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and ligation at rates 10-fold faster than that for the G8U variant. Physical evi-
dence for the proximity of G8 to the active center of the ribozyme was pro-
vided by Thomas and Perrin,
86
who found a transfer of the alkyl group to G8
from bromoacetamide attached at the position of the 2
0
-OH nucleophile.
The proximity of G8 to the catalytic center was shown crystallograph-
ically in a structure of the ribozyme in which the scissile phosphate was rep-
laced by vanadate as a model of a pentacoordinate phosphorane transition
state (
Fig. 3.9
).
38
G8 was found to be hydrogen bonded to the 2
0
-O and
the
proS
nonbridging O of the scissile phosphate, and was well positioned
to participate in the catalytic chemistry. The structure also revealed the pres-
ence of a second nucleotide juxtaposed with the scissile phosphate. The
nucleobase of A38 (contributed by loop B) was found to form hydrogen
bonds to the 5
0
-O and the
proR
O. Removal of this nucleobase resulted
in a 10,000-fold loss of activity,
87
while ligation activity was shown to be
sensitive to substitution by inclusion of modified nucleotides in NAIM
experiments.
88
2.9. The catalytic mechanism of the hairpin ribozyme
is it
—
same as that of the VS ribozyme?
There is a striking similarity between the active sites of the VS and hairpin
ribozymes in our current view. Both are formed through the interaction of
Figure 3.9 Parallel-eye stereo image of the crystal structure of the active site region of a
transition state analog of the hairpin ribozyme.
38
The ApG segment containing the reac-
tion site is highlighted green, but in this structure the phosphate is replaced by a vana-
dium atom to mimic a pentacovalent phosphorane transition state. Note that G8 is
hydrogen bonded to the 2
0
-O and A38 is hydrogen bonded to the 5
0
-O, consistent with
the proposed mechanism of general acid
base catalysis.
-
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