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Ribozyme
3D structure of a hammerhead ribozyme

Ribozymes (ribonucleic acid enzymes) are

RNA molecules that have the ability to
catalyze specific biochemical reactions,
including RNA splicing in gene expression,
similar to the action of protein enzymes.
The 1982 discovery of ribozymes
demonstrated that RNA can be both
genetic material (like DNA) and a
biological catalyst (like protein enzymes),
and contributed to the RNA world
hypothesis, which suggests that RNA may
have been important in the evolution of
prebiotic self-replicating systems.[1] The
most common activities of natural or in
vitro-evolved ribozymes are the cleavage
or ligation of RNA and DNA and peptide
bond formation.[2] Within the ribosome,
ribozymes function as part of the large
subunit ribosomal RNA to link amino acids
during protein synthesis. They also
participate in a variety of RNA processing
reactions, including RNA splicing, viral
replication, and transfer RNA biosynthesis.
Examples of ribozymes include the
hammerhead ribozyme, the VS ribozyme,
Leadzyme and the hairpin ribozyme.

Investigators studying the origin of life

have produced ribozymes in the laboratory
that are capable of catalyzing their own
synthesis from activated monomers under
very specific conditions, such as an RNA
polymerase ribozyme.[3] Mutagenesis and
selection has been performed resulting in
isolation of improved variants of the
"Round-18" polymerase ribozyme from
2001. "B6.61" is able to add up to 20
nucleotides to a primer template in 24
hours, until it decomposes by cleavage of
its phosphodiester bonds.[4] The "tC19Z"
ribozyme can add up to 95 nucleotides
with a fidelity of 0.0083
mutations/nucleotide.[5]

Attempts have been made to develop

ribozymes as therapeutic agents, as
enzymes which target defined RNA
sequences for cleavage, as biosensors,
and for applications in functional
genomics and gene discovery.[6]

Discovery
Schematic showing ribozyme cleavage of RNA.

Before the discovery of ribozymes,

enzymes, which are defined as catalytic
proteins,[7] were the only known biological
catalysts. In 1967, Carl Woese, Francis
Crick, and Leslie Orgel were the first to
suggest that RNA could act as a catalyst.
This idea was based upon the discovery
that RNA can form complex secondary
structures.[8] These ribozymes were found
in the intron of an RNA transcript, which
removed itself from the transcript, as well
as in the RNA component of the RNase P
complex, which is involved in the
maturation of pre-tRNAs. In 1989, Thomas
R. Cech and Sidney Altman shared the
Nobel Prize in chemistry for their
"discovery of catalytic properties of
RNA."[9] The term ribozyme was first
introduced by Kelly Kruger et al. in 1982 in
a paper published in Cell.[1]
It had been a firmly established belief in
biology that catalysis was reserved for
proteins. However, the idea of RNA
catalysis is motivated in part by the old
question regarding the origin of life: Which
comes first, enzymes that do the work of
the cell or nucleic acids that carry the
information required to produce the
enzymes? The concept of "ribonucleic
acids as catalysts" circumvents this
problem. RNA, in essence, can be both the
chicken and the egg.[10]
In the 1980s Thomas Cech, at the
University of Colorado at Boulder, was
studying the excision of introns in a
ribosomal RNA gene in Tetrahymena
thermophila. While trying to purify the
enzyme responsible for the splicing
reaction, he found that the intron could be
spliced out in the absence of any added
cell extract. As much as they tried, Cech
and his colleagues could not identify any
protein associated with the splicing
reaction. After much work, Cech proposed
that the intron sequence portion of the
RNA could break and reform
phosphodiester bonds. At about the same
time, Sidney Altman, a professor at Yale
University, was studying the way tRNA
molecules are processed in the cell when
he and his colleagues isolated an enzyme
called RNase-P, which is responsible for
conversion of a precursor tRNA into the
active tRNA. Much to their surprise, they
found that RNase-P contained RNA in
addition to protein and that RNA was an
essential component of the active
enzyme. This was such a foreign idea that
they had difficulty publishing their findings.
The following year, Altman demonstrated
that RNA can act as a catalyst by showing
that the RNase-P RNA subunit could
catalyze the cleavage of precursor tRNA
into active tRNA in the absence of any
protein component.

Since Cech's and Altman's discovery, other

investigators have discovered other
examples of self-cleaving RNA or catalytic
RNA molecules. Many ribozymes have
either a hairpin – or hammerhead –
shaped active center and a unique
secondary structure that allows them to
cleave other RNA molecules at specific
sequences. It is now possible to make
ribozymes that will specifically cleave any
RNA molecule. These RNA catalysts may
have pharmaceutical applications. For
example, a ribozyme has been designed to
cleave the RNA of HIV. If such a ribozyme
were made by a cell, all incoming virus
particles would have their RNA genome
cleaved by the ribozyme, which would
prevent infection.

Structure and mechanism

Despite having only four choices for each
monomer unit (nucleotides), compared to
20 amino acid side chains found in
proteins, ribozymes have diverse
structures and mechanisms. In many
cases they are able to mimic the
mechanism used by their protein
counterparts. For example, in self cleaving
ribozyme RNAs, an in-line SN2 reaction is
carried out using the 2’ hydroxyl group as a
nucleophile attacking the bridging
phosphate and causing 5’ oxygen of the
N+1 base to act as a leaving group . In
comparison, RNase A, a protein that
catalyzes the same reaction, uses a
coordinating histidine and lysine to act as
a base to attack the phosphate
backbone.[2]
Like many protein enzymes metal binding
is also critical to the function of many
ribozymes.[11] Often these interactions use
both the phosphate backbone and the
base of the nucleotide, causing drastic
conformational changes.[12] There are two
mechanism classes for the cleavage of
phosphodiester backbone in the presence
of metal. In the first mechanism, the
internal 2’- OH group attacks phosphorus
center in a SN2 mechanism. Metal ions
promote this reaction by first coordinating
the phosphate oxygen and later stabling
the oxyanion. The second mechanism also
follows a SN2 displacement, but the
nucleophile comes from water or
exogenous hydroxyl groups rather than
RNA itself. The smallest ribozyme is UUU,
which can promote the cleavage between
G and A of the GAAA tetranucleotide via
the first mechanism in the presence of
Mn2+. The reason why this trinucleotide
rather than the complementary tetramer
catalyze this reaction may be because the
UUU-AAA pairing is the weakest and most
flexible trinucleotides among the 64
conformations, which provides the binding
site for Mn2+.[13]

Phosphoryl transfer can also be catalyzed

without metal ions. For example,
pancreatic ribonuclease A and hepatitis
delta virus(HDV) ribozymes can catalyze
the cleavage of RNA backbone through
acid-base catalysis without metal
ions.[14][15] Hairpin ribozyme can also
catalyze the self-cleavage of RNA without
metal ions but the mechanism is still
unclear.[15]

Ribozyme can also catalyze the formation

of peptide bond between adjacent amino
acid by lowering the activation entropy.[14]

Image showing the diversity of ribozyme structures

Image showing the diversity of ribozyme structures.
From left to right: leadzyme, hammerhead ribozyme,
twister ribozyme

Activity
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A ribosome is a biological machine that utilizes
protein dynamics on nanoscales to translate RNA into
proteins

Although most ribozymes are quite rare in

the cell, their roles are sometimes
essential to life. For example, the
functional part of the ribosome, the
biological machine that translates RNA
into proteins, is fundamentally a ribozyme,
composed of RNA tertiary structural
motifs that are often coordinated to metal
ions such as Mg2+ as cofactors.[16] In a
model system, there is no requirement for
divalent cations in a five-nucleotide RNA
catalyzing trans-phenylalanation of a four-
nucleotide substrate with 3 base pairs
complementary with the catalyst, where
the catalyst/substrate were devised by
truncation of the C3 ribozyme.[17] RNA
may catalyze folding of the pathological
protein conformation of a prion in a
manner similar to that of a chaperonin,[18]
and may be involved in the viral
concatemer cleavage that precedes the
packing of viral genetic material into some
virons.

RNA can also act as a hereditary molecule,

which encouraged Walter Gilbert to
propose that in the distant past, the cell
used RNA as both the genetic material and
the structural and catalytic molecule rather
than dividing these functions between
DNA and protein as they are today; this
hypothesis is known as the "RNA world
hypothesis" of the origin of life.[19]
Evidence that ribozymes were the first
molecular machines used by early life
suggests that they are in effect "molecular
fossils".

Artificial ribozymes
Since the discovery of ribozymes that exist
in living organisms, there has been interest
in the study of new synthetic ribozymes
made in the laboratory. For example,
artificially-produced self-cleaving RNAs
that have good enzymatic activity have
been produced. Tang and Breaker[20]
isolated self-cleaving RNAs by in vitro
selection of RNAs originating from
random-sequence RNAs. Some of the
synthetic ribozymes that were produced
had novel structures, while some were
similar to the naturally occurring
hammerhead ribozyme. In 2015,
researchers at Northwestern University
and the University of Illinois at Chicago
have engineered a tethered ribosome that
works nearly as well as the authentic
cellular component that produces all the
proteins and enzymes within the cell.
Called Ribosome-T, or Ribo-T, the artificial
ribosome was created by Michael Jewett
and Alexander Mankin.[21] The techniques
used to create artificial ribozymes involve
directed evolution. This approach takes
advantage of RNA's dual nature as both a
catalyst and an informational polymer,
making it easy for an investigator to
produce vast populations of RNA catalysts
using polymerase enzymes. The
ribozymes are mutated by reverse
transcribing them with reverse
transcriptase into various cDNA and
amplified with error-prone PCR. The
selection parameters in these experiments
often differ. One approach for selecting a
ligase ribozyme involves using biotin tags,
which are covalently linked to the
substrate. If a molecule possesses the
desired ligase activity, a streptavidin
matrix can be used to recover the active
molecules.

Lincoln and Joyce developed an RNA

enzyme system capable of self replication
in about an hour. By utilizing molecular
competition (in vitro evolution) of a
candidate RNAmixture, a pair of ribozymes
emerged, in which each synthesizes the
other by joining synthetic oligonucleotides,
with no protein present.[22]
Although not true catalysts, the creation of
artificial self-cleaving riboswitches, termed
aptazymes, has also been an active area
of research. Riboswitches are regulatory
RNA motifs that change their structure in
response to a small molecule ligand to
regulate translation. While there are many
known natural riboswitches that bind a
wide array of metabolites and other small
organic molecules, only one ribozyme
based on a riboswitch has been described,
glmS.[23] Early work in characterizing self-
cleaving riboswitches was focused on
using theophylline as the ligand. In these
studies an RNA hairpin is formed which
blocks the ribosome binding site, thus
inhibiting translation. In the presence of
the ligand, in these cases theophylline, the
regulatory RNA region is cleaved off,
allowing the ribosome to bind and
translate the target gene. Much of this
RNA engineering work was based on
rational design and previously determined
RNA structures rather than directed
evolution as in the above examples. More
recent work has broadened the ligands
used in ribozyme riboswitches to include
thymine pyrophosphate (2). Fluorescence-
activated_cell_sorting has also been used
to engineering aptazymes.[24]

Applications
Ribozymes have been proposed and
developed for the treatment of disease
through gene therapy (3). One major
challenge of using RNA based enzymes as
a therapeutic is the short half-life of the
catalytic RNA molecules in the body. To
combat this, the 2’ position on the ribose
is modified to improve RNA stability. One
area of ribozyme gene therapy has been
the inhibition of RNA-based viruses.

A type of synthetic ribozyme directed

against HIV RNA called gene shears has
been developed and has entered clinical
testing for HIV infection.[25][26]
Similarly, ribozymes have been designed
to target the hepatitis C virus RNA, SARS
coronavirus (SARS-CoV),[27] Adenovirus[27]
and influenza A and B virus
RNA.[28][29][30][27] The ribozyme is able to
cleave the conserved regions of the virus’s
genome which has been shown to reduce
the virus in mammalian cell culture.[31]
Despite these efforts by researchers, these
projects have remained in the preclinical
stage.
Known ribozymes
Well validated naturally occurring ribozyme
classes:

GIR1 branching ribozyme[32]

glmS ribozyme
Group I self-splicing intron
Group II self-splicing intron -
Spliceosome is likely derived from
Group II self-splicing ribozymes.[33]
Hairpin ribozyme
Hammerhead ribozyme
HDV ribozyme
rRNA - Found in all living cells and links
amino acids to form proteins.
RNase P
Twister ribozyme
Twister sister ribozyme
VS ribozyme
Pistol ribozyme
Hatchet ribozyme
Viroids
See also
Deoxyribozyme
Spiegelman Monster
Catalysis
Enzyme
RNA world hypothesis
Peptide nucleic acid
Nucleic acid analogues
PAH world hypothesis
SELEX
 OLE RNA

Notes and references

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Further reading
Sigel A, Sigel H, Sigel RK (2011). Structural
and catalytic roles of metal ions in RNA. Metal
Ions in Life Sciences. 9. RSC Publishing.
pp. vii–ix. doi:10.1039/9781849732512 .
ISBN 978-1-84973-251-2. PMID 22010266 .
Johnson-Buck AE, McDowell SE, Walter NG
(2011). Metal ions: supporting actors in the
playbook of small ribozymes. Metal Ions in
Life Sciences. 9. pp. 175–96.
doi:10.1039/9781849732512-00175 .
ISBN 978-1-84973-094-5. PMC 3365584 .
PMID 22010272 .
Donghi D, Schnabl J (2011). Multiple roles of
metal ions in large ribozymes. Metal Ions in
Life Sciences. 9. pp. 197–234.
doi:10.1039/9781849732512-00197 .
ISBN 978-1-84973-094-5. PMID 22010273 .
Trappl K, Polacek N (2011). The ribosome: a
molecular machine powered by RNA. Metal
Ions in Life Sciences. 9. pp. 253–75.
doi:10.1039/9781849732512-00253 .
ISBN 978-1-84973-094-5. PMID 22010275 .
Suga H, Futai K, Jin K (2011). Metal ion
requirements in artificial ribozymes that
catalyze aminoacylation and redox reactions.
Metal Ions in Life Sciences. 9. pp. 277–97.
doi:10.1039/9781849732512-00277 .
ISBN 978-1-84973-094-5. PMID 22010276 .
Wedekind JE (2011). Metal ion binding and
function in natural and artificial small RNA
enzymes from a structural perspective. Metal
Ions in Life Sciences. 9. pp. 299–345.
doi:10.1039/9781849732512-00299 .
ISBN 978-1-84973-094-5. PMID 22010277 .
Doherty EA, Doudna JA (2001). "Ribozyme
structures and mechanisms". Annual Review
of Biophysics and Biomolecular Structure. 30:
457–75.
doi:10.1146/annurev.biophys.30.1.457 .
PMID 11441810 .
Joyce GF (2004). "Directed evolution of
nucleic acid enzymes". Annual Review of
Biochemistry. 73: 791–836.
doi:10.1146/annurev.biochem.73.011303.07
3717 . PMID 15189159 .
Ikawa Y, Tsuda K, Matsumura S, Inoue T
(September 2004). "De novo synthesis and
development of an RNA enzyme" .
Proceedings of the National Academy of
Sciences of the United States of America. 101
(38): 13750–5.
 Bibcode:2004PNAS..10113750I .
doi:10.1073/pnas.0405886101 .
PMC 518828 . PMID 15365187 .

External links
Tom Cech's Short Talk: "Discovering
Ribozymes"

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title=Ribozyme&oldid=932818356"
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