1 / 39

RNA catalysis

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

tahlia
Télécharger la présentation

RNA catalysis

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  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

More Related