Hammerhead ribozyme
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Hammerhead Ribozyme
Stylized rendering of the full-length
hammerhead ribozyme RNA molecule
Scientific classification
Species:
Catalytic RNA
Hammerhead RNAs are RNAs that self-cleave via a small conserved secondary
structural motif termed a hammerhead because of its shape[1]. Most hammerhead RNAs
are subsets of two classes of plant pathogenic RNAs: the satellite RNAs of RNA viruses
and the viroids. The self-cleavage reactions, first reported in 1986[2][3], are part of a
rolling circle replication mechanism. The hammerhead sequence is sufficient for selfcleavage[4] and acts by forming a conserved three-dimensional tertiary structure.
Contents
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1 Autocatalyst or ribozyme?
2 Species distribution
3 Chemistry of catalysis
o 3.1 Cleavage by phosphodiester isomerization, not hydrolysis
o 3.2 Requirement for divalent metal ions
o 3.3 Not a metallo-enzyme
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4 Primary and secondary structure of the hammerhead ribozyme
o 4.1 Minimal hammerhead ribozyme
 4.1.1 Type I & type III hammerhead RNA
o 4.2 Full-length hammerhead ribozyme
5 Tertiary structure of the minimal hammerhead ribozyme
6 Ensuing structure-function dilemma
7 Tertiary structure of the full-length hammerhead ribozyme
8 Structure and catalysis
9 Therapeutic applications
10 References
11 External links
[edit] Autocatalyst or ribozyme?
In the natural state, a hammerhead RNA motif is a single strand of RNA, and although
the cleavage is autocatalytic and takes place in the absence of protein enzymes, the
hammerhead RNA itself is not a true enzyme in its natural state, as it cannot catalyze
multiple turnovers.
In vitro hammerhead constructs can be engineered such that they consist of two RNA
strands. The strand that gets cleaved can be supplied in excess, and multiple turnover can
be demonstrated and shown to obey Michaelis-Menten kinetics, typical of protein
enzyme kinetics. Such constructs are typically employed for in vitro experiments, and the
term "hammerhead RNA" has become in practice synonymous with the more frequently
used "hammerhead ribozyme".
The minimal hammerhead ribozyme sequence that is catalytically active consists of three
base-paired stems flanking a central core of 15 conserved (mostly invariant) nucleotides,
as shown. The conserved central bases, with few exceptions, are essential for ribozyme’s
catalytic activity. Such hammerhead ribozyme constructs exhibit a turnover rate (kcat) of
about 1 molecule/minute and a Km on the order of 10 nanomolar.
The hammerhead ribozyme is arguably the best-characterized ribozyme. Its small size,
thoroughly-investigated cleavage chemistry, known crystal structure, and its biological
relevance make the hammerhead ribozyme particularly well-suited for biochemical and
biophysical investigations into the fundamental nature of RNA catalysis.
Hammerhead ribozymes may play an important role as therapeutic agents; as enzymes
which tailor defined RNA sequences, as biosensors, and for applications in functional
genomics and gene discovery.[5]
[edit] Species distribution
Hammerhead ribozymes are found in a wide range of plant viroids and helmenths such as:
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Peach latent mosaic viroid
Eggplant latent viroid
Avocado sunblotch viroid
Velvet tobacco mottle virus Satellite RNA
Schistosoma mansoni Satellite DNA [6]
Dianthus caryophyllus viroid-like DNA
Cherry small circular viroid-like RNA
Newt
Dolichopoda cave cricket
[edit] Chemistry of catalysis
The hammerhead ribozyme carries out a very simple chemical reaction that results in the
breakage of the substrate strand of RNA, specifically at C17, the cleavage-site nucleotide.
Although RNA cleavage is often referred to as hydrolysis, the mechanism employed does
not in fact involve the addition of water. Rather, the cleavage reaction is simply an
isomerization that consists of rearrangement of the linking phosphodiester bond. It is the
same reaction, chemically, that occurs with random base-mediated RNA degradation,
except that it is highly site-specific and the rate is accelerated 10,000-fold or more.
[edit] Cleavage by phosphodiester isomerization, not hydrolysis
In-line transition-state for the hammerhead ribozyme reaction. A general base (B, red)
abstracts a proton from the 2'-O, and a general acid (A, blue), supplies a proton to the 5'O leaving group as negative charge accumulates. The bonds breaking and forming
(dotted lines) must be in the axial positions and reside approximately 180° apart, as
shown. The reaction product is a 2',3'-cyclic phosphate.
The cleavage reaction is a phosphodiester isomerization reaction that is initiated by
abstraction of the cleavage-site ribose 2’-hydroxyl proton from the 2’-oxygen, which then
becomes the attacking nucleophile in an “in-line” or SN2(P)-like reaction, although it is
not known whether this proton is removed prior to or during the chemical step of the
hammerhead cleavage reaction. (The cleavage reaction is technically not bimolecular, but
behaves in the same way a genuine SN2(P) reaction does; it undergoes inversion of
configuration subsequent to forming an associative transition-state consisting of a
pentacoordinated oxyphosphrane.) The attacking and leaving group oxygens will both
occupy the two axial positions in the trigonal bipyramidal transition-state structure as is
required for an SN2-like reaction mechanism.
The 5’-product, as a result of this cleavage reaction mechanism, possesses a 2’,3’-cyclic
phosphate terminus, and the 3’-product possesses a 5’-OH terminus, as with nonezymatic
alkaline cleavage of RNA. The reaction is therefore, in principle, reversible, as the
scissile phosphate remains a phosphodiester, and may thus act as a substrate for
hammerhead RNA-mediated ligation without a requirement for ATP or a similar
exogenous energy source. The hammerhead ribozyme-catalyzed reaction, unlike the
formally identical non-enzymatic alkaline cleavage of RNA, is a highly sequence-specific
cleavage reaction with a typical turnover rate of approximately 1 molecule of substrate
per molecule of enzyme per minute at pH 7.5 in 10 mM Mg2+ (so-called “standard
reaction conditions” for the minimal hammerhead RNA sequence), depending upon the
sequence of the particular hammerhead ribozyme construct measured. This represents an
approximately 10,000-fold rate enhancement over the nonezymatic cleavage of RNA.
[edit] Requirement for divalent metal ions
All ribozymes were originally thought to be metallo-enzymes, in the sense that they were
assumed to require the presence of divalent cations, such as Mg2+, for both folding and
catalysis. It was presumed that hexahydrated magnesium ions, which exist in equilibrium
with magnesium hydroxide, could play the roles of general acid and general base, in a
way analogous to those played by two histidines in RNase A. An additional role for
divalent metal ions has also been proposed in the form of electrostatic stabilization of the
transition-state.
[edit] Not a metallo-enzyme
In 1998 it was discovered[7]that the hammerhead ribozyme, as well as the VS ribozyme
and hairpin ribozyme, do not require the presence of metal ions for catalysis, provided a
sufficiently high concentration of monovalent cation is present to permit the RNA to fold.
This discovery suggested that the RNA itself, rather than serving as an inert, passive
scaffold for the binding of chemically active divalent metal ions, is instead itself
intimately involved in the chemistry of catalysis. The latest structural results, described
below, indeed confirm that two invariant nucleotides, G12 and G8, are positioned
consistent with roles as the general base and general acid in the hammerhead cleavage
reaction.
Strictly speaking, therefore, the hammerhead ribozyme cannot be a metallo-enzyme.
[edit] Primary and secondary structure of the
hammerhead ribozyme
Secondary structures and sequences of the minimal (A) and full-length (B) hammerhead
ribozymes. Conserved and invariant nucleotides are shown explicitly. Watson-Crick
base-paired helical stems are represented as ladder-like drawings. The red arrow depicts
the cleavage site, 3' to C17, on each construct.
[edit] Minimal hammerhead ribozyme
The minimal hammerhead sequence that is required for the self-cleavage reaction
includes approximately 13 conserved or invariant "core" nucleotides, most of which are
not involved in forming canonical Watson-Crick base-pairs. The core region is flanked
by Stems I, II and III, which are in general made of canonical Watson-Crick base-pairs
but are otherwise not constrained with respect to sequence. The catalytic turnover rate of
minimal hammerhead ribozymes is ~ 1/min (a range of 0.1/min to 10/min is commonly
observed, depending upon the nonconserved sequences and the lengths of the three
helical stems). Much of the experimental work carried out on hammerhead ribozymes has
used a minimal construct.
[edit] Type I & type III hammerhead RNA
Hammerhead ribozyme (type I)
Type:
Gene; ribozyme;
2° structure:
Published; PubMed
Seed alignment: Bateman A
Avg length:
45.70 nucleotides
Avg identity:
76.00%
Hammerhead ribozyme (type III)
Type:
Gene; ribozyme;
2° structure:
Published[8]
Seed alignment: Bateman A
Avg length:
54.2 nucleotides
Avg identity:
77%
Structurally the hammerhead ribozyme is composed of three base paired helices,
separated by short linkers of conserved sequences. These helices are called I, II and III.
Hammerhead ribozymes can be classified into three types based on which helix the 5' and
3' ends are found in. If the 5' and 3' ends of the sequence contribute to stem I then it is a
type I hammerhead ribozyme, and if the and 3' ends of the sequence contribute to stem III
then it is a type III hammerhead ribozyme. Of the three possible topological types both
type I and type III are common. It is not known if examples of the type II topology are
found in nature.
[edit] Full-length hammerhead ribozyme
The full-length hammerhead ribozyme consists of additional sequence elements in stems
I and II that permit additional tertiary contacts to form. The tertiary interactions stabilize
the active conformation of the ribozyme, resulting in cleavage rates up to 1000-fold
greater than those for corresponding minimal hammerhead sequences. [9]
[edit] Tertiary structure of the minimal hammerhead
ribozyme
The crystal structure of a minimal hammerhead ribozyme
The minimal hammerhead ribozyme has been exhaustively studied by biochemists and
enzymeologists as well as by X-ray crystallographers, NMR spectroscopists, and other
practitioners of biophysical techniques. The first detailed three-dimensional structural
information for a hammerhead ribozyme appeared in 1994 in the form of an X-ray crystal
structure of a hammerhead ribozyme bound to a DNA substrate analogue, published in
Nature by Pley, Flaherty and McKay. Subsequently, an all-RNA minimal hammerhead
ribozyme structure was published by Scott, Finch and Klug in Cell in early 1995.
The minimal hammerhead ribozyme is composed of three base paired helices, separated
by short linkers of conserved sequence as shown in the crystal structure.[10] These helices
are called I, II and III. The conserved uridine-turn links helix I to helix II and usually
contains the sequence CUGA. Helix II and III are linked by a sequence GAAA. The
cleavage reaction occurs between helix III and I, and is usually a C.
The structure of a full length ribozyme shows that there are extensive interactions
between the loop of stem II and stem I.[11]
Hammerhead ribozymes can be divided into three classes according to which of the three
stems is formed from the 5' and 3' end of the sequence region. If stem III is formed from
the 5' and 3' most parts of the sequence they are known as class III.
[edit] Ensuing structure-function dilemma
Despite the observations of hammerhead ribozyme catalysis in a crystal of the minimal
hammerhead sequence in which the crystal lattice packing contacts by necessity confined
the global positions of the distal termini of all three flanking helical stems, many
biochemical experiments designed to probe transition-state interactions and the chemistry
of catalysis appeared to be irreconcilable with the crystal structures.
For example, the invariant core residues G5, G8, G12 and C3 in the minimal
hammerhead ribozyme were each observed to be so fragile that changing even a single
exocyclic functional group on any one of these nucleotides results in a dramatic reduction
or abolition of catalytic activity, yet few of these appeared to form hydrogen bonds
involving the Watson-Crick faces of these nucleotide bases in any of the minimal
hammerhead structures, apart from a G-5 interaction in the product structure.
A particularly striking and only recently observed example consisted of G8 and G12,
which were identified as possible participants in acid/base catalysis. Once it was
demonstrated that the hammerhead RNA does not require divalent metal ions for
catalysis, it gradually became apparent that that the RNA itself, rather than passively
bound divalent metal ions, must play a direct chemical role in any acid-base chemistry in
the hammerhead ribozyme active site. It was however completely unclear how G12 and
G8 could accomplish this, given the original structures of the minimal hammerhead
ribozyme.
Other concerns included an NOE between U4 and U7 of the cleaved hammerhead
ribozyme that had also been observed during NMR characterization, which suggested that
these nucleotide bases must approach one another closer than about 6 Å, although close
approach of U7 to U4 did not appear to be possible from the crystal structure. Finally, as
previously discussed, the attacking nucleophile in the original structures, the 2’-OH of
C17, was not in a position amenable to in-line attack upon the adjacent scissile phosphate.
Perhaps most worrisome were experiments that suggested the A-9 and scissile phosphates
must come within about 4 Å of one another in the transition-state, based upon double
phosphorothioate substitution and soft metal ion rescue experiments; the distance
between these phosphates in the minimal hammerhead crystal structure was about 18 Å,
with no clear mechanism for close approach if the Stem II and Stem I A-form helices
were treated as rigid bodies. Taken together, these results appeared to suggest that a fairly
large-scale conformational change must have take place in order to reach the transitionstate within the minimal hammerhead ribozyme structure.
For these reasons, the two sets of experiments (biochemical vs. crystallographic)
appeared not only to be at odds, but to be completely and hopelessly irreconcilable,
generating a substantial amount of discord in the field. No compelling evidence for
dismissing either set of experimental results was ever made successfully, although many
claims to the contrary were made in favor of each.
[edit] Tertiary structure of the full-length hammerhead
ribozyme
Three-dimensional structure of the full-length hammerhead ribozyme
In 2006 a 2.2 Å resolution crystal structure of the full-length hammerhead ribozyme was
obtained. This new structure (shown on the right) appears to resolve the most worrisome
of the previous discrepancies. In particular, C17 is now positioned for in-line attack, and
the invariant residues C3, G5, G8 and G12 all appear involved in vital interactions
relevant to catalysis. Moreover, the A9 and scissile phosphates are observed to be 4.3 Å
apart, consistent with the idea that, when modified, these phosphates could bind a single
thiophilic metal ion. The structure also reveals how two invariant residues, G-12 and G-8,
are positioned within the active site consistent with their previously proposed role in
acid/base catalysis. G12 is within hydrogen bonding distance to the 2’–O of C17, the
nucleophile in the cleavage reaction, and the ribose of G8 hydrogen bonds to the leaving
group 5’-O. (see below), while the nucleotide base of G8 forms a Watson-Crick pair with
the invariant C3. This arrangement permits one to suggest that G12 is the general base in
the cleavage reaction, and that G8 may function as the general acid, consistent with
previous biochemical observations. G5 hydrogen bonds to the furanose oxygen of C17,
helping to position it for in-line attack. U4 and U7, as a consequence of the base-pair
formation between G8 and C3, are now positioned such that an NOE between their bases
is easily explained.
The crystal structure of the full-length hammerhead ribozyme thus clearly addresses all of
the major concerns that appeared irreconcilable with the previous crystal structures of the
minimal hammerhead ribozyme.
[edit] Structure and catalysis
The active site of the full-length hammerhead ribozyme. G12 is positioned consistent
with a role as a general base in the cleavage reaction, and the 2'-OH of G8 is positioned
for acid catalysis. Potentially "active" hydrogen bonds are shown as orange dotted lines.
The 2'-O of C17 is shown to be aligned for nucleophilic attack along the blue dotted line
trajectory.
The tertiary interactions in the full-length hammerhead ribozyme stabilize what strongly
appears to be the active conformation. The nucleophile, the 2'-oxygen of the cleavage-site
nucleotide, C17, is aligned almost perfectly for an in-line attack (the SN2(P) reaction).
G12 is positioned within hydrogen bonding distance of this nucleophile, and therefore
would be able to abstract a proton from the 2'-oxygen if G12 itself becomes deprotonated.
The 2'-OH of G8 forms a hydrogen bond to the 5'-leaving group oxygen, and therefore
potentially may supply a proton as negative charge accumulates on the 5'-oxygen of the
ribose of A1.1.
The most likely explanation is then that G12, in the deprotonated form, is the general
base, and the ribose of G8 is the general acid. The apparent kinetic pKa of the
hammerhead ribozyme is 8.5, whereas the pKa of guanosine is about 9.5. It is possible
that the pKa of G12 is perturbed from 9.5 to 8.5 in the hammerhead catalytic core; this
hypothesis is currently the subject of intense investigation.
Possible transition-state interactions as extrapolated from the crystal structure.
If the invariant G8 is changed to C8, hammerhead catalysis is abolished. However, a G8C
+ C3G double-mutant that maintains the G8-C3 base pair found in the full-length
hammerhead restores most of the catalytic activity. The 2'-OH of G8 has also been
observed to be essential for catalysis; replacement of G8 with deoxyG8 greatly reduces
the rate of catalysis, suggesting the 2'-OH is indeed crucial to the catalytic mechanism.
The close approach of the A9 and scissile phosphates requires the presence of a high
concentration of positive charge. This is probably the source of the observation that
divalent metal ions are required at low ionic strength, but can be dispensed with at higher
concentrations of monovalent cations.
The reaction thus likely involves abstraction of the 2'-proton from C17, followed by
nucleophilic attack upon the adjacent phosphate. As the bond between the scissile
phosphorus and the 5'-O leaving group begins to break, a proton is supplied from the
ribose of G8, which then likely reprotonates at the expense of a water molecule observed
to hydrogen bond to it in the crystal structure.
[edit] Therapeutic applications
Modified hammerhead ribozymes are being tested as therapeutic agents.[12] Synthetic
RNAs containing sequences complementary to the mutant SOD1 mRNA and sequences
necessary to form the hammerhead catalytic structure are being studied as a possible
therapy for amyotrophic lateral sclerosis. Work is also underway to find out whether they
could be used to engineer HIV resistant lines of T-cells.
The therapeutic use of trans-cleaving hammerhead ribozymes has been severely
hampered by its low-level activity in vivo. The true catalytic potential of trans-cleaving
hammerhead ribozymes may be recouped in vivo and therapeutic derivatives are likely to
complement other nucleic acid hybridizing therapeutic strategies. Already there are
hammerhead ribozymes which are close to clinical application.[5]
[edit] References
1. ^ Forster AC, Symons RH (1987). "Self-cleavage of plus and minus RNAs of a virusoid
and a structural model for the active sites". Cell 49 (2): 211–220.
2. ^ Prody GA, Bakos JT, Buzayan JM, Schneider IR, Bruening G (1986). "Autolytic
Processing of Dimeric Plant Virus Satellite RNA". Science 231 (4745): 1577–1580.
doi:10.1126/science.231.4745.1577.
3. ^ Hutchins CJ, Rathjen PD, Forster AC, Symons RH (1986). "Self-cleavage of plus and
minus RNA transcripts of avocado sunblotch viroid". Nucleic Acids Res. 14 (9): 3627–
3640.
4. ^ Forster AC, Symons RH. (1987). "Self-cleavage of virusoid RNA is performed by the
proposed 55-nucleotide active site". Cell 50 (1): 9–16.
5. ^ a b Hean J and Weinberg MS (2008). "The Hammerhead Ribozyme Revisited: New
Biological Insights for the Development of Therapeutic Agents and for Reverse
Genomics Applications". RNA and the Regulation of Gene Expression: A Hidden Layer
of Complexity. Caister Academic Press. ISBN 978-1-904455-25-7.
http://www.horizonpress.com/rnareg.
6. ^ Ferbeyre G, Smith JM, Cedergren R. (1998). "Schistosome satellite DNA encodes
active hammerhead ribozymes". Mo. Cell. Biol. 18 (7): 3880–3888. PMID 9632772.
7. ^ J.B. Murray, A.A. Seyhan, N.G. Walter, J.M. Burke and W.G. Scott (1998). "The
hammerhead, hairpin and VS ribozymes are catalytically proficient in monovalent cations
alone.". Chem Biol 5: 587-595.
8. ^ Pley, HW; Flaherty KM, McKay DB (1994). "Three-dimensional structure of a
hammerhead ribozyme". Nature 372: 68–74. doi:10.1038/372068a0. PMID 7969422.
9. ^ Khvorova A, Lescoute A, Westhof E, Jayasena SD. (2003). "Sequence elements
outside the hammerhead ribozyme catalytic core enable intracellular activity". Nat Struct
Biol. 10 (9): 708–712. doi:10.1038/nsb959. PMID 12881719.
10. ^ Scott WG, Finch JT, Klug A. (1995). "The crystal structure of an all-RNA hammerhead
ribozyme: a proposed mechanism for RNA catalytic cleavage". Cell 81 (7): 991–1002.
doi:10.1016/S0092-8674(05)80004-2. PMID 7541315.
11. ^ Martick M, Scott WG (2006). "Tertiary contacts distant from the active site prime a
ribozyme for catalysis". Cell 126 (2): 309–320. doi:10.1016/j.cell.2006.06.036.
PMID 16859740.
12. ^ Citti L, Rainaldi G. (2005). "Synthetic hammerhead ribozymes as therapeutic tools to
control disease genes". Current Gene Therapy 5 (1): 11–24. PMID 15638708.