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.
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