The Hammerhead Ribozyme

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The Hammerhead Ribozyme
Peter J. Mikulecky
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What is a Ribozyme?
1) Enzyme
2) Ribonucleic Acid
NOT PROTEIN
1989 Nobel Prize In Chemistry
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Structure
As with proteins, we consider...
Primary
GGCCGAACUGGUA
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Primary Structure
Limited to ribonucleotides (base ribose
phosphate)
Common Bases
Uncommon Bases
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Secondary Structure
Watson-Crick Base Pairing
Helix Formation
B-DNA
A-DNA
Small pore along helical axis
Rungs stack obliquely to axis
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Secondary Structure
Conserved base-pairing interactions result in...

• Three stem regions

• Uridine-containing turn

• An augmenting helix
• joining stems II and III

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Ribozyme vs. tRNAPhe
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Tertiary Structure
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Catalytic Mechanism
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Catalytic Mechanism
New crystal structure shows five Mg2 sites
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Ribozymes in Our Past
?
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The Future of Ribozymes
In Vitro Molecular Evolution of RNA

High Throughput Screening
Ribozyme-Based Therapies
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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
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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
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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

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References
Pley HW, Flaherty KM, and McKay DB
Three-dimensional structure of a hammerhead
ribozyme Nature 372 (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 Cell 81 (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 Science 274 (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
Biochemistry 37 (1998) 14765-75. Tuschl T, and
Eckstein F Hammerhead ribozymes importance of
stem-loop II for activity Proceedings of the
National Academy of Sciences 90 (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
Biology 217 (1991) 113-24. Ruffner DE, Stormo
GD, Uhlenbeck OC Sequence requirements of the
hammerhead RNA self-cleavage reaction
Biochemistry 29 (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
Chemistry 272 (1997) 26822-6.
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Internet References
http//www.tulane.edu/biochem/nolan/lectures/rna/
frames/hambtx.htm http//unisci.com/stories/08039
84.htm http//ndbserver.ebi.ac.uk5700/NDB/NDBATL
AS/indexes/ribozyme.html http//www.actupgg.org/B
AR/art082897.html http//ull.chemistry.uakron.edu
/biochem/animations/index.html http//www.horizon
press.com/gateway/ribozyme.html http//www.rpi.co
m/ http//www.cup.org/Journals/JNLSCAT/RNA/abstra
cts/rna1355838296026024h.html http//www.chemistr
y.wustl.edu/Faculty/Bashkin/jkbhomeu1.html