1. Ribozymes and Functions
Biochemistry
Ribozymes and Functions
Presented by MCHF19E008
NIMRA FIRDOS

2. Structure and Mechanism Characteristic Features
Content
Introduction
and History
Structure and Mechanism
Characteristic Features
Applications
Classification

3. Introduction
The term ribozyme was first discovered by Kelly kurger et al. in 1982.
It is an RNA molecule.
It is also known as catalytic RNA or RNA zyme or ribonucleic acid enzyme.
It has the ability to perform specific biochemical reactions, similar to the actions of the protein enzymes.
Ribozymes are found in the ribosome and here they form protein chains by joining amino acids together.

4. Introduction
Ribozymes function as part of the large subunit ribosomal RNA, within the ribosome.
Cleavage or ligation of RNA and DNA in a sequence specific, catalytic manner and peptide bond formation are common activities of natural ribozymes.
It also takes part in a variety of reactions like viral replication, RNA splicing and transfer RNA biosynthesis.

5. History
In 1967, Carl Woese, Francis Crick, and Leslie Orgel were the first who suggested that RNA could act as a catalyst.
In 1970s, Thomas Cech, at the University of Colorado, proposed that phosphodiester bonds could be broke and reformed by the intron sequence protein of the RNA.
In , ribozyme was discovered.
In 1982, ribozyme term was introduced by Kelly Kruger et al.
In 1989, Thomas Cech and Sidney Altman shared the noble prize for demonstrating that RNA could act as an enzyme.

6. Characteristic features
It uses RNA as substrate.
It has enzymatic activity.
It catalyzes the association between large and small ribosomal subunits.
It synthesizes RNA as part of the transcription process.
It synthesizes RNA primers during DNA replication.

7. Structure and Mechanism
Ribozymes have diverse structure. The active center of many ribozymes is either hairpin – or hammerhead – shaped.
They also have a unique secondary structure by which they cleave other RNA molecules at specific sequences.
Now there are ribozyme that are made specifically to cleave a specific RNA molecule. These ribozymes can have pharmaceutical applications.
For example, to cleave the RNA of HIV, a ribozyme has been already designed.

8. Structure and Mechanism
Structure of ribozyme

9. Structure and Mechanism
Metal binding is critical to the function of many ribozymes. Both phosphate backbone and base of the nucleotide are used in these interactions. As a result, drastic conformational changes occur. For the cleavage of phosphodiester backbone in the presence of metal, two mechanisms are proposed.
In a SN2 mechanism, phosphorus center is attacked by the internal 2’- OH group.
As for the second mechanism, it also follows a SN2 displacement, but water or exogenous hydroxyl groups provide nucleophile rather than RNA itself.

10. Structure and Mechanism
The self-cleavage of RNA without metal ions can be catalyzed by Hairpin ribozyme.
The formation of peptide bond between adjacent amino acid can also be catalyzed by ribozymes by lowering the activation entropy.

11. Examples of naturally occurring ribozymes
RNase P
Hairpin ribozyme
Peptidyl transferase 23S rRNA
Hammerhead ribozyme
Leadzyme
HDV ribozyme
Group I and Group II introns
VS ribozyme

12. Classification
Based on the size and reaction mechanisms, catalytic RNAs are broadly classified into two classes.
Group I and group II introns and RNase P fall under the category of large catalytic RNAs. These are also called self-splicing ribozymes. These are relatively larger ribozymes and range in size from a few hundred nucleotides to around 3000.
The small catalytic RNAs comprises the hairpin, hepatitis delta and hammerhead ribozyme. These are also known as self-cleaving ribozymes and are generally small in size and range in size from ∼35 to ∼155 nucleotides.

13. Classification Group I intron
Group I introns are large self-splicing ribozymes.
Size range is from a few hundred nucleotides to around 3000.
Various group I introns are characterized by four short conserved sequence elements, called P, Q, R, and S.
It is found in bacteria, lower eukaryotes, higher plants, fungal and plant mitochondria, in nuclear rRNA genes, chloroplast DNA (ctDNA), bacteriophage, eukaryotic viruses, and in the tRNA of ctDNA and eubacteria is also found inserted into genes of a wide variety of bacteriophage of Gram-positive bacteria .

14. Classification
MECHANISM The group I splicing reaction requires the guanine residue cofactor. The 3’ OH group of guanosine is used as a nucleophile. The new phosphodiester bond is formed as 3’ OH group attacks the 5’ phosphate of the intron. The 3’ OH of then exon that is displaced now acts as a nucleophile in a similar reaction at the 3’ end of the intron. So the intron is precisely excised and exons are joined together.

15. Classification Group II intron
It is a large class of self-catalytic ribozymes.
Size range for group II introns is from several hundred to around 2500 nucleotides.
They are found in chloroplasts of plants, in algae, fungal and plant mitochondria, in eubacteria and in the chloroplasts of protist Euglena gracilis and in bacteria, in mitochondrial and chloroplast genomes of fungi, plants, protists, and an annelid worm.

16. Classification
Mechanism The 2’ OH of a specific adenosine attacks the 5’ splice site creating a branched intron structure. Here, 2’ OH of that specific adenosine acts as nucleophile. The 3’ OH of the 5’ exon attacks the 3’ splice site, ligating the exon end releasing the intron as the lariat structure.

17. Classification RNase P
Ribonuclease P (RNase P)is a ribonucleoprotein. It is an essential tRNA processing enzyme found in all living organisms.
Mechanism
In ribonuclease –P, protein component facilitates binding between RNase and t-RNA substrate. It requires the divalent metal ions (like Mg2+) for its activity

18. Classification
5’ end of matured tRNA molecules is generated by endo-ribonuclease. Cleavage occurs via nucleophillic attack on the phosphodiester bond leaving a 5’- phosphate and 3’ –hydroxyl at the cleavage site.

19. Classification Hammerhead ribozyme
Hammerhead ribozymes (HHRZs) are tiny autocatalytic RNAs.
They are found in nature as a part of virusoids.
They cleave single-stranded RNA.
The HHRZs is named as because its secondary structure is similar to that of a hammerhead. But its tertiary is more like “Y” shaped.

20. Classification Mechanism
Autocatalytic cleavage occurs via nucleophillic attack by the 2’ OH of a specific core nucleotide on its adjacent phosphodiester bond, producing, 2’, 3’-cyclic phosphate and 5’- OH termini. A long strand of multiple copies of the virusoid RNA is produced. Each copy contains a hammerhead motif. By virtue of HHRZ motifs, the long strand breaks itself in to many individual molecules as the hammerhead motif catalyzes strand breakage between itself and the next copy in the transcript.

21. Classification Hairpin ribozyme The hairpin ribozyme is an RNA motif.
It catalyzes RNA processing reactions.
Ribozyme motif mediates both cleavage and end joining reactions. This mechanism does not require direct coordination of metal cations to the phosphate or water oxygen.

22. Classification Hepatitis delta virus ribozyme
Hepatitis delta virus (HDV) ribozyme is a non -coding RNA.
The hepatitis delta virus (HDV) ribozyme is generally found in satellite virus of hepatitis B virus.
Hepatitis delta virus (HDV) ribozyme is the fastest known self-cleaving RNA that is also naturally occurring.
It is found in hepatitis delta virus that is necessary for viral replication.

23. Applications
1. Therapeutic applications of ribozymes (Treatment of cancer, infectious diseases and genetic disorders).
Ribozymes can be specifically tailored for the suppression of particular genes by modifying the substrate recognizing sequences.
Ribozymes are of great help in the treatment of cancer, infectious diseases and genetic disorders. For example there are few ribozymes that are under clinical trials for application to human diseases such as for cancer and AIDS.

24. Applications 2.Ribozymes as a cofactor
RNA can act as cofactor for amino acid residues.
3.Chaperon like ribozymes
Like chaperon, RNA catalyzes protein folding. Such RNAs are called chaperon like ribozymes.
4.Ribozymes as tool to study gene function and in target validation
They are used to study the function, regulation and expression of genes.

25. Applications
They provide a unique tool for understanding gene functions as they allow one to assess cellular responses to a rapid ablation of target gene expression.
They inactivate specific gene expression, and thereby can be used to help identify the function of a protein or the role of a gene in a functional cascade.
5.Riboswitches as antimicrobial agents
Many antimicrobial compounds have RNA as their primary target. For example antibiotics pyrithiamine is metabolized into pyrithiamine pyrophosphate when it enters the cell.

26. Applications
Pyrithiamine pyrophosphate binds and activates the TPP (Thiamine pyrophosphate) ribowitch. It causes the cell to cease the synthesis and import of TPP. Because riboswitch pyrophosphate does not substitute for the TPP as coenzyme, cell dies.
6.Riboswitch as tool for regulated gene expression
Inducers (such as IPTG) are too expensive. Natural riboswitches may represent an affordable alternative for such applications as they are activated by amino acids
7.Riboswitch-based control of bacterial behavior
Ribozymes can be used to reprogram a variety of bacterial behaviors, because riboswitches are versatile tools for controlling

27. Applications
gene expression. Synthetic riboswitches can be engineered to repress or activate any gene expression in a ligand-dependent fashion, just as natural riboswitches can regulate gene expression in response to small-molecule ligands during transcription or translation.