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The Hammerhead Ribozyme

Learn about the intricate structure, catalytic mechanism, and pharmaceutical potential of the Hammerhead Ribozyme, with insights into ribonucleotide bases, secondary and tertiary structures, and its role in targeting diseases like HCV and HIV. References and internet resources included.

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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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