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In-Depth Information
is little apparent sequence variation, the contact appears to be present in most
if not all hammerhead sequences. Although the minimal hammerhead has a
turnover rate of approximately 1 min
1
, full-length sequences that include
a tertiary contact, described in
Section 3
, are up to 1000-fold more
active.
19-21
2.1. Enzymology
2.1.1 Rate enhancement
The rate of non-site-specific, spontaneous decay of RNA is highly depen-
dent upon the secondary structural context, but is on average about
10
6
min
1
.
22
Hence, the rate enhancement provided by an optimized
minimal hammerhead is on the order of 10
6
, and for the full-length natural
hammerhead can be as much as 10
9
. To achieve this magnitude of rate
enhancement, not to mention site specificity, the hammerhead ribozyme
must adopt several effective catalytic strategies simultaneously. Each of these
is separated perhaps somewhat artificially and analyzed below.
2.1.2 Metal ions and catalysis
Originally, it was believed that all ribozymes, including the hammerhead
ribozyme, were obligate metalloenzymes.
23,24
Mg
2
þ
ion is assumed to be
the biologically relevant divalent cation, although the hammerhead is active
in the presence of a variety of divalent cations.
25
Proposed roles for Mg
2
þ
ion in catalysis included both acid and base catalysis components
24,26
(with
Brønsted and Lewis variants of this proposal articulated) as well as direct
coordination of the
pro-R
nonbridging phosphate oxygen of the scissile
phosphate for transition-state stabilization. Mg
2
þ
ion has also been impli-
cated in structural roles that facilitate formation of the active ribozyme.
27-35
In 1998, it was demonstrated that the hammerhead, along with the hair-
pin and VS ribozymes (but not the HDV ribozyme) could also function in
the absence of divalent metal ions as long as a high enough concentration of
positive charge was present (molar quantities of Li
þ
,Na
þ
, or even the non-
metallic NH
4
þ
ion permit cleavage to take place). The study suggested that
ribozymes were not strictly metalloenzymes.
36,37
Considering the volume of research devoted to understanding the mech-
anistic roles of divalent metal ions in hammerhead ribozyme catalysis and the
fundamental tenet of ribozyme enzymology that all ribozymes are
metalloenzymes, it was unexpected to find that at least three of the four small
naturally occurring ribozymes can function reasonably efficiently in the
absence of divalent metal ions; a fact that was discovered in the course of
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