ANTIBIOTIC INHIBITION of CATALYTIC RNA FUNCTION by Jeff
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ANTIBIOTIC INHIBITION OF CATALYTIC RNA FUNCTION by Jeff Rogers B.Sc. (Honours), University of Regina, 1991 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (DEPARTMENT OF MICROBIOLOGY AND IMMUNOLOGY) We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA August 1996 ©Jeff Rogers, 1996 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department The University of British Columbia Vancouver, Canada DE-6 (2/88) ii ABSTRACT A number of compounds inhibit group I intron splicing. Competitive inhibitors include deoxyguanosine, dideoxyguanosine, arginine and streptomycin; the non-competitive inhibitors include members of the aminoglycoside family of antibiotics. Further screening of a collection of antibiotics for their ability to inhibit group I intron splicing identified several novel compounds. In particular, the pseudodisaccharide antibiotic lysinomicin, the peptide antibiotics netropsin and distamycin, and the tetracycline analog chelocardin, were found to inhibit group I intron splicing at concentrations of 250 pM or lower. Inhibition of group I intron splicing by pseudodisaccharide antibiotics was studied in detail. Lysinomicin and three closely related compounds were found to inhibit the self splicing reaction of the Tetrahymena, Bacillus phage SP01 and T4 phage td and sun 7 group I introns at concentrations less than 50 u.M. Lysinomicin competitively inhibited sunY intron splicing with a Kj of 8.5 pM (+/- 5 u,M). The pseudodisaccharides were also shown to interact at the A-site on the ribosome, as Escherichia coli strains resistant to neomycin, which binds to the ribosomal A- site, were also resistant to the pseudodisaccharides. To further examine antibiotic/ribozyme interactions, antibiotic inhibition of a second ribozyme system, the human hepatitis delta virus (HDV) ribozyme, was examined. The small size (150 nucleotides) of this ribozyme and the fact that it lacks a guanosine binding site (the proposed site of interaction of inhibitors of group I intron splicing) made it a good candidate for detailed studies of antibiotic/ribozyme interactions. The antibiotics that have been shown to inhibit group I intron splicing were found to inhibit the HDV genomic and antigenomic iii ribozymes. Kinetic analysis showed that neomycin competes with magnesium binding to the ribozyme with a Kj of 28 uM (+/- 10 uM). Lead cleavage also suggested that neomycin inhibits the self-cleavage reaction of the HDV ribozyme by competing with divalent cation binding. Footprint analysis also supported this hypothesis as neomycin binds HDV RNA near the cleavage site. I propose that the binding of neomycin to several different RNAs (Rev Responsive Element, 16S rRNA, and the hammerhead, group I intron and HDV ribozymes) may be due to neomycin recognition of divalent cation binding site(s) in these RNAs. iv TABLE OF CONTENTS ABSTRACT TABLE OF CONTENTS LIST OF TABLES LIST OF FIGURES vii ABBREVIATIONS ix ACKNOWLEDGMENTS x INTRODUCTION 1 MATERIALS AND METHODS 12 a. Bacterial strains and growth conditions 12 b. Antibiotic inhibition assays 12 c. Plasmids 12 d. DNA manipulations 15 e. In vitro transcription 15 f. Polyacrylamide gel electrophoresis 16 g. RNA purification and elution 17 h. Group I intron splicing assay and antibiotic screening 17 i. Kinetic analysis of group I intron inhibition by antibiotics 18 j. Hepatitis Delta Virus cleavage assay and antibiotic screening 19 k. The kinetics of antibiotic inhibition of the HDV ribozyme 19 self-cleavage reaction 1. 5' -(a-thio)triphosphate incorporation transcription and 20 iodine cleavage m. 5' end labeling of HDV ribozyme RNA 21 n. 3'end labeling of HDV ribozyme RNA 21 o. Pb++ cleavage of the HDV ribozyme 21 p. Chemical modification of HDV ribozyme RNA 22 q. Reverse transcription of HDV RNA 23 RESULTS 24 Chapter 1. Identification of antibiotics which inhibit group I intron splicing 24 a. Screening for antibiotics which inhibit group I intron splicing 24 b. Netropsin 27 c. Chelocardin 33 d. Lysinomicin 36 V Chapter 2. Analysis of pseudodisaccharide inhibition of group I intron splicing 37 a. Pseudodisaccharides competitively inhibit group I intron 37 splicing in vitro b. The effect of lysinomicin on other group I introns 40 c. Antimicrobial activity of lysinomicins 44 Chapter 3. Competitive inhibition of group I intron splicing by the 47 tuberactinomycin antibiotics a. Viomycin inhibits group I intron splicing 47 b. Peptide antibiotics of the tuberactinomycin family inhibit 47 group I intron splicing c. Structure/function relationships of tuberactinomycin inhibition 51 of group I intron splicing Chapter 4. Inhibition of the self-cleavage reaction of the hepatitis delta virus 52 ribozyme a. Specific antibiotics inhibit HDV self cleavage 52 b. Kinetic analysis of antibiotic inhibition of self cleavage 55 c. Effect of pH on antibiotic inhibition of self cleavage 58 d. Lead cleavage analysis of the HDV ribozyme 62 e. Footprint analysis of neomycin binding to the HDV ribozyme 68 DISCUSSION 75 a. Specificity of antibiotic inhibition of ribozyme function 75 b. Competitive inhibition of group I intron splicing by lysinomicin 78 c. Antibiotic inhibition of the HDV ribozyme 79 d. Searching for the divalent cation binding site(s) of the HDV 80 ribozyme e. A model for neomycin inhibition of ribozyme function 82 f. Antibiotics and their interactions with RNA: evolutionary and 85 clinical implications REFERENCES 88 vi LIST OF TABLES Table 1. Plasmids 14 Table 2. Compounds screened for ability to inhibit group I intron splicing 28 Table 3. Error in the slopes of the lines from the Lineweaver-Burk plot 42 of Fig. 7a Table 4. Antimicrobial activity of the lysinomicins 46 Table 5. Tuberactinomycin antibiotics and group I intron splicing 49 Table 6. Comparison of the effect of antibiotics on group I and HDV 54 ribozymes Table 7. Effect of pH on antibiotic inhibition of the HDV ribozyme 61 Table 8. Analysis of HDV RNA treated with dimethyl sulfate (followed 72 by aniline cleavage) in the presence and absence of neomycin and paromomycin Table 9. The eight classes of ribozyme inhibitors 76 Vll LIST OF FIGURES Figure 1. Group I introns 4 a. Group I intron splicing mechanism b. Group I intron secondary structure Figure 2. The hammerhead ribozyme self-cleavage pathway 9 Figure 3. Screening antibiotics for inhibition of group I intron splicing a. Tetrahymena group I intron 25 b. Bacillus phage group I intron 26 Figure 4. a. Structure of chelocardin 34 b. Structure of neomycin and related antibiotics • . 34 c. Structure of lysinomicin and related antibiotics 35 Figure 5. Lysinomicin inhibits group I intron splicing 38 Figure 6. Pseudodisaccharides inhibit group I intron splicing 39 Figure 7. Kinetic analysis of lysinomicin inhibition of group I intron 41 splicing a. Lineweaver-Burk plot b. Slopes of Lineweaver-Burk plot vs. concentration of lysinomicin Figure 8. Lysinomicins inhibit different group I introns 43 Figure 9. Structures of the tuberactinomycin antibiotics 48 Figure 10. Peptide antibiotics of the tuberactinomycin family inhibit 50 group I intron splicing Figure 11. The secondary structure of the HDV genomic and antigenomic 53 ribozymes Figure 12. Several antibiotics inhibit the human hepatitis delta virus 56 self-cleavage reaction a. Antibiotics and the genomic HDV ribozyme at 37° b. Antibiotics and the genomic HDV ribozyme at 95° c. Antibiotics and the antigenomic HDV ribozyme at 95 ° Figure 13. The effect of magnesium on HDV self-cleavage and neomycin 57 inhibition of HDV self-cleavage Figure 14. Determination of the Kj for neomycin inhibition of the HDV 59 ribozyme Figure 15. The effect of pH on neomycin, viomycin and chelocardin 60 inhibition of HDV self-cleavage Figure 16. Lead cleavage of the HDV genomic ribozyme 63 a. Competition with divalent cations 64 b. Competition with antibiotics 65 Figure 17. Three dimensional model of the proposed divalent cation 67 binding sites in HDV RNA Figure 18. Reverse transcription of dimethyl sulfate and kethoxal treated 70 HDV RNA (in the presence and absence of neomycin and paromomycin) Figure 19. Three dimensional representation of chemical modification 74 viii studies in HDV RNA (in stereo) Figure 20. Summary of the lead cleavage and footprinting experiments 84 with the HDV ribozyme (secondary structure) ix ABBREVIATIONS ATP adenosine triphosphate cpm counts per minute CTP cytidine triphosphate DEPC diethyl pyrocarbonate DMS dimethylsulfate DNA deoxyribonucleic acid DTT dithiothreitol El 5' exon E1-E2 ligatedexons E2 3' exon EDTA ethylenediaminetetraacetic acid EtOH ethanol F fraction of HDV ribozyme cleaved GTP guanosine triphosphate HDV hepatitis delta virus HEPES (N-[2-Hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid]) HIV human immunodeficiency virus I linear intron I-E2 intron-3' exon k reaction rate constant (min"1) K; inhibition constant KM Michaelis-Menten constant KMg the magnesium concentration where the HDV self-cleavage reaction is proceeding at 1/2 its maximal rate LB Luria-Bertani