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In-Depth Information
It might be expected that the VS ribozyme would be a dynamic structure
in solution, but there is less direct information on this compared to the hair-
pin ribozyme. The folding of the complete ribozyme could be followed by
its compaction (revealed as a reduction of radius of gyration) with an increase
of Mg
2
þ
ionic concentration, fitting to a two-state model with a [Mg
2
þ
]
1/2
of 330
m
M and a near-unit Hill coefficient.
46
However, the transition is not
complete with less than millimolar concentrations of Mg
2
þ
, and the best
X-ray scattering profiles were obtained in the presence of high metal ion
concentration. We think that the transverse ridge in the structure is occupied
by helix I, but it is not certain to be 100% occupancy. The interaction with
helix I is not especially strong; kinetic analysis of the cleavage of helix I by
ribozyme in
trans
gives an apparent affinity of
K
app
1
m
M.
20
Single-
molecule FRET experiments using ribozyme (lacking helix VII) carrying
fluorophores on the 5
0
termini of helices I and VI indicated a dynamic struc-
ture in the presence of 50 mM K
þ
and 35 mM Mg
2
þ
. The results were
interpreted to indicate that one-third of the species had undocked substrate
at any given time in these conditions.
52
Taken together, the data suggest that
there is probably a reversible association between the substrate helix I and
the core of the VS ribozyme that is similar to the hairpin ribozyme. Further-
more, it has been suggested that
in vivo
the ribozyme could act on a more
distant substrate stem-loop in
cis.
53
Consistent with these ideas, we demon-
strated a
trans
cleavage reaction between two complete VS ribozymes.
54
This
reaction clearly requires that helix I of one ribozyme must be able to displace
that of another ribozyme and dock in its place. From these experiments, we
estimated
K
app
¼
¼
1.7
m
M.
2.4. Identification of the active site of the VS ribozyme
The key catalytic components of the VS ribozyme have been identified by
nucleotide substitution. Most sequence changes in the ribozyme that affect
catalytic activity do so because they alter the structure. These are typically
located in the helical junctions or the bulges.
39,40
Changes in the lengths
of critical helices III and V are also deleterious because they affect the relative
orientation of helices II and V or the loop-loop interaction between helices
I and V, respectively.
40
However, sequence changes in the internal loop of
helix VI, termed the A730 loop, have no discernible effect on folding, yet
most single-base changes introduced into this loop result in significant loss of
cleavage activity (generally 50-fold or more) in
trans
20
and in
cis.
55
The A730
loop had earlier been found to be sensitive to ethylation interference, and it
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