The Hammerhead Ribozyme: Structure, Catalysis, and Gene Regulation - Progress in Molecular Biology and Translational Science - page 8

anticodon loop of tRNA, and the remaining conserved residues augment or
extend stem II via stacked sheered GA pairs. 14,15 Together, these residues
form a three-strand junction, in which the augmented stem II stacks upon
stem II, and stem(s) I branches out via the uridine turn and the cleavage-site
nucleotide.
The first hammerhead ribozyme structure, solved by McKay and
coworkers in 1994, 13 was that of a minimal hammerhead RNA enzyme
strand bound to a DNA substrate-analogue inhibitor, and in 1995 a different
all-RNA hammerhead construct having a 2 0 -OMe inhibitory substitution of
the nucleophilic 2 0 -OH of C17 appeared. 14,15 Subsequently, structures of
minimal hammerheads without modified nucleophiles appeared in various
precatalytic conformational states, 44 and finally a structure of the cleavage
product appeared 53 in 2000, providing the opportunity to construct the first
“molecular movie” of ribozyme catalysis.

2.3. Experimental discord
It was immediately apparent from the first hammerhead crystal structures 13
that a conformational change would need to take place to position the
attacking nucleophile in line for activation of the cleavage reaction. The
requirement for this conformational change motivated subsequent crystallo-
graphic freeze-trapping experiments. 36,37
Meanwhile, a growing list of discrepancies between the minimal ham-
merhead ribozyme structure and mechanistic biochemical experiments
designed to probe transition-state interactions began to accumulate. 17
The observed hydrogen-bonding patterns within the minimal hammerhead
crystal structures could not explain the immutability of G8, G12, G5, C3,
and a number of other core residues. 16 Even more concerning was evidence
that the phosphate of A9 and the scissile phosphate, separated by 18 ˚ in the
minimal hammerhead crystal structures, might bind a single metal ion in the
transition state of the reaction. 54 Such an interaction would require the two
phosphates to approach each other within about 4.4 ˚ , but this requirement
could be demonstrated to be incompatible with the minimal hammerhead
crystal structure unless significant unwinding or base-unpairing were to take
place in one or more of the helices. 55

3. THE FULL-LENGTH SEQUENCE
When the hammerhead RNA was first discovered, it was observed to
be embedded within an 370 nucleotide single-stranded genomic satellite