biotechnology, ribozymes

1. RIBOZYMES
PRESENTED BY:
KRITI KUMARI
FGB-MB1-03
ICAR-CENTRAL INSTITUTE OF FISHERIES
EDUCATION
PRESENTATION ON:

2. RIBOZYME?
Ribozyme ( Ribonucleic acid enzyme) is an RNA
molecule that is capable to performing specific
biochemical reactions, similar to the action of
protein enzymes.
RNA possessing catalytic activity
Increases the rate and specificity of:
• Phosphodiester bond cleavage
• Peptide bond synthesis

3. • The first ribozyme discovered was in the 1980s
by Thomas R Cech and Sidney Altman.
• The term Ribozyme was first introduced by
Kelly Kurger et al. in 1982.

4. Characteristics
• An enzyme that uses RNA as a substrate
Or An RNA with enzymatic activity
• An enzyme that catalyses the association
between larger and smaller ribosomal
subunits
• An enzyme that synthesises RNA as a part of
the transcription process
• An enzyme that synthesises RNA primers
during DNA replication.

5. Classification
1. On the basis of number of nucleotides:
• Small ribozyme -
30-150 nucleotides
eg. Hammerhead, hairpin, and HDV ribozymes.
• Large ribozyme-
100-3000 nucleotides
eg. Introns ( Group I and Group II ), Rnase P

6. 2. On the basis of occurrence :
Naturally occurring ribozyme-

7. Artificial ribozymes-
• Leadzyme
• Ligase ribozyme
• Allosteric ribozyme

8. TYPES OF RIBOZYMES
• Group I and Group II intron splicing ribozymes
• Rnase P
• Hammerhead Ribozyme
• Hairpin Ribozyme
• Ribozome
• Glms ribozyme ( Glucosamine 6 phosphate
activated ribozyme)
• HDV ribozyme ( Hepatitis delta virus ribozyme )

9. Group I intron splicing ribozymes
• One of the main class
• 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 T-7 like bacteriophages.

10. Mechanism
• The group I splicing reaction requires a
guanine residue cofactor, the 3’ OH group
attacks the 5’ phosphate of the intron and the
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 introns is
precisely excised and exons are joined
together.

11. -No need of energy or any protein molecule
Autosplicing

12. GENE:

13. Mechanism

14. Group II intron splicing
• Group II intron are found in bacteria and in the
mitochondrial and chloroplast genomes of fungi,
plants, protists and an annelid worm.
• The 2’-OH of an 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.

16. Rnase P
• Ribonuclease p, a ribonucleoprotein is an essential t
RNA processing enzyme found in all living organisms.
• Research on Rnase P has led to the discovery of
catalytic properties of RNA, and of only known,
naturally occuring RNA enzymes.
• All Rnase P enzyme are ribonucleoproteins
(bacteria: 1 RNA + 1 protein subunit ;
eukaryotes: 1 RNA + many protein subunits (11 in human)

17. • In ribonuclease P, protein component 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 t RNA molecules
• Cleavage via nucleophilic attack on the
phosphodiester bond leaving a 5’ phosphate and
3’ hydroxyl at the clevage site.

20. Hammerhead ribozyme
• 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
• This is so named because its secondary structure
is similar to that of a hammerhead, but its
tertiary structure is more like ‘Y’ shaped.

22. Mechanism

23. • Autocatalytic cleavage occurs via nucleophilic attack by
the 2’-hydroxyl of a specific core nucleotide on its
adjacent phosphodiester bond, producing 2’-3’- cyclic
phosphate and 5’ hydroxyl termini.
• Rolling circle replication initially produces a long strand
of multiple copies of the virusoid RNA.
• Each copy contain a hammerhead motif that catalyses
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.

24. Hairpin ribozymes
• The Hairpin ribozyme is an RNA motif that catalyses
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 meditated 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.

25. Structure

27. The glms ribozyme
• It is a ribozyme that is also a riboswitch.
• The regulatory effector of the ribozyme,
glucosamine 6-phosphate is actually a
functional group that binds to the ribozyme
active site and participates in the acid base
catalysis of RNA self cleavage.
• Riboswitch: a regulatory segment of a messenger RNA
molecule that binds a small molecule, resulting in a change in
production of the proteins encoded by mRNA.

28. Structure

29. Hepatitis delta virus ribozyme
• The hepatitis delta virus ribozyme is a non
coding RNA and is thought to be the only
catalytic RNA known to be required for
viability of a human pathogen.
• This is the fastest known naturally occuring
self-cleaving RNA.

30. Structure

31. Leadzyme
• Small ribozyme
• For in vitro selection techniques
• Cleaves RNA in presence of lead
• Structure determined by x ray crystallography

32. Structure

33. Ligase ribozymes
• They are important class of ribozymes because
they catalyse the assembly of RNA fragments
into RNA polymers by forming phosphodiester
bond
• A reaction required of all extant nucleic acid
polymerases and thought to be required for
any self replicating molecule in a prebiotic
RNA world
• 2 types ligase 1 and 2

34. • Micheal robertson and Andrew elington
evolved a ligase ribozyme that perform the
desired 5’-3’ RNA assembly reaction and this
one L1 ligase

35. Allosteric ribozyme
• Act as allosteric enzyme
• Enhances the catalytic rates of certain
ribozymes
• Molecular switches
• Eg novel biosensors and controllable
biosynthetic enzymes.

38. APPLICATIONS
• Ribozymes as therapeutic agents
• Treatment of diseases and cancer
• To study gene function and gene therapy for disease
• The property of ribozyme make them suitable in the research of
genetics and developmental biology
• Develop new drugs
• Ribozyme therapy
• Molecular scissors for investigating gene function
• Riboaptdb – a comprehensive database of ribozymes and aptamers.
• It is useful in genetic disorders
• Sickle cell anaemia
• Myotonic dystrophy
• Ribozymes are being tested for the potential use in the treatment
of immunological disorder.