MAGNESIUM AND CATALYTIC RNA 297
information on function and mechanism of catalysis. In contrast to the success
of this method for other systems, analyses of hammerhead structure – function
relationships has led to disagreement and controversy.
Hammerhead crystal structures (identifi ed by reference and PDB accession
number in Table 6.3 ) have identifi ed two important domains: (1) domain I
consists of the substrate ’ s cleavage site (C 17 and X 1.1 , where X stands for any
nucleotide) and the ribozyme ’ s C 3 U 4 G 5 A 6 loop (called the CUGA turn) and
(2) domain II consists of 10 residues connecting helices II and III — U 16.1 , U 7 ,
G 8 , A 9 , G 10.1 , C 11.1 , G 12 , A 13 , A 14 , A 15.1. All of these residues are conserved and
essential for all hammerheads, except for U 7. In the domain II group, there are
four adjacent non - Watson – Crick base pairs including two mismatch pairs:
A 13 – G 8 , A 9 – G 12 that appear next to each other (as is the case for many ribo-
somal RNA secondary structures). The two domains are connected at a three -
strand junction comprised of nucleotides A 6 , U 7 , U 16.1 , and C 17. Overall, the
hammerhead is Y - shaped with coaxial helices II and III and helices (stems) I
and II alongside each other and roughly parallel.
Many hydrogen bonding and interaction characteristics in the hammerhead
crystal structures as described by Blount and Uhlenbeck in reference 61 relate
to possible functional details. Residues in domain II form non - Watson – Crick
base pairs whereas one pair, A15.1 – U16.1, forms only one H - bond, widening
the major groove to facilitate formation of the three - strand junction. Cleavage
site residue C 17 interacts with residues in the CUGA turn, possibly having
implications for the catalytic mechanism. In the initial all - RNA hammerhead
structure, PDB: 1MME, published in 1995, the 2 ′ - oxygen nucleophile is 90 °
away from the proper alignment to carry out the S N 2(P) cleavage mechanism.
Since then, as described in the previous discussion, the Scott group has changed
crystallization conditions and adjusted the hammerhead construct to increase
the attack angle — achieving a O2 ′ (C 17 ) – P (A 1.1 ) – O5 ′ angle of 135 ° , a O2 ′
(C 17 ) – P (A 1.1 ) distance of 2.24 Å , and an “ inline fi tness ” parameter of 1.6 in the
PDB: 1Q29 structure.
The important presence of metal ions has also been addressed in crystal
structure determinations. Since magnesium ions had been found to enhance
the cleavage reaction, it was important to fi nd where these ions were placed
in the structure. Soaking crystals in Mg 2+ ions positioned fi ve Mg 2+ ions in the
hammerhead ribozyme, as has been described above in the discussion of PDB:
1MME. Importantly, ions were found to be interacting with nucleotides near
the catalytic site and in domain II with residues A 9 and G 10.1. Analysis of metal
ion placement in the Scott group hammerhead crystal structures has been
collected in Table 6.3. Many, but not all, structures where the pH was main-
tained at 8.5 show close metal ion interactions with important domain II resi-
dues A 9 and G 10.1. The points of metal ion attachment at the pro - R p - oxygen of
A 9 and N 7 of G 10.1 agree well with the essentiality of these atoms found in
biochemical and spectroscopic experiments (see especially Figure 4 of refer-
ence 61 ). Close metal ion interactions are often seen for the G 5.0 residue that
is part of the CUGA loop of domain I, although few of them connect the metal