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. Allrightsreserved. U-M-I 300N.ZecbRd. Ann Arbor, MI48106 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: P. A. Fuerst Approved by L. F. Johnson —A. M. Lambowitz P. S. Perlman * Adviser 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, 1980 1960 ............. Born - Andhra Pradesh, India ................... B«S., Osnania University, Hyderabad, India 1980 - 1982 1983 - 1986 ................... M.S., Andhra University, Waltair, Andhra Pradesh .................... 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 I.B. Yeast mitochondrialDNA I.B.l. Mosaic genes .......................... ......... 122356............................... I.B.2. Maturases I.B.3. Group I introns I.B.3.a. Discovery of RNA catalysis I.B.3.b. Tetrahymena rRNA intron self-splices by a two step transesterification pathway I.B.3.C. Tetrahymena rRNA intron is a group I intron 79 I.B.3.d. Mitochondrial group I introns self-splice in vitro 10 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 I.B.3.g. Active site model 12 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 novel.......................... 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 ....... 22 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 29 II.A. Plasmid constructions ............................... 29 MATERIALS AND METHODS II.B. Plasmid preparation............... 30 II.C. Transcription and Purification of RNA..............30 II.D. All Splicing reactions............ II.E. Site-directed mutagenesis.......... II.F. Purification and end-labeling of 30 31 oligonucleotides ....................................... 31 II.G. RNA sequencing..................................,....32 II.H. Northern Blots....................................... 32 <ul style="display: flex;"><li style="flex:1">II.I. 3* end-labeling of RNA....................... </li><li style="flex:1">32 </li></ul>II.J. Limit T1 digest...................................... 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................. III.B.3. All RNA has a higher threshold for »........37 magnesium than aI5g................................ 38 III.B.4. NH4C1 as the standard splicing buffer.................................. III.B.5. All reactions have a temperature optimum of 4QoC............................ III.B.6 . The reaction is unimolecular and time dependent . ..... 39 40 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*exon........... III.C.2. First test of the validity of the assignments................... 43 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 reactionproducts in KC1 III.D.1. Further analysis of KC1 effects on 49 the all reaction ............................. 50 III.D.2. Characterization of novel KC1 products ..........................51 III.D.3 A proposed pathway for the KC1 reaction....... 54 55 55 57 58 III.E. Spliced exon reopening (SER) III.E.l. All does not reopen spliced exons......... III.E.2. SER is sequence specific................... III.F. Much of the intron ORF can bedeleted III.F.l. Location of the branch site in the <ul style="display: flex;"><li style="flex:1">shortened IVS-LAR.......... </li><li style="flex:1">-</li></ul>........... 60 62 62 I1I.G. Studies using portions of all III.G.1. Trans-splicing ................. III.G.2. The conserved 5*boundary sequence is needed for trans-splicing...... III.H. Summary ............ .64 66 III.I. Studies of aI5g self-splicing 111.1.1. Introduction........... <ul style="display: flex;"><li style="flex:1">-</li><li style="flex:1">67 </li></ul>67 111.1.2. Domain 5 is required, in cis, for the second step of splicing........................68 111.1.3. 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 SER.....................................75 111.1.4. Domain 6 is not required for splicing in vitro.......... 76 <ul style="display: flex;"><li style="flex:1">77 </li><li style="flex:1">III.J. Molecular dissection of domain 5 </li></ul>III.J.l. Introduction................................. 77 III.J.2. Heterologous experiments...... III.J.3. Mutations of the highly conserved unpaired regions of domain 5 77 ...... 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 88 IV.A. All self-splices in vitro 88 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 KC1..... ........................ IV.A.4. All does not carry out apliced exon reopening ......... ........................ .IV.A.5. Much of the intron open reading frame can be deleted....................... IV.B. 5* end of the intron is not required for 5' exon release but plays a role in branch formation IV.C.. 5* end of the intron plays a role in SER IV.D. Lariat formation is not required for efficient splicing in vitro .. 93 94 96 96 97 IV.E. Domain 5 is also required for the second step in splicing 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 ............................ 100 IV.F. Future experiments IV.F.l. Point of interaction..................... IV.F.2. Transformation experiments

In Vitro Studies of Self-Splicing Group II Introns

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