Nucleic Acids Research, Vol 27, Issue 2 403-410, Copyright © 1999 by Oxford University Press
F Nishikawa, M Roy, H Fauzi and S Nishikawa
To determine the stem I structure of the human hepatitis delta virus (HDV)
ribozyme, which is related to the substrate sequence in the trans -acting
system, we kinetically studied stem I length and sequences. Stem I
extension from 7 to 8 or 9 bp caused a loss of activity and a low amount of
active complex with 9 bp in the trans -acting system. In a previous report,
we presented cleavage in a 6 bp stem I. The observed reaction rates
indicate that the original 7 bp stem I is in the most favorable location
for catalytic reaction among the possible 6-8 bp stems. To test base
specificity, we replaced the original GC-rich sequence in stem I with
AU-rich sequences containing six AU or UA base pairs with the natural +1G.U
wobble base pair at the cleavage site. The cis -acting AU-rich molecules
demonstrated similar catalytic activity to that of the wild-type. In trans
-acting molecules, due to stem I instability, reaction efficiency strongly
depended on the concentration of the ribozyme-substrate complex and
reaction temperature. Multiple turnover was observed at 37 degreesC,
strongly suggesting that stem I has no base specificity and more efficient
activity can be expected under multiple turnover conditions by substituting
several UA or AU base pairs into stem I. We also studied the substrate
damaging sequences linked to both ends of stem I for its development in
therapeutic applications and confirmed the functions of the unique
structure.
ARTICLES
Detailed analysis of stem I and its 5' and 3' neighbor regions in the trans-acting HDV ribozyme
National Institute of Bioscience and Human Technology, AIST, MITI, 1-1 Higashi, Tsukuba Science City,Ibaraki 305-8566, Japan. nisikawa@nibh.go.jp
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