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The Hammerhead Ribozyme

The Hammerhead Ribozyme. Peter J. Mikulecky. Sid Altman. Tom Cech. What is a Ribozyme?. 1) Enzyme. 2) Ribonucleic Acid. NOT PROTEIN. 1989 Nobel Prize In Chemistry. Secondary:. Tertiary:. Structure. As with proteins, we consider. Primary:. GGCCGAACUGGUA.

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The Hammerhead Ribozyme

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  1. The Hammerhead Ribozyme Peter J. Mikulecky

  2. Sid Altman Tom Cech What is a Ribozyme? 1) Enzyme 2) Ribonucleic Acid NOT PROTEIN 1989 Nobel Prize In Chemistry

  3. Secondary: Tertiary: Structure As with proteins, we consider... Primary: GGCCGAACUGGUA

  4. A G C U Pseudouridine Inosine etc... Primary Structure Limited to ribonucleotides (base + ribose + phosphate): Common Bases: Uncommon Bases:

  5. RNA RNA usually assumes A-form helices… Secondary Structure Watson-Crick Base Pairing Helix Formation B-DNA A-DNA Small pore along helical axis “Rungs” stack obliquely to axis

  6. Secondary Structure Conserved base-pairing interactions result in... • Three “stem” regions • Uridine-containing turn • An “augmenting helix” • joining stems II and III

  7. folding Ribozyme vs. tRNAPhe

  8. Tertiary Structure

  9. Intact Phosphodiester Mg2+Coordinated Transition Cleaved Phosphodiester Catalytic Mechanism

  10. Catalytic Mechanism New crystal structure shows five Mg2+ sites:

  11. Ribozymes in Our Past ?

  12. The Future of Ribozymes In Vitro Molecular Evolutionof RNA + High Throughput Screening Ribozyme-Based Therapies

  13. In Clinical Trial... ANGIOZYME TM Ribozyme designed to inhibit Vascular Endothelial Growth Factor (VEGF)... ANGIOGENESIS HEPTAZYME TM Ribozyme targets highly conserved sequences of the Hepatitis C Virus... HCV DRUG RESISTANCE

  14. In Clinical Trial... HIV Gene Therapy... Bone Marrow Sample Treat Stem Cells with Retroviral Vector Encodes Gene for anti-HIV Ribozyme Re-Implant Treated Cells

  15. In Summary... • Hammerhead Ribozyme Challenges • the “Central Dogma” • Tertiary Structure Stabilized by • Conserved H-Bonding Regions • Divalent Cations Are Required for Catalysis • Ribozymes Offer Great Pharmaceutical Promise

  16. References Pley HW, Flaherty KM, and McKay DB “Three-dimensional structure of a hammerhead ribozyme” Nature372 (1994) 68-74. Scott WG, Finch JT, and Klug A “The Crystal Structure of an All-RNA Hammerhead Ribozyme: A Proposed Mechanism for RNA Catalytic Cleavage” Cell81 (1995) 991-1002. Scott WG, Murray JB, Arnold JRP, Stoddard BL, and Klug A “Capturing the Structure of a Catalytic RNA Intermediate: The Hammerhead Ribozyme” Science274 (1996) 2065-69. Peracchi A, Karpeisky A, Maloney L, Beigelman L, and Herschlag D “A Core Folding Model for Catalysis by the Hammerhead Ribozyme Accounts for Its Extraordinary Sensitivity to Abasic Mutations” Biochemistry37 (1998) 14765-75. Tuschl T, and Eckstein F “Hammerhead ribozymes: importance of stem-loop II for activity” Proceedings of the National Academy of Sciences90 (1993) 6991-4. Heus HA, and Pardi A “Nuclear magnetic resonance studies of the hammerhead ribozyme domain. Secondary structure Formation and magnesium ion dependence” Journal of Molecular Biology217 (1991) 113-24. Ruffner DE, Stormo GD, Uhlenbeck OC “Sequence requirements of the hammerhead RNA self-cleavage reaction” Biochemistry29 (1990) 10695-702. Peracchi A, Beigelman L, Scott EC, Uhlenbeck OC, Herschlag D “Involvement of a specific metal ion in the transition of the Hammerhead ribozyme to its catalytic conformation” Journal of Biological Chemistry272 (1997) 26822-6.

  17. Internet References http://www.tulane.edu/~biochem/nolan/lectures/rna/frames/hambtx.htm http://unisci.com/stories/0803984.htm http://ndbserver.ebi.ac.uk:5700/NDB/NDBATLAS/indexes/ribozyme.html http://www.actupgg.org/BAR/art082897.html http://ull.chemistry.uakron.edu/biochem/animations/index.html http://www.horizonpress.com/gateway/ribozyme.html http://www.rpi.com/ http://www.cup.org/Journals/JNLSCAT/RNA/abstracts/rna1355838296026024h.html http://www.chemistry.wustl.edu/Faculty/Bashkin/jkbhomeu1.html

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