Bridging the Gap Between Theory and Experiment to Derive a Detailed Understanding of Hammerhead Ribozyme Catalysis - Progress in Molecular Biology and Translational Science - page 29

X-ray crystal structure of mHHR, an RNA/DNA hybrid, was published in
1994 by Pley et al., 87 followed shortly thereafter by an all-RNA mHHR
structure published in 1995 by Scott et al. 88 In a pioneering series of
papers, 70-72 crystallographic structures of the mHHR were solved at differ-
ent stages along the reaction coordinate, including unbound and Mg 2 þ -
bound systems, early and late intermediates, and a ribozyme product
structure.
These mHHR crystal structures were the departure point for many ini-
tial theoretical investigations, and initiated a significant amount of effort
toward detailed understanding of HHR mechanism. On the other hand,
it also ignited numerous mechanistic debates among research groups 89 that
had conflicting interpretations of structural and biochemical data. As the
crystal structures did provide the global positions of the distal termini of
all three flanking helical stems, many biochemical experiments designed
to probe transition-state interactions and the chemistry of catalysis appeared
to be irreconcilable with the crystal structures. 61,90 Molecular simulations
based on the mHHR only served to fuel speculation and further debate
without providing new insights. Some of the key issues that remained unre-
solved are summarized in the next sections.
2.1.1 Positions of the A9 and scissile phosphates were inconsistent
with interpretations from thio/rescue effect experiments
Thio-substitution and soft metal ion rescue experiments 63,91,92 suggest that
both the two oxygen atoms (A9:O2P) and the scissile phosphate oxygen
(C1.1:O2P) acquire key direct metal ion interactions in the transition state
for catalysis, and may be bound to a single active Mg 2 þ ion simultaneously in
the transition state, requiring these residues to be in close proximity. In the
mHHR structure, however, the distance between these two oxygen atoms is
almost 20 ˚ . Hence, it is not possible to explain the related thio-substitution
data if only one Mg 2 þ ion is considered, 93 unless a fairly large-scale confor-
mational change takes place or unless two Mg 2 þ ions are involved and bind
to A9:O2P and C1.1:O2P separately.
2.1.2 Crystal structures did not provide straightforward
interpretation of mutagenesis data
It has been shown that four totally conserved residues, G5, G8, G12, and C3,
were critical for the HHR catalysis, and they are extremely sensitive to any
modification. Even a small chemical modification of a single exocyclic func-
tional group on any one of these nucleotides could result in the nearly total