NMR structural investigations of the U4 snRNA kink-turn and of a lariat-forming ribozyme [Elektronische Ressource] / vorgelegt von Melanie Falb

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NMR Structural Investigationsof the U4 snRNA kink-turn and of alariat-forming ribozymeINAUGURAL-DISSERTATIONzurErlangung der Doktorwürdeder Naturwissenschaftlich-Mathematischen Gesamtfakultätder Ruprecht-Karls-UniversitätHeidelbergvorgelegt vonDipl.-Ing. BiotechnologieMelanie Falbaus Heilbad Heiligenstadt(Eichsfeld)Heidelberg, 2009Tag der mündlichen Prüfung: 27.01.2010NMR Structural Investigations of the U4 snRNA kink-turnand of a lariat-forming ribozymeGutachter: Dr. Anne-Claude GavinProf. Dr. Irmgard SinningBetreuer: PD Dr. Teresa CarlomagnoFürmeineFamilieErwirb Dir Gold so viel du brauchstund Weisheit so viel du kannst.Kuppelinschrift der Moschee in SchwetzingenContentsSummary 1Zusammenfassung 51 Introduction 91.1 Biological system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91.1.1 Pre-mRNA splicing . . . . . . . . . . . . . . . . . . . . . . . . 91.1.2 The splicing reaction . . . . . . . . . . . . . . . . . . . . . . . 101.1.3 The machinery . . . . . . . . . . . . . . . . . . . . . 111.2 State of research . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131.2.1 The U4/U6 snRNP specific proteins 15.5K and hPrp31 . . . 131.2.2 The Kink-turn motif of the U4 snRNA . . . . . . . . . . . . . 191.2.3 The role of U2, U5 and U6 snRNA in the first step of splicing 211.2.4 The lariat-forming ribozyme -a model system for the first splicing step . . . . . . . . . . . 261.2.5 NMR Spectroscopy in RNA research . . . . . .

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NMR Structural Investigations
of the U4 snRNA kink-turn and of a
lariat-forming ribozyme
INAUGURAL-DISSERTATION
zur
Erlangung der Doktorwürde
der Naturwissenschaftlich-Mathematischen Gesamtfakultät
der Ruprecht-Karls-Universität
Heidelberg
vorgelegt von
Dipl.-Ing. Biotechnologie
Melanie Falb
aus Heilbad Heiligenstadt
(Eichsfeld)
Heidelberg, 2009
Tag der mündlichen Prüfung: 27.01.2010NMR Structural Investigations of the U4 snRNA kink-turn
and of a lariat-forming ribozyme
Gutachter: Dr. Anne-Claude Gavin
Prof. Dr. Irmgard Sinning
Betreuer: PD Dr. Teresa CarlomagnoFürmeineFamilieErwirb Dir Gold so viel du brauchst
und Weisheit so viel du kannst.
Kuppelinschrift der Moschee in SchwetzingenContents
Summary 1
Zusammenfassung 5
1 Introduction 9
1.1 Biological system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
1.1.1 Pre-mRNA splicing . . . . . . . . . . . . . . . . . . . . . . . . 9
1.1.2 The splicing reaction . . . . . . . . . . . . . . . . . . . . . . . 10
1.1.3 The machinery . . . . . . . . . . . . . . . . . . . . . 11
1.2 State of research . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
1.2.1 The U4/U6 snRNP specific proteins 15.5K and hPrp31 . . . 13
1.2.2 The Kink-turn motif of the U4 snRNA . . . . . . . . . . . . . 19
1.2.3 The role of U2, U5 and U6 snRNA in the first step of splicing 21
1.2.4 The lariat-forming ribozyme -
a model system for the first splicing step . . . . . . . . . . . 26
1.2.5 NMR Spectroscopy in RNA research . . . . . . . . . . . . . . 27
1.3 Aims of this work . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
2 Methods 31
2.1 RNA synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
2.1.1 T7 polymerase expression and purification . . . . . . . . . . 32
2.1.2 Transcription optimization . . . . . . . . . . . . . . . . . . . 32
2.1.3 Large scale RNA synthesis . . . . . . . . . . . . . . . . . . . 33
2.1.4 Protein and DNA removal in RNA samples . . . . . . . . . . 34
2.1.5 RNA purification . . . . . . . . . . . . . . . . . . . . . . . . . 35
2.1.6 Determination of nucleic acid concentration . . . . . . . . . 36
2.1.7 Hammerhead cleavage . . . . . . . . . . . . . . . . . . . . . . 37
2.2 Effects of RNA mutations upon lariat formation . . . . . . . . . . . 38
2.3 Protein synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
152.3.1 Expression of unlabeled and N-labeled proteins . . . . . . 40
2.3.2 Expression in 99% deuterated media . . . . . . . . . . . . . . 40
2.3.3 Purification of the GST-tagged 15.5K protein . . . . . . . . . 41
2.3.4 of the His-tagged 15.5K protein . . . . . . . . . 41
2.3.5 Purification of the hPrp31 protein . . . . . . . . . 42
iiiii
2.3.6 Determination of protein concentration . . . . . . . . . . . . 42
2.3.7 Electrophoretic Mobility Shift Assay . . . . . . . . . . . . . . 43
2.4 NMR sample preparation . . . . . . . . . . . . . . . . . . . . . . . . 44
2.4.1 Preparation of U4 snRNA samples . . . . . . . . . . . . . . . 44
2.4.2 Dimeric complex U4-15.5K . . . . . . . . . . . . . . . . . . . 45
2.4.3 Lariat-forming ribozyme . . . . . . . . . . . . . . . . . . . . . 45
2.5 NMR methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
2.5.1 Spectral assignment of isotope-labeled RNA . . . . . . . . . 48
2.5.2 Identification of base to sugar spin system . . . . . . . . . . 52
2.5.3 Correlation of base to sugar spin system . . . . . . . . . . . . 52
2.5.4 Assignment of the sugar spin system . . . . . . . . . . . . . 53
2.5.5 of base spin system . . . . . . . . . . . . . . . . 54
2.5.6 Sequential assignment . . . . . . . . . . . . . . . . . . . . . . 55
2.5.7 Assignment of exchangeable protons . . . . . . . . . . . . . 56
2.5.8 Restraints for RNA structure determination . . . . . . . . . . 57
3 Results: Structural studies of the U4 snRNA 5’ stem loop 59
3.1 RNA synthesis and hammerhead cleavage . . . . . . . . . . . . . . 59
3.2 Spectral assignments of the U4 snRNA 5’ stem loop construct . . . 61
3.2.1 Identification of base and sugar protons . . . . . . . . . . . . 61
3.2.2 Correlation of the base spin system to the sugar spin system 62
3.2.3 Assigning the sugar spin system . . . . . . . . . . . . . . . . 63
3.2.4 the base spin system . . . . . . . . . . . . . . . . . 64
3.2.5 Sequential assignment . . . . . . . . . . . . . . . . . . . . . . 66
3.2.6 Assignment of exchangeable base protons . . . . . . . . . . 67
3.3 NMR data analysis of the U4 snRNA 5’ stem loop . . . . . . . . . . 69
3.3.1 Structural restraints from Nuclear Overhauser Effect . . . . 69
3.3.2 Base pairing . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71
3.3.3 Torsion angles . . . . . . . . . . . . . . . . . . . . . . . . . . . 72
3.3.4 Residual dipolar couplings . . . . . . . . . . . . . . . . . . . 75
3.4 Structure calculation of U4 5’ stem loop . . . . . . . . . . . . . . . . 76
3.4.1 Structure of the unbound U4-Kt RNA . . . . . . . . . . . . . 78
3.4.2 Verification of the NMR structure of U4-Kt2 by SANS . . . . 80
3.5 Ion dependency of the U4-Kt2 fold . . . . . . . . . . . . . . . . . . . 83
3.6 Dimeric U4 snRNA-15.5K complex . . . . . . . . . . . . . . . . . . . 84
3.7 Ternary U4 snRNA-15.5K-hPrp31 complex . . . . . . . . . . . . . . 87
4 Discussion: The U4 snRNA 5’ stem loop 89