HDV Family of Self-Cleaving Ribozymes - Progress in Molecular Biology and Translational Science - page 127

these data suggest that the ribozymes are properly folded and highly active
under in vivo -like conditions, but can sample many inhibitory conformations
resulting from alternative base-pairing interactions.

4. HDV RIBOZYMES ' REACTION MECHANISM

Like other small self-cleaving RNAs, the HDV ribozymes promote
self-scission through a nucleophilic attack by a 2 0 hydroxyl group of the

1 nucleotide on the adjacent phosphate. The reaction yields a 2 0 -3 0 -cyclic
phosphate on the upstream (

1) nucleotide and a 5 0 OH moiety on the
downstream product (formally the first nucleotide of the ribozyme).

4.1. Active-site groups
The mechanism of HDV ribozyme self-scission has been one of the most
intensely studied enzymatic reactions of the last decade. Initial mutagenesis,
crosslinking, and phosphorothioate interference studies implicated J4/2,
particularly the genomic C75 (C76 in antigenomic ribozyme) residue,
J1/4, and P3/L3 as key regions for catalysis. 40,48,64,65 Crystal structure of
the genomic ribozyme product afforded the first glimpse of the active site
at atomic resolution and provided two surprises: lack of electron density
corresponding to a magnesium ion in the active site, and a nucleobase
(C75) located within potentially hydrogen bonding distance of the G1 5 0
hydroxyl group, which is the leaving group of the reaction. 34 The authors
thus concluded “that C75 acts as the general base that activates the 2 0 -
hydroxyl group of nucleotide the negative

electrostatic potential in the region of C75 (resulting from the trefoil turn),
could perturb the p K a values of C75, making it basic.” 34 Besides C75, the
active site consists of the L3 ribose-phosphate backbone between C22 and
U23 and the U20-G25 pair, which in the most recent and highest resolution
crystal structure of the inhibited ribozyme precursor forms a reverse wobble
pair and coordinates a divalent metal ion. 36
The proposal that a nucleobase could act directly in proton transfer dur-
ing a transesterification reaction motivated a large number of studies aimed at
deciphering the catalytic mechanism of these ribozymes. All of the studies
implicated the cytosine in proton transfer during the rate-limiting step of
the mechanism. This was the case for both cis - and trans -cleaving constructs,
as well as ribozymes acting on nonnatural substrates including 2 0 -5 0 pho-
sphodiesters and 5 0 phosphorothiolates. 66,67 The arguments for a direct role
of C75 or C76 in the self-scission of genomic or antigenomic ribozymes,

1 for nucleophilic attack ...