Binding of IRE-BP to Its Cognate RNA Sequence: SFM Studies on a Universal RNA Backbone for the Analysis of RNA-Protein Interaction
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Michael Bonin
Abstract
We have used an RNA consisting of the potato spindle tuber viroid (PSTVd) and 240 bp of doublestranded RNA derived from the GUS gene as a backbone for scanning force microscope (SFM) studies on RNA binding proteins. The in vitro transcribed RNA forms a rodlike structure of apparent 130 nm in length with a completely base paired central part flanked by the incompletely paired viroid helix with bulges on both sides. The termini of the molecule consist of loops such that no blunt or staggered RNA ends are exposed. Suitable, asymmetrical restriction sites in the construct allow for the insertion of sequences of interest, e. g. protein binding sites. We have inserted the IRE (iron responsive element) sequence into the construct and have used in vitro transcripts to study binding of IREBP. Relative binding frequencies show that 70% of the protein binds to the expected site in the molecule while only a slightly enhanced binding is observed at the termini. In the GUSPSTVdIRE backbone, the orientation of the molecule is easily determined by IREBP binding. It thus provides a versatile tool to study specific as well as preferential interaction of other proteins with sequences or structures inserted into a different part of the molecule.
Copyright © 2001 by Walter de Gruyter GmbH & Co. KG
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Articles in the same Issue
- Roger D. Kornberg Felix Hoppe-Seyler Lecturer 2001
- The Eukaryotic Gene Transcription Machinery
- Paper of the Year 2000: Award to Eva Estébanez-Perpiñá and Pablo Fuentes-Prior
- GABAC Receptors: A Molecular View
- Recent Advances in Plant MAP Kinase Signalling
- The Chemical Biology of Ras Lipidation
- This Is the End: Processing, Editing and Repair at the tRNA 3-Terminus
- Binding of IRE-BP to Its Cognate RNA Sequence: SFM Studies on a Universal RNA Backbone for the Analysis of RNA-Protein Interaction
- PAF67, a Novel Protein That Is Associated with the Initiation-Competent Form of RNA Polymerase I
- Characterization of RNase P Holoenzymes from Methanococcus jannaschii and Methanothermobacter thermoautotrophicus
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- Biophysical Characterization of Lipopolysaccharide and Lipid A Inactivation by Lactoferrin
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- Functional Dissection of Trigger Factor and DnaK: Interactions with Nascent Polypeptides and Thermally Denatured Proteins
- A Persulfurated Cysteine Promotes Active Site Reactivity in Azotobacter vinelandii Rhodanese
- The Catechol 1,2 Dioxygenase System of Acinetobacter radioresistens: Isoenzymes, Inductors and Gene Localisation
- A Structural Role for Asp83 in the Photoactivation of Rhodopsin
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