Identification of the Catalytic Subdomain of the VS Ribozyme and Evidence for Remarkable Sequence Tolerance in the Active Site Loop

We show here that the ribozyme domain of the Neurospora VS ribozyme consists of separable upper and lower subdomains. Deletion analysis demonstrates that the entire upper subdomain (helices III/IV/V) is dispensable for site-specific cleavage activity, providing experimental evidence that the active site is contained within the lower subdomain and within the substrate itself. We demonstrate an important role in cleavage activity for a region of helix VI called the 730 loop. Surprisingly, several loop sequences, sizes, and structures at this position can support site-specific cleavage, suggesting that a variety of non-Watson–Crick structures, rather than a specific loop structure, in this region of the ribozyme can contribute to formation of the active site.