250 GROUP I AND II METALS IN BIOLOGICAL SYSTEMS: GROUP II
present a model for the 5 ′ splice site based on previous functional group sub-
stitution experimental data and the structure of the PDB: 1GRZ ribozyme.
The P1 duplex is hypothesized to fi t into a concave binding site provided in
the ribozyme ’ s core. The “ sides ” of the pocket would be J4/5 and P3, while the
“ fl oor ” would be J8/7. (See Figure 6.4 .) It is known that J8/7 is involved in
binding P1, and its other interactions with the backbone of P3 and helix P4,
for instance, defi nes a space in which the phosphate at the 5 ′ splice site is well
placed with respect to the guanosine nucleophile. The authors also speculate
as to which residues might coordinate the divalent metal ions required for
catalytic activity. Four phosphate positions are named — those at positions
A306, A261, A207, and C208 — that span the “ gateway ” to the G site. The
phosphate groups of these nucleotides have been previously identifi ed as sites
of phosphorothioate interference.^26 Briefl y, substitution of sulfur at phosphate
oxygen positions thought to be important metal - ion binding sites involved in
ribozyme folding or catalysis causes a change in catalytic activity that confi rms
or denies the importance of the substituted site. This type of experiment has
been described earlier in Section 6.2.2.
Other research groups have conducted biochemical experiments intended
to place metal ions in the catalytic site of ribozymes. Groups led by Piccirilli
and Herschlag proposed a three - metal model based on the difference in Mn 2+
concentrations needed to rescue different sulfur or amino substitutions of
substrate functional groups.^27 The experiments were intended to quantitatively
determine metal ion affi nities that in turn would allow individual metal ions
to be distinguished from one another. The functional group substitution also
identifi ed the important groups involved in catalysis. In the three - metal ion
model for theTetrahymena ribozyme, two different metals (M B and M C ) were
proposed to coordinate to the O2 ′ and O3 ′ sites of ω G with M C also coordinat-
ing to thepro - S p O of the 3 ′ - exon terminus. A third metal (M A ) was proposed
to coordinate thepro - S p oxygen of the 3 ′ - exon terminus as well as to the O3 ′
of the 5 ′ - exon residue. (dt − 1 in the PDB: 1U6B structure, see Figure 6.7 ). In
this model, none of these three metal ions bridge between the scissile phos-
phate and the leaving group — the O3 ′ of ω G. Figure 8 of reference 27b illus-
trates the three - metal models in two - and three - dimensional forms.
In the reference 15 conclusions, the Cech group mentions the good agree-
ment between the group I intron ribozyme crystal structures achieved as of
this date (PDB: 1GID and 1GRZ) and the constructed model of Michel and
Westhof. The latter model was based mainly on comparative sequence analysis,
stereochemical constraints, and biochemical testing of proposed ribozyme
structural interactions.^28 The reference 15 researchers were concerned that
the crystallization construct may deviate from the catalytically signifi cant
structure, a concern that applies to any RNA crystal structure, especially since
RNA enzymes appear to be much more dynamic in solution (and perhaps in
the solid state as well) compared to their protein enzyme counterparts. In
contrast to the hammerhead ribozyme to be discussed in Section 6.2.4 , the