1. 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.  

2. Ribozyme discovery (open with media player)

3. 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 dimensional structure for activity

4. 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

5. 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

6. RNA cleavage: alkaline pH1
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

7. RNA cleavage: alkaline pH1
RNA at pH 10
Base catalysis 2
2’ OH
deprotonation 3
Nucleophilic
oxyanion 4
RNA breaks
& 2’,3’ cyclic P 5
base returns proton

8. 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.

9. Ribozymes: RNase P
The RNA component of bacterial Rnase P has  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

10. 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

11. 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.

12. RNase P Eucaryal RNase P Bacterial RNase P class A
Bacterial RNase P class B
Archaeal RNase P
Eucaryal RNase P

13. 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.

14. Peptide transfer mechanism

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

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

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

18. 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.

19. Group I self-splicing of Tetrahymena 26S rRNA precursor

20. Mechanism of Group I Intron

21. The mechanism of Group II intron splicing
pre-mRNA

22. 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

23. Mechanism of Group II Intron

24. Group I self-splicing of Tetrahymena 26S rRNA precursor

25. Group I self-splicing of Tetrahymena 26S rRNA precursor

26. Tetrahymena ribozyme: self splicing

27. 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,

28. 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

29. Enzymatic activity of the L-19 IVS

30. 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

31. 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

32. Hammer head ribozyme

33. 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.

34. hairpin ribozyme
Transition state
Ruppert et al, Science 2002

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

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

37. The hairpin ribozyme (plant virus)
From Lilley TIBS (2003)

38. The hepatitis delta ribozyme (human virus)

39. 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

40. 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

41. Ribozyme-based Biosensors
Reagentless biosensor that produces a signal upon binding a target
Fluorescence-Signaling Nucleic Acid-Based Sensors