Ribozyme by KK Sahu sir

Introduction
History
Definition
Types of RNA catalytic activity
1.Intermolecular catalysis
2.Intramolecular catalysis
RNA splicing
Types of ribozymes
1. Group 1 introns
2. Group 2 introns
3. Spliceosomes
4. Hammerhead ribozymes
5. RNase P ribozyme
6. Ribosomes
Clinical application of ribozyme
Conclusion
Reference

1. ‘RIBOZYMES’
By
KAUSHAL KUMAR SAHU
Assistant Professor (Ad Hoc)
Department of Biotechnology
Govt. Digvijay Autonomous P. G. College
Raj-Nandgaon ( C. G. )

2. Synopsis
Introduction
History
Definition
Types of RNA catalytic activity
1.Intermolecular catalysis
2.Intramolecular catalysis
RNA splicing
Types of ribozymes
1. Group 1 introns
2. Group 2 introns
3. Spliceosomes
4. Hammerhead ribozymes
5. RNaseP ribozyme
6. Ribosomes
Clinical application of ribozyme
Conclusion
Reference

3. INTRODUCTION
Ribozymeis derived from the word ‘RNA’ and
‘enzyme’.
Term ribozymeis used to describe an RNA with
catalytic activity.
Ribozymeactivity is based on transesterification
and phosphodiesterbond hydrolysis.
Ribozymesare inactivated by heating above
their melting temperature and if essential
nucleotides are changed.

4. HISTORY
In 1967, Carl Woese, Francis Crick & Leslic
Orgel were the first to suggest that RNA
could act as catalyst.
In 1880, Sidney Altman discovered first
ribozyme.
The term ribozyme were first introduced by
Kelly Krugger et al in 1982.

5. DEFINITION
A ribozyme is RNA molecules that is
capable of catalyzing RNA cleavage in a
sequence specific way and they may
catalyze self cleavage as well as
cleavage of external substrates.

6. TYPES OF RNACATALYTIC ACTIVITY
RNA has two types of catalytic activities:-
1.Intermolecular catalysis:
In catalytic activity of RNA is directed against a separate substrate.
Eg:-RNaseP
2. Intramolecularcatalysis:
Catalysis within themselves. It is of 2 types-
SELF CLEAVAGE
It involves cleavage of RNA molecules by the catalytic activity of RNA
itself
Eg: small plant pathogenic RNA of the virusoidclass
SELF SPLICING
It is the ability possessed by certain Introns to splice themselves out of
the RNA that contains them.
Eg: group I introns

8. RNASPLICING
The primary transcript
contains exons & Introns
are spliced out. The
process of removal of
Introns is known as
“RNA Splicing”.
The Introns of nuclear
pre-mRNA transcripts
are spliced out in 2 step
reaction carried out by
complex ribo-
nucleoprotein particles
spliceosomes

9. Types of ribozymes:
1) Group I Introns (Autocatalytic splicing)
2) Group II introns
3) RNAaseP Ribozyme(tRNAprecursor splicing)
4) Spliceosomes(pre mRNAsplicing)
5) Hammerhead ribozyme(self cleavage)
6) Hairpin ribozymes
7) Ribosomes

10. RIBOSOMES
Ribosomesare complex RNA particles made up of two dissimilar subunits
each of which contains RNA & many proteins. Ribosomesplay role as a
ribozyme.
In prokaryotes the active site of ribosome is on the 50S subunit. There are
no protein functional groups to catalyze peptide bond formation. Instead,
residues within 23SrRNA helps transfer a H
+
during peptide bond
synthesis.
Large subunit is a complex ribozymein which peptide bond formation is
an RNA catalyzed reaction.
rRNAare more highly conserved throughout evolution than are ribosomal
proteins.
Most mutations that confer resistance to antibiotics that inhibit protein
synthesis occurs in genes encoding rRNAsrather than ribosomal proteins

11. Fig: binding of A site tRNA to P site tRNA

12. Fig: Ribosomes with RNA and proteins

13. GROUP I INTRONS
Group I Introns are found in diverse location they occur in genes
coding for rRNAin the nuclei of the lower eukaryotes-
Tetrahymenathermophila(a ciliate) and Physarumpolycephalum
(a slime mould).
They have intrinsic ability to splice themselves. This is called as
“SELF SPLICING” or “AUTOCATALYTIC SPLICING”.
REQUIREMENTS:-
Cofactor –
A guanine nucleoside or nucleotide with free 3’OH
group (GTP,GDP, GMP) as a cofactor.
Cation –
A divalent cation& a monovalentcation.

14. MECHANISM
In group I splicing reaction
the 3’-hydroxyl group of
guanosineforms a normal
3’,5’-phosphodiester bond
with the 5’end of intron.
The 3’hydroxyl of the exon
that is displaced in 1
st
step
then acts as a nucleophile
in a similar reaction at the
3’end of the intron.
The final result is precise
excision of the intronand
ligation of the exons. MECHANISM OF GROUP I INTRONS

15. GROUP II INTRONS
Group II introns are generally found in the
primary transcripts of mitochondrial or
chloroplast mRNAs in fungi, algae, and
plants.

16. MECHANISM
In group II introns the
nucleophile is the 2’-
hydroxyl group of an
adenylate residue
withinthe intron.
A branched lariat is
formed as an
intermediate.
Mechanism of group II introns

17. RNASEP RIBOZYMES
RibonucleaseP (RNaseP)is a type of
ribonucleasewhich cleaves RNA. RNaseP is
unique from other RNasesin that it is a ribozyme–
a ribonucleic acid that acts as a catalyst in the
same way that a protein based enzyme would.
Its function is to cleave off an extra, or precursor,
sequence of RNA on tRNAmolecules

18. Splicing mechanism of RnaseP ribozyme

19. SPLICEOSOMES
A spliceosomeis a complex of specialized RNA and
protein subunits that removes intronsfrom a
transcribed pre-mRNA segment. This process is
generally referred to as splicing.
Each spliceosomeis composed of five small nuclear
RNA proteins, called snRNPs, (pronounced "snurps")
and a range of non-snRNPassociated protein
factors.
The snRNPsthat make up the nuclear spliceosome
are named U1, U2, U4, U5, and U6, and participate in
several RNA-RNA and RNA-protein interactions. The
RNA component of the snRNPis rich in uridine(the
nucleoside analog of the uracilnucleotide).

20. MECHANISM
Several snRNPs assemble to
form a spliceosome.
A specific adenine nucleotide
attacks 5’ end of intron, breaking
RNA.
The 5’end of the intron becomes
attached to the A nucleotide,
forming a loop of RNA.
The free 3’end of one exon
attacks the 5’ end of the other.
3’ and 5’ ends of adjacent exons
bond covalently, releasing the
intron which will then degrade.

21. HAMMERHEAD RIBOZYMES
Another example of the ability of
RNA to function as an
endonucleaseis provided by
some small plant RNAs that
undertake a self cleavage
reaction.
These small plant RNAs falls
into two general groups:
Viroids
Virusoids
Certain virus like elements
called “virusoids” has small RNA
genomes & usually requires
another virus to assist in their
replication or packaging.
Fig:Hammerhead ribozymes

22. .
Some virusoidRNA includes
small segments that promote site
specific RNA cleavage reactions
associated with replication,these
segments are called as hammer
head ribozymebecause their
secondary structure are shaped
like the head of a hammer.
Hammerhead ribozymeis a
metalloenzyme, Mg
++
ions are
required for activity. The
phosphodiesterbond at the site
of self cleavage is indicated by an
arrow.

23. HAIRPIN RIBOZYMES
The hairpin ribozymeis a
small section of RNA that can
act as an enzyme known as
a ribozyme.
The hairpin ribozymedoes
not require a metal ion for the
reaction.
The hairpin ribozymehas
been identified in only 2
naturally occurring
sequences:
1.satellite RNA of
tobacco ringspotvirus
(sTRSV)
2.satellite RNA of arabis
mosaic virus (sARMV) HAIRPIN RIBOZYME

24. APPLICATIONS OF RIBOZYME
The ribozyme binds to an RNA (substrate)
having complementary nucleotide sequences
& catalyze the reaction that cleaves the
backbone of the substrate RNA.
Ribozyme binds to mRNA and inhibits its
expression, thus helps in many diseases
therapy eg: genetical diseases. Mechanism
is as follows:

26. 3.TO HIVINFECTION
Hammerhead ribozyme which is
normally in active state when binds
to Rev protein, becomes inactive
and cause disease.
Alternatively, an inactive
hammerhead ribozyme was
constructed to use as a protein-
dependent ribozyme
The modified ribozyme possesses
an additional oligonucleotide region
that serves as a part of the binding
site for the Rev protein.
This design allows the substrate to
bind to the ribozyme due to the
displacement of the inhibitor region
by the protein.
In this design, the ribozyme is
allosterically activated via a specific
interaction between protein and
RNA.

28. Books Author
Gene 8 Benjamin Lewin
Cell and molecular biologyGerald Karp
Principle of genetics Simmons,Snustad&
Gardener
Biochemistry (IIIedition) Nelson& Cox
Reference
Websites:
www.wikipedia.com