The contribution of 2′-hydroxyls to the cleavage activity of the Neurospora VS ribozyme

We have used nucleotide analog interference mapping and site-specific substitution to determine the effect of 2′-deoxynucleotide substitution of each nucleotide in the VS ribozyme on the self-cleavage reaction. A large number of 2′-hydroxyls (2′-OHs) that contribute to cleavage activity of the VS ribozyme were found distributed throughout the core of the ribozyme. The locations of these 2′-OHs in the context of a recently developed helical orientation model of the VS ribozyme suggest roles in multi-stem junction structure, helix packing, internal loop structure and catalysis. The functional importance of three separate 2′-OHs supports the proposal that three uridine turns contribute to local and long-range tertiary structure formation. A cluster of important 2′-OHs near the loop that is the candidate region for the active site and one very important 2′-OH in the loop that contains the cleavage site confirm the functional importance of these two loops. A cluster of important 2′-OHs lining the minor groove of stem–loop I and helix II suggests that these regions of the backbone may play an important role in positioning helices in the active structure of the ribozyme.