Binding of IRE-BP to its cognate RNA sequence: SFM studies on a universal RNA backbone for the analysis of RNA-protein interaction

We have used an RNA consisting of the potato spindle tuber viroid (PSTVd) a nd 240 bp of double-stranded RNA derived from the GUS gene as a backbone fo r scanning force microscope (SFM) studies on RNA binding proteins. The in v itro transcribed RNA dforms a rod-like structure of apparent 130 nm in leng th with a completely base paired central part flanked by the incompletely p aired viroid helix with bulges on both sides. The termini of the molecule c onsist of loops such that no blunt or staggered RNA ends are exposed. Suita ble, asymmetrical restriction sites in the construct allow for the insertio n of sequences of interest, e. g. protein binding sites. We have inserted t he IRE (iron responsive element) sequence into the construct and have used in vitro transcripts to study binding of IRE-BP. Relative binding frequenci es 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 G US-PSTVd-IRE backbone, the orientation of the molecule is easily determined by IRE-BP binding. It thus provides a versatile tool to study specific as well as preferential interaction of other proteins with sequences or struct ures inserted into a different part of the molecule.