Biology Reference
In-Depth Information
The hammerhead ribozyme sequence derived from Schistosoma Sm a 1is
arguably the most extensively characterized of full-length hammerhead
sequences. The cleavage kinetics and internal equilibrium have been thor-
oughly investigated, revealing significant surprises. The apparent cleavage
rate at pH 8.5 in 200 mM Mg 2 þ is at least 870 min 1 , which in actuality
is a lower bound as there is also a significant rate of ligation under these con-
ditions. In contrast to minimal hammerheads that show a log-linear depen-
dence of rate on pH up to about pH 8.5, the Sm a 1 hammerhead has a lower
apparent p K a that is dependent upon Mg 2 þ concentration. At 100 mM
Mg 2 þ , the apparent p K a is about 7.5-8. 49 The Sm a 1 hammerhead is also
a
ligase, 59
rather efficient
revealing internal equilibrium constants
( K int
¼
[EP]/[ES]) as small as 0.5 in the presence of high concentrations
of Mg 2 þ , and as small as 1.3 under physiological concentrations of Mg 2 þ .
Cleavage and ligation reaction rates are also highly dependent upon the
identity of the divalent cation present, with Mn 2 þ accelerating the reaction
almost two orders of magnitude relative to Mg 2 þ . This suggests that the abil-
ity to coordinate soft ligands (perhaps including the N7 of G10.1) optimizes
catalysis, whereas simply folding the RNA is only weakly dependent upon
the identity of the divalent cation present. 60
3.3. Crystal structure
The full-length hammerhead structure ( Fig. 1.1 D) reveals how tertiary
interactions occurring remotely from the active site prime the ribozyme
for catalysis. G12 and G8, two invariant residues previously identified in bio-
chemical studies to be potential acid-base catalysts, are in fact positioned in a
way that is consistent with their suggested roles. In contrast to the minimal
hammerhead structure, the nucleophile in the full-length structure is aligned
with the scissile phosphate which in turn is positioned proximal to the A9
phosphate, and previously unexplained roles of other conserved nucleotides
become apparent within the context of a distinctly new fold that nonetheless
accommodates the previous structural studies. These interactions allow us to
explain the previously irreconcilable sets of experimental results in a unified,
consistent, and unambiguous manner. 41
Figure 1.2 A is a close-up of the active site. The light blue dotted lines are
conventional hydrogen-bonding interactions. The other dotted lines repre-
sent interactions that may be relevant to the catalytic mechanism. The struc-
ture includes an introduced modification, a 2 0 -OMeC at the cleavage site, to
prevent abstraction of the 2 0 -H from the nucleophilic oxygen. G12 is
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