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contained sites of interference by phosphorothioate incorporation and sup-
pression by thiophilic metal ions.
56
Sequence changes in the loop also
affected ligation activity,
41
and the loop was also very sensitive to the intro-
duction of nucleotide analogs.
45
In our SAXS-derived model of the ribozyme,
46
the scissile phosphate is
readily juxtaposed with the A730 loop. Docking the substrate into the cleft
between helices II and VI naturally facilitates a close interaction between the
cleavage site and the A730 loop. Considering the major sensitivity of ribo-
zyme activity to changes in this region, the results suggest that the A730 loop
is likely to be a significant part of the active site of the ribozyme.
Within the A730 loop, one particular nucleotide stands out. Substitution
of A756 by G, C, or U leads to
300-fold loss of cleavage
20
and ligation
activity.
41
These changes had only a small effect on substrate binding affinity
(
△
K
d
fivefold), and most of the effect arose from a reduced rate of central
conversion of the substrate into product.
20
In NAIM experiments studying
ligation rate, the 756 position was the most sensitive nucleotide to substitu-
tion by a variety of analogs.
45
UV cross-linking data placed A756 physically
close to the cleavage site in the substrate.
57
Removal of the 2
0
-hydroxyl
group of A756 resulted in a small reduction in observed cleavage rate,
whereas removal of the nucleobase (creating an abasic site at position
756) lowered the activity
1000-fold. Removal of the exocyclic amine
from the 6 position, translocation to the 2 position, or addition of a
2-NH
2
group all led to 1000-fold loss of cleavage. By contrast, replacement
of N7 by CH (7-deazaadenosine) had a negligible effect on activity. Thus,
the Watson-Crick edge of the nucleobase of A756 is important for catalytic
activity. While activity of the A756 abasic VS ribozyme could not be
restored by high concentrations of imidazole in the medium,
21
a variant
ribozyme with a covalently linked imidazole at position 756 demonstrated
significant rates of cleavage and ligation.
58
The activity of the A756 imidazole ribozyme suggested that A756 might
be involved in general acid-base catalysis. This was also supported by other
substitution experiments at this position.
45
Consequently, we thought it
likely that A756 might have a partner in general acid-base catalysis that could
be another nucleobase somewhere in the RNA. The nucleobase was
unlikely to be located within the main body of the ribozyme, but we iden-
tified G638 as a strong candidate for the role.
22
Substitution of G638 with
any other nucleotide resulted in a reduction of cleavage rate by four orders of
magnitude, whereas substrate folding was not perturbed. The variant sub-
strates bound the ribozyme with similar affinity, acting as competitive
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