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In Vitro Analysis of the Self-Cleaving Satellite RNA of Barley Yellow Dwarf Virus Stanley Livingstone Silver Iowa State University

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In Vitro Analysis of the Self-Cleaving Satellite RNA of Barley Yellow Dwarf Virus Stanley Livingstone Silver Iowa State University Iowa State University Capstones, Theses and Retrospective Theses and Dissertations Dissertations 1993 In vitro analysis of the self-cleaving satellite RNA of barley yellow dwarf virus Stanley Livingstone Silver Iowa State University Follow this and additional works at: https://lib.dr.iastate.edu/rtd Part of the Biochemistry Commons, Molecular Biology Commons, and the Plant Pathology Commons Recommended Citation Silver, Stanley Livingstone, "In vitro analysis of the self-cleaving satellite RNA of barley yellow dwarf virus " (1993). Retrospective Theses and Dissertations. 10274. https://lib.dr.iastate.edu/rtd/10274 This Dissertation is brought to you for free and open access by the Iowa State University Capstones, Theses and Dissertations at Iowa State University Digital Repository. It has been accepted for inclusion in Retrospective Theses and Dissertations by an authorized administrator of Iowa State University Digital Repository. For more information, please contact [email protected]. _UMI MICROFILMED 1993 | INFORMATION TO USERS This manuscript has been reproduced from the microfilm master. UMI films the text directly from the original or copy submitted. Thus, some thesis and dissertation copies are in typewriter face, while others may be from any type of computer printer. The quality of this reproduction is dependent upon the quality of the copy submitted. Broken or indistinct print, colored or poor quality illustrations and photographs, print bleedthrough, substandard margins, and improper alignment can adversely affect reproduction. In the unlikely event that the author did not send UMI a complete manuscript and there are missing pages, these will be noted. Also, if unauthorized copyright material had to be removed, a note will indicate the deletion. Oversize materials (e.g., maps, drawings, charts) are reproduced by sectioning the original, begiiming at the upper left-hand corner and continuing from left to right in equal sections with small overlaps. Each original is also photographed in one exposure and is included in reduced form at the back of the book. Photographs included in the original manuscript have been reproduced xerographically in this copy. Higher quality 6" x 9" black and white photographic prints are available for any photographs or illustrations appearing in this copy for an additional charge. Contact UMI directly to order. University Microfilms International A Bell & Howell Information Company 300 North Zeeb Road, Ann Arbor, Ml 48106-1346 USA 313/761-4700 800/521-0600 Order Number 9335023 In vitro analysis of the self-cleaving satellite RNA of barley yellow dwarf virus Silver, Stanley Livingstone, Ph.D. Iowa State University, 1993 UMI 300 N. ZeebRd. Ann Arbor, MI 48106 In vitro analysis of the self-cleaving satellite RNA of barley yellow dwarf virus by Stanley Livingstone Silver A Dissertation Submitted to the Graduate Faculty in Partial Fulfillment of the Requirements for the Degree of DOCTOR OF PHILOSOPHY Department: Plant Pathology Interdepartmental Major; Molecular, Cellular, and Developmental Biology Approved: Signature was redacted for privacy. In Charge of Major Work Signature was redacted for privacy. or/the InterdeHartmental Major Signature was redacted for privacy. De^rtapiept Signature was redacted for privacy. For the dM^^Co^ege Iowa State University Ames, Iowa 1993 ii DEDICATION This dissertation is dedicated to my parents, Stewart and Jean Silver iii TABLE OF CONTENTS Page GENERAL INTRODUCTION 1 Group I Intron Splicing 1 Ribonuclease P; The First True RNA Enzyme 4 Group II Intron Splicing 6 Nuclear, Pre-mRNA Splicing 6 Small Catalytic RNAs 8 Reaction Mechanism 15 Target-specific Ribozymes 16 Barley Yellow Dwarf Virus 19 Satellite RNAs 20 Satellite RNA of Barley Yellow Dwarf Virus 2 6 Project Goals 27 Explanation of Dissertation Format 27 PAPER I. ALTERNATIVE TERTIARY STRUCTURE ATTENUATES SELF-CLEAVAGE OF THE RIBOZYME IN THE SATELLITE RNA OF BARLEY YELLOW DWARF VIRUS 3 0 ABSTRACT 32 INTRODUCTION 33 MATERIALS AND METHODS 38 Synthesis of wildtype and mutant self-cleavage structures 38 Self-cleavage assays 39 Synthesis of end-labeled RNA 41 Nuclease digestion 42 RESULTS 43 Self-cleavage of wildtype and mutant RNAs 43 Nuclease sensitivity 46 iv Page DISCUSSION 53 Effects of mutations on self-cleavage rate 53 Nuclease sensitivity 54 Comparison with other hammerheads 57 REFERENCES 61 PAPER II. NONCONSENSUS BASES IN THE sBYDV (+) RNA RNA AFFECT THE RATE OF HAMMERHEAD FORMATION AND SELF-CLEAVAGE EFFICIENCY 64 INTRODUCTION 66 MATERIALS AND METHODS 70 Synthesis of wildtype and mutant self-cleavage structures 70 Self-cleavage assays 72 Synthesis of end-labeled RNA and nuclease digestion 73 RESULTS 74 Self-cleavage of mutant RNAs 74 Nuclease sensitivity 79 DISCUSSION 84 Effects of mutations on self-cleavage rate 84 Nuclease sensitivity 86 Other hammerheads 88 REFERENCES 91 PAPER III. REPLICATION OF TRANSCRIPTS FROM A cDNA CLONE OF BARLEY YELLOW DWARF VIRUS SATELLITE RNA IN OAT PROTOPLASTS 94 ABSTRACT 96 INTRODUCTION 97 V Page Self-cleavage of dimeric sBYDV RNA 100 Dependence of sBYDV RNA replication on BYDV genomic RNA 100 Replication of both strands of sBYDV RNA by a rolling circle mechanism 105 REFERENCES 109 PAPER IV. BIMOLECULAR CLEAVAGE REACTION AND THE DESIGN OF TARGET-SPECIFIC RIBOZYMES 113 INTRODUCTION 115 MATERIALS AND METHODS 123 Construction of ribozymes 123 In vitro transcriptions and cleavage assays 126 RESULTS AND DISCUSSION 129 Bimolecular cleavage 129 Gene targeted ribozymes 132 REFERENCES 138 GENERAL SUMMARY 140 LITERATURE CITED 143 1 GENERAL INTRODUCTION A ribozyme is defined as an RNA molecule that has the inherent ability to catalyze cleavage or ligation of covalent bonds (Kruger et al., 1982). The term ribozyme was coined initially to describe the mechanism by which the intervening sequence (IVS) of Tetrahymena thermophilia processes the pre-rRNA to form mature rRNA. However, the IVS RNA is not truly an enzyme since it does not exhibit turnover ability or remain unchanged during the splicing reaction. Since the initial discovery of ribozymes in 1982, many other RNAs have been observed to exhibit autocatalysis. Such varied RNAs as Group II mitochondrial introns, pre- tRNAs, nuclear mRNA introns, several small, pathogenic RNAs, transcripts of Neurospora plasmid DNA, and transcript II from newt all demonstrate the capability of autocatalysis. Group I Intron Splicing Group I introns were the first RNA molecules demonstrated to have at least quasi-catalytic activity (Kruger et al., 1982). The splicing mechanism involves two transesterifications (Cech et al., 1981; Fig. 1). The first involves a free guanosine or a 5'- phosphorylated form of guanosine serving as the nucleophile that attacks the phosphorous atom at the 5' splice site, resulting in the 2 A. Group I intron splicing pQqh i Ujp —Gpi ? < UIOH + pQp—Qp[ Exon Intron Exon i V|P| k + pQp—QQH B. RNase P cleavage Excised intron - c{^ 5—J 1—3" 5- _0H J L_3' iWtRNA C. Group II intron self-splicing, nuclear pre-mRNA splicing r~ IpQU A PI J I I OH C3,^A^p[ A^p Lariat structure D. Self-cleaving, small pathogenic RNAs —p §• SL 3' A+ OH 2'-3'-cyclic phosphate Figure l. Four major types of RNA-catalyzed RNA cleavage. The rectangles with one jagged end represents a less-than- full- length exon. The pre-mRNA and Group II intron splicing mechanisms are shown together; even though both RNAs most likely use a different mechanism to form an efficient cleavage structure (Symons, 1991b). 3 formation of a 3' 5' phosphodiester bond between the nucleotide cofactor and the 5' end of the intron. The free 3' OH of exon 1 then attacks the 3' end splice site displacing the intron and ligating exon 1 and 2 (Cech, 1990). Efficient Group I intron splicing, in addition to the nucleotide cofactor, requires specific secondary and tertiary interactions within the RNA. The secondary structure consists of a complex of nine stem-loops collectively called the Michel-Davies model (Davies et al., 1982; Michel et al., 1982). Molecular modelling analysis, sequence comparisons, mutational analysis, and enzymatic probing have verified several of the proposed secondary features of the IVS RNA (Been & Cech, 1986; Cech, 1988; Ehrenmann et al., 1989). The tertiary structure is believed to form a cavity which facilitates GTP binding, whereas the primary structure of the IVS RNA confers RNA specificity via an internal guide sequence located near the splice site (Davies et al., 1982; Waring et al., 1986). In addition to RNA secondary and tertiary interactions, proteins called mRNA maturases enhance RNA splicing in Neurospora crassi (Garriga & Lambowitz, 1986) and yeast mitochondria (Lazowaska et al., 1980). Finally, the IVS RNA also undergoes a secondary reaction that involves the cleavage of 19 nucleotides from the 5' end to form the molecule L-19 IVS (Zaug et al., 1986). The 4 L-19 IVS has recently spurred great interest in the concept of using RNA molecules as target-specific endonucleases. Ribonuclease F: The First True RNA Enzyme Ribonuclease P (RNase P) was the first and only RNA molecule shown thus far to have true catalytic activity without prior modification (Zaug et al., 1986). In prokaryotes a single gene may code for several copies of a tRNA; therefore, excision of a single, mature tRNA requires an endoribonucleolytic cleavage on both the 5' and 3' sides of each pre-tRNA. This discussion will be limited only to the events involved with the 5' pre-tRNA cleavage since the enzymes(s) involved in 3' processing have not been identified clearly. The 5' pre-tRNA sequence that undergoes cleavage is not conserved and thus, RNase P must recognize a structure common to all tRNA molecules. After recognition, the enzyme specifically cleaves the RNA by base hydrolysis to produce the 5' terminus (Fig. IB). The RNase P holoenzyme consists of one basic protein with a molecular mass of 14 kilodaltons (kD) and one single-stranded RNA of 377 nts in Escherichia coli (Baer et al., 1990) or 401 nts in Bacillus subtilis (Pace & Smith, 1990).
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