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In Vitro Studies of Self-Splicing Group II Introns

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In Vitro Studies of Self-Splicing Group II Introns Order Number 8913650 In vitro studies of self-splicing group II introns Hebbar, Sharda Kattingeri, Ph.D. The Ohio State University, 1989 Copyright ©1989 by Hebbar, Sharda Kattingeri. All rights reserved. U-M-I 300N.ZecbRd. Ann Arbor, MI 48106 IN VITRO STUDIES OF SELF-SPLICING GROUP II INTRONS DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of the Ohio State University By Sharda Kattingeri Hebbar, B.S., M.S. ***** The Ohio State University 1989 Dissertation Committee: Approved by P. A. Fuerst L. F. Johnson — A. M. Lambowitz * Adviser P. S. Perlman Department of Molecular Genetics Copyright by Sharda Kattingeri Hebbar 1989 To My Parents, and To My Husband, Raja. ACKNOWLEDGEMENTS I would like to thank Dr. P.S. Perlman for his assistance and guidance. Special thanks go to Kevin Jarrell and Rosemary Dietrich not only for their technical advice but also for their friendship. To my husband, Raja, I would like to express my sincere gratitude for his encouragement, support and endurance throughout my endeavors. iii VITA February 21, 1960 ............. Born - Andhra Pradesh, India 1980 ................... B«S., Osnania University, Hyderabad, India 1980 - 1982 ................... M.S., Andhra University, Waltair, Andhra Pradesh 1983 - 1986 .................... Graduate Teaching Associate, MCDB Program, The Ohio State University, Columbus, Ohio 1986 - 1988 ................. Graduate Research Associate, Department of Molecular Genetics, The Ohio Sta+e University, Columbus, Ohio FIELDS OF STUDY Major Field: Molecular Genetics TABLE OF CONTENTS INTRODUCTION ................................................. 1 I.A. Catalytic RNA .......................... ......... 1 I.B. Yeast mitochondrial DNA ............................... 2 I.B.l. Mosaic genes 2 I.B.2. Maturases 3 I.B.3. Group I introns 5 I.B.3.a. Discovery of RNA catalysis 6 I.B.3.b. Tetrahymena rRNA intron self-splices by a two step transesterification pathway 7 I.B.3.C. Tetrahymena rRNA intron is a group I intron 9 I.B.3.d. Mitochondrial group I introns self-splice in vitro 1 0 I.B.3.e. Not all group I introns are self­ splicing 11 I.B.3.f. The intervening sequence RNA of Tetrahymena is an Enzyme 12 I.B.3.g. Active site model 13 I.B.4. Group II intron splicing ...................... 14 I.B.4.a. Conserved sequence elements and secondary structure ........................... 14 I.B.4.b. Group II introns in yeast mitochondria ................................... 16 I.B.4.C. Group II introns encode proteins related to reverse transcriptase..................................,, 17 I.B.4.d. AI5g self-splices in vitro by a n o v e l .......................... 18 I.B.4.e. Role of branch formation ................ 19 I.B.4.f. Group II intron splicing resembles nuclear pre-mRNA splicing .......... 20 I.B.4.g. Alternative reaction conditions ....... 2 2 I.B.4.h. Trans-splicing .......................... 23 I.B.4.i. Dependance on 5’ exon sequences............................... 25 I.B.4.j. Multiple 5’exon binding sites .......... 26 I.B.4.k. Domain 4 is dispensible ................. 26 I.B.4.1. Domain 5 is required for 5’ exon release....................................27 I.C. Dissertation Goals ......... 28 v MATERIALS AND METHODS 29 II.A. Plasmid constructions ............................... 29 II.B. Plasmid preparation............... 30 II.C. Transcription and Purification of R N A . ........... 30 II.D. All Splicing reactions............ 30 II.E. Site-directed mutagenesis.......... 31 II.F. Purification and end-labeling of oligonucleotides ....................................... 31 II.G. RNA sequencing.................................. ,....32 II.H. Northern B l o t s ....................................... 32 II.I. 3* end-labeling of R N A ....................... 32 II.J. Limit T1 d i g e s t ...................................... 32 U.K. Other Methods........................................ 33 II. L. Enzyme reagents........ 33 RESULTS........................................... 34 III.A. Test of the generality of group II self­ splicing .............................. 34 III.A.I. Cloning of intron 1 of the COX I gene ............................................. 35 III.B. Features of the all self-splicing reaction ....... *................................. 36 III.B.1. All RNA is inactive in the standard aI5g splicing buffer ............................... 36 III.B.2. All has an absolute requirement for monovalent cations................. »........ 37 III.B.3. All RNA has a higher threshold for magnesium than aI5g ................................ 38 III.B.4. NH4C1 as the standard splicing b u f f e r .................................. 39 III.B.5. All reactions have a temperature optimum of 4QoC ............................ 40 III.B.6 . The reaction is unimolecular and time dependent . ..... 40 III.B.7. Optimum reaction conditions ................ 41 III.C. Characterization of the NH4C1 reaction products 42 III.C.l. Identification of products containing the 3 * ex o n ........... 43 III.C.2. First test of the validity of the assignments................... 44 IiI.C.3. Accurate ligation of exons ................. 45 III.C.4. The slowly migrating RNA species is excised intron lariat (IVS-LAR) .................. 46 III.C.5. Accurate 5 ’ Cleavage ........................ 47 III.C.6 . Technical problems in mapping the branch point ....................................... 48 III.C.7. Summary and implications of product characterization ............................. 48 vi III.D. All yields some novel reaction products in KC1 49 III.D.1. Further analysis of KC1 effects on the all reaction ............................. 50 III.D.2. Characterization of novel KC1 products .......................... 51 III.D.3 A proposed pathway for the KC1 reaction....... 54 III.E. Spliced exon reopening (SER) 55 III.E.l. All does not reopen spliced exons ......... 55 III.E.2. SER is sequence specific................... 57 III.F. Much of the intron ORF can be deleted 58 III.F.l. Location of the branch site in the shortened IVS-LAR..........-........... 60 I1I.G. Studies using portions of all 62 III. G. 1. Trans-splicing ................. 62 III.G.2. The conserved 5*boundary sequence is needed for trans-splicing...... ............ 64 III.H. Summary 6 6 III.I. Studies of aI5g self-splicing - 67 111.1.1. Introduction........... 67 111.1.2. Domain 5 is required, in cis, for the second step of splicing........................ 6 8 111.1.3. Mutational analysis of the conserved 5* end of the intron ....... 71 111.1.3.a. The first 7 nt of the intron are essential for branch formation ............ 71 111.1.3.b. 5’ end of the intron plays a role in S E R ..................................... 75 111.1.4. Domain 6 is not required for splicing in v i t r o .......... 76 III.J. Molecular dissection of domain 5 77 III.J.l. Introduction................................. 77 III.J.2. Heterologous experiments...... 77 III.J.3. Mutations of the highly conserved unpaired regions of domain 5 ...... 78 III.J.3.a. The 4 base pair loop ................. 78 III.J.3.b. RNA truncated at the Rsa I site in domain 5 is inactive.................. 80 III.J.3.0. The CG bulge ........................... 81 III.J.4. Bottom helix ................................. 83 III.J.5. 7 nt in the 5* half of domain 5 can form a perfect match with 7nt in domain 1 .................................................... 85 DISCUSSION 8 8 IV.A. All self-splices in vitro 8 8 IV.A.l. All self-splices under conditions different from those of aI5g and bll ............. 90 IV.A.2. All reaction pathways ........................ 91 IV.A.3. All undergoes a post-splicing vii reaction in K C 1 ..... ........................ IV.A.4. All does not carry out apliced exon reopening ................................. IV.A.5. Much of the intron open reading frame can be deleted.......... ............. .. 93 IV.B. 5* end of the intron is not required for 5' exon release but plays a role in branch formation 94 IV.C.. 5* end of the intron plays a role in SER 96 IV.D. Lariat formation is not required for efficient splicing in vitro 96 IV.E. Domain 5 is also required for the second step in splicing 97 IV.E.l. 4 base GAAA loop is not critical for domain 5 function ........................... IV.E.2. The 2 base bulge is crucial for domain 5 function ............................ IV.F. Future experiments 100 IV.F.l. Point of interaction..................... IV. F. 2. Transformation experiments ............ .... viii LIST OF FIGURES Figure 1. Conserved Sequences in Nuclear, Group I and Group II introns ................ 102 Figure 2. Mosaic Genes in Yeast mtDNA...................104 Figure 3. Transesterification Mechanism for the Tetrahymena IVS.......... 106 Figure 4. The Internal Guide Sequence (IGS)........ 108 Figure 5. The Single "Active Site" or the "Guide Sequence" Model....... 110 Figure 6 . Secondary Structure Model of a Group II intron (aI5g)............................. 112 Figure 7. Splicing Mechanism of Nuclear mRNA Precursors. ............ 114 Figure 8 . Comparison of Group I, Group II and Nuclear Intron Splicing...................... 116 Figure 9. Group II Intron Splicing Pathway............. 118 Figure 10. Plasmid Construction. ...................... 120 Figure 11. In Vitro Reactivity of the all Precursor RNA................................ 122 Figure 12. Effect of Different Salts on the In Vitro reaction............................... 124 Figure 13. Concentration of Magnesium Chloride......................................126
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