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We have used an RNA consisting of the potato spindletuber viroid (PSTVd) and 240 bp of doublestranded RNA derived from the GUS geneas a backbone for scanning force microscope (SFM) studies on RNA bindingproteins. The in vitro transcribed RNA forms a rodlike structure of apparent130 nm in length with a completely base paired central part flanked by theincompletely paired viroid helix with bulges on both sides. The termini of themolecule consist of loops such that no blunt or staggered RNA ends are exposed.Suitable, asymmetrical restriction sites in the construct allow for theinsertion of sequences of interest, e. g. protein binding sites. We haveinserted the IRE (iron responsive element) sequence into the construct and haveused in vitro transcripts to study binding of IREBP. Relative bindingfrequencies show that 70% of the protein binds to the expected site in themolecule while only a slightly enhanced binding is observed at the termini. Inthe GUSPSTVdIRE backbone, the orientation of the molecule is easily determinedby IREBP binding. It thus provides a versatile tool to study specific as wellas preferential interaction of other proteins with sequences or structuresinserted into a different part of the molecule.

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