MAGNESIUM AND CATALYTIC RNA 281
(or both) site(s), A 9 /G 10.1 or G 1.1 /C 17 , had a deleterious effect on substrate cleav-
age rate (at least 10^4 - fold decrease in rate). Catalysis could be rescued by
addition of Cd 2+ ions. Extensive kinetic studies reported by the reference 45
researchers led to the conclusion that a single rescuing metal ion bound to the
P9/G10.1 site acquired a pro - R p phosphoryl oxygen at the P1.1 cleavage site
as an additional ligand in the transition state. The single metal ion was pro-
posed to cause a large conformational change into an “ active conformation ”
and then into a transition state in which cleavage would take place. As shown
in Figure 4 of reference 45 , helix II, containing the P9/G10.1 site in domain II,
is pulled toward the substrate cleavage site at P1.1. In this model, the domain
II conformation remains relatively unchanged while domain I of the ribozyme
undergoes the most extensive conformational change to form the catalytically
active structure. The authors hypothesize that the docking of domain I onto
domain II forms a catalytic pocket in which cleavage of the scissile phosphate
group could be carried out.
Another paper in this series of three studied the behavior of the same two
sites observed spectroscopically using^31 P NMR.^20 The reference 20 researchers
used the same site - specifi c phosphorothioate labeling of the hammerhead
RNA at the A9/G10.1 and P1.1 (G1.1/C17) sites as described above. However,
they used a DNA substrate or 2 ′ - OMe - substituted nucleotide at the cleavage
site in the RNA substrate (RNA - OMe) so that catalytic cleavage would not
proceed. The PDB: 1HMH X - ray crystallographic structure was used as a basis
for visualizing the three - dimensional structure of the two sites. Figure 1B of
reference 20 and Figure 6.19 show a close - up view of the two sites.
For an RNA/RNA - OMe hybrid, substitution of the A 9 pro - R p phosphoryl
oxygen with sulfur showed upfi eld shifts of 2 – 3 ppm in the^31 P NMR A 9 reso-
nance with increasing Cd 2+ concentration. Addition of only 0.5 equivalents of
Cd2+ was suffi cient to cause the upfi eld shift indicating relatively high Cd 2+
affi nity for this site. The authors also believe that the upfi eld (rather than the
expected downfi eld shift on metal binding) shift indicated the more covalent
nature of the Cd 2+ – S bond. Essentially the same behavior was observed for
the A 9 pro - S p phosphoryl oxygen. Behavior at the cleavage site differed con-
siderably. For an RNA/DNA hybrid, substitution at the P1.1 phosphate site
caused the following behaviors: (1) For the P1.1 pro - R p phosphoryl oxygen
substitution by sulfur, addition of up to 10 equivalents of Cd 2+ was required
to shift the^31 P resonance upfi eld by ≤ 0.6 ppm; and (2) for the P1.1 pro - S p
phosphoryl oxygen substitution by sulfur, addition of up to 10 equivalents of
Cd2+ shifted the^31 P resonance downfi eld by ≤ 0.6 ppm. The reference 20 authors
believed that the downfi eld shift in the latter case indicated Cd 2+ coordination
to the pro - R p oxygen in the S p - substituted phosphorothioate. In other words,
Cd2+ coordinated to the pro - R p position at the P1.1 site (G 1.1 in the hammer-
head construct used in these experiments) whether that position was taken by
a sulfur atom or an oxygen atom. The much larger concentration of Cd 2+
required to obtain shifts at the P1.1 site argued for a weaker metal – ligand
interaction at this site. These authors found their NMR results for the substrate