Dokument: FRET restrained high-precision structural modeling of RNA junctions

Titel:	FRET restrained high-precision structural modeling of RNA junctions
URL für Lesezeichen:	https://docserv.uni-duesseldorf.de/servlets/DocumentServlet?id=31244
URN (NBN):	urn:nbn:de:hbz:061-20141029-083715-4
Kollektion:	Dissertationen
Sprache:	Englisch
Dokumententyp:	Wissenschaftliche Abschlussarbeiten » Dissertation
Medientyp:	Text
Autor:	Dr. Vardanyan, Hayk [Autor]
Dateien:	[Dateien anzeigen] Adobe PDF [Details] 18,36 MB in einer Datei [ZIP-Datei erzeugen] Dateien vom 27.10.2014 / geändert 27.10.2014
Dewey Dezimal-Klassifikation:	500 Naturwissenschaften und Mathematik » 540 Chemie
Beschreibung:	RNA studies Naturally occurring RNA systems mostly exhibit a complex structure, containing several structural elements like junctions, loops and bulges at a time [1, 2]. Hence investigation of helical junctions and the influence of the separate structural elements in the folding procedure is a key step towards understanding the functionality of the molecules. In this work, structural modelling of large RNA four- and three-way junction molecules (35-45 kD) based on FRET experiments, Molecular dynamics and coarse-grained simulations are presented. In total 173 independent single-molecule FRET (smFRET) measurements were performed in order to obtain the structures of RNA four-way junction and three RNA three-way junction molecules (fully paired, with bulge with two or five cytosine (C) bases in the junction region). One major and two minor coexisting conformers were resolved for four-way junction molecule. For three-way junctions structural determination of only major populations was performed as the minor populations was shown to be due to incompletely hybridized molecules or acceptor photophysics. For each dataset distances and corresponding uncertainties were extracted. In the case of four-way junction three distances and corresponding uncertainties were extracted and successfully assigned to the three conformers of the RNA four-way junction using their distinct Mg2+-affinities. For three-way junctions distance assignment to the conformers was straightforward. FRET positioning and screening toolkit (FPS), which combines experimental and computational techniques in a hybrid approach, was applied to derive accurate structural models [3]. Assuming the helices of the RNA molecules to be a rigid double stranded RNAs, rigid body models of the conformers were obtained. The models were then refined by MD simulations and coarse grained RNA folding, resulting in meaningful all-atom structural models for the studied molecules. Confidence levels of the obtained models were estimated from cluster analysis. Quality assessment was done via rigorous error estimation. Resulting precisions are significantly better than the uncertainty of dye position with respect to macromolecules. Furthermore, this is the first that coexisting transient minor conformers of an RNA four-way junction (J(abcd)) molecule were structurally solved. Noteworthy, structural determination of fully paired RNA four- and three-way junction molecules, without tertiary contacts between helices or with other molecules, was never done before. Also, I succeeded to investigate the influence of the single structural element (bulge) on the overall structure of the three-way junction molecules. Hayk Vardanyan 1. Lescoute, A. and E. Westhof, Topology of three-way junctions in folded RNAs. RNA, 2006. 12(1): p. 83-93. 2. Lilley, D.M.J., Structures of helical junctions in nucleic acids. Quarterly Reviews of Biophysics, 2000. 33(2): p. 109-159. 3. Kalinin, S.P., T.; Sindbert, S.; Rothwell, P. J.; Berger, S.; Restle, T.; Goody, R. S.; Gohlke, H.; Seidel, C. A. M., A toolkit and benchmark study for FRET-restrained high-precision structural modeling. Nature Methods, 2012. 9: p. 1218-1225.
Lizenz:	Urheberrechtsschutz
Fachbereich / Einrichtung:	Mathematisch- Naturwissenschaftliche Fakultät » WE Chemie » Physikalische Chemie und Elektrochemie
Dokument erstellt am:	29.10.2014
Dateien geändert am:	29.10.2014
Promotionsantrag am:	03.07.2014
Datum der Promotion:	15.10.2014