The Hammerhead Ribozyme

Peter J. Mikulecky

What is a Ribozyme?

1) Enzyme

2) Ribonucleic Acid

NOT PROTEIN

1989 Nobel PrizeIn Chemistry

Sid Altman Tom Cech

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Structure

As with proteins, we consider...

Primary: GGCCGAACUGGUA

Secondary:

Tertiary:

Primary Structure

Limited to ribonucleotides (base + ribose + phosphate):

Common Bases:

Uncommon Bases:

N

N

N

N

O

N

N

N

N

N

N

N

N

O

N

N

N

O

O

A G C U

N

N

O

O

N

N

N

N

O

Pseudouridine Inosine etc...

Secondary Structure

Watson-Crick Base Pairing Helix Formation

B-DNA RNA

RNA usually assumesA-form helices…

Small pore along helical axis

“Rungs” stack obliquely to axis

A-DNA

Secondary Structure

Conserved base-pairing interactions result in...

• Three “stem” regions

• Uridine-containing turn

• An “augmenting helix” joining stems II and III

Ribozyme vs. tRNAPhe

folding

Tertiary Structure

Catalytic Mechanism

IntactPhosphodiester

Mg2+CoordinatedTransition

CleavedPhosphodiester

Catalytic MechanismNew 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

HEPTAZYME TM

Ribozyme designed to inhibit Vascular EndothelialGrowth Factor (VEGF)...

ANGIOGENESIS

Ribozyme targets highly conserved sequences of theHepatitis C Virus... HCV DRUG RESISTANCE

In Clinical Trial...

HIV Gene Therapy...Bone Marrow Sample

Treat Stem Cells with Retroviral Vector

Re-Implant Treated Cells

Encodes Gene for anti-HIV Ribozyme

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 NationalAcademy of Sciences 90 (1993) 6991-4.

Heus HA, and Pardi A “Nuclear magnetic resonance studies of the hammerhead ribozyme domain. Secondary structureFormation 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