The Hammerhead Ribozyme
Peter J. Mikulecky
What is a Ribozyme?
1) Enzyme
2) Ribonucleic Acid
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NOT PROTEIN
1989 Nobel Prize
In Chemistry
Sid Altman
Tom Cech
Structure
As with proteins, we consider...
Primary:
GGCCGAACUGGUA
Secondary:
Tertiary:
Primary Structure
Limited to ribonucleotides (base + ribose + phosphate):
Common Bases:
N
N
N
O
N
N
N
N
N
N
N
A
N
N
G
Uncommon Bases:
N
N
O
C
O
N
O
N
O
U
O
N
O
Pseudouridine
N
N
N
N
Inosine
etc...
Secondary Structure
Watson-Crick Base Pairing
B-DNA
A-DNA
Helix Formation
RNA
RNA usually assumes
A-form helices…
Small pore along helical axis
“Rungs” stack obliquely to axis
Secondary Structure
Conserved base-pairing interactions result in...
• Three “stem” regions
• Uridine-containing turn
• An “augmenting helix”
joining stems II and III
Ribozyme
vs.
folding
Phe
tRNA
Tertiary Structure
Catalytic Mechanism
Intact
Phosphodiester
Mg2+Coordinated
Transition
Cleaved
Phosphodiester
Catalytic Mechanism
New crystal structure shows five Mg2+ sites:
Ribozymes in Our Past
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?
The Future of Ribozymes
In Vitro Molecular Evolution of RNA
+
High Throughput Screening
Ribozyme-Based Therapies
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
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
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
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.
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