MAGNESIUM AND CATALYTIC RNA 251
group I intron crystal structures exhibit much more agreement between bio-
chemical and spectroscopic studies in solution and the structure observed in
the solid state. The researchers believe that the difference may lie in the fact
that, in contrast to the hammerhead, group I introns appear to be largely pre-
organized to bind their substrates and carry out the catalyzed reaction upon
them.
Next, the Cech group studied mutants of the Tetrahymena thermophila
intron P4 – P6 domain that formed a more stable tertiary structure in solution,
hoping that this would lead to more easily crystallized ribozymes and more
stable crystals. One mutant, Δ C209 with the C209 residue deleted, formed
crystals suitable for X - ray crystallography that diffracted to a resolution of
2.25 Å (PDB: 1HR2).^29 The Δ C209 RNA construct contained 159 nucleotides
with 27 octahedrally coordinated magnesium ions, 15 additional magnesium
cations, and 129 water molecules distributed in two molecules in the asym-
metric unit of the crystal. The better - diffracting crystals allowed assignment of
innersphere and outersphere coordination contacts for 27 magnesium ions and
established the positions of numerous coordinated water molecules found on
the inside rather than at the surface of the folded RNA domain. The authors
proposed that the water molecules might stabilize noncanonical base - pair
interactions — U – G or A – G pairs, for instance — in the ribozyme. The PDB:
1HR2 global structure was relatively unchanged compared to the wild - type
structure (PDB: 1GID), and the two molecules in the asymmetric unit were
essentially equivalent. However, changes were noted in the immediate vicinity
of the deleted residue. Removal of the C209 residue in helix P4 causes A210
(bulging out in the wild - type ribozyme) to move into the helix and make a
mismatch base pair with G111. The purine – purine base pair pushes the phos-
phate backbone out slightly and the helix is widened. The base pair itself is
somewhat twisted, but hydrogen bonding distances are still maintained — for
instance, G111 O 6 – A210 N 6 = 2.9 Å. The G – A motif has been observed in other
RNAs. In the same P4 region, neighboring hydrogen - bonding base pairs form
between G112 – C208 and G110 – C211. The authors believe that the elimination
of the A210 P4 helix bulge and formation of the G111 – A210 base pair caused
the increased stability of theΔ C209 mutant. However, the catalytic activity
of the intron for self - splicing decreases 91 - fold with the Δ C209 mutation
because the C209 – G111 – U305 base triple that is part of the ribozyme ’ s active
site can no longer form.
The PDB: 1HR2 crystal structure showed electron density corresponding
to well - occupied binding sites for several magnesium ions. Inner - sphere metal
ion coordination distances were defi ned to be within 1.9 – 2.5 Å of appropriate
ligands, and outer - sphere coordination distances to be 2.5 – 3.3 Å from coordi-
nating water molecules or other intervening ligands. The coordination sphere
for the innersphere magnesium ions agreed with those found in the PDB:
1GID structure both in nucleotides atoms involved in bonding and in bonding
distances although in most cases, bond lengths are 0.1 – 0.2 Å longer for the