Latest documents http://publikationen.ub.uni-frankfurt.de/index/index/ Tue, 22 Jan 2013 10:44:14 +0100 Tue, 22 Jan 2013 10:44:14 +0100 http://publikationen.ub.uni-frankfurt.de/frontdoor/index/index/docId/27851 While many different RNA aptamers have been identified that bind to a plethora of small molecules only very few are capable of acting as engineered riboswitches. Even for aptamers binding the same ligand large differences in their regulatory potential were observed. We address here the molecular basis for these differences by using a set of unrelated neomycin-binding aptamers. UV melting analyses showed that regulating aptamers are thermally stabilized to a significantly higher degree upon ligand binding than inactive ones. Regulating aptamers show high ligand-binding affinity in the low nanomolar range which is necessary but not sufficient for regulation. NMR data showed that a destabilized, open ground state accompanied by extensive structural changes upon ligand binding is important for regulation. In contrast, inactive aptamers are already pre-formed in the absence of the ligand. By a combination of genetic, biochemical and structural analyses, we identified a switching element responsible for destabilizing the ligand free state without compromising the bound form. Our results explain for the first time the molecular mechanism of an engineered riboswitch. Julia Weigand; Sina R. Schmidtke; Tristan J. Will; Elke Duchardt-Ferner; Christian Hammann; Jens Wöhnert; Beatrix Suess article http://publikationen.ub.uni-frankfurt.de/frontdoor/index/index/docId/27851 Tue, 22 Jan 2013 10:44:14 +0100 http://publikationen.ub.uni-frankfurt.de/frontdoor/index/index/docId/23230 Eukaryotic ribosome biogenesis requires the concerted action of numerous ribosome assembly factors, for most of which structural and functional information is currently lacking. Nob1, which can be identified in eukaryotes and archaea, is required for the final maturation of the small subunit ribosomal RNA in yeast by catalyzing cleavage at site D after export of the preribosomal subunit into the cytoplasm. Here, we show that this also holds true for Nob1 from the archaeon Pyrococcus horikoshii, which efficiently cleaves RNA-substrates containing the D-site of the preribosomal RNA in a manganese-dependent manner. The structure of PhNob1 solved by nuclear magnetic resonance spectroscopy revealed a PIN domain common with many nucleases and a zinc ribbon domain, which are structurally connected by a flexible linker. We show that amino acid residues required for substrate binding reside in the PIN domain whereas the zinc ribbon domain alone is sufficient to bind helix 40 of the small subunit rRNA. This suggests that the zinc ribbon domain acts as an anchor point for the protein on the nascent subunit positioning it in the proximity of the cleavage site. Thomas Veith; Roman Martin; Jan Philip Wurm; Benjamin L. Weis; Elke Duchardt-Ferner; Charlotta Safferthal; Raoul Hennig; Oliver Mirus; Markus Bohnsack; Jens Wöhnert; Enrico Schleiff article http://publikationen.ub.uni-frankfurt.de/frontdoor/index/index/docId/23230 Mon, 11 Jun 2012 14:37:27 +0200 http://publikationen.ub.uni-frankfurt.de/frontdoor/index/index/docId/6015 Using (13)C spin relaxation NMR in combination with molecular dynamic (MD) simulations, we characterized internal motions within double-stranded DNA on the pico- to nano-second time scale. We found that the C-H vectors in all cytosine ribose moieties within the Dickerson-Drew dodecamer (5´-CGCGAATTCGCG-3´) are subject to high amplitude motions, while the other nucleotides are essentially rigid. MD simulations showed that repuckering is a likely motional model for the cytosine ribose moiety. Repuckering occurs with a time constant of around 100 ps. Knowledge of DNA dynamics will contribute to our understanding of the recognition specificity of DNA-binding proteins such as cytosine methyltransferase. Elke Duchardt; Lennart Nilsson; Jürgen Schleucher article http://publikationen.ub.uni-frankfurt.de/frontdoor/index/index/docId/6015 Wed, 12 Nov 2008 13:14:14 +0100 http://publikationen.ub.uni-frankfurt.de/frontdoor/index/index/docId/5854 In a combined NMR/MD study, the temperature-dependent changes in the conformation of two members of the RNA YNMG-tetraloop motif (cUUCGg and uCACGg) have been investigated at temperatures of 298, 317 and 325 K. The two members have considerable different thermal stability and biological functions. In order to address these differences, the combined NMR/MD study was performed. The large temperature range represents a challenge for both, NMR relaxation analysis (consistent choice of effective bond length and CSA parameter) and all-atom MD simulation with explicit solvent (necessity to rescale the temperature). A convincing agreement of experiment and theory is found. Employing a principle component analysis of the MD trajectories, the conformational distribution of both hairpins at various temperatures is investigated. The ground state conformation and dynamics of the two tetraloops are indeed found to be very similar. Furthermore, both systems are initially destabilized by a loss of the stacking interactions between the first and the third nucleobase in the loop region. While the global fold is still preserved, this initiation of unfolding is already observed at 317 K for the uCACGg hairpin but at a significantly higher temperature for the cUUCGg hairpin. Jan Ferner; Alessandra Villa; Elke Duchardt; Elisabeth Widjajakusuma; Jens Wöhnert; Gerhard Stock; Harald Schwalbe article http://publikationen.ub.uni-frankfurt.de/frontdoor/index/index/docId/5854 Thu, 02 Oct 2008 15:58:58 +0200 http://publikationen.ub.uni-frankfurt.de/frontdoor/index/index/docId/2812 The 5'-terminal cloverleaf (CL)-like RNA structures are essential for the initiation of positive- and negative-strand RNA synthesis of entero- and rhinoviruses. SLD is the cognate RNA ligand of the viral proteinase 3C (3Cpro), which is an indispensable component of the viral replication initiation complex. The structure of an 18mer RNA representing the apical stem and the cGUUAg D-loop of SLD from the first 5'-CL of BEV1 was determined in solution to a root-mean-square deviation (r.m.s.d.) (all heavy atoms) of 0.59 A (PDB 1Z30). The first (antiG) and last (synA) nucleotide of the D-loop forms a novel ‘pseudo base pair’ without direct hydrogen bonds. The backbone conformation and the base-stacking pattern of the cGUUAg-loop, however, are highly similar to that of the coxsackieviral uCACGg D-loop (PDB 1RFR) and of the stable cUUCGg tetraloop (PDB 1F7Y) but surprisingly dissimilar to the structure of a cGUAAg stable tetraloop (PDB 1MSY), even though the cGUUAg BEV D-loop and the cGUAAg tetraloop differ by 1 nt only. Together with the presented binding data, these findings provide independent experimental evidence for our model [O. Ohlenschläger, J. Wöhnert, E. Bucci, S. Seitz, S. Häfner, R. Ramachandran, R. Zell and M. Görlach (2004) Structure, 12, 237–248] that the proteinase 3Cpro recognizes structure rather than sequence. Yvonne Ihle; Oliver Ohlenschläger; Sabine Häfner; Elke Duchardt; Martin Zacharias; Simone Seitz; Roland Zell; Ramadurai Ramachandran; Matthias Görlach article http://publikationen.ub.uni-frankfurt.de/frontdoor/index/index/docId/2812 Thu, 04 May 2006 15:22:22 +0200