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is further stabilized by helical stacking in which the P1, P1.1, and P4 helices
form one coaxial stack and the P2 and P3 helices form a second. Helix
length for the P1 and P3 regions is identical between the genomic and anti-
genomic forms of the ribozyme; however, the P2 helix is several base pairs
longer in the antigenomic ribozyme. Less sequence conservation is observed
for the P4 helix, and mutational studies indicate that this region can be
completely removed with only a 100-fold loss of catalytic activity. 37 This
feature was exploited to first crystallize the ribozyme by replacing the P4
helix with a protein binding stem-loop to improve crystal quality. 38
Linking the helical structures is single-stranded regions that are crucial for
the catalytic activity. These regions are named according to the helices
that are linked (e.g., J1/2 refers to the “joining” region between the P1
and P2 helices). Whereas sequence conservation between the genomic
and antigenomic forms of the ribozyme within the helical regions of the
molecule is low, several positions in the joining regions are highly con-
served, and mutational studies have shown them to be essential for catalysis
( Fig. 4.2 ). 39,40
The catalytic core of the ribozyme is formed around the P1.1 and P3
helices and is composed of the L3 and J4/2 regions. In the genomic structure
of the ribozyme, the P3 helix is extended by invariant U20 and G25 nucle-
otides that form a reverse wobble pair and stack on another conserved nucle-
otide, C24. 36 Substitutions to either U20 or G25 that force Watson-Crick
base pairing result in ribozymes with markedly decreased activity. 39 Some
variability is permitted in the L3 core region, as the number and composition
of nucleotides flanking the C24 and G25 residues differs between the geno-
mic and antigenomic HDV ribozymes.
The J4/2 region of the ribozyme can range somewhat in length, but
requires a “CNRA” motif within it to support self-cleavage. 41-44 When this
motif is properly positioned in the genomic ribozyme, the N4 of the con-
served C75 residue forms two hydrogen bonds, one with a nonbridging
oxygen atom of the phosphate backbone linking the C21 and C22 residues
that form the 5 0 side of the P1.1 helix, and a second to the 2 0 OH of
U20. 34-36 These interactions lock the J4/2 region into the active site of
the ribozyme. Mutating the genomic C75 or antigenomic C76 residue to
a U or G results in a loss of catalytic activity. 39,40,44-46
The J4/2 strand is further stabilized by the A78 residue of the genomic
ribozyme via an A-minor tertiary interaction with the C18 and G29 residues
that form the middle base pair of the P3 helix. 34-36,47 A purine nucleotide is
required 5 0 to A78 to allow for hydrogen bonding between its Watson-
Crick face and the 2 0 OH of the C19 nucleotide at the base of P3. 34-36
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