RIBOZYMES

VIPIN MOHAN2011-09-112College of Agriculture , vellayani, TVM

Sidney Altman Thomas R Cech

1989 Nobel

Prize

In Chemistr

y

RIBOZYMES are RNA possessingcatalytic activity.

The first Ribozymes were discovered in the 1980s by Thomas R. Cech and Sidney altman.

The term “ribozyme” was first introduced by kelly kurger et al. in 1982

RIBOZYMES

RIBOSOME IS A RIBOZYME

PEPTIDYL TRANSFERASE = RIBOZYME

General Mechanism of Ribozyme

Catalytic Mechanism

IntactPhosphodiester

Mg2+CoordinatedTransition

CleavedPhosphodiester

Catalytic efficiency, condition

- ribozyme follows a Michaelis-Menten kinetics

E + S ES E + P k1 k2

k-1

- all ribozyme need cations for activity (Mg2+ ,Mn2+)

Km= k-1+ k2

k1

= 10-5-10-7 M kcat= 0.5-2 min-1

kcat/ Km= 103-106 M-1.min-1 Good catalytic efficiency!!

NATURALLY OCCURRING RIBOZYMES ARE,

PEPTIDYL TRANSFERASE 23S RRNARNASE PGROUP 1 & 2 INTRONESHAIRPIN RIBOZYMEHAMMERHEAD RIBOZYMEHDV RIBOZYMEMAMMALIAN CPEB3 RIBOZYMEVS RIBOZYMEGLMS RIBOZYME

CLASSES OF RIBOZYMES

Catalytic rnas are broadly grouped in to 2 classes based on their size and reaction mechanisms,large and small ribozymes.

The group 1 and group 2 intrones, rnase p ribozymes are relatively large ribozymes(100s of nucleotides).

The hammerhead ribozyme, hairpin ribozyme, hdv ribozymes are substantially smaller(less than 100 nucleotides).

CLASSIFICATION

SMALL RBZYM LARGE RBZYM

• 100 – 3000 nucleotides

Eg: Introns(GROUP I & GROUP II) , RNase P

•30- 150 nucleotides

Eg: Hammer head , Hair pin , and HDV Ribozymes

HAMMERHEAD RIBOZYMEThe hammerhead ribozyme is a RNA  that catalyzes

reversible cleavage and joining reactions at a specific site within an RNA molecule

It has been found in several satellite RNAs of plant viruses,viroids and transcripts of a nuclear satellite DNA.

Like protein-based enzymes,ribozyme catalysis obeys michaelis-menten principles,with some exceptions.

CIS ACTING HAMMER HEAD RIBOZYME

Self splicing happens after the sequence guc between helix i and helix iii , g and u are conserved and crucial for the catalysis.

TRANS ACTING HAMMER HEAD RIBOZYME

Enginnered to cleave rna in trans by separating the catalytic domain from ribozyme & attaching recognition sequence to target the substrate

CATALYTIC ACTIVITY

Capable of cleaving in cis or in trans

RNASE P

Ribonuclease p (RNAse p) was the first true RNA enzyme identified.

An rna–protein complex, the catalytic subunit of bacterial rnase p is composed entirely of RNA.

It processes precursor trnas and other RNAs required for cellular metabolism.

Act as an endonuclease to generate the mature 5’ end of tRNA precursors

Exists as ribonucleoprotein complex

Process the 5’ leader sequence of precursor tRNA

Human nuclear RNAse p is required for the normal and efficient transcription of tRNA , 5s rRNA, u6 snRNA

Require divalent cations for cleaving

Mechanism of clevage is hydrolysis rather than transesterification

Lack the requirement for specific nucleotide sequence for cleavage

MAMMALIAN CPEB3 RIBOZYME

The mammalian cpeb3 ribozyme is a self cleaving non-coding RNA

Located in the second intron of the cpeb3gene which belongs to a family of genes regulating messenger RNA polyadenylation.

This ribozyme is highly conserved and found only in mammals

The cpeb3 ribozyme is structurally and biochemically related to the human hepatitis delta virus ribozyme

GROUP I INTRONIt was demonstrated to be a true enzyme.

Whose splicing acquires a free guanine nucleoside.

They possess a secondary structure different from that of grp 2 introns.

There are several hundred examples of grp 1 introns,including those found in plant &fungal mitochondria,bacteriophage,eubacteria,&chloroplastis dna chloroplast tRNA.

They have phylogenetically conserved secondary structures and a common reaction mechanism.

- Self splicing by Trans esterification process First transfer: 3’ OH end of G attacks

5’ end of intron Second transfer: 3’ –OH of exon A

attacks 5’ end of exon B .

Self splicing of Group I intron

Commonly found in mitochondrial genes in plants , fungi, yeast ,bacteria, archeal genus methanosarcina

Do not require a free guanosine cofactor

GROUP II INTRONS

GROUP II INTRON SELF SPLICING STEPS

STEP 1: The bulged adenosine in the domain 6 attacks the 5’ splice site resulting in the cleavage of 5’ exon and formation of lariate intermediate

STEP 2: .5’ , 3’ exons ligated together and introns is released as a lariate

HAIRPIN RIBOZYMEFound in satelite RNA of pathogenic plant viruses(tobacco

ring spot virus )

Require mg 2+, sr 2+ , & ca 2+ metal ions

Function both in cis and in trans

The hairpin ribozyme (plant virus)

Hair pin contains 4 helical regions & 2 single stranded loops

Consists of 2 domains , each with 2 helical regions separated by an internal loop , connected by a hinge region

STRUCTURE

Developed mainly for antiviral applications ( Anti HIV Ribozyme)

- Anti HIV ribozyme vector – HIV infected patients – virus replication delayed by 2- 3 weeks

HDV RIBOZYMESThe hepatitis delta virus(HDV) single stranded circular

RNA virus of approximately 1700 nucleotides.HDV infection requers the presence of the hepatitis B

virion either coincident with HDV infection or as a pre-existing infection.

HDV is designated as asatellite virus of hepatitia B.HDV ribozyme are 85 nucleotides in length.Mutagenesis experiments and chemical studies are

shows that 4 helical regions ,in many of which the conserved nucleotides are located in helix2 &it’s hairpin.

The structural motif resembles neither the hammerhead nor the hairpin ribozymes.

The hepatitis delta ribozyme displays high resistance to denaturing agents like urea or formamide.

The self cleaving activity of HDV RNA is enhanced in the presence of denaturents

Substrate recognition requires formation of the p1 stem.

P1 ribozyme is a circular ribozyme and a hybrid of both genomic and anti genomic HDV ribozymes

The hepatitis delta ribozyme (human virus)

VS RIBOZYME

The Varkud Satellite Ribozyme is a 154-nucleotide long catalytic entity.

That is transcribed from a plasmid discovered in the mitochondria of certain stains of Neurospora.

The VS ribozyme is the largest of the known nucleolytic ribozymes.

Binding of the substrate is determined by teritiary interactions.

The glmS ribozyme is a recently discovered ribozyme.

That is unique in the world of naturally occurring ribozymes in two respects.

First, it is a ribozyme that is also a riboswitch. Second, the regulatory effector of the ribozyme, glucosamine-6-phosphate (GlcN6P), is actually a

functional group that binds to the ribozyme active site and participates in the acid-base catalysis of RNA self-cleavage.

THE GLMS RIBOZYME

The glmS ribozyme is derived from a self-cleaving RNA sequence found in the 50-untranslated region (50-

UTR) of the glmS message;

It cleaves itself, inactivating the message, when the cofactor GlcN6P binds.

GlcN6P production is thus regulated in many Gram-positive bacteriavia this ribozyme-mediated negative-feedback mechanism.

NATURALLY OCCURRING RIBOZYMES

ARTIFICIAL RIBOZYMES

LEADZYMELIGASE RIBOZYMESALLOSTERIC RIBOZYMES

LEADZYMES

small ribozymeFor in vitro selection techniques,Cleave RNA in presence of lead.Structure determined by x-ray crystallography.it has been proposed that a naturally occurring

leadzyme in the 5s rRNA &that this may be an important mechanism in lead toxicity.

LIGASE RIBOZYME

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 pre-biotic RNA world.

2 types ligase1 &2MICHAEL ROBERTSON & ANDREW ELINGTON

evolved a ligase ribozyme that performs the desired 5’-3’ RNA assembly reaction ,&called this is L1 ligase.

ALLOSTERIC RIBOZYMES

Act as allosteric enzyme.Enhance the catalytic rates of certain ribozymes.Molecular switches.eg: Novel biosensors & controllable therapeutic

enzymes.

APPLICATIONSRibozymes as therapeutic agents.

• treatment of diseases and cancer.

• To study gene function & gene therapy for disease.

• The property of ribozyme make them suitable in the research of genetics & developmental biology.

• Develop new drugs.

• Ribozyme therapy.

Ribozymes-molecular scissors for investigating gene function.

Riboaptdb-a comprehensive data base of ribozymes and aptamers.

THANK YOU