242 GROUP I AND II METALS IN BIOLOGICAL SYSTEMS: GROUP II
Reference 16 discusses biochemical methods to predict specifi c ribozyme
metal - binding sites and spectroscopic methods to identify and examine specifi c
sites.
It is known that low concentrations of monovalent ions (Na + , K + ) do not,
by themselves, catalyze ribozyme reactions. For instance, in “ small ” ribozymes
such as hammerhead, hairpin, Neurospora VS, and hepatitis delta virus (HDV),
which catalyze a single site - specifi c phosphodiester bond cleavage reaction,
∼ 1 M concentrations of monovalent ions will not promote catalytic activity
unless micromolar concentrations of Mg 2+ or certain other divalent cations are
also present. Very high concentrations of monovalent ions can catalyze the
activity of the small ribozymes but not that of “ large ” group I and II introns
or ribonuclease P (RNase P). Figure 1a of reference 16 illustrates the cleavage
reactions catalyzed by the small ribozymes as well as RNase P. Figure 1b shows
the splicing reactions catalyzed by group I intron ribozymes such asTetrahy-
mena pre - rRNA using an exogenous guanosine as a nucleophile. Group II
intron ribozymes catalyze a reaction similar to that of group I using a different
extrinsic nucleophile.
A major challenge in studying the role of metal ions in ribozymes is decid-
ing whether a cation is necessary only for structural purposes, only for catalytic
purposes, or for both purposes. In other words, one would like to correlate
metal ion sites with either the folding, or the activity, of the ribozyme. It is
possible to fi nd out that metal ions have structural purposes only by using
ribozymes prohibited from undergoing their catalytic reaction by single atom
changes at the active site. Ion - dependent structural changes in ribozymes have
been illuminated using fl uorescence resonance energy transfer (FRET),^17
hydroxyl radical footprinting,^18 small - angle X - ray scattering (SAXS),^19 and
other experimental techniques. These techniques usually do not place the
metal ion at a specifi c binding site but do explain the ion ’ s overall affect on
ribozyme folding. Conversely, few experimental techniques can report on the
metal ion ’ s effect on ribozyme activity without the metal ion concurrently
causing structural alterations.
The main technique used by researchers in attempting to isolate catalytic
activity from structural changes is one called the phosphorothioate metal -
rescue (PS - rescue) experiment. This experiment tests the hypothesis that a
metal ion binds to a particular phosphate oxygen on a specifi c nucleotide and
that the metal ion is important to ribozyme catalytic activity. A successful PS -
rescue experiment does the following: (1) substitutes one nonbridging phos-
phate oxygen with a sulfur atom creating a stereospecifi c thiophosphate; (2)
tests that the substituted site has lost affi nity for the “ hard ” magnesium cation,
Mg2+ , but has enhanced affi nity for “ soft ” cations such as Cd 2+ , Zn 2+ , or Tl + (see
Section 1.4 ); and (3) tests that Mg 2+ - dependent activity, is lost but that the soft
cations can “ rescue ” the activity thus predicting a functional catalytic cation
site. The PS - rescue experiment can be successful in predicting sites of catalytic
activity, although the situation is complicated by the fact that ribozyme active
sites undergo several conformational changes between ground, pre - transition,