Nucleic Acids Research, 2002, Vol. 30, No. 5 1132-1138
© 2002 Oxford University Press
The contribution of 2'-hydroxyls to the cleavage activity of the Neurospora VS ribozyme
Department of Molecular and Medical Genetics, University of Toronto, 1 King’s College Circle, Toronto, Ontario M5S 1A8, Canada
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.
* To whom correspondence should be addressed. Tel: +1 416 978 3541; Fax: +1 416 978 6885; Email: rick.collins{at}utoronto.ca Present address:Soraya Yekta, Department of Biology, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139, USA
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