Biology Reference
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state are k dock
0.1) s 1 ; these rates are
significantly slower than the junction dynamics under the same conditions. 31
Data from many single molecules give the internal conversion rates for the
hairpin ribozyme. The cleavage rate ( k C )is
0.1) s 1 and k undock
¼
¼
2.5 (
2.3 (
0.6 min 1 in the presence of
1mMMg 2 þ , while the ligation rate ( k L : the rate of ligation in the docked
ribozyme) is
0.3 s 1 . Thus the reaction is biased toward ligation, with an
internal equilibrium constant of K int
34.
The hinged form of the ribozyme is similarly biased toward ligation, but
overall the cleavage rate is limited by docking and undocking rates. 74 The
bias to ligation maintains the integrity of the circular (
¼
k L / k C
¼
) strand allowing it to
act as a template for (
) strand synthesis. In the junction form, however,
cleavage of the concatenated product of replication is facilitated by the rapid
undocking that follows cleavage. Thus, the four-way junction confers the
properties required for the biological function of the ribozyme.
þ
2.8. Candidate catalytic components in the hairpin ribozyme
Like the VS ribozyme, there is no evidence for a direct role of site-bound
metal ions in catalysis. Significant levels of activity can be maintained in both
ribozymes in the presence of high concentrations of monovalent ions such as
Li þ and NH 4 þ . 67 The hairpin ribozyme was also shown to remain active
when Mg 2 þ ions were replaced by substitutionally inert hexamminecobalt
(III) ions 75-77 or aminoglycoside antibiotics. 78 This excludes a requirement
for a site-bound metal ion acting as a Lewis acid to activate nucleophilic
attack, or ion-bound water molecules performing general acid-base cataly-
sis. In addition, cleavage rates measured in single-molecule experiments
were found to be almost independent of Mg 2 þ ion concentration. 73
There is, however, evidence for nucleobase involvement in catalysis by
the hairpin ribozyme. The first to be suggested was G8, located on the oppo-
site strand from the scissile phosphate in loop A. Substitution by other nucle-
otides led to rates of cleavage being reduced by two orders of magnitude in
both the hinged form 79-81 and the junction form of the ribozyme, 82 without
significantly affecting ion-induced folding in the latter. Complete ablation of
the nucleobase to leave an abasic site also led to a major loss of ribozyme
activity. 83 The cleavage rate was found to decrease with pH when DAP
was substituted at this position, showing that catalysis was dependent on
proton transfer involving a group with a p K a of 6.9. 80 We introduced the
C 0 -linked imidazole nucleoside 84 at position 8. 85 The substitution did not
perturb folding, and the modified ribozyme was active in both cleavage
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