Presentation on theme: "Ribozymes RNA molecules that act as enzymes are called ribozymes."— Presentation transcript:
1Ribozymes RNA molecules that act as enzymes are called ribozymes. Thomas CzechSidney AltmanRNA molecules that act as enzymes are called ribozymes. This property of some RNAs was discovered bySidney Altman and Thomas Czech,who were awarded theNobel Prize in Chemistry in 1989.
3Ribozymes RNA molecules capable of catalyzing biochemical reactions Earliest known examples:RNase PGroup I and II intronsRibosomeshammerhead ribozymesPrincipal reactions:RNA transesterificationRNA cleavage (hydrolysis of phosphodiester bonds)Substrate aligned into the active site using a guide sequence which is complimentary to the substrateAll ribozymes depend absolutely on the assumption of correct dimensional structure for activity
4TransesterificationTransesterification is the process in which an ester group is exchanged with that of another, alcohol to form a new ester.CleavagesiteTarget sequenceGuide sequence
5RNA 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
8RNase PMajor types of endoribonucleasesRNase A is an RNase that is commonly used in research.RNase H is a ribonuclease that cleaves the RNA in a DNA/RNA duplexRNase 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 cellRNase P is a ribozyme – a ribonucleic acid that acts as a catalyst. Its function is to cleave off an extra on tRNA moleculesRNase PhyM is sequence specific for single-stranded RNAs.RNase T1 is sequence specific for single-stranded RNAs.
9Ribozymes: RNase PThe RNA component of bacterial Rnase P has nucleotides. It has:a specificity domain anda catalytic domain.Bacterial RNase P contains a single protein subunit of about 120 amino acid residues.Zn & Mg needed as cofactor
10tRNA Processing: 5 steps Removal of the 5’ leader sequence by RNase PRemoval of the 3’ trailer sequenceAddition of CCA to the 3’ endSplicing of introns in some tRNAsNumerous modifications at multiple residues
11RNase 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.
12RNase P Eucaryal RNase P Bacterial RNase P class A Bacterial RNase P class BArchaeal RNase PEucaryal RNase P
13Ribosome 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.
15The ribosome is a ribozyme Steitz et al. (Aug.2000) applied pioneering atomic-resolution viewing techniques to completely visualize a (bacterial) ribosome.
16peptidyl transfer reaction: 5S rRNAA-site tRNAproteins23S rRNApeptidyl transfer reaction:P-site tRNA
17RNA Processing Intron removal By spliceosomes Group I Self splicing Introns:RibozymesGroup II
18Self-splicing introns Self splicing intrins: two types:group Igroup IIGroup I intronsG-OH needed (GMP, GDP GTP).Found in protozoa, fungal mitochondria, bacteriophage T4 and bacteriaGroup II intronsThe lariat pathway is used.G-OH not needed.Found in fungal mitochondria, higher plant mitochondria, plastids.
19Group I self-splicing of Tetrahymena 26S rRNA precursor
27Enzymic RNA: L19 RNAA 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 instancewhere,C= and for example,
28Nucleotidyl 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:TransesterificationNucleotidyl transferaseExoribonucleaseLigase &Phosphatase
30A new concept: the Ribozyme: enzymic RNA Exactly following the definition of an enzyme, the L-19 IVS RNAaccelerates the reaction by a factor of around 1010is regenerated after each reaction each enzyme molecule can react with many substrate molecules.specificity exists
31hammerhead ribozymeThe hammerhead ribozyme is a RNA module that catalyzes reversible cleavage and joining reactions at a specific site within an RNA moleculeThe minimal catalytic sequence active consists of three base-paired stems flanking a central core of 15 conserved nucleotides.Hammerhead ribozymes play an important role astherapeutic agentsbiosensors, andits applications in functional genomics and gene discovery
33Hairpin ribozymeThe 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.
34hairpin ribozymeTransition stateRuppert et al, Science 2002
35Structure of the hairpin ribozyme Ruppert et al, Nature 2001, Science 2002
36hairpin ribozyme Ground state Transition state Ruppert et al, Nature Ruppert et al, Science 2002
37The hairpin ribozyme (plant virus) From Lilley TIBS (2003)
39Application of ribozyme Ribozyme based therapeutics RNA containing short EGS injected into host cell for destruction of RNA of mumps virus, influenza, human papilloma virus etcInverse Genomics to find out the function of geneRibozyme as biosensor: oligonucleotide- regulated ribozymes, also known as aptazymes
40Ribozyme-based therapeutics (targeted gene silencing) Ribozymes are being designed to fight viral diseasesAIDS (HIV-1)Viral hepatitis (HBV)And cellular diseasesCancerDiabetesRheumatoid arthritisthis is an alternative approach to “designer transcription factors”, suchas polydactyl zinc finger proteins (C. Barbas), and RNA interference (RNAi,lecture 15) for altering gene expression
41Ribozyme-based Biosensors Reagentless biosensor that produces a signal upon binding a targetFluorescence-Signaling Nucleic Acid-Based Sensors