Nucleic Acids Research, Vol 26, Issue 24 5551-5561, Copyright © 1998 by Oxford University Press
DJ Earnshaw and MJ Gait
The hairpin ribozyme is a small catalytic RNA that achieves an active
configuration by docking of its two helical domains in an antiparallel
fashion. Both docking and subsequent cleavage are dependent on the presence
of divalent metal ions, such as magnesium, but there is no evidence to date
for direct participation of such ions in the chemical cleavage step. We
show that aminoglycoside antibiotics inhibit cleavage of the hairpin
ribozyme in the presence of metal ions with the most effective being
5-epi-sisomicin and neomycin B. In contrast, in the absence of metal ions,
a number of aminoglycoside antibiotics at 10 mM concentration promote
hairpin cleavage with rates only 13-20-fold lower than the
magnesium-dependent reaction. We show that neomycin B competes with metal
ions by ion replacement with the postively charged amino groups of the
antibiotic. In addition, we show that the polyamine spermine at 10 mM
promotes efficient hairpin cleavage with rates similar to the
magnesium-dependent reaction. Low concentrations of either spermine or the
shorter polyamine spermidine synergize with 5 mM magnesium ions to boost
cleavage rates considerably. In contrast, at 500 microM magnesium ions, 4
mM spermine, but not spermidine, boosts the cleavage rate. The results have
significance both in understanding the role of ions in hairpin ribozyme
cleavage and in potential therapeutic applications in mammalian cells.
ARTICLES
Hairpin ribozyme cleavage catalyzed by aminoglycoside antibiotics and the polyamine spermine in the absence of metal ions
Medical Research Council, Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK.
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