The Hammerhead Ribozyme: Structure, Catalysis, and Gene Regulation - Progress in Molecular Biology and Translational Science - page 13

3.5. Mechanistic proposals
Based upon the arrangement of invariant nucleotides in the hammerhead
active site, as well as the solvent structure in a combined crystallographic
and molecular dynamics investigation, we have formulated a testable
hypothesis for how the chemical mechanism of cleavage works. Our pro-
posal is that a specifically bound water molecule accepts a proton from
G12. G12 must ionize to function as the general base, and the proton is rep-
laced by that from the 2 0 -OH of C17. The original G12 proton can then be
relayed directly to the 2 0 -OH of G8 to replace a proton that must be donated
to the 5 0 -O leaving group of C1.1 as the phosphodiester backbone is
cleaved. This mechanism ( Fig. 1.2 B) conserves the number of protons dur-
ing the phosphodiester isomerization. It is testable in that it predicts that
altering the p K a of either the purine base at position 12 or the 2 0 -OH at posi-
tion 8 will alter the cleavage rate without inducing gross structural pertur-
bations. There are also opportunities for transition-state stabilization of the
accumulating negative charges in the pentacoordinate oxyphosphorane. We
propose that either the exocyclic amine of A9 or a divalent cation can per-
form this function.
The roles of G12 and G8 in general base and general acid catalysis, respec-
tively, have been examined using chemical modification strategies in a ham-
merhead RNA sequence closely resembling that of the crystal structure.
To test the hypothesis that G12 is the general base, an affinity label was
synthesized to identify the relevant functionality. The full-length hammer-
head ribozyme was titrated with a substrate analogue possessing a 2 0 -
bromoacetamide group at C17. The electrophilic 2 0 -bromoacetamide group
alkylated the general base, which was then identified as N1 of G12 by
footprinting analysis. In addition, the experiment provided evidence that
the p K a of G12 is perturbed downward to about 8.5 in the context of the
hammerhead active site structure relative to unstructured RNA. 61
To test the hypothesis that the 2 0 -OH of G8 participates in general acid
catalysis, either by itself or accompanied by a divalent metal ion, a bridging
phosphorothioate substrate analogue, in which the leaving group oxygen
atom is replaced by a sulfur atom, was synthesized and characterized in a
full-length hammerhead ribozyme self-cleavage reaction. 62 Cleavage of
the unmodified substrate, unlike the modified leaving group, was inhibited
by modification of the G8 2 0 -OH, and evidence for involvement of a diva-
lent metal ion assisting in p K a perturbation of the general acid was also
obtained.