290 GROUP I AND II METALS IN BIOLOGICAL SYSTEMS: GROUP II
by RNA folding they may not appear on the assay or will appear at lower
concentration. Reaction products are assayed through electrophoresis on a
15% (w/v) acrylamide – 8 M urea gel. The hydroxyl radical footprinting method
can be used to accurately defi ne RNA folding rate constants slower than 6 – 8/
min and to probe the structure of the entire hammerhead construct at the
individual nucleotide level, because inaccessible nucleotides will not be visual-
ized. Results can then be compared to experiments using base change or
functional - group substituted mutants that have diminished catalytic activity.
First, Hampel and Burke described hammerhead secondary structure as
determined by fl uorescence resonance energy transfer (FRET) experiments.
Metal ion titrations using FRET signals demonstrated RNA folding to the
typical hammerhead secondary structure Y - shape. (See Figures 6.10 , 6.11 , and
6.12 .) The FRET technique reveals that folding occurs with two discrete transi-
tions: (1) At < mM Mg 2+ the domain II stack forms, and (2) at low mM Mg 2+
the domain I motif assumes a specifi c structure that orients stem (helix) I close
to stem (helix) II. Substitution of the ribose 2 ′ - deoxy nucleotide at the con-
served position G 5 alters the geometry of the three - way junction such that
stems I and II are not close together while the domain II structure is main-
tained. From previous studies, it is known that cations are required for folding
to the hammerhead global structures described (Figures 6.10 , 6.11 , and 6.12 ).
The principal metal ion that has been used in folding studies is Mg 2+. In con-
trast, many cations have been shown to promote catalysis including divalent
cations Mg 2+ , Mn 2+ , Ca 2+ , Sr 2+ , Cd 2+ , and Co 2+ and monovalent cations Na + ,
NH 4 + , and Li +. Although catalytic cleavage rates are enhanced by these
cations — some requiring high ionic concentration — catalysis does not have an
absolute requirement for divalent metal ions or for inner - sphere coordination
by metal ions.
Hampel and Burke observed that protection of hammerhead backbone
sites in Mg 2+ solutions required assembly of the full ribozyme – substrate
complex. In other words, testing of ribozyme or substrate separately in the
hydroxyl footprinting assay showed essentially complete hydrolysis of all
nucleotides (Figure 2B of reference 56 ). In contrast, the fully assembled
ribozyme – substrate complex showed protection of nucleotides structurally
near the densely packed three - helix junction of hammerhead constructs
HH16, HH α 1, and RNA 6. Two of the ribozyme group of protected nucleotides
(G 5 , A 6 ) are part of the conserved uridine U - turn seen in all known hammer-
head constructs. (See Figures 6.10 , 6.11 , and 6.12 .) The footprinting results are
collected in Table 6.5.
The three hammerhead constructs display nearly identical solvent -
protected (folded) sites except that HH α 1 and RNA 6 show an additional
protected site at position 15.3. This is an important site since in the hammer-
head crystal structures this site in stem III is approached by domain I, part of
the catalytic pocket. The authors believe that a single structure gives rise to
the observed pattern of hydroxyl radical protection of nucleotides since pro-
tection at individual nucleotides varies in the same manner with Mg 2+ concen-