Nucleic Acids Research, Vol 25, Issue 23 4715-4722, Copyright © 1997 by Oxford University Press
C Hammann, R Hormes, G Sczakiel and M Tabler
The catalytic activity of the trans cleaving hammerhead ribozyme 2as- Rz12,
with long antisense flanks of 128 and 278 nt, was tested under a wide range
of different reaction conditions for in vitro cleavage of a 422 nt RNA
transcript derived from human immunodeficiency virus type 1 (HIV-1).
Depending on the reaction conditions, in vitro cleavage rates varied by a
factor of approximately 100. Increasing concentrations of magnesium up to 1
M were found to enhance the reaction. Sodium when added simultaneously with
magnesium showed an inhibitory effect on the cleavage reaction. Addition of
sodium during pre-annealing, however, produced a stimulating effect. It was
found that the additional inclusion of spermidine during pre-annealing
further increased the reaction rate markedly. In accordance with
accelerated cleavage, it was possible to identify a distinct,
spermidine-induced conformer of the ribozyme-substrate complex. Under the
most favourable conditions cleavage rates of 1/min were obtained, which are
in the range of rates obtained for conventional hammerhead ribozymes with
short antisense flanks. A comparison of thermodynamic data for short- and
long-armed hammerhead ribozymes suggested that the activation entropy
became unfavourable when helices I and III formed a long chain ribozyme-
substrate complex. We conclude that in the absence of spermidine folding
into the active conformation is impaired by increased friction of long
helices, resulting in relatively low cleavage rates in vitro.
ARTICLES
A spermidine-induced conformational change of long-armed hammerhead ribozymes: ionic requirements for fast cleavage kinetics
Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology Hellas, PO Box 1527, GR-71110 Heraklion, Crete, Greece.
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