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Structural Features Affecting the Cleavage Rate And STRUCTURAL FEATURES AFFECTING THE CLEAVAGE RATE AND MAGNESIUM OPTIMUM IN THE NEUROSPORA VS RIBOZYME Alan Hing-Lun Poon A thesis submitted in conformity with the requirements for the degree of Master of Science Graduate Department of Molecular and Medical Genetics University of Toronto O Copyright by Alan Hing-Lun Poon (2001) nie author has granteci a non- L'auteur a accordé une licence non exclusive licence aliowing the exclusive permettant ii la Natiod Library of Canada to BibliotMque nationale du Canada de reproduce, Io-, distnie or sefl reproduire, prêter, distnûuer ou copies of this thesis in microform, vendre des copies 6 cette thèse sous paper or electronic formats. la forme de microfiche/film, de reproduction sur papier ou sur format Clectronique. The author retains ownersbip of the L'auteur conserve la propriété du copyright in this thesis. Neikthe droit d'auteur qui protège cette thèse. thesis nor substantial extracts fkom it Ni la thèse ni des extraits substantiels may be printed or otherwise de celle-ci ne doivent être imprimés reproduced without the author's ou autrement reproduits sans son permission. autorisation. ABSTRACT Structural Fwhves Affécting the Cleavage Rate and Magnesium Optimum in the Neurospom VS Ribozyme Alan Hing-Lun Poon Master of Science, 2001 Department of Molecular and Medical Genetics, University of Toronto Varkud Satellite (VS) RNA catalyzes a magnesium-dependent, site-specific, self- cleavage reaction. It has been previousl y detemined that a base-paired region, called helix la, adjacent to the site of self-cleavage, is a structural element that limits the rate of self-cleavage. Mutants with a disrupted helix Ia not only display an increase in cleavage rate, but also are active at much lower concentrations of magnesium. By measuring the cleavage kinetics of a series of linker-insertion mutants with or without a disrupted helix Ia, 1 have been able to identify separate structural causes for the slow cleavage rate and high magnesium optimum of the wild-type RNA. The slow cleavage rate is due to a steric constraint imposed by the natural RNA sequence. The high magnesium optimum reflects the instability of an alternative secondary structure that must fom near the cleavage site dunng folding into the active conformation of the RNA. ACKNOWLEDGMENTS 1 extend my deepest thanks to Dr. Richard Collins for his advice, support and encouragement, and for making the last couple of years a great and memorable learning experience. In addition, 1 would like to thank my supervisory cornmittee members, Dr. Alan Cochrane and Dr. Barbara Funnell, as they have been very helpful in guiding and supporting me thmughout my project. 1 am also grateful to the members of the Collins lab, both past and present: Angela Andersen, Shawna Hiley, Jonathon Maguire, Joan Olive, Vanita Sood, Elisabeth Tillier and Ricardo Zarnel. Finally, 1 am deeply thankful to my wonderful parents, Ching-Po and Tai-Kwan, my sister Grace and brother Kevin, for their never-ending love, support and encouragement. iii TABLE OF CONTENTS TABLE OF CONTENTS. .. .. .. .. .. iv LIST OF FIGURES. .. .. .. .. , .. vi INTRODUCTION ............ .... .... ......... ................. ...... ....... ......... .,... 1 1. Role of Metal Ions in RNA Folding and Stability 3 II. Role of Metal Ions in RNA Catalysis 7 m.Neurospora VS Ribozyme 12 IV. Research Rationale 14 MATERIALS AND METHODS .. .. .. 17 1. DNA Cloning 17 II. Synthesis of RNA by ln Vitro Transcription For CIeavage Reactions 20 III. Measurement of Self-Cleavage Rate Constants 2 1 IV. Measurement of k,, and [M~'+]ln 23 VI. Chernical Modification Structure Probing DEPC modification DMS modification Flexible Single Stranded Linker Insertion Into GI1 - Longth Does Matter 26 Linker Insertion Mutants Have High Magnesiwn Optima Sirnilar 28 to that of GI 1 Disrupting HelLr la Lowers Magnesiurn Optimum 29 Mutationally Pre-Shijfing Helix Ib Lowen Magnesium Optimum 30 Chernical Modification Structure Probing of ANI6-Pre 32 DISCUSSSON ...... ..... ................ ................................................ .... 35 1. Linker insertion Increases Cleavage Rate 35 II. Dismption of Helix Ia Lowers Magnesium Optimum 4 t III. Re-Shifting Helix b Lowers Magnesium Optimum 44 REFERENCES. .. .. , . .. 64 LIST OF FIGüRJ3S Figure 1. Chemistry of Small Ribozyme Cleavage Figure 2. Secondary Structure Mode! of G11 in the Absence and Presence of Magnesium Figure 3. G1 I and H3 Figure 4. Secondary Structure Mode1 of Linker insertion Mutants Figure 5. Linker Insertion Mutants and Their Effect on Self-Cleavage Figure 6. Summary of Linker Insertion Mutants (25 mM M$+) Figure 7. Cleavage is Independent of Ribozyme Concentration Fipre 8. Increasing Linker Length Causes an Increase In Cleavage Rate With Little Effect on Magnesiurn Optimum Figure 9. Secondary Structure Model of AN 16-H3 Figure 10. Dismption of Helix Ia bersthe Magnesiurn Optimum With Little Effect on the Maximal Cleavage Rate Figure 1 1. Secondary Structure Model of Mutants With Constitutively Shifted Helix Ib (MCS) Figure 12. Mutationally Pre-Shifting Helix Ib Lowers the Magnesium Optimum With Little Effect on the Maximal Cleavage Rate Figure 13. DEPC and DMS Structure Probing of AN 16 Figure 14. Summary of k,, and mgt+]In Fipre 15. Alternative Representation of the Secondary Structure of O1 1 Figure 16. Model of Putative Coaxial Stacking of Helix Ia on Helix Il INTRODUCTION --- For the longest tirne, proteins were thought of as the only molecules capable of efficiently catalyzing chernical reactions. Then in the 1980's, the revolutionary discovery of catalytic RNAs in the laboratories of Sidney Altman and Thomas R. Cech changed this scientific view (Guemer-Takada et al., 1983; Kruger et al., 1982). These findings indicated that, in addition to carrying genetic information, RNA molecules were also capable of catalyzing phosphodiester bond cleavage in the absence of proteins. These findings made previous proposals of an "RNA world", with RNA king the ancestral molecule that gave rise to both DNA and proteins, more plausible (Gesteland and Atkins, 1993). Since the initiai discovery of catalytic RNAs, better known as ribozymes, a number of naturally occumng ribozyme motifs have been discovered In addition, in vitro evolution methods (Lorsch and Szostak, 1994; Tsang and Joyce, 1996) have been very successhil in generating new ribozymes. Artificial ribozymes, which now exceed the number of known natural ribozymes, have significantly extended the number of chemical reactions thought previously to be only performed by proteins. These chemical reactions include nucleophilic attack at phosphoryl, carbonyl and alkyl halide centers, as well as metdation and isomenzation react ions (reviewed by Jaeger 1997). Seven nanually occumng catalytic RNAs have ken identified to date and they have been divided into two groups according to size: large and small. Group 1 and II introns and ribonuclease P (RNase P) are grouped together as the "large ribozymes" because they are several hundred nucleotides in length and more stnicturally complex than the small ribozymes. Secondly, al1 three ribozymes cleave RNA to generate 5' phosphate and 3' hydroxyl termini (reviewed by Cech 1993). The hammerhead, hepatitis delta virus (HDV), hairpin and Neurospom Varkud satellite (VS) ribozyme comprise the "small - -- ribozymes". These RNAs are between 50-150 nucleotides and are found in viral, virusoid, or satellite RNA genomes where they process the products of rolling circle replication into genome-length strands (Branch and Robertson, 1984). The general mechanism of these ribozymes is a 2' oxygen nucleophile attacking the adjacent phosphate in the RNA backbone, resulting in cleavage products with 2'.3'-cyclic phosphate and 5' hydroxyl termini (Figure 1 ; Symons, 1992). Just like the protein-folding problem, scientists have ken searching for rules to predict the threedimensional structures of RNA from their primary sequences. By cornparison, RNA folding is much simpler since only four nucleotides, each made of a base, a ribose, and a phosphate, are used as building blocks of the structure. Nevertheless, little is known about the relationship between structure and function. Although secondary structures have ken elucidated for many of the known ribozymes, less is known about the interactions goveming tertiary structure. Ribozymes have therefore proven to be powerfbl tools in studying RNA structure-function relationships and RNA folding since functionally important changes in structure can often be detected by changes in cataiytic properties. As a result. much effort has ken focused towards elucidating elements of RNA structure that exist in ribozymes as well as in how these structural elements contribute to the catalytic function of the ribozyme. in general, an RNA molecule can be thought of as possessing a hierarchical structure in which the pnmary sequence determines the secondas, structure, which. in mm, determines its tertiary folding, whose formation alters the secondary structure only marginally (Conn and Draper, 1998). The pnmary structure of each ribonucleic acid is - characterized by the sequence of its four nucleosides: adenosine (A), guanosine (G), cytidine (C) and uridine (U). The folding of the RNA chain, depending on its sumundings, produces its secondary and tertiary structure. Short range base pairing establishes the secondary structure of RNA while long range interactions detemine elements of tertiary structure. There are four basic secondary stmcture elements in RNA and include helices,
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