Identification of structural elements critical for inter-domain interactions in a group II self-splicing intron

Thus far, conventional biophysical techniques, such as NMR spectroscopy or X-ray crystallography, allow the determination, at atomic resolution, of only structural domains of large RNA molecules such as group I introns. Determination of their overall spatial organization thus still relies on modeling. This requires that a relatively high number of tertiary interactions are defined in order to get sufficient topological constraints. Here, we report the use of a modification interference assay to identify structural elements involved in interdomain interactions. We used this technique, in a group II intron, to identify the elements involved in the interactions between domain V and the rest of the molecule. Domain V contains many of the active site components of these ribozymes. In addition to a previously identified 11 nucleotide motif involved in the binding of the domain V terminal GAAA tetraloop, a small number of elements were shown to be essential for domain V binding. In particular, we show that domain III is specifically required for the interaction with sequences encompassing the conserved 2 nucleotide bulge of domain V.