MAGNESIUM AND CATALYTIC RNA 279
First, the researchers found that the rate of substrate turnover in the talo -
5 ′ - C - methyl modifi ed RNA was reduced well over 300 - fold. The experiments
were carried out in the presence of Co 2+ as a divalent ion slightly more cata-
lytically active than Mg 2+. Further study found that the modifi ed RNA ground -
state crystal structure at pH 6 (both in the presence and absence of Co 2+ ) was
indistinguishable from the unmodifi ed hammerhead structures reported and
discussed previously (PDB: 299D and 300D). The same was found for the
structure at pH 8.5 in the presence of Co 2+ in comparison to the structure seen
for Mg 2+ - soaked crystals at pH 8.5 (PDB: 301D). The Co 2+ - soaked crystals
(100 mM CoCl 2 , 30 minutes at pH 6 and 50 mM CoCl 2 , 30 minutes
at pH 8.5) served as controls for the current research because they indicated
little or no ground - state structure disturbance with inclusion of the talo - methyl
modifi cation. In these “ ground state ” structures, the A - form RNA conforma-
tion at the cleavage site puts the C 17 O2 ′ oxygen maximally out of register
(90 ° ) with the A 1.1 phosphorus and O5 ′ oxygen for an inline attack
mechanism.
Following these fi ndings, the hammerhead ribozyme – substrate complex
crystals were soaked for 2.5 hours in 50 mM Co 2+ at pH 8.5. Under these condi-
tions, cleavage in an unmodifi ed RNA would have taken place in the crystal-
line state even faster than the rate in solution. However, cleavage did not take
place in thetalo - 5 ′ - C - methyl modifi ed RNA. Instead, extensive conforma-
tional changes were noted in the positions of C 17 and at the scissile phosphate
of A 1.1. The base and ribose of C 17 rotated about 60 ° , causing the base of C 17
to move over 8.7 Å (relative to its position in the comparable ground - state
structure PDB: 301D) and to stack onto the A 6 nucleobase. These and other
changes at the cleavage site pulled the scissile phosphate away from its stan-
dard helical geometry and toward a position more relevant to the inline S N 2
mechanism. Essentially the scissile phosphate is rendered more susceptible to
nucleophilic attack from the cleavage site C 17 2 ′ - hydroxyl that has also been
repositioned in thetalo - 5 ′ - C - methyl - modifi ed RNA crystal structure — the 5 ′
O atom of A 1.1 (leaving group) and the 2 ′ O atom of C 17 (nucleophile) are
3.1 Å apart in the PDB: 379 structure, whereas the same atoms are 4.0 Å apart
in the PDB: 301D structure. Modeling different phosphorus coordination
spheres at the scissile phosphate site does not unequivocally place the phos-
phorus atom in the pentacoordination conformation necessary for the S N 2
reaction mechanism. This is due to the inability to visualize the necessary
atoms (the crucial phosphate group of A 1.1 , for instance) at the 3.0 - Å resolu-
tion of the crystal structure. Despite the changes in conformation at the cleav-
age site, other stacking interactions within the catalytic pocket and in its
vicinity are conserved in the activated structure displayed in this work (PDB:
379D). This led the reference 44 authors to conclude that global conforma-
tional changes do not take place in the hammerhead ribozyme during cleavage
and to describe the PDB: 379D structure as a “ late intermediate ” in the reac-
tion pathway.