294 GROUP I AND II METALS IN BIOLOGICAL SYSTEMS: GROUP II
during crystallization creates a kinetic bottleneck preventing cleavage but
allowing a conformational change (at C 17 ) that precedes catalytic cleavage.
Next, the researchers raised the pH in the crystal from 6 to 8.5, the pH
found previously to be slightly above the apparent p Ka of the cleavage reac-
tion. It was hoped that this would drive most of the RNA molecules in the
crystal into their catalytically active conformation. Crystals that formed were
treated by fl ash - freezing and diffraction data were collected at 100 K as above.
These data were deposited as PDB: 1Q29.^58 This crystal structure showed that
the C – G hydrogen - bonding tether remained intact, although possibly more
strained. At the cleavage site in the PDB: 1Q29 crystal, conformational changes
had taken place to achieve an intermediate that had been activated for cata-
lytic cleavage but unable to cleave because of the stem I – stem II tether. The
conformation was found to have an “ inline fi tness ” parameter measuring 1.6.
This compared to an inline fi tness parameter calculated for PDB: 1NYI crystal
structure measuring 0.06. As discussed previously, the inline parameter (ideal
value 1.0) assesses the ability of the conformation to achieve a cleavage site
alignment proper for the S N 2 mechanism — lining up the O2 ′ (C 17 ) – P (A 1.1 ) – O5 ′
(A1.1 ) atoms at the correct 180 ° angle and bringing the O2 ′ (C 17 ) – P (A 1.1 ) dis-
tance to approximately 3.0 Å. Table 6.6 collects the data for PDB: 1NYI and
PDB: 1Q29 and compares them to other X - ray crystallographic structures
published by the Scott group.
If the reader is using PDB data to visualize these structures, the following
nucleotide numbering system will be useful. For PDB: 1NYI, Chain A = ribo-
zyme and Chain B = substrate, C 17 = C120, A 1.1 = A121, and a C – G hydrogen -
bonding pair forms between C26 and G500: N 4 – O 6 = 3.30 Å , N 1 – N 3 = 3.12 Å ,
O 2 – N 2 = 2.91 Å. For PDB: 1Q29, Chain A = ribozyme and Chain B = substrate,
C 17 = C120, A 1.1 = A121, and a C – G hydrogen - bonding pair forms between
C26 and G10: N 4 – O 6 = 3.10 Å , N 1 – N 3 = 3.11 Å , O 2 – N 2 = 3.14 Å. Fruitful com-
parison can be made between the PDB: 1Q29 - unmodifi ed but tethered RNA
“ late intermediate ” structure (angle = 135 ° , O2 ′ (C 17 ) – P (A 1.1 ) = 2.24 Å and
fi tness = 1.6) and that previously published — PDB: 379D with talo - 5 ′ - C - methyl
modifi ed leaving group at A 1.1 (angle = 111 ° , O2 ′ (C 17 ) – P (A 1.1 ) = 2.51 Å and
fi tness = 0.84). Obviously, the tether is preventing the PDB: 1Q29 structure
from achieving the ideal 170 – 180 ° angle required for cleavage. Additional dif-
ferences can be noted in comparison to the hairpin ribozyme ligation site
where the 172 ° angle is almost ideal and the O2 ′ – P distance equal to 2.70 Å
indicates atoms approaching for bond formation. Comparison of the tethered
late intermediate to the cleaved product PDB: 488D shows that the angle
between stem I and stem II widens signifi cantly during cleavage. The reference
58 authors believe that the tether prevents stem I from moving relative to stem
II and thus prevents cleavage by restricting the helical motion and unwinding
that stem I would undergo during cleavage. They believe that the crystalline
lattice also prevents unwinding and that the stem I helical motion and unwind-
ing would be much greater in solution. Finally, they hypothesize that structural
interactions between stem I and stem II in hammerhead self - cleaving RNA