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.
Sood
Vanita D
VD
Department of Molecular and Medical Genetics, University of Toronto, 1 King's College Circle, Toronto, Ontario M5S 1A8, Canada.
Yekta
Soraya
S
Collins
Richard A
RA
D001483:Base Sequence; D003854:Deoxyribonucleotides / Q000737:chemistry; D004722:Endoribonucleases / Q000737:chemistry* / Q000378:metabolism*; D006878:Hydroxides / Q000737:chemistry; D008969:Molecular Sequence Data; D009491:Neurospora / Q000201:enzymology*; D009690:Nucleic Acid Conformation; D016337:RNA, Catalytic / Q000737:chemistry* / Q000378:metabolism*; D013873:Thionucleotides / Q000737:chemistry
D003854:Deoxyribonucleotides; D006878:Hydroxides; D016337:RNA, Catalytic; D013873:Thionucleotides; D004722:Endoribonucleases; C438195:varkud satellite ribozyme