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catalyzed by the ribozymes, although significantly greater rate enhancements
have been demonstrated recently for specific forms of the hammerhead and
VS ribozymes.
7,8
Understanding just how RNA molecules can generate significant accel-
eration of these phosphoryl transfer reactions is a major goal. Inspection of
the chemical mechanism (
Fig. 3.1
) suggests a number of possible strategies.
Many enzymes lower activation barriers by differential stabilization of the
transition state structure. For the nucleolytic ribozymes, the transition state
will strongly resemble the pentacoordinate phosphorane, which might be
stabilized by hydrogen bonding or juxtaposition of positive charge. The
reaction requires the nucleophile to attack in-line with the 5
0
-O leaving
group, and if the structure can facilitate this trajectory it might provide a rate
enhancement of perhaps 10-100-fold. Looking at the protein world suggests
another possible source of catalysis. The enzyme pancreatic ribonuclease
A catalyzes essentially the same reaction as the nucleolytic ribozymes using
general acid-base catalysis. A general base is used to deprotonate the
attacking nucleophile; an alkoxide ion is a stronger nucleophile than a
hydroxyl by many orders of magnitude. In parallel, a general acid is used
to protonate the oxyanion leaving group, facilitating its departure. In RNase
A, the imidazole side chains of two histidine residues provide the general
acid and base. These residues are ideally suited to the task because they have
p
K
a
values close to physiological pH. By contrast, RNA is limited to
nucleobases, hydroxyl groups, and hydrated metal ions, all of which have
p
K
a
values that are significantly shifted from physiological pH. This lowers
the effective concentration of active catalyst. For example, if a general acid
has a p
K
a
of 5 then only 1% will be protonated at neutral pH. However, the
low effective concentration of acid will be offset to some degree by its
greater reactivity. The observed rates for nucleolytic ribozymes are generally
slow, but turnover is not required for their biological function.
2. THE HAIRPIN AND VS RIBOZYMES
Both the hairpin and VS ribozymes process replication products that
are transcribed from RNA and DNA, respectively. The tobacco ringspot
virus has a circular single-stranded satellite RNA of 359 nt. In its replication
cycle, the negative strand of the satellite RNA is produced as a concatameric
transcript that is processed into monomeric circular molecules by sequential
cleavage and ligation reactions catalyzed by the hairpin ribozyme within the
RNA. The VS RNA is an abundant transcript from a plasmid found in the
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