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RNA catalysis

RNA catalysis. Understand the basics of RNA/DNA catalysts - what functional groups used for catalysis? structures formed? Know about transesterification & cleavage reactions

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RNA catalysis

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  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?

  13. RNA Catalysts • 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 2451 in 23S rRNA • Proposed acid-base mechanism for peptide bond formation • Recent evidence showssubstrate positioningaccounts 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. 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

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