RYBOZYMES: its strucure, types and applications.

1. RIBOZYME
BY
MUMTHAS P K
2nd MSc. MICROBIOLOGY
KANNUR UNIVERSITY CAMPUS,
PALAYAD

2. HISTORY
Before the discovery of the ribozyme, the enzymes are defined as the
catalytic protein.
 1967 : Carl Woese, Francis Crick, and Leslie Orgel were the first
to suggest that RNA could act as a catalyst.
 1970s : Thomas Cech, at 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.

3.  Cech proposed that the intron sequence portion of the RNA could
break and reform phosphodiester bonds.
 Sidney Altman, a professor at Yale University, was studying the
way 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 enzyme
 1982 : Ribozyme term was introduced by Kelly Kurger etal. In a
paper published in ‘The Cell’.

4.  1989 : Thomas Cech and Sidney Altman shared the Nobel
Prize for demonstrating that RNA could act as an enzyme.
Figure: 1

5. RIBOZYME
The ribozyme (Ribonucleic acid enzyme ) is
an RNA molecule that is capable of
performing specific biochemical reactions,
similar to the action of protein enzyme.
Ribozyme
Figure : 2

6. CHARACTERISTIC FEATURES OF RIBOZYME
1. An enzyme that uses RNA as a substrate.
2. It is an RNA with enzyme activity.
3. An enzyme that catalyzes the association between the large and
small ribosomal subunits.
4. An enzyme that synthesizes RNA as part of the transcription
process.
5. It is an enzyme that synthesizes RNA primers during DNA
replication.
6. Investigators studying the origin of life have produced ribozymes in
the laboratory that are capable of catalysing their own synthesis
under specific conditions, such as an RNA polymerase ribozyme.

7. 7. Some ribozymes may play an important role as therapeutic
agents, as enzyme which target defined RNA sequences for
cleavage, as biosensors and for applications in functional
genomics and gene discovery.
8. Ribozyme molecule that have the ability to catalyse specific
biochemical reactions, including RNA splicing in gene expression,
similar to the action of protein enzyme.
9. The most common activities of natural or in vitro evolved
ribozymes are the cleavage or ligation of RNA and DNA , peptide
bond formation.
10. Within the ribosome, ribozyme function as part of the large
subunits ribosomal RNA to link amino acid during protein
synthesis.
11. They also participate in a variety of RNA processing reactions

8. MECHANISM AND FUNCTIONS
 Despite having only four choices for each monomer unit (nucleotides
) compared to 20 amino acids found in the proteins, ribozymes have
diverse structure and mechanisms.
 Like many protein enzymes, metal binding is critical to the function
of many ribosomes.
 These interaction may use both the phosphate backbone and the base
of the nucleotide, causing drastic conformational changes.
 There are two mechanisms are involved in it :
1. The internal 2’OH group attacks phosphorus centre in SN2
mechanism. Metal ion promote this reaction.

9. 2. The SN2 mechanism occur but the nucleophile comes from water
or oxogenous hydroxyl groups rather than RNA if self.
 The smallest ribozyme UUU, which can promote the cleavage
between G and A of the GAAA tetra nucleotide via the first
mechanism in the presence of Mn2+
 The hairpin ribozyme can catalyze the self cleavage of RNA with
out the presence of metal ion.
 Ribozyme is the functional part of the ribosome, that is the
biological machine which translate RNA into proteins. It composed
of RNA tertiary structural motifs.

10. TYPES OF RIBOZYMES
1. GROUP I INTRON SPLICING
 Group 1 intron ribozymes constitute one of the main classes of
ribozymes.
 Found in bacteria, lower eukaryotes and wide variety of plants.
 It also found inserted in to genes of a wide variety of
bacteriophage of Gram positive bacteria.
 However , their distribution in the phage of Gram negative
bacteria is mainly limited to the T4, T7 like bacteriophages.

11. MECHANISM
The group 1 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 displaced now
and acts as nucleophile in a similar reaction
at the 3’end of the intron.
So the intron is precisely excised and exons
are joined together.
Figure : 3
GROUP I INTRON SPLICING

12. 2. GROUP II INTRON
SPLICING
 Group II intron splicing have been found in bacteria and in
the mitochondrial and chloroplast genome of fungi, plants,
protists and an annelid worm.
MECHANISM
 The 2’OH of a specific adenosine act 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.

13. FIGURE : 4GROUP II INTRON
SPLICING

14. 3. HAMMERHEAD RIBOZYME
 Hammerhead ribozymes (HHRZs) are tiny autocatalytic RNAs, that
cleave single stranded RNA .
 They are found in nature as part of certain virus-like elements called
virusoids, which use a ‘rolling- circle mechanism’ to reproduce their
small, circular RNA genomes.
 The HHRZ is so named because its secondary structure, it is similar
to that of a hammerhead, but actually its tertiary structure is more
like ‘y’ shaped.

15. MECHANISM
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 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
[FIGURE :5]
HAMMERHEAD RIBOZYME

16. 4. RNase- P
 Ribonuclease P (RNase P), a ribonucleoprotein is essential tRNA
processing enzyme found in all living organisms.
 Research on RNase P has led to the discovery of the catalytic
properties of RNA, and of the only known naturally occurring RNA
enzymes.MECHANISM
All Rnase-P enzymes are ribonucleoproteins (in bacteria: 1RNA +
protein subunit, in eukaryotes : 1RNA + many protein subunits )
The protein component facilitates binding between RNase and
tRNA substrate.
Requires divalent metal ions for the activity.

17. Endonucleases 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.
FIGURE :6
RNase- P

18. 5. HAIRPIN RIBOZYME
 The hairpin ribozyme is an RNA motif that catalyzes RNA
processing reactions essential for replication of the satellite RNA
molecule in which it is embedded.
 These reactions are self processing that is 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 cation to phosphate or water oxygens .

19. FIGURE :7
HAIRPIN RIBOZYME

20. RIBOSOME
 Ribosome is a large and complex
molecular machine, found within all
living cells, that serves as the priming
site of biological protein synthesis.
 Consists of two subunits large and
small.
 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 acid together.
 That is why ribosome is also a
ribozyme.
FIGURE : 8

21. APPLICATIONS
 Ribozyme have been proposed and developed for the treatment of
disease through gene therapy. It’s stability improved by 2’ position
on the ribose is modified to improve RNA stability.
 Type of synthesis ribozyme directed against HIV RNA, called gene
shears has been developed and has entered clinical testing for HIV
infection.
 It also designed to target the Hepatitis C virus RNA, SARS Corona
virus (SARS-COV), Adenovirus and Influenza A and B virus RNA.
 The ribozyme able to cleave the conserved region of the virus’s
genome which have been shown to reduce the virus in mammalian
cell culture.

22. CONCLUSION
 The best studied ribozymes are probably those that cut themselves or
RNAs, as in the original discovery by Cech and Altman.
 The ribozymes can be designed to catalyze a range of reactions, many
of which may occur in life but have not been discovered in the cell.
 The RNA also act as a hereditary molecules, the presence of ribozyme
enzyme used to prove the RNA hypothesis which act as the first
replicator.
 Artificial ribozymes are also developed for improving their quality.
 The naturally occurring ribozymes include GIR 1 branching
ribozyme, glms ribozyme, Group 1 self splicing intron, hairpin
ribozyme, hammerhead ribozyme, rRNA ect.

23. REFERENCE
 Understanding Enzymes – An introdutory text, By Adithya Arya, Amit
Kumar
 The Fundamentals of Enzymology By Nicholas C Price.
 Ribozymes – Methods and Products- Jory S Hartig
 Ribozyme- Overview/science direct

24. THANK YOU