Ribozymes RNA molecules that act as enzymes are called ribozymes. This property of some RNAs was...

Ribozymes

RNA molecules that act as enzymes are called ribozymes. 

This property of some RNAs was discovered by

Sidney Altman and Thomas Czech,

who were awarded the

Nobel Prize in Chemistry in 1989.  

Sidney AltmanThomas Czech

Ribozyme discovery (open with media player)

RibozymesRNA molecules capable of catalyzing

biochemical reactions• Earliest known examples:

RNase PGroup I and II intronsRibosomeshammerhead ribozymes

• Principal reactions:RNA transesterificationRNA cleavage (hydrolysis of phosphodiester bonds)

• Substrate aligned into the active site using a guide sequence which is complimentary to the substrate

• All ribozymes depend absolutely on the assumption of correct 3-dimensional structure for activity

Transesterification

Guide sequence

Target sequence

Cleavagesite

Transesterification is the process in which an ester group is exchanged with that of another, alcohol to form a new ester.

RNA cleavage at alkaline pH

alkaline pH

RNA undergoes spontaneous hydrolytic cleavage about one hundred times faster than DNA. This is believed due to intramolecular attack of the 2'-hydroxyl group on the neighboring phosphate diester, yielding a 2',3'-cyclic phosphate

RNA cleavage: alkaline pH

RNA at pH 10Base catalysis

2’ OH deprotonation

Nucleophilicoxyanion

RNA breaks2’,3’ cyclic P

base returns proton

1

3

2

4

5

RNA cleavage: alkaline pH

RNA at pH 10Base catalysis

2’ OH deprotonation

Nucleophilicoxyanion

RNA breaks& 2’,3’ cyclic P

base returns proton

1

3

2

4

5

Major types of endoribonucleases• RNase A is an RNase that is commonly used in research. • RNase H is a ribonuclease that cleaves the RNA in a DNA/RNA

duplex • RNase III is a type of ribonuclease that cleaves rRNA (16s

rRNA and 23s rRNA) • RNase L is an interferon-induced nuclease which, destroys all

RNA within the cell

RNase P is a ribozyme – a ribonucleic acid that acts as a catalyst. Its function is to cleave off an extra on tRNA molecules

• RNase PhyM is sequence specific for single-stranded RNAs. • RNase T1 is sequence specific for single-stranded RNAs.

RNase P

Ribozymes: RNase P

1. The RNA component of bacterial Rnase P has 350-400 nucleotides. It has: • a specificity domain and • a catalytic domain.

2. Bacterial RNase P contains a single protein subunit of about 120 amino acid residues.

3. Zn & Mg needed as cofactor

tRNA Processing: 5 steps

1.Removal of the 5’ leader sequence by RNase P

2.Removal of the 3’ trailer sequence

3.Addition of CCA to the 3’ end

4.Splicing of introns in some tRNAs

5.Numerous modifications at multiple residues

RNase P & tRNAThe interaction of the

leader sequence in the pre-tRNA (bold, dashed line) with the RPP (RNaseP Protein) is indicated.

Mg2+-activated hydroxide nucleophile (arrow), which attacks the phosphorus atom in the scissile bond.

RNase P

Bacterial RNase P class A

Bacterial RNase P class B Archaeal RNase P Eucaryal RNase P

Ribosome is a Ribozymes

The three-dimensional structure of the large (50S) subunit shows that formation of the peptide bond is catalyzed by the 23S RNA (& 28S RNA) molecule in the large subunit. The 31 proteins in the subunit probably provide the scaffolding needed to maintain the tertiary structure of the RNA.

Peptide transfer mechanism

Steitz et al. (Aug.2000) applied pioneering atomic-resolution viewing techniques to completely visualize a (bacterial) ribosome.

The ribosome

is a ribozyme

proteins

23S rRNA

peptidyl transfer reaction:

P-site tRNA

5S rRNA

A-site tRNA

Intron removal

By spliceosomes

Self splicingIntrons:

Ribozymes

Group I

Group II

RNA Processing

Self-splicing introns• Self splicing intrins: two types:

group Igroup II

Group I introns • G-OH needed (GMP, GDP GTP).

• Found in protozoa, fungal mitochondria, bacteriophage T4 and bacteria

Group II introns • The lariat pathway is used.• G-OH not needed.• Found in fungal

mitochondria, higher plant mitochondria, plastids.

Group I self-splicing of Tetrahymena 26S rRNA precursor

Mechanism of Group I Intron

The mechanism of Group II intron splicing

pre-mRNA

The lariat is formed by

transesterification between the 5’-

end of the intron sequence and

the 2’-OH of the branch site adenosine

Group II intron & Lariat formation

Mechanism of Group II Intron

Group I self-splicing of Tetrahymena 26S rRNA precursor

Group I self-splicing of Tetrahymena 26S rRNA precursor

Tetrahymena ribozyme: self splicing

Enzymic RNA: L19 RNA

• A shortened form of a rRNA of Tetrahymena has been shown to be an enzyme (Cech&Zaug, 1986).

• It catalyzes the cleavage and ligation of various nucleotide chains, for instance

where,

C= and for example,

n

n

pC

pC

CCCCpCdef

4

1

1

n

n

pC

pCcatalyst

catalyst

• The enzyme binds its substrate (pyrimidines) at the binding site, by Watson-Crick base-pairing (steps 1-2).

• A cytosine (C) molecule is detached by the G-end (step 3), and used for subsequent substrates (step 4).

Nucleotidyl transfer activity of the L-19 IVSL-19 IVS RNA (intervening sequence lacking 19 Ns) (414-19=395 N

long RNA)

This L-19 can be used for:• Transesterification• Nucleotidyl transferase• Exoribonuclease• Ligase &• Phosphatase

Enzymatic activity of the L-19 IVS

A new concept: the Ribozyme: enzymic RNA

• Exactly following the definition of an enzyme, the L-19 IVS RNA

• accelerates the reaction by a factor of around 1010

• is regenerated after each reaction - each enzyme molecule can react with many substrate molecules.

• specificity exists

hammerhead ribozyme

The hammerhead ribozyme is a RNA module that catalyzes reversible cleavage and joining reactions at a specific site within an RNA molecule

The minimal catalytic sequence active consists of three base-paired stems flanking a central core of 15 conserved nucleotides.

Hammerhead ribozymes play an important role as • therapeutic agents• biosensors, and • its applications in functional genomics and gene discovery

Hammer head ribozyme

Hairpin ribozyme• The hairpin ribozyme of plant

viruses is 50 nucleotides long, and can cleave itself internally, or, can cleave other RNA strands in a transesterification reaction. 

• The structure consists of two domains, stem A required for binding (self or other RNA molecules) and stem B, required for catalysis.

• Self-cleavage in the hairpin ribozyme occurs in stem A between an A and G bases when the 2' OH on the A attacks the phosphorous in the phosphodiester bond connecting A and G.

hairpin ribozyme

Ruppert et al, Science 2002

Transition state

Structure of the hairpin ribozyme

hairpin ribozyme

Ruppert et al, Nature 2001, Science 2002

hairpin ribozyme

Ruppert et al, Nature 2001 Ruppert et al, Science 2002

Transition stateGround state

The hairpin ribozyme (plant virus)

From Lilley TIBS (2003)

The hepatitis delta ribozyme (human virus)

Application of ribozyme•Ribozyme based therapeutics RNA

containing short EGS injected into host cell for destruction of RNA of mumps virus, influenza, human papilloma virus etc

• Inverse Genomics to find out the function of gene

•Ribozyme as biosensor: oligonucleotide-regulated ribozymes, also known as aptazymes

Ribozyme-based therapeutics (targeted gene silencing)

• Ribozymes are being designed to fight viral diseases

AIDS (HIV-1)

Viral hepatitis (HBV)• And cellular diseases

Cancer

Diabetes

Rheumatoid arthritis

• this is an alternative approach to “designer transcription factors”, such as polydactyl zinc finger proteins (C. Barbas), and RNA interference (RNAi, lecture 15) for altering gene expression

Ribozyme-based Biosensors

Reagentless biosensor that produces a signal upon binding a target

Fluorescence-Signaling Nucleic Acid-Based Sensors