Ribozymes RNA molecules that act as enzymes are called ribozymes. Thomas Czech Sidney Altman 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.  

Ribozyme discovery (open with media player)  

Ribozymes RNA molecules capable of catalyzing biochemical reactions Earliest known examples: RNase P Group I and II introns Ribosomes hammerhead ribozymes Principal reactions: RNA transesterification RNA 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 Transesterification is the process in which an ester group is exchanged with that of another, alcohol to form a new ester. Cleavage site Target sequence Guide sequence

RNA cleavage at 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 1 RNA cleavage: alkaline pH RNA at pH 10 Base catalysis 3 5 2’ OH deprotonation RNA breaks 2’,3’ cyclic P 2 Nucleophilic oxyanion base returns proton 4

RNA cleavage: alkaline pH 1 RNA at pH 10 Base catalysis 2 2’ OH deprotonation 3 Nucleophilic oxyanion 4 RNA breaks & 2’,3’ cyclic P 5 base returns proton

RNase P 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.

Ribozymes: RNase P The RNA component of bacterial Rnase P has 350-400 nucleotides. It has: a specificity domain and a catalytic domain. Bacterial RNase P contains a single protein subunit of about 120 amino acid residues. Zn & Mg needed as cofactor

tRNA Processing: 5 steps Removal of the 5’ leader sequence by RNase P Removal of the 3’ trailer sequence Addition of CCA to the 3’ end Splicing of introns in some tRNAs Numerous modifications at multiple residues

RNase P & tRNA The 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 Eucaryal 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

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

peptidyl transfer reaction: 5S rRNA A-site tRNA proteins 23S rRNA peptidyl transfer reaction: P-site tRNA

RNA Processing Intron removal By spliceosomes Group I Self splicing Introns: Ribozymes Group II

Self-splicing introns Self splicing intrins: two types: group I group 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

Group II intron & Lariat formation The lariat is formed by transesterification between the 5’-end of the intron sequence and the 2’-OH of the branch site adenosine

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,

Nucleotidyl transfer activity of the L-19 IVS L-19 IVS RNA (intervening sequence lacking 19 Ns) (414-19=395 N long RNA) 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). 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 Transition state Ruppert et al, Science 2002

Structure of the hairpin ribozyme Ruppert et al, Nature 2001, Science 2002

hairpin ribozyme Ground state Transition state Ruppert et al, Nature 2001 Ruppert et al, Science 2002

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