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Structural Investigation of the Effect of Magnesium-Ion Binding On STRUCTURAL INVESTIGATION OF THE EFFECT OF MAGNESIUM-ION BINDING ON STEM-LOOP V OF THE NEUROSPORA VS RIBOZYME by DEAN O. CAMPBELL (Under the direction of Pascale Legault) ABSTRACT An essential step in the substrate recognition of the Neurospora VS ribozyme is the formation of a loop-loop interaction between the terminal loops of stem-loop I and stem-loop V. Site-specific substitution and mutagenesis have suggested that this interaction is facilitated by the presence of a U-turn in the loop of stem-loop V. We have determined the structure of stem-loop V by nuclear magnetic resonance (NMR) spectroscopy and show that it adopts a U-turn conformation, a common motif found in RNA. Structural comparisons indicate that the U-turn of stem-loop V (SL5) fulfills some but not all of the structural characteristics found in canonical U- turn motifs. The formation of the stem-loop I / stem-loop V interaction is magnesium-ion dependent. Chemical shift mapping indicated that the addition of magnesium ions to SL5 resulted in a conformational change in loop. We have determined the structure of SL5 in the presence of magnesium ions and show that the binding of magnesium ions to the loop causes a conformational change. This conformational change results in the U-turn motif of SL5 having more characteristics of a canonical U-turn motif in the presence of magnesium ions. We used paramagnetic line broadening effect of manganese ions to localize four divalent metal ion binding sites in the loop of stem-loop V. Three of these divalent metal ion binding sites are specific to the U-turn motifs and have been found in structures of other U-turn motifs. We also show that the U-turn conformation in stem-loop V allows the bases involved in the stem loop I / stem-loop V interaction (G697, A698, and C699) to be accessible, by exposing their Watson- Crick faces to the solvent. This study brings some light on the mechanism of substrate recognition in the VS ribozyme and expands our understanding of the role of U-turn motifs in RNA structure and function. INDEX WORDS: U-turn motif, Neurospora VS ribozyme, Magnesium-ion binding, Chemical-shift mapping, RNA structure. STRUCTURAL INVESTIGATION OF THE EFFECT OF MAGNESIUM-ION BINDING ON STEM-LOOP V OF THE NEUROSPORA VS RIBOZYME by DEAN O. CAMPBELL B.Sc., The University of the West Indies, Jamaica, 1998 A Dissertation Submitted to the Graduate Faculty of The University of Georgia in Partial Fulfillment of the Requirements for the Degree DOCTOR OF PHILOSOPHY ATHENS, GEORGIA 2004 © 2004 Dean O. Campbell All Rights Reserved STRUCTURAL INVESTIGATION OF THE EFFECT OF MAGNESIUM-ION BINDING ON STEM-LOOP V OF THE NEUROSPORA VS RIBOZYME by DEAN O. CAMPBELL Major Professor: Pascale Legault Committee: Sidney Kushner Kojo Mensa-Wilmot Michael Pierce Michael Terns Electronic Version Approved: Maureen Grasso Dean of the Graduate School The University of Georgia August 2004 DEDICATION This is dedicated to Beverly Dale iv ACKNOWLEDGEMENTS I would like to thank Drs. Bao Nguyen and Paola Di Lello for their many helpful tips and instruction. And also for making my stay in the “downstairs crew” a memorable one. Also to the members of the Leagult/Omichinksi labs, past and present for all the fun and excitement that allowed the years to go by so quickly. v TABLE OF CONTENTS Page ACKNOWLEDGEMENTS.................................................................………..................…..........v CHAPTER 1 LITERATURE REVIEW........................................................................................1 The Neurospora VS Ribozyme................................................................... 1 U-Turn Motifs in the VS Ribozyme ........................................................... 6 Metal-Ions Binding in the Neurospora VS Ribozyme ............................... 8 Study of RNA by NMR Spectroscopy...................................................... 10 Purpose of Study....................................................................................... 11 References................................................................................................. 20 2 RESONANCE ASSIGNMENT AND STRUCTURAL RESTRAINTS OF SL5...................................................................................................... 28 Introduction............................................................................................... 28 13 15 1 PART A: Resonance assignment of the C, N, and H chemical shifts of SL5 .............................................................................. 29 A.1 Atomic composition of stem-loop V (SL5) ........................... 29 A.2 Assignment procedure for SL5 .............................................. 29 vi PART B: Obtaining the structure calculation restraints for the structure calculation of SL5...................................................................... 45 B.1. Restraints for structure calculation........................................ 45 B.2 NOE distance restraints.......................................................... 45 B.3. Torsion angle restraints ......................................................... 50 B.4. Hydrogen bonds .................................................................... 54 References................................................................................................. 94 3 NMR STRUCTURE OF STEM-LOOP V OF THE VS RIBOZYME AND MAGNESIUM-ION BINDING FROM CHEMICAL-SHIFT MAPPING………………………………...................................………..98 Abstract..................................................................................................... 99 Introduction............................................................................................... 99 Materials and Methods............................................................................ 102 Results..................................................................................................... 108 Discussion............................................................................................... 118 Acknowledgement .................................................................................. 123 Figures and Tables .................................................................................. 124 References............................................................................................... 137 Supplementary Material.......................................................................... 147 4 NMR STRUCTURE OF THE VS RIBOZYME STEM-LOOP V IN THE PRESENCE OF MAGNESIUM IONS AND LOCALIZATION OF DIVALENT METAL ION BINDING SITES………………….............150 Abstract................................................................................................... 151 vii Introduction............................................................................................. 151 Materials and Methods............................................................................ 155 Results..................................................................................................... 161 Discussion............................................................................................... 172 Acknowledgement .................................................................................. 177 Figures and Tables .................................................................................. 178 References............................................................................................... 189 Supplementary Material.......................................................................... 197 5. CONCLUSIONS….……………………………………………………………202 Summary of study................................................................................... 202 Impact of research and future directions................................................. 203 References............................................................................................... 207 viii CHAPTER 1 LITERATURE REVIEW The Neurospora VS Ribozyme Ribozymes were discovered in the 1980s independently in the labs of Drs. Sydney Altman and Thomas R. Cech (1,2). Ribozymes are ribonucleic acid enzymes, and naturally-occurring ribozymes are capable of a variety of chemical reactions, most notably phosphoryl transfer reactions and peptide bond formation (3). Synthetic ribozymes have been developed that are capable of a wide range of chemical activity, such as aminoacylation, alkylation, and carbon- carbon bond formation (4,5). There are many types of naturally-occurring ribozymes including the self-splicing group I and group II introns (2,6), ribonuclease P (1), the hepatitis delta virus (7), hairpin (8), hammerhead (9), and the Neurospora VS ribozymes (10). The ribosome (11,12) is a ribonucleoprotein complex which has been postulated to have RNA-catalyzed chemistry. The hammerhead, hairpin, HDV and Neurospora VS ribozymes belong to the group of small self-cleaving nucleolytic ribozymes (13). The small nucleolytic ribozymes produce 2', 3'-cyclic phosphate and 5'-hydroxyl termini after cleaving their substrate (14,15). The Neurospora VS RNA was isolated from the Varkud Satellite (VS) plasmid of the Neurospora mitochondria in the laboratory Dr. Richard A. Collins at the University of Toronto (10). In the presence of divalent metal ions, the VS RNA is capable of site-specific cis- and 1 trans-cleavage and ligation reactions (in vitro and in vivo) (16,17). The physiological role of the VS RNA is that it is a step in the replication cycle of the double-stranded VS plasmid DNA. The double-stranded plasmid
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