Nucleic Acids Research, Vol 26, Issue 14 3379-3384, Copyright © 1998 by Oxford University Press
M Araki, Y Okuno, Y Hara and Y Sugiura
An allosteric ribozyme has been designed using the hammerhead ribozyme as
the active site and aflavin-specific RNA aptamer as a regulatory site. We
constructed six variants with a series of base pairs in the linker region
(stem II). Under single turnover conditions, kinetic studies were carried
out in the absence and presence of flavin mononucleotide (FMN).
Interestingly, FMN addition did not influence the cleavage rate of
constructs with a 5-6 bp linker but stimulated the catalytic activity of
those bearing a shorter linker. In particular, the apparent k cat of Rz3
increases by approximately 10-fold upon addition of saturating amounts of
FMN. To determine the rate constants( K m4and k cat), the ribozyme
regulated most effectively by FMN was further investigated. FMN mainly
affected the k cat value, reflecting the rate limiting conformational
change step of the overall cleavage reaction, depending on helix formation
in stem II. Probably, FMN influences the orientation of structures
necessary for the cleavage reaction through stem II formation. The result
of chemical modification revealed that binding of FMN to the aptamer domain
induced the helix formation in stem II required for catalytic activity.
Therefore, a specific FMN-mediated allosteric interaction seems to promote
a conformational alteration from an open to a closed structure in stem II.
The concept of conformational modification in the allosteric effect is
consistent with other allosteric enzymes, suggesting that such a
conformational change is a fundamental feature of allosteric enzymes in
biological systems.
ARTICLES
Allosteric regulation of a ribozyme activity through ligand-induced conformational change
Institute for Chemical Research, Kyoto Universitiy, Uji, Kyoto 611- 0011, Japan.
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