A long‐range pseudoknot is required for activity of the Neurospora VS ribozyme.

Four small RNA self‐cleaving domains, the hammerhead, hairpin, hepatitis delta virus and Neurospora VS ribozymes, have been identified previously in naturally occurring RNAs. The secondary structures of these ribozymes are reasonably well understood, but little is known about long‐range interactions that form the catalytically active tertiary conformations. Our previous work, which identified several secondary structure elements of the VS ribozyme, also showed that many additional bases were protected by magnesium‐dependent interactions, implying that several tertiary contacts remained to be identified. Here we have used site‐directed mutagenesis and chemical modification to characterize the first long‐range interaction identified in VS RNA. This interaction contains a 3 bp pseudoknot helix that is required for tertiary folding and self‐cleavage activity of the VS ribozyme.