MAGNESIUM AND CATALYTIC RNA 291
tration over a range of Mg 2+ or Li + concentrations from 0.1 to 100 mM. The
importance of the 2 ′ - deoxy substitution at G 5 , a substituted residue that alters
hammerhead global folding according to other experimental techniques (it
blocks formation of the stem I/stem II catalytic motif in FRET analyses), is
also demonstrated in the hydroxyl radical footprinting experiment. In a test
of a ribozyme – substrate complex containing a 2 ′ - deoxy G 5 , the nucleotide
pattern indicates that the ribozyme – substrate complex does not fold into its
solvent - protected form. Looking at PDB: 299D, 300D, and 301D crystal struc-
tures, one can fi nd several potential hydrogen bonding partners between G 5 ’ s
2 ′ - O position and atoms in the nucleotide at position 15.3.
Up to the experimental limit where both folding and catalytic effect could
be measured, the researchers always found that folding was faster than cataly-
sis. They also found a log - linear dependence of cleavage rate on pH in agree-
ment with other experimental techniques. In almost all of their experiments,
Hampel and Burke^56 used a substrate containing a 2 ′ - deoxy substitution at C 17 ,
the cleavage site, to prevent substrate cleavage. Only in one experiment with
HH16 at pH 6 were the workers able to use an intact substrate. They found
that the solvent protection of the ribozyme strand, residues G 5 – A 9, was
unchanged and concluded that the 2 ′ - deoxy C 17 substitution does not affect
global folding. The hydroxyl footprinting results showing protection from
hydroxyl radical attack near the hammerhead three - stem junction are also in
agreement with the crystal structures in which residues G 5 , A 6 , G 8 , and C 17
show reduced solvent accessibility. However, four residues that are protected
in the hydroxyl radical attack experiments, U 7 and A 9 in the ribozyme and A 1.1
TABLE 6.5 Protected Nucleotides in Hydroxyl Footprinting Assay as Adapted
from Reference 57
Hammerhead Construction Ribozyme Strand Substrate Strand
Additional
Ribozyme SiteHH16 A 9 U 1.2
G 8 G 1.1
U 7 C 17
A 6
G 5
HH α 1 A 9 A 1.2 G 15.3
G 8 U 1.1
U 7 C 17
A 6
G 5
RNA 6 A 9 C 1.2 U 15.3
G 8 A 1.1
U 7 C 17
A 6
G 5