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only recently observed example consisted of G8 and G12, which had been
identified as possible participants in acid-base catalysis. After we demon-
strated that the hammerhead ribozyme does not require divalent metal ions
for catalysis, it gradually became apparent that theRNA itself, rather than pas-
sively bound divalent metal ions, must play a direct chemical role in any acid-
base chemistry within the hammerhead active site. However, it was
completely unclear howG12 andG8 could accomplish this, given the original
structures of the minimal hammerhead ribozyme. In addition, the attacking
nucleophile in the original structures, that is, the 2
0
-OH of C17, was not in a
position amenable to in-line attack upon the adjacent scissile phosphate.
16
Perhaps most worrisome were experiments that suggested the A9 and scissile
phosphates must come within about 4
˚
of one another in the transition state
based upon double phosphorothioate substitution and soft metal ion rescue
experiments.
54
The distance between these phosphates in the crystal structure
was about 18
˚
, with no clear mechanism for close approach if the stem II-
and stem(s) I A-form helices were treated as rigid bodies. Taken together,
these results appeared to suggest that a fairly large-scale conformational
change must take place to reach the transition state within the minimal ham-
merhead ribozyme structure. For these reasons, results from the two sets of
experiments (biochemical vs. crystallographic) appeared not only to be at
odds, but completely and hopelessly irreconcilable, and they generated a sub-
stantial amount of discord in the field. No compelling evidence for dismissing
either set of experimental results was ever successfully made, although some
claims to the contrary were made in favor of each.
The resolution of this vexing conundrum came only with the crystal
structure of the full-length hammerhead ribozyme in which C17 is posi-
tioned for in-line attack, and the invariant residues C3, G5, G8, and G12
all appear involved in vital interactions relevant to catalysis. Moreover,
the A9 and scissile phosphates are observed to be 4.3
˚
apart, which is con-
sistent with the idea that these phosphates when modified could bind a single
thiophilic metal ion. The structure also reveals how two invariant residues,
G12 and G8, are positioned within the active site in a manner consistent
with their previously proposed roles in acid-base catalysis. G12 is within
hydrogen-bonding distance to the 2
0
-O of C17, the nucleophile in the
cleavage reaction, and the ribose of G8 hydrogen bonds to the leaving group
5
0
-O, while the nucleobase of G8 forms a Watson-Crick pair with the
invariant C3. The crystal structure of the full-length hammerhead ribozyme
thus clearly addressed the major concerns that appeared irreconcilable with
earlier minimal hammerhead structures.
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