HDV Family of Self-Cleaving Ribozymes - Progress in Molecular Biology and Translational Science - page 135

Co 2 þ . 2 However, no reactivity was observed in the presence of monovalent
cations, even at high concentrations (3 M Li þ ), or in the presence of cobalt
hexamine. Folding of the CPEB3 ribozyme into an HDV-like secondary
structure is further supported by data from phosphorothioate interference
mapping. In HDV, several key interactions are made between the non-
bridging oxygen atoms and the nucleotides essential to catalysis. 34-36 When
the CPEB3 ribozyme sequence is fit to an HDV-like secondary structure,
the phosphorothioate substitutions that disrupt its scission correspond to
the same interference positions in the HDV ribozyme. 2,65,80 The CPEB3
sequence also exhibits a flat pH profile around pH 7, as is observed in both
HDV ribozymes under similar conditions. 2,45
Sequence-based searches using the human CPEB3 sequence revealed a
remarkable conservation of this motif across mammalian CPEB3 genes
( Fig. 4.5 ). 2 Corresponding motifs were not observed among analogs of
the CPEB3 gene in nonmammalian eukaryotic organisms.
Although the mammalian CPEB3 ribozymes share a high degree of
sequence conservation, the variations present have profound effects on
the reaction. All mammals other than humans contain a C-G base-pair at
the top of the P1 helix, whereas humans have a C A mismatch at this loca-

tion ( Fig. 4.5 ). This might at least in part explain the slower cleavage rate
observed for the human CPEB3 ribozyme. The ribozyme found in Canis
familiaris is most likely inactive because it contains a cytosine in lieu of
the requisite guanosine at the cleavage site. This isolate also contains a single
base-pair mismatch in the P2 helix and a U-G wobble pair at the top of P3.
In the bovine ribozymes, a mutation at the base of the P3 helix transforms a
G-C base pair into an A C mismatch that would be expected to destabilize

the overall structure and significantly decrease the self-cleavage rate. Several
other mutations are observed among other mammalian CPEB3 ribozymes;
however, most of them occur in regions that are known to be nonessential
for catalysis or that covary in support of the secondary structure ( Fig. 4.5 ).
Despite sharing a common secondary structure, the catalytic rates
between the human CPEB3 and viral HDV ribozymes are highly divergent.
HDV ribozyme catalyzes its self-scission with a t 1/2 approximately 3.5 min
in 2 mMMg 2 þ at 37 C, whereas the human CPEB3 ribozyme has a t 1/2 on
the order of 1 h in 5 mM Mg 2 þ at 37 C. 2,51 This is at least partially attrib-
utable to the relatively weak P1.1 found in the CPEB3 sequences. In all
isolates, this helix consists of a single G-C base pair and a U U mismatch.

Mutating this U U mismatch into a canonical U-A base pair increases the

catalytic rate of the ribozyme by an order of magnitude. 2