 The T box Riboswitch ............................................................................................. 35 1.5.1 Discovery of the T Box Riboswitch ................................................................. 35 1.5.2 T box Riboswitch in Bacillus Subtilis glyQS Gene ......................................... 36 1.5.3 The Natural Ligand of The T box Riboswitch: tRNA ..................................... 45 1.5.4 T box Riboswitch as a Drug Target ................................................................. 47 1.5.5 Transcription Termination ............................................................................... 48 1.6 Principle of Experimental Methods ........................................................................ 50 1.6.1 Fluorescence Resonance Energy Transfer ....................................................... 50 8 1.6.2 Fluorescence Quenching .................................................................................. 51 1.6.3 Molecular Beacon ............................................................................................ 51 1.6.4 In Silico Prediction of Nucleic Acid Folding and Hybridization .................... 52 1.6.5 Fluorescence-monitored Thermal Denaturation Analysis ............................... 53 1.6.6 Radioisotopic Labeling of RNA by 32P............................................................ 54 1.6.7 Autoradiography .............................................................................................. 54 1.6.8 In-line Probing of RNA Structure .................................................................... 55 1.6.9 Enzymatic Probing of RNA Structure ............................................................. 56 1.6.10 EC50, IC50 and Kd ........................................................................................... 57 Chapter 2. Development of the Fluorescently-monitored Antitermination Assay ........... 59 2.1 Scheme of the Assay Mechanism ........................................................................... 59 2.2 Design of Probes ..................................................................................................... 60 2.2.1 Terminator Probes: TERprbRNA, TERprbDNA, TERprbCHIM .................................. 61 2.2.2 Readthrough Probe: RTprb ................................................................................ 63 2.2.3 Upstream Probe: UPprb ..................................................................................... 71 2.3 Evaluation and Optimization of the Assay ............................................................. 76 2.3.1 Melting Curves and Preliminary Tests of the Probes ...................................... 76 2.3.2 Structural Integrity and Fluorophore Photostability of the Probes................... 87 2.3.3 Target Sensitivity of the Probes ....................................................................... 88 2.3.4 Optimize RNAP Concentration ....................................................................... 91 2.3.5 Selection of the Background Control ..............................................................
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