INTRODUCTION RIBOZYME CATALYSIS SMALL SELF CLEAVING RIBOZYME HAMMERHEAD HAIRPIN HDV COMPLEX RIBOZYME GROUP I INTRON GROUP II INTRON RNaseP 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
RIBOZYME CATALYSIS
SMALL SELF CLEAVING RIBOZYME
HAMMERHEAD
HAIRPIN
HDV
COMPLEX RIBOZYME
GROUP I INTRON
GROUP II INTRON
RNaseP
CONCLUSION
REFERENCE

3. INTRODUCTION
First discovered in the cellular RNA splicing activity
and its machinery as self splicing.
Catalytic RNAs are generally found in the
genomes of RNA viruses and in some virus
related RNAs.
Classes of catalytic RNA has been discovered
which cleavage or ligation of the RNA backbone
by transesterification or hydrolysis of
phosphate group

4. 1) Ribonucleic Acid
2)) Enzyme
NOT PROTEIN
1989 Nobel Prize
In Chemistry
Sir Altman Tom Cech
HISTORY

5. How many ribozyme
- the hammerhead ribozyme (plant virus)
SMALL SELF CLEAVING
- the hairpin ribozyme (plan virus)
- hepatitis delta ribozyme (human virus)
COMPLEX RIBOZYMES
-group I and
-group II intron ribozyme
- RNAse P (tRNA maturation)

6. SMALL SELF CLEAVING RIBOZYME
carry 50-150 nucleotide sequence that performs
site specific cleavage (1-7).
Hammer head
Hairpin
Hepatitis delta virus

7. HAMMERHEAD RIBOZYME STRUCTURE
These are known to cleave any NUH triplets (where H is any
nucleotide except guanosine) with AUC and GUC triplets being
processed most efficiently.

8. Minimised hammerhead ribozyme is less than 40
nucleotides long and consists of two substrate binding
arms and a catalytic domain.
The hammerhead ribozyme carries out a very simple
chemical reaction that results in the breakage of the
substrate strand of RNA, specifically at C17, the
cleavage-site nucleotide.
This CUGA turn is strikingly similar to that found in
anticodon loop of tRNA which serve as metal binding
pocket.

9. The reaction rate for catalysis increases linearly
with pH, indicating that the nucleophile is activated
by a hydroxide ion .
Three helices arranged in Y- shape as predicted by
fluorescence and native gel electrophoresis.

10. THE HAIRPIN RIBOZYME
From Lilley TIBS (2003)
359 nucleotide present,50 nucleotide responsible for
cleaving

11. A minimal catalytic domain for this RNA molecule has
been identified, which consist of a 50-base RNA catalyst
that efficiently cleaves an RNA substrate containing 14
bases of satellite RNA sequence.
Cleaving depend upon the binding of complementry

12. THE HEPATITIS DELTA virus
This animal RNA virus undergoes autocatalytic self-
cleavage as part of its replication cycle.
Presence of divalent cation is required for
catalysis,self-cleavage.
P1 is stacked upon P1.1,P4 while P2 is stacked upon
P3.
The structure is surrounded by L3, J1/3, J4/2.

14. HDV is a 1700-nucleotide, covalently closed
circular RNA that is associated with hepatitis B virus
infection in certain patient.
Stable at temp 65 C
The crystal structure of this ribozyme has been
solved using X-ray crystallography and shows five
helical segments connected by a double pseudoknot.

15. LARGE COMPLEX RIBOZYME
which carry several hundred nucleotides performing site
specific cleavage from 9-11.
Group I introns
Group II introns
RNase P

16. Group I introns are considerably larger and more structurally
complex than any of the self-cleaving RNAs.
 They are found in precursor mRNA, tRNA, and Rrna
transcripts in a variety of organisms and self-splice in two
steps to ligate flanking
50 and 30 exons, producing mature RNA transcripts.
Group I & ii introns

18.  the exon-intron boundary at
the 3’ end of the intron is
called 3’ splicing site
 the exon-intron boundary at
the 5’ end of the intron is
called 5’ splicing site
 Branch point site: the third
sequences necessary for
splicing found entirely within
the intron ,usually close to its
3’end ,and is followed by a
polypyrimidine tract(Py tract)

19. Step 1
The OH of the
conserved A at the
branch site attacks the
phosphoryl group of
the conserved G in the
5’ splice site. As a result,
the 5’ exon is released
and the 5’-end of the
intron forms a three-
way junction structure.

20. The OH of the 5’ exon
attacks the phosphoryl
group at the 3’ splice site.
As a consequence, the 5’
and 3’ exons are joined
and the intron is
liberated in the shape of
a lariat.

21. RNASE P
RNase P is found in both prokaryotic and eukaryotic cells,
where it catalyzes the removal of the 5’ ladder sequences from
the variety of precursor tRNAs.
 M1 RNA are able to cleave tRNA precursors with multiple
turnover in the presence of high concentrations of magnesium
in the test tube.
RNase P cleaves substrate RNA by hydrolysis to generate
5’-phosphate and 3’-hydroxyl termini.

22. Made by 377 nucleotide 119 protein

23. The Future of Ribozymes
In Vitro Molecular Evolution of RNA
High Throughput Screening
Ribozyme-Based Therapies
+

24. Reference
MOLECULER BIOLOGY OF THE GENE 5th Edition by
James D. Watson, et.al.
WIKIPEDIA
PDF-Annu Rev. Biophysics, biomol struct.
2001