Detecting protein-induced folding of the U4 snRNA kink-turn by single-molecule multiparameter FRET measurements
- ANNA K. WOŹNIAK1,
- STEPHANIE NOTTROTT2,4,
- EVA KÜHN-HÖLSKEN2,
- GUNNAR F. SCHRÖDER3,
- HELMUT GRUBMÜLLER3,
- REINHARD LÜHRMANN2,
- CLAUS A.M. SEIDEL1, and
- FILIPP OESTERHELT1
- 1Heinrich-Heine-Universität Düsseldorf, Institut für molekulare Physikalische Chemie, Universitütsstraße 1, 40225 Düsseldorf, Germany
- 2Max-Planck-Institut für biophysikalische Chemie, Zelluläre Biochemie, Am Faßberg 11, D-37077 Göttingen, Germany
- 3Max-Planck-Institut für biophysikalische Chemie, Theoretische und computergestützte Biophysik, Am Faßberg 11, D-37077 Göttingen, Germany
Abstract
The kink-turn (k-turn), a new RNA structural motif found in the spliceosome and the ribosome, serves as a specific protein recognition element and as a structural building block. While the structure of the spliceosomal U4 snRNA k-turn/15.5K complex is known from a crystal structure, it is unclear whether the k-turn also exists in this folded conformation in the free U4 snRNA. Thus, we investigated the U4 snRNA k-turn by single-molecule FRET measurements in the absence and presence of the 15.5K protein and its dependence on the Na+ and Mg2+ ion concentration. We show that the unfolded U4 snRNA k-turn introduces a kink of 85° ± 15° in an RNA double helix. While Na+ and Mg2+ ions induce this more open conformation of the k-turn, binding of the 15.5K protein was found to induce the tightly kinked conformation in the RNA that increases the kink to 52° ± 15°. By comparison of the measured FRET distances with a computer-modeled structure, we show that this strong kink is due to the k-turn motif adopting its folded conformation. Thus, in the free U4 snRNA, the k-turn exists only in an unfolded conformation, and its folding is induced by binding of the 15.5K protein.
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Footnotes
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↵4 Present address: Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, MA 01605, USA.
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Article and publication are at http://www.rnajournal.org/cgi/doi/10.1261/rna.2950605.
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- Accepted June 29, 2005.
- Received April 27, 2005.
- Copyright 2005 by RNA Society