Background
A hammerhead ribozyme is a small catalytic RNA motif that catalyzes self-cleavage reaction. Its name comes from its secondary
structure which resembles a carpenter's hammer. The hammerhead ribozyme is involved in the replication of a type of viroid
and some satellite RNAs.
Viroids are small RNA agents (up to 400 nucleotides) of infectious plant diseases.
They exist as naked RNAs so that, unlike RNA viruses, they are not encapsidated by a protein coat. The viroid genome is
a single-stranded circular RNA that contains self-complementary sequences so it can form extensive double-stranded regions.
In their native form, viroids assume a rod-like conformation that is very stable against ribonucleases. It appears
that the viroids do not encode any proteins because the viroid genomes do not contain open-reading frame. Since the viroids
do not encode a viroid-specific RNA polymerase, it is proposed that host RNA-dependent RNA polymerases may be involved in
replication. Some satellite RNAs are referred to as virusoids. Virusoids are similar to viroids except that the virusoids
require helper viruses for the genome replication or encapsidation into a protein coat. Viroids and satellite RNAs are plant-specific,
however, there is an exception. Hepatitis delta agent (or sometimes referred to as hepatitis delta virus) is a human pathogen
that has striking similarities to viroids and satellite RNAs. Like virusoids, the hepatitis delta agent is associated with
a helper virus, Hepatitis B virus in natural infections.
Viroid replication
The mode of viroid replication is proposed to be a rolling-circle mechanism similar
to the one that produces the plus strand of the bacteriophage fX174. Since the viroid
genome (a plus strand) is a single-stranded
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Figure 6. Rolling circle replication of viroid RNAs.
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circular genome, a host RNA polymerase can synthesize a complementary strand (a minus strand) by going through the circular
genome more than once. This results in the minus strand that is a longer-than-unit-length (oligomeric) compared to the original
viroid genome. The new, longer plus strand is then synthesized by the host RNA polymerase. The cleavage of the longer positive
strand to an individual unit is catalyzed by the hammerhead motifs. Circularization of the plus strand can be accomplished
by the reverse reaction of hammerhead catalysis although this may be an inefficient process. Circularization of the plus
strand may be catalyzed by a host RNA ligase. It has been demonstrated that RNA ligase purified from wheat germ can readily
circularize linear, potato spindle tuber viroid RNA. It should be noted that hammerhead motifs are not the only self-cleaving
RNA motif found in viroids and satellite RNAs.
Pathogenesis
Pathogenesis of viroids and satellite RNAs is still unclear. It has been proposed that viroids
may cause harm in host plants by disrupting host RNA processing. In theory, if the viroid RNA contains a complementary sequence
to a host cellular RNA, it can form a complex and prevent it from participating in normal cellular function. Potato spindle
tuber viroid (PSTV) contains a complementary sequence to that of host 7S RNA. 7S RNA, along with other proteins, is involved
in protein translocation. Formation of a hybrid between PSTV and host 7S RNA would disrupt the protein translocation in
host plants. It is proposed that the viroid transmission occurs either with a help of insects or by mechanical damages to
plants by farming tools.
Catalytic mechanism
The hammerhead ribozyme catalyzes a transesterification reaction in which the 3',5'-phosphodiester bond
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Figure 7.
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between nucleotides 17 and 1.1 is cut. The reaction yields a cyclic 2',3'- phosphodiester on nucleotide 17 and a free 5'-hydroxyl
on nucleotide 1.1. The mode of catalysis is called an in-line mechanism. The reaction requires divalent cations such as
Mg2+. It is proposed that a metal hydroxide abstracts a hydrogen from 2'-hydroxyl of the nucleotide 17. The resulting
nucleophile (2' O-) attacks the scissile phosphate. The configuration of the reaction intermediate is a
trigonal bipyramid in which the apical positions are occupied by the leaving group (5'-OH) and the attacking group (2'-OH).
Therefore, the free 2'-OH on nucleotide 17 and divalent cations are essential for the catalysis.
Tertiary structures
The hammerhead motifs can be prepared in vitro by synthesizing two separate strands and combining
them together to observe the self-cleavage reaction. In this system, a strand that is cleaved is defined as a substrate
and the other strand is defined as an enzyme. Using this system, it has been possible to study the structure and the function
of hammerhead motifs through kinetic and mutational experiments. A number of crystal structures of hammerhead ribozymes
are now available. The first crystal structure of the hammerhead ribozyme (PDB: 1HMH) is a RNA-DNA hybrid in which a substrate
strand is replaced with the DNA strand (an inhibitor because the deoxyribonucleotide at the position 17 lacks 2'-hydroxyl,
so the cleavage reaction can not occur). If the nucleotide 17 is replaced with a single ribonucleotide, the catalysis can
take place. Therefore, the overall structure was considered to be quite similar to the all-RNA hammerhead ribozyme (This
was later confirmed by the crystal structure of all-RNA hammerhead).
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Figure 8. Tertiary structure of a hammerhead ribozyme.
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The tertiary structure of the hammerhead ribozyme resembles a wishbone. Three stem regions are A-form helices that are stabilized
mainly through Watson-Crick base pairs. The region where the catalysis takes place is formed by a hairpin loop that is very
similar to the U-turn (uridine turn) observed in the tertiary structure of the transfer RNAPhe. It was not possible
to determine the positions of the essential divalent metal ions from this model. The crystal structures of the all-RNA hammerhead
ribozymes in the presence of divalent metals (either Mg2+
or Mn2+) were later solved. Since the crystals were destroyed after a period of time (due to the cleavage of
the substrate strands), it was necessary to work around this problem. The crystals were first grown in the absence of the
divalent metals. The divalent metals were later added to the crystals at either pH 5 or pH 8.5. The catalysis of the hammerhead
ribozyme is dependent on the pH. At pH 5, the catalysis is very inefficient and slow, so the structure is considered to
be at the ground state (PDB: 300D). At pH 8.5, the hammerhead ribozyme is fully active, so the crystal was frozen in a liquid
nitrogen at 4 minutes after the addition of the divalent metals to trap a reaction intermediate (PDB: 301D).
The comparison of the two structures at different pH reveals that the structural
difference is localized to the scissile bond and the nucleotides between it. Except for the approximately 2 angstroms shift
of the scissile bond, the overall structure is unchanged and very similar for the both models. An additional Mg2+
binding site was observed in the structure at pH 8.5. This Mg2+
is located close to the pro-Rp oxygen of the scissile phosphate between nucleotides 17 and 1.1. This Mg2+ is
proposed to abstract 2'OH of the nucleotide 17. Because the conformational change observed in the structure at pH 8.5 is
still not enough to allow the in-line mechanism, an additional conformational change is expected to occur.
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Figure 9A (left). The inhibited structure at pH 5.0.
Enzyme strand: red; Substrate strand: yellow; Scissile phosphate: blue; Divalent ions: green. Note:
Only the divalent ions close to the cleavage site are shown.
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Figure 9B (right). The structure at pH 8.5.
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Hammerhead ribozyme
1. Branch, A. D., Robertson, H. D., and Dickson, E. Longer-than-unit-length viroid minus strands are present in RNA from
infected plants. Proc. Natl. Acad. Sci. USA (1981) 78, 6381-6385.
2. Branch, A. D., Robertson, H. D., Greer, C., Gegenheimer, P, Peebles, C., and Abelson, J. Cell-free circularization
of viroid progeny RNA by an RNA ligase from wheat germ. Science (1982) 217, 1147-1149.
3. Buzayan, J. M., Gerlach, W. L., and Gruening, G. Satellite tobacco ringspot virus RNA: A subset of
the RNA sequence is sufficient for autolytic processing. Proc. Natl. Acad. Sci. USA (1986) 83, 8859-8862.
4. Dahm, S. C., Derrick, W. B., and Uhlenbeck, O. C. Evidence of the role of solvated metal hydroxide in the
hammerhead cleavage mechanism. Biochemistry (1993) 32, 13040-13045.
5. Diener, T. O., Viroids and satellites: molecular parasites at the frontier of life, Ch. 1 in Maramorosch,
K., ed., CRC press, Inc., Boca Raton, (1991).
6. Pan, T., Long, D. M., and Uhlenbeck, O. C., Ch. 12 in The RNA World, Gesteland, R. F. and Atkins,
J. F., ed., Cold Spring Harbor Laboratory Press (1993)
7. Pely, H. W., Flasherty, K. M., and McKay, D. B. Three-dimentional structure of a hammerhead ribozyme:
Nature (1994) 372, 68-74.
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Last updated: 3:11pm on 9/26/08 by pgegen@ku.edu