Binding of IRE-BP to Its Cognate RNA Sequence: SFM Studies on a Universal RNA Backbone for the Analysis of RNA-Protein Interaction

Binding of IRE-BP to Its Cognate RNA Sequence: SFM Studies on a Universal RNA Backbone for the... 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. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Biological Chemistry de Gruyter

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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Publisher
de Gruyter
Copyright
Copyright © 2001 by the
ISSN
1431-6730
DOI
10.1515/BC.2001.145
pmid
11592396
Publisher site
See Article on Publisher Site

Abstract

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.

Journal

Biological Chemistryde Gruyter

Published: Aug 28, 2001

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