 Ribonuclease P
Until about 20 years ago,
 Group I Introns
all known enzymes were
 Group II Introns
proteins. But then it was
 Spliceosomes
discovered that some RNA
 Viroids
molecules can act as
 An RNA World?
enzymes; that is, catalyze
o The Ribosome is a Ribozyme
covalent changes in the
o RNA polymerization by RNA
structure of substrates
 Ribozymes for Human Therapy
(most of which are also
RNA molecules). Catalytic
RNA molecules are called ribozymes.
Most classes of RNA
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transfer RNA (tRNA)
ribosomal RNA (rRNA)
messenger RNA (mRNA)
are transcribed as precursors that are larger than the final product.
These precursors often contain
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"head" (5') and "tail" (3') sequences
intron sequences
that must be removed to make the final product. Some of the
processing steps employ other RNA molecules (always associated
with proteins).
Ribonuclease P
Almost all living things synthesize an enzyme — called Ribonuclease
P (RNase P) — that cleaves the head (5') end of the precursors of
transfer RNA (tRNA) molecules.
In bacteria, ribonuclease P is a heterodimer containing
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
a molecule of RNA and one of
protein
Separated from each other, the RNA retains its ability to catalyze
the cleavage step (although less efficiently than the intact dimer),
but the protein alone cannot do the job.
Group I Introns
Some ribosomal RNA (rRNA) genes
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in the mitochondrial genome of certain fungi (e.g., yeast)
in some chloroplast genomes
in the nuclear genome of some "lower" eukaryotes, for
example
o the ciliated protozoan Tetrahymena thermophila)
o the plasmodial slime mold Physarum polycephalum
contain introns that must be spliced out to make the final product.
The splicing reaction is self-contained; that is, the intron — with the
help of associated proteins — splices itself out of the precursor
RNA.
Once excision of the intron and splicing of the adjacent exons are
completed, the story is over. In other words, although the action is
catalyzed by the RNA, only a single molecule of substrate is
involved (unlike protein enzymes that repeatedly catalyze a
reaction).
However, synthetic versions of Group I introns made in the
laboratory can — in vitro — act repeatedly; that is, like true
enzymes.
The DNA of some Group I introns includes an open reading frame
(ORF) that encodes a transposase-like protein that can make a copy
of the intron and insert it elsewhere in the genome.
Link to a discussion of transposons.
All the Group I introns share a characteristic secondary structure
and mode of action that distinguishes them from the next group.
Group II Introns
Some messenger RNA (mRNA) genes
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in the mitochondrial genome of yeast and other fungi
(encoding the proteins cytochrome b and subunits of
cytochrome c oxidase)
in some chloroplast genomes
also contain self-splicing introns.
Because their secondary structure and the details of the splicing
reaction differ from the rRNA introns discussed above, these are
called Group II introns.
The DNA of some Group II introns also includes an open reading
frame (ORF) that encodes a transposase-like protein that can make
a copy of the intron and insert it elsewhere in the genome.
Spliceosomes
Spliceosomes remove introns and splice the exons of most nuclear
genes. They are composed of 5 kinds of small nuclear RNA (snRNA)
molecules and a large number of protein molecules. It is the snRNA
— not the protein — that catalyzes the splicing reactions.
The molecular details of the reactions are similar to those of Group
II introns, and this has led to speculation that this splicing
machinery evolved from them.
Viroids
Viroids are
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RNA molecules that infect plant cells as conventional viruses
do, but
o are far smaller (one has only 246 nucleotides)
o are naked; that is, they are not encased in a capsid.
Some viroidlike molecules get into the cell as passengers
inside a conventional plant virus. These are called virusoids or
viroidlike satellite RNAs.
In both cases, the molecules consists of
o single-stranded RNA whose
ends are covalently bonded to form a circle.
o There are several regions where base-pairing occurs
across adjacent portions of the molecule.
New viroids and virusoids are synthesized by the host cell as
long precursors in which the viroid structure is tandemly
repeated.
These repeats must be cut out and ligated to form the final
product.
Most virusoids and at least one viroid are self-splicing; that is,
they can cut themselves out of the precursor and ligate their
ends without the aid of any host enzymes.
Thus they represent another class of ribozyme.
o
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Both viroids and virusoids are responsible for a number of serious
diseases of economically important plants; e.g. the coconut palm
and chrysanthemums. (The problem is so severe with
chrysanthemums that all growers in the U.S. now secure their stock
from a few companies that raise the plants in "clean" rooms using
stringent precautions to prevent infection by the viroid.)
An RNA World?
The discovery that RNA molecules can act as catalysts provides a
possible solution to a long-standing dilemma:


DNA encodes the genetic information of proteins but
DNA replication and transcription requires proteins.
So which came first in the evolution of life?
But if RNA can serve both as a


repository of information (in its sequence of nucleotides) and
as a
catalyst,
then it has both properties needed for life. This provides the basis
for the notion that life began as RNA — the so-called "RNA World".
However, all the reactions described above


involve RNA acting on RNA (not protein)
and (except for Ribonuclease P) are self-limited.
Is there evidence that RNA can catalyze the synthesis of proteins?
Yes, the ribosome turns out to be a ribozyme.
The Ribosome is a Ribozyme
Ribosomes are huge aggregates containing 3 (4 in eukaryotes) rRNA
molecules and scores of protein molecules.
The three-dimensional structure of the large (50S) subunit of a
bacterial ribosome was published in August 2000. It clearly shows
that formation of the peptide bond that links each amino acid to the
growing polypeptide chain is catalyzed by the 23S RNA molecule in
the large subunit. The 31 proteins in the subunit probably provide
the scaffolding needed to maintain the three-dimensional structure
of the RNA.
Link to discussion of ribosome structure and function.
RNA polymerization by RNA
In today's world, RNA polymerases — made of protein — make the
RNA molecules (using the antisense strand of DNA as a template
[View]). Could RNA alone have done it?
It can be done in the laboratory. Wochner, A. et al. report in
Science, 332:209, 8 April 2011, their creation of a synthetic RNA
molecule that when presented with single-stranded RNA templates,
polymerizes ribonucleotide triphosphates into strands of RNA
complementary to the template. Their synthetic RNA polymerase
was able to faithfully incorporate up to 95 nucleotides into
complementary strands of RNA. One product was a functional
endonuclease ribozyme. (By the autumn of 2013, they were able to
copy a template of 206 nucleotides.)
Ribozymes for Human Therapy
The ability of ribozymes to recognize and cut specific RNA
molecules makes them exciting candidates for human therapy.
Already, a synthetic ribozyme that destroys the mRNA encoding a
receptor of Vascular Endothelial Growth Factor (VEGF) is being
readied for clinical trials. VEGF is a major stimulant of
angiogenesis, and blocking its action may help starve cancers of
their blood supply.
A ribozyme (from ribonucleic acid enzyme, also called RNA enzyme
or catalytic RNA) is an RNA molecule that catalyzes a chemical
reaction. Many natural ribozymes catalyze either the hydrolysis of
one of their own phosphodiester bonds, or the hydrolysis of bonds in
other RNAs, but they have also been found to catalyze the
aminotransferase activity of the ribosome.
Investigators studying the origin of life have produced ribozymes in
the laboratory that are capable of catalyzing their own synthesis
under very specific conditions, such as an RNA polymerase
ribozyme. 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 hydrolysis of its
phosphodiester bonds.
Some 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.
Further Reading
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Ribozyme Discovery
Ribozyme Activity
Known Ribozymes
Artificial Ribozymes
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This article is about the chemical. For the rock band, see Ribozyme
(band).
Structure of hammerhead ribozyme
A ribozyme (ribonucleic acid enzyme) is an RNA molecule that is
capable of performing specific biochemical reactions, similar to the
action of protein enzymes. The 1981 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. Also
termed catalytic RNA, ribozymes function within the ribosome (as
part of the large subunit ribosomal RNA) to link amino acids during
protein synthesis, and 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 and the hairpin ribozyme.
Investigators studying the origin of life have produced ribozymes in
the laboratory that are capable of catalyzing their own synthesis
under very specific conditions, such as an RNA polymerase
ribozyme.[1] 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.[2] The "tC19Z" ribozyme can add up to 95
nucleotides with a fidelity of 0.0083 mutations/nucleotide.[3]
Some ribozymes may play an important role as therapeutic agents,
as enzymes which target defined RNA sequences for cleavage, as
biosensors, and for applications in functional genomics and gene
discovery.[4]
Contents
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1
2
3
4
5
6
7
8
Discovery
Activity
Known ribozymes
Artificial ribozymes
Applications
See also
References
Further reading

9 External links
Discovery
Schematic showing ribozyme cleavage of RNA.
Before the discovery of ribozymes, enzymes, which are defined as
catalytic proteins,[5] 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.[6]
The first ribozymes were discovered in the 1980s by Thomas R.
Cech, who was studying RNA splicing in the ciliated protozoan
Tetrahymena thermophila and Sidney Altman, who was working on
the bacterial RNase P complex. 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 won the Nobel Prize in
chemistry for their "discovery of catalytic properties of RNA."[7] The
term ribozyme was first introduced by Kelly Kruger et al. in 1982 in
a paper published in Cell.[8]
It had been a firmly established belief in biology that catalysis was
reserved for proteins. In retrospect, catalytic RNA makes a lot of
sense. This is based on 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.[9]
In the 1970s 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 splicing reaction, he found that 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.
Activity
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 molecular 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. There is no requirement for divalent cations in a fivenucleotide RNA that can catalyze trans-phenylalanation of a fournucleotide substrate which has three base complementary
sequence with the catalyst. The catalyst and substrate were
devised by truncation of the C3 ribozyme.[10]
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 became known as the
"RNA world hypothesis" of the origin of life.
If ribozymes were the first molecular machines used by early life,
then today's remaining ribozymes—such as the ribosome
machinery—could be considered living fossils of a life based
primarily on nucleic acids.
A recent test-tube study of prion folding suggests that an RNA may
catalyze the pathological protein conformation in the manner of a
chaperone enzyme.[11]
Ribozymes have been shown to be involved in the viral concatemer
cleavage that precedes the packing of viral genetic material into
virions.[12][13][14]
Known ribozymes
Naturally occurring ribozymes include:
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Peptidyl transferase 23S rRNA - Found in all living cells
RNase P
Group I and Group II introns
GIR1 branching ribozyme[15]
Leadzyme - Although initially created in vitro, natural
examples have been found
Hairpin ribozyme
Hammerhead ribozyme
HDV ribozyme
Mammalian CPEB3 ribozyme
VS ribozyme
glmS ribozyme
CoTC ribozyme
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 selfcleaving RNAs that have good enzymatic activity have been
produced. Tang and Breaker[16] 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.
The techniques used to create artificial ribozymes involve
Darwinian 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 mutagenic 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 enzyme mixture, a pair of RNA
enzymes emerged, in which each synthesizes the other from
synthetic oligonucleotides, with no protein present.[17]