HDV Family of Self-Cleaving Ribozymes - Progress in Molecular Biology and Translational Science - page 131

activated. 67 A recent crystal structure of an inhibited precursor of the geno-
mic ribozyme provided electron density at sufficiently high resolution so
that the hydration of an active-site Mg 2 þ ion could be modeled. The struc-
ture shows that the metal ion does form inner sphere contacts, although not
to the oxygens on the scissile phosphate, but rather to the phosphate
between C22 and U23. 36 This structure was determined using a 2 0 deoxy
form of the 1 nucleotide, which together with the scissile phosphate is dis-

ordered in the crystal. Therefore, the ligands of the active-site metal ion
could not be fully described. A previous crystal structure of a precursor ribo-
zyme contained a ribose at the

1 position, but an active-site mutation cau-
sed rearrangement in the network of hydrogen bonds around the scissile
phosphate. 35 Metal ions observed in the active site included Sr 2 þ , which
made inner sphere contacts with the phosphates of C22 and U23, the base
of U20, and with the 5 0 oxygen of G1. Tl þ ,aK þ isostere, was observed
making an inner sphere contact with both the 2 0 hydroxyl of the 1 nucle-

otide and the scissile phosphate. 81 The active site contained two other Tl þ
ions, which were displaced by a single Co NH ð Þ 6 3 þ when it was soaked into
the crystals. In the crystal structure of the ribozyme product, the active site
appears to accommodate two different weakly bound metal ions at slightly
distinct locations. 82 All together, the data support a model for the active site
capable of binding several different metal ions that most likely activate the 2 0
nucleophile by promoting its deprotonation.
Crystal structures of the precursor forms of the genomic HDV ribozyme
show a poorly ordered leader sequence (the 5 0 product), with only the sugar-
phosphate backbone electron density observable in the most ordered
case. 35,36,81 It is clear that the RNA strand has to make a sharp turn at
the cleavage site to accommodate the leader, which makes no base-specific
contacts with the ribozyme. This observation correlates well with the lack of
sequence conservation upstream of the cleavage site among HDV-like
ribozymes, although the identity of the

1 nucleotide affects the metal
ion preference of the active site, suggesting that the base may affect the pre-
cise orientation of the scissile phosphate in the active site. 83 Terbium(III)
footprinting of the genomic ribozyme revealed that the antigenomic ribo-
zyme undergoes a conformational change between the precursor and prod-
ucts states and that the identity of the 1 nucleotide affects the metal ion

accessibility of the groups lining the active site, suggesting that the active-
site conformation is influenced by the 5 0 sequence. 77,84,85 The models
derived from the crystal structures show that the ribozyme does not undergo
significant conformational change during the reaction. In solution, how-
ever, a FRET-based assay measured a significant change in the overall shape