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inhibitors of the natural sequence substrate. In other words, the G638A sub-
strate bound to the ribozyme in an apparently normal manner, but was cat-
alytically incompetent. This strongly suggested that G638 plays a direct role
in the function of the ribozyme. Since its 2
0
-hydroxyl is dispensable,
59
it is
probable that the guanine nucleobase is the key participant, and nucleotide
substitution experiments revealed that the key functionalities lie on theWat-
son-Crick edge.
22
We also explored the cleavage of a substrate in which
G638 was replaced by an imidazole nucleoside. Initially, we used the same
compound that has been shown to be active when replacing A756,
58
but this
was found to be inactive.
22
However, in this species, the imidazole ring is
bonded directly to the C1
0
atom of the ribose (C
0
-linked imidazole), so that
it is not possible to superimpose either ring N with N1 of guanine. We
therefore made a new derivative in which two methylene carbon atoms link
imidazole to ribose (C
2
-linked imidazole), and which exhibited measurable
activity
60
and provided further evidence supporting general acid-base
catalysis.
These studies collectively point to an involvement of two key
nucleobases, A756 and G638 in the catalytic mechanism of the VS ribo-
zyme. An intimate association of the A730 loop and the internal loop of sub-
strate helix I would bring these nucleotides and the scissile phosphate into
juxtaposition. In the NMR structure of the substrate RNA,
51
N1 of
G638 is 6
˚
from the 2
0
-OH group of G620 (the nucleophile). We suspect
that the loop-loop interaction with the A730 loop leads to structural reor-
ganization that generates the active conformation.
2.5. A new crystal structure of the VS ribozyme
A crystal structure of the VS ribozyme was solved that confirms many of the
features described above (Suslov, N., Huang, H., Lilley, D. M. J., Rice, P.,
and Piccirilli, J. A., unpublished data). The structure has a resolution of
3.07
˚
in the best direction and the electron density is readily interpretable
for the whole molecule. The first point to note is that in the crystal the RNA
had dimerized in exactly the manner we had deduced from our earlier com-
plementation experiments.
54
Thus the substrate stem-loop I of one ribo-
zyme molecule docked into the cleft between helices VI and II of the
other in a mutual manner like the number 69. The junction of helices
III, IV, and Vwas almost exactly as previously deduced,
39
although the junc-
tion of helices II, III, and VI was slightly different from our earlier model
40
in
that helices II and III are coaxial. The loop I-loop V interaction occurs as
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