1. RNA catalysis
Understand the basics of RNA/DNA catalysts - what functional groups used for catalysis? structures formed?
Know about transesterification & cleavage reactions
Know four types of natural catalytic RNAs (group I introns, group II introns, RNase P, small self-cleaving), what reactions they perform, know basics of their secondary and tertiary structure, requirements for cofactors/metals/proteins/ATP
Know details of glmS ribozyme self-cleavage
Understand use of ribozymes as therapeutics
In vitro selection - understand the process
Know some of the ribozymes and deoxyribozymes that have been discovered using in vitro selection

2. Outline • RNA transesterification • Naturally occurring catalysts
• Catalytic functions
• Catalytic mechanisms

3. RNA transesterification
• Exchange one phosphate ester for another
• Free energy change is minimal (reversible)

4. RNA transesterification
• Nucleophile can be either the adjacent 2´ hydroxyl or another ester
• Referred to as hydrolysis when water serves as the nucleophile

5. RNA transesterification
• Nucleophilic attack on the phosphorus center leads to a penta-coordinate intermediate
• Ester opposite from the nucleophile serves as the leaving group (in-line attack)

6. General mechanisms • Substrate positioning
• Transition state stabilization
• Acid-base catalysis
• Metal ion catalysis

7. RNA Catalysts

8. Naturally occurring catalysts
• RNA cleavage
glmS ribozyme (crystal structure) hammerhead ribozyme (crystal structure) hairpin ribozyme (crystal structure) Varkud satellite (VS) ribozyme (partial NMR structure) hepatitis delta virus (HDV) ribozyme (crystal structure) M1 RNA (RNase P) (partial crystal structure)
• RNA splicing
group I introns (crystal structure) group II introns (crystal structure) *** U2-U6 snRNA (spliceosome) (partial NMR structure) ***
• Peptide bond formation
ribosome (crystal structure)

9. Small self-cleaving ribozymes
• Hammerhead, hairpin, VS, HDV ribozymes
• Derivative of viral, viroid, or satellite RNAs
• Involved in RNA processing during rolling circle replication
• RNA transesterification via 2´ hydroxyl
• Reversible: cleavage and ligation (excepting HDV)

10. Hammerhead ribozyme • Three-stem junction with conserved loop regions
• Coaxial stacking of stems II and III through extended stem II structure containing canonical Watson-Crick and non-canonical base pairs
• Metal-ion catalysis

11. Hammerhead ribozyme • In nature is self-cleaving (not a true enzyme)
• Can be manipulated to function as a true catalyst
• Biotechnological and potential therapeutic applications for target RNA cleavage

12. Hammerhead ribozyme Separation of catalytic and substrate strands
Strand with hairpin is the enzyme
Single strand is substrate
KM = 40nM; kcat = ~1 min-1; kcat/KM = ~107 M -1 min -1 (catalytic efficiency)
Compare to protein enzymes?
Proteins typically have much greater kcat and much higher Km. The 40 nM Km of the hamemrhead indicates an extremely stable enzyme-substrate complex which will not dissociate product readily. This slow release of product is the reason for the low kcat. A protein enzyme with a Km/kcat of 10-7 might have a kcat of 105/min and Km of 10-2M

13. RNA Catalysts RNase A Protein enzyme Hammerhead ribozyme
• basics of catalytic reactions (cleavage)
RNase A
Protein enzyme
Hammerhead
ribozyme

14. Hairpin ribozyme • In nature is part of a four-stem junction
• Ribozyme consists of two stems with internal loops
• Stems align side-by-side with 180 degree bend in the junction (hence ‘hairpin’)
• Internal loops interact to form active site

15. Hairpin ribozyme
• Crystal structure reveals interactions between stems
• Nucleobases position and activate scissile phosphodiester linkage
• Combination of transition state stabilization and acid-base catalysis?

16. HDV ribozyme • Genomic and antigenomic ribozymes
• Nested pseudoknot structure
• Very stable
• Cleaves off 5´ leader sequence

17. HDV ribozyme

18. HDV ribozyme
• Active site positions an important cytidine near the scissile phosphodiester bond

19. RNase P
• True enzyme
• Cleaves tRNA precursor to generate the mature 5´ end
• Composed of M1 RNA and C5 protein (14 kD)
• RNA is large and structurally complex
• Protein improves turnover
• Hydrolysis

20. Group I introns
• Large family of self-splicing introns usually residing in rRNA and tRNA
• Two step reaction mechanism

21. Group I intron structure
• Crystal structure of ‘trapped’ ribozyme before second transesterification reaction
• Metal ion catalysis

22. Group I intron structure
Ribose zipper P1
J8/7

23. Group II introns

24. Group II introns
• Usually found in organelles (e.g. plant chloroplasts, mitochondria)
• mechanism proceeds through a branched lariat intermediate structure which is produced by the attack of a 2’-OH of an internal A on the phosphodiester of the 5’-splice site
• proteins thought to stabilize structure but not necessary for catalysis
• no ATP or exogenous G needed

25. Summary of splicing reactions

26. The ribosome is a ribozyme
• Ribosome is 2/3 RNA and 1/3 protein by mass
• Crystal structures prove that RNA is responsible for decoding and for peptide bond formation

27. Peptidyl transferase
Crystal structure of 50S subunit shows no protein within 20 Å of peptidyl transferase center
Closest component to aa-tRNA is adenosine in 23S rRNA
Proposed acid-base mechanism for peptide bond formation
Recent evidence shows substrate positioning accounts for catalysis

28. Glucosamine 6-phosphate riboswitch/ribozyme
• Glucosamine-6-phosphate (GlcN6P)-dependent self-cleaving ribozyme
• Regulates biosynthesis of amino sugars used in bacterial cell wall synthesis

29. glmS is a metabolite-responsive ribozyme
Effects of [glcN6P] on the rate constant. M

30. Optimization of catalysis by the glmS ribozyme

31. Glucosamine 6-phosphate ribozyme self-cleavage RNA transesterification

32. Glucosamine 6-phosphate ribozyme self-cleavage RNA transesterification
Might glucosamine 6-phosphate serve as the general acid-base (coenzyme) for self-cleavage?

33. Ribozyme exhibits self-cleavage activity in
TRIS buffer in the absence of ligand
McCarthy, T.J., Plog, M.A., Floy, S.A., Jansen, J.A., Soukup, J.K. & Soukup, G.A. "Ligand requirements for glmS ribozyme self-cleavage." Chemistry & Biology 12:1-6 (2005).

34. importance of amine functionality
Ligand specificity -
importance of amine functionality
McCarthy, T.J., Plog, M.A., Floy, S.A., Jansen, J.A., Soukup, J.K. & Soukup, G.A. "Ligand requirements for glmS ribozyme self-cleavage." Chemistry & Biology 12:1-6 (2005).

35. Observed rate constants and apparent binding of ligand analogs
McCarthy, T.J., Plog, M.A., Floy, S.A., Jansen, J.A., Soukup, J.K. & Soukup, G.A. "Ligand requirements for glmS ribozyme self-cleavage." Chemistry & Biology 12:1-6 (2005).

36. pH-reactivity profiles
• GlcN and serinol are lower affinity ligands
• Apparent pKa for ligand-dependent self-cleavage approximates the solution pKa of ligand
• Suggest the amine functionality of the ligand functions as a general acid/base in catalysis
McCarthy, T.J., Plog, M.A., Floy, S.A., Jansen, J.A., Soukup, J.K. & Soukup, G.A. "Ligand requirements for glmS ribozyme self-cleavage." Chemistry & Biology 12:1-6 (2005).

37. RNA/DNA Catalysts
RNA/DNA catalysis & evolution
• in vitro selection

38. RNA/DNA Catalysts RNA/DNA catalysis & evolution
• increasing numbers of examples of reactions catalyzed by nucleic acids

39. DNA Catalysts