DARWIN Digitale Dissertationen German Version Strich

FU Berlin
Digitale Dissertation

Marco K. Vallazza :
Ways to the Thermus flavus 5S rRNA crystal
Molecular design, RNA-protein complex formation and structural fragments
Wege zum Thermus flavus 5S rRNA Kristall

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Abstract

Functions of RNA molecules which are structurally determined can be explained by highly resoluted crystal structures. At the example of Thermus flavus 5S rRNA which represents a scaffold of the peptidyl transferase three ways has been analyzed concerning their suitability to improve RNA crystallization: the molecular design (1), the complex formation of RNA with the protein TflL18 (2) and the subdivision of 5S rRNA into structural domains (3). The optimization of synthesis and downstream processing of the RNA as well as the protein components is included in this thesis.

By adjusting several reaction parameters up to 1350 copies of rRNA could be produced from a single DNA template during in-vitro transcription. The subsequent 3'-processing of the heterogeneous transcript pool has been performed first in the E. coli S100 fraction containing various RNases and secondly by means of an optimized DNA enzyme originated from the catalytic motif 10-23. In both cases a homogeneous RNA product was generated. A pushing-away-hypothesis specific for 5S rRNA molecules is presented explaining the appearance of homogeneous transcripts in-vitro. Crystallization studies revealed that crystallization slot of the engineered molecules is not be optimized in comparison with the 5S rRNA wildtype.

Two ribosomal binding proteins of Th. flavus 5S rRNA termed TflL18 and TflL25 have been cloned and characterized. Probably, the sequence variability observed in the protein centre of different L18 species causes a thermo-stabilizing 3D folding. The comparison of TflL18 overexpression in E. coli with the synthesis in the protein bioreactor showed at least 20-fold higher yields for the in-vitro system. The native 5S rRNA domain B has been proved to be essential for the binding of TflL18. A well-shaped crystal of the RNA-protein-complex consisting of the Th. flavus 5S rRNA wildtype and the protein TflL18 was obtained.

The systematical optimization of the chemical and physical influences in the crystallization trial resulted in the derivation of diffusion kinetics for the hanging drop as well as in effective buffer compositions for RNA crystallization. The positive effect of microgravity on the crystal growth and the diffraction ability has been confirmed. Fragments of Th. flavus 5S rRNA domains B, C and E which were partly mutated and modified with heavy atoms could successfully be crystallized. Data sets have been collected to max. resolutions of 1.5 Å. The structure solution of the 8 bp helix of the domain E revealed important RNA features. G:U wobble base pairs are stabilized by highly conserved structural water molecules. A novel G:C base pair appears in non-Watson-Crick formation enabled by the protonation of cytidine.

Table of Contents

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Titelblätter
Inhaltsverzeichnis
1. Einleitung 1
1.1 RNA-Welt 1
1.2 Ribosomale 5S RNA 2
1.3 RNA-Strukturaufklärung 8
1.4 Kristallisation & Diffraktion der Thermus flavus 5S rRNA 9
1.5 Kristallstruktur gegen Lösungsstruktur 10
1.6 Grundlagen der Kristallographie 11
2. Problemstellung 16
3. Material und Methoden 17
3.1 Material 17
3.2 Methoden 23
3.2.1 Nukleinsäure-Aufreinigung 23
3.2.2 Gelelektrophorese 26
3.2.3 Konzentrationsbestimmung von Nukleinsäuren 30
3.2.4 DNA-Amplifikation und Mutagenese 32
3.2.5 in-vitro Rekombination und Klonierung 35
3.2.6 in-vitro Transkription 41
3.2.7 Prozessierung von RNA 42
3.2.8 Proteinexpression von TflL18 45
3.2.9 Chemische Synthese von RNA-Oligonukleotiden 49
3.2.10 Kristallisation 50
4. Ergebnisse 56
4.1 Thermus flavus 5S rRNA Mutanten 56
4.2 Charakterisierung thermophiler 5S rRNA Bindungsproteine 72
4.3 Strukturaufklärung von Thermus flavus 5S rRNA Domänen 82
5. Diskussion 104
5.1 RNA-Kristallisation 104
5.2 Kristallisation von Oligoribonukleotiden 106
5.3 RNA-Strukturmotive 108
5.4 in-vitro Transkription und Prozessierung 111
5.5 Ribosomale Bindungsproteine der 5S rRNA 112
6. Ausblick 114
7. Zusammenfassung / Summary 115
8. Literaturverzeichnis 117
9. Anhang 125
9.1 Abkürzungsverzeichnis 125
9.2 Sequenzen, ALKABRID, Kristallisationsscreen 127
9.3 Eigene Publikationen 130
9.4 Lebenslauf 132
9.5 Danksagung 133

More Information:

Online available: http://www.diss.fu-berlin.de/2001/275/indexe.html
Language of PhDThesis: german
Keywords: ribosome, 5S rRNA, RNA processing, crystallization, microgravity, structural water
DNB-Sachgruppe: 30 Chemie
Date of disputation: 09-Nov-2001
PhDThesis from: Fachbereich Biologie, Chemie, Pharmazie, Freie Universität Berlin
First Referee: Prof. Dr. Volker A. Erdmann
Second Referee: Prof. Dr. Christian Betzel
Contact (Author): vallazza@chemie.fu-berlin.de
Contact (Advisor): erdmann@chemie.fu-berlin.de
Date created:19-Dec-2001
Date available:20-Dec-2001

 

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