MAGNESIUM AND CATALYTIC RNA 269
attachment. If the added ligands arose from additional interactions with RNA
nucleobases, riboses, or phosphate oxygens, this might signal that the metal
ion may have participated in a conformation change preceding hammerhead
substrate cleavage.
In 1996, the W. G. Scott group published three hammerhead ribozyme
structures describing them in aScience magazine article (PDB: 299D, 300D,
and 301D).^39 While both previously published structures described modifi ed
hammerheads, these structural studies were carried out on the unmodifi ed
ribozyme – substrate complex. The researchers used time - resolved crystallog-
raphy to observe structural changes that occur during the ribozyme - catalyzed
reaction. (See Section 3.7.2.3 .) The reaction is arrested chemically or physically
by adjusting the pH, fl ash - freezing the crystal, or a combination of both
methods. In the PDB: 299D structure (containing no divalent metal ions) the
crystal was formed in a solution held at pH 6, fl ash - frozen, and then main-
tained at 100 K. At pH 6, the cleavage reaction takes place at a slower rate in
solution and at a yet slower rate in the crystalline state. The PDB: 299D struc-
ture is quite similar to that of the 2 ′ - O - methyl C17 modifi ed hammerhead
described above (PDB: 1MME), especially for residues in or interacting with
the active site (see Figure 3B of reference 39 ). The PDB: 300D structure was
obtained at pH 5 in the presence of Mn 2+ ions. Six manganese ions appear in
the crystal structure; however, none are close enough to oxygen atoms at cleav-
age site (C 17.0 – A 1.1 ) for bonding interactions. In the A 9.0 – G 10.1 region, Mn 2+ – O
(Mn2+ – O distance to a non - bridging phosphate oxygen of A 9.0 = 2.31 Å ) and
Mn2+ – N (Mn 2+ – N distance to the N 7 nitrogen atom of G 10.1 = 2.24 Å ) interac-
tions are found. Selected bonding distances for these structures are included
in Table 6.4.
The overall structure resembles that of PDB: 299D (no divalent metal ions
present) and PDB: 1MME (modifi ed C 17.0 ). The scissile phosphodiester bond
(A1.1 5 ′ O – A 1.1 P) and the adjacent ribose (that of C 17.0 ) are still in the approxi-
mate A - form helical conformation. This places the C 17.0 2 ′ - OH within 3.5 Å of
the A 1.1 phosphate phosphorus atom but on the wrong side for an S N 2 nucleo-
philic attack to occur. If crystals of the Mn 2+ - modifi ed ribozyme – substrate
complex (PDB: 300D) are dissolved after being soaked in 100 mM MnSO 4 at
pH 7 for 30 minutes, a denaturing polyacrylamide gel assay shows residual
intact 25 - nt substrate as well as 20 - nt cleaved product, indicating that some
cleavage has taken place at pH 7.
The third crystal (PDB: 301D) was formed by freeze - trapping the RNA
four minutes after Mg 2+ was added at pH 8.5. At this pH the hammerhead
RNA cleaves itself in the crystalline state, with the cleavage reaction reaching
90% completion within 15 minutes. This structure contained fi ve co -
crystallized magnesium ions, and it revealed signifi cant conformational change
when compared to both 299D and 300D structures. The change is most evident
at the scissile phosphodiester bond located between C 17.0 and A 1.1. This phos-
phodiester bond moves approximately 2.9 Å into the catalytic cleft when com-
pared to the same region for the other crystallographic structures. Its change