A ribozyme is a ribonucleic acid (RNA) enzyme that catalyzes a chemical reaction. The ribozyme catalyses specific reactions in a similar way to that of protein enzymes. Also called catalytic RNA, ribozymes are found in the ribosome where they join amino acids together to form protein chains.
2. WHAT IS 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.
3. HISTORY
• 1967: Carl Woese, Francis Crick, and Leslie Orgel were the first to
suggest that RNA could act as a catalyst.
• 1970s: Thomas Cech, at the University of Colorado, 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.
4. • 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.
• 1981-82: Discovery of Ribozyme
• 1982: Ribozyme term was introduced by Kelly Kruger et al. in
a paper published in The Cell
• 1989: Thomas Cech and Sidney Altman shared the Nobel
Prize fo demonstrating that RNA could act as an enzyme.
5. TYPES OF RIBOZYMES
• Group I and group II intron splicing ribozymes
• RNase P
• Hammerhead Ribozyme
• Hairpin ribozyme
• Ribosome
6. GROUP 1 INTRON SPLICING
Group I intron ribozymes constitute one of the main classes of
ribozymes.
Found in bacteria, lower eukaryotes and higher plants.
Group I introns are also found inserted into genes of a wide variety
of bacteriophages of Gram-positive bacteria.
However, their distribution in the phage of Gram-negative bacteria
is mainly limited to the T4, T-even and T7-like like bacteriophages.
The group I splicing reaction requires a
guanine residue cofactor, the 3’ OH
group of guanosine is used as a
nucleophile. The 3’ OH group attacks
the 5’ phosphate of the intron and a
new phosphodiester bond is formed.
The 3’ OH of the exon that is displaced
now acts as the nucleophile in a similar
reaction at the 3’ end of the intron. So
the intron is precisely excised and
exons are joined together.
7. GROUP 2 INTRON SPLICING
Group II introns have been found
in bacteria and in the
mitochondrial and chloroplast
genomes of fungi, plants,
protists, and an annelid worm.
Mechanism:
The 2’OH of a specific adenosine
acts as a nucleophile and attacks the
5’ splice site creating a branched
intron structure. The 3’ OH of the 5’
exon attacks the 3’ splice site,
ligating the exons and releasing the
intron as a lariat structure.
8. Rnase P : Ribonuclease P (RNaseP), a ribonucleoprotein, is an
essential tRNA processing enzyme found in all living
organisms.
Mechanism:
• All RNase P enzymes are ribonucleoproteins [bacteria: 1RNA + 1 protein subunit;
eukaryotes: 1 RNA + many protein subunits (11 in human)],
• In Ribonuclease – P, protein component is facilitates binding between RNase and t-RNA
substrate.
• Requires divalent metal ions (like Mg2+) for its activity.
• Endo-ribonuclease responsible for generating 5’ end of matured tRNA molecules.
• Cleavage via nucleophilic attack on the phosphodiester bond leaving a 5’-phosphate
and 3’-hydroxyl at the cleavage site.
9. HAMMERHEAD
RIBOZYME
Hammerhead ribozymes (HHRZs) are tiny autocatalytic RNAs that cleave
single-stranded RNA. They are found in nature as a part of certain virus-like
elements called virusoids, which use a "rolling-circle replication" mechanism to
reproduce their small, circular RNA genomes.
• The HHRZ is so named because its secondary structure is similar to that of a
hammer head, but actually its tertiary structure is more like ‘Y’ shaped.
Rolling-circle replication initially produces a long
strand of multiple copies of the virusoid RNA.
Each copy contains a hammerhead motif that
catalyzes strand breakage between itself and the
next copy in the transcript. Thus, by virtue of
HHRZ motifs, the long strand breaks itself into
many individual molecules.
10. HAIRPIN RIBOZYME
The hairpin ribozyme is an RNA
motif that catalyzes RNA
processing reactions essential for
replication of the satellite RNA
molecules in which it is embedded.
These reactions are self processing,
i.e. a molecule
rearranging its own structure. Both
cleavage and end joining reactions
are mediated by the ribozyme
motif. In contrast to the hammerhead
and Tetrahymena ribozyme
reactions, hairpin-mediated
cleavage and ligation proceed
through a catalytic mechanism that
does not require direct
coordination of metal cations to
phosphate or water oxygens.
11. RIBOSOMES
Ribosome is a large and complex
molecular machine, found within all
living cells, that serves as the
primary site of biological protein
synthesis (translation). It consists of
two sub-units, one large and one
small. The large(50s) subunit has 5s
and 23s rRNAs as its core.
After the determination of the high-resolution
structure of ribosome, it
was clear that the 23s subunit is
responsible for the catalytic peptidyl
transferase activity that links amino
acids together.
That is why ribosome is also a
ribozyme.
The red region indicates the site
where mRNA interacts with the tRNA
anticodons.
13. • Genetic regulation by RNA is widespread in bacteria.
• One common form of riboregulation in bacteria is the use of
ribonucleic acid sequences encoded within mRNA that directly
affect the expression of genes encoded in the full transcript
(called cis-acting elements because they act on the same
molecule they're coded in).
• These regulatory elements are known as riboswitches and are
defined as mRNA elements that bind metabolites or metal ions
as ligands and regulate mRNA expression by forming alternative
structures in response to this ligand binding.
14. Most known riboswitches
occur in bacteria, but
functional riboswitches of
one type (the TPP
riboswitch) have been
discovered in plants and
certain fungi.
15. Mechanism :
Riboswitch-mediated changes in gene expression can
occur either transcriptionally or translationally. The
expression platform for a riboswitch that acts during
transcription typically involves the ligand-dependent
formation of an intrinsic terminator or anti-terminate
structure.
16. APPLICATION
• Riboswitches as tools for regulated gene expression
• Ligand-inducible expression systems are important genetic tools for common laboratory organisms
such as E. coli and B. subtilis. However, inducers (such as IPTG) are too expensive. Natural riboswitches
that are activated by amino acids may therefore represent an affordable alternative for such
applications. Toward this goal, a tandem glycine riboswitch from B. subtilis was used for glycine
inducible production of β-galactosidase in B. subtilis cells.
• Just as natural riboswitches can regulate gene expression in response to small-molecule ligands
during transcription or translation, synthetic riboswitches can be engineered to repress or activate any
gene expression in a ligand-dependent fashion. This feature should enable RNA switches to play an
increasingly important role as chemical biologists seek to modulate many types of cellular behavior in
response to a broad range of chemical signals.
