Dokument: Untersuchungen zur Struktur und Funktion der bakteriellen 6S RNA und Charakterisierung der 6S RNA Wechselwirkung mit RNA-Polymerase
| Titel: | Untersuchungen zur Struktur und Funktion der bakteriellen 6S RNA und Charakterisierung der 6S RNA Wechselwirkung mit RNA-Polymerase | |||||||
| Weiterer Titel: | Structural and functional studies on bacterial 6S RNA and characterization of the 6S RNA-RNA polymerase interaction | |||||||
| URL für Lesezeichen: | https://docserv.uni-duesseldorf.de/servlets/DocumentServlet?id=28030 | |||||||
| URN (NBN): | urn:nbn:de:hbz:061-20140109-123200-6 | |||||||
| Kollektion: | Dissertationen | |||||||
| Sprache: | Englisch | |||||||
| Dokumententyp: | Wissenschaftliche Abschlussarbeiten » Dissertation | |||||||
| Medientyp: | Text | |||||||
| Autor: | Dr. Steuten, Benedikt [Autor] | |||||||
| Dateien: |
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| Beitragende: | Prof. Dr. Wagner, Rolf [Gutachter] Prof. Dr. Ernst, Joachim F. [Gutachter] Prof. Dr. Franz Narberhaus [Gutachter] | |||||||
| Stichwörter: | ncRNA, Transkriptionsregulation, RNA-Polymerase, E. coli | |||||||
| Dewey Dezimal-Klassifikation: | 500 Naturwissenschaften und Mathematik » 570 Biowissenschaften; Biologie | |||||||
| Beschreibungen: | Die Transkription von Genen ist ein grundlegender Prozess in allen biologischen Systemen. In prokaryotischen Zellen wird die gesamte Transkription durch lediglich eine RNA Polymerase (RNAP) katalysiert, welche daher einer komplexen Regulation unterliegt. Die Aktivität der RNAP wird durch verschiedenste, zelluläre Komponenten, wie Proteine, niedermolekulare Verbindungen und Nukleinsäuren, kontrolliert. Obwohl Ribonukleinsäure (RNA) das eigentliche Produkt der Transkription ist, stellt die bakterielle 6S RNA ein Paradebeispiel für die Regulation der Transkription durch eine kleine, nicht-kodierende RNA (ncRNA) dar. In dieser Dissertation wurden funktionelle und strukturelle Details der 6S RNA-RNAP Interaktion genauer untersucht.
Neben der 6S RNA aus E. coli wurden die Sekundärstrukturen verschiedener 6S RNAs aus phylogenetisch entfernten Cyanobakterien analysiert. Aufgrund der strukturellen Ähnlichkeit wurden diese 6S RNAs für heterologe in vitro Studien mit der RNAP aus E. coli verwendet. Trotz des Fehlens größerer Sequenz-Konservierung weisen die 6S RNAs aus E. coli und den cyanobakteriellen Spezies sehr ähnliche Eigenschaften auf. Dazu gehören die spezifische Bindung an die RNAP sowie das pRNA-vermittelte Ablösen von der RNAP. Die einheitliche Struktur der 6S RNA erlaubt eine präzise Lokalisierung im aktiven Zentrum der RNAP, wodurch in einer ungewöhnlichen, RNA-abhängigen Transkription kurze product RNAs (pRNAs) synthetisiert werden können. Eine strukturelle Analyse der resultierenden 6S-pRNA Hybride hat gezeigt, dass die 6S RNA einer Konformationsänderung unterliegt, welche das Ablösen von der RNAP fördert und dadurch den intrazellulären Abbau der 6S RNA einleitet. Die Bedeutung der strukturellen Veränderung der 6S RNA nach pRNA Transkription in vivo konnte durch eine chemische footprint Analyse untermauert werden. In dieser Arbeit wurden darüber hinaus Regionen der 6S RNA-RNAP Interaktion genauer charakterisiert. Dazu wurde die räumliche Nachbarschaft zwischen funktionellen Domänen der RNAP σ70 Untereinheit und der 6S RNA durch Anwendung der chemischen Nuklease FeBABE bestimmt. Die räumliche Zuordnung der 6S RNA Spaltungspositionen sowie die Zuhilfenahme von Tertiärstrukturvorhersagen für 6S RNA Fragmente erlaubten die Modellierung einer drei-dimensionalen 6S RNA Struktur relativ zu der hoch-aufgelösten Kristallstruktur der RNAP aus E. coli. Dieses Modell gibt die größtenteils helikale Topologie der 6S RNA wider und beschreibt einen plausiblen Pfad der RNA entlang der RNAP. Eine Erweiterung der Analysen mittels 6S RNA Mutanten hebt die Bedeutung von asymmetrischen bulge loops innerhalb der 6S RNA internal stem Struktur als potentielle Interaktionsstellen für die RNAP hervor.Transcription of genes is a fundamental process in all biological systems. In prokaryotic cells the transcription is catalysed by a single RNA polymerase (RNAP), which is thus subject to a complex regulatory network. The activity of RNA polymerase is controlled by a variety of molecules including proteins, low-molecular compounds and nucleic acids. Although RNA is the actual product of transcription the bacterial 6S RNA is a prime example for the regulation of transcription via a small, non-coding RNA. In this PhD thesis structural and functional implications of the 6S RNA-RNAP interaction were investigated. In addition to the well-studied 6S RNA from E. coli the secondary structures of several 6S RNAs from the phylogenetically distant cyanobacteria were analysed in detail. Due to the structural similarity those RNAs were used for heterologous in vitro studies with E. coli RNAP. Despite the lack of major sequence conservation the 6S RNAs from E. coli and the cyanobacterial species exhibit the same characteristics, comprising RNAP binding and pRNA-mediated release from RNAP. The common 6S RNA structure allows the positioning in the RNAP active site resulting in an unusual RNA-templated transcription of short product RNAs (pRNA). A structural probing of the 6S-pRNA hybrid revealed that 6S RNA undergoes a conformational rearrangement, which facilitates the release from RNAP and probably induces the intracellular degradation of 6S RNA. The significance of this structural transition of 6S RNA in vivo was underscored by a chemical footprint technique. Additionally, the 6S RNA-RNAP interaction sites were further scrutinized in this work. Therefore the spatial neighborhood between functional domains of the RNAP σ70 subunit and 6S RNA was mapped by applying the chemical nuclease FeBABE. The spatial assignment of the generated 6S RNA cleavage sites and tertiary structure predictions of 6S RNA fragments allowed the modelling of a three-dimensional 6S RNA structure relative to the high resolution crystal structure of RNAP. This model nicely reflects the helical topology of the RNA and proposes a reliable path of 6S RNA across the RNAP. Moreover, extension of the studies with 6S RNA mutants emphasize the importance of asymmetric bulge loops within the 6S RNA internal stem as potential interaction sites for RNAP. | |||||||
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| Lizenz: |  Urheberrechtsschutz | |||||||
| Fachbereich / Einrichtung: | Mathematisch- Naturwissenschaftliche Fakultät » WE Biologie » Molekularbiologie der Prokaryoten | |||||||
| Dokument erstellt am: | 09.01.2014 | |||||||
| Dateien geändert am: | 09.01.2014 | |||||||
| Promotionsantrag am: | 01.10.2013 | |||||||
| Datum der Promotion: | 13.12.2013 |
