25 Years of Ribozymes

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25 Years of Ribozymes

• Thomas R. Cech, Ph.D
• Howard Hughes Medical Institute

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(No Transcript)
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Tetrahymena thermophila
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An Unexpected Result (1980)
Unspliced RNA
Nuclear Extract
MgCl2, GTP
Gel Electrophoresis
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Its a Protein, Its Not
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Self-Splicing Mechanism of Group I Introns
1st reaction
2nd reaction
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Cleavage of RNA Substrate by a Group I Ribozyme
GOH
0
.17
.33
.5
1
2
5
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Time (min)
pCUUAAAA
pCUU
Each ribozyme cleaves 400 RNA substrates/hour
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Just Like an Enzyme
Reaction rate increases with guanosine
concentration
0.1 mM pG
0.5 mM pG
3.0 mM pG
and then saturates at high concentration
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Transition State Stabilization by the
Tetrahymena Group I Ribozyme
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Secondary Structure of the Tetrahymena Group I
Intron
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Crystal of the P4-P6 Domain RNA
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Crystal Structure of the P4-P6 Domain
Cate et al. Science 273 1678 (1996)
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Specific H-bonding to a Penta-hydrated Mg2
Juneau, Podell, Harrington and Cech Structure 9
221 (2001)
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Hexahydrated Mg2 Bridges Both Halves of P4-P6
RNA
Juneau, Podell, Harrington and Cech Structure 9
221 (2001)
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First Crystals of an Active Group I Ribozyme
(247 nt)
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5-Å Resolution Crystal Structure of the
Tetrahymena Ribozyme Core
Golden et al. Science 282 259 (1998)
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2004 the Year of the Intron
Twort/td (IA2-Golden) 3.6Å-NSMB, December
Azoarcus (IC3-Strobel) 3.1Å-Nature, July
Tetrahymena (IC1-Cech) 3.8Å-Mol Cell, November
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Guanosine Bound in a Base-Triple Sandwich
Guo, Gooding, and Cech Molecular Cell (2004)
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Functional non-coding
Informational coding mRNA genome
Lots!
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RNA is Transcribed from DNA
DNA
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RNA Can Spontaneously Fold
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RNA Can Spontaneously Fold
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Spectrum of Non-coding RNAs
Importance of RNA component Complexity of RNA
1. Ribozymes, other functional RNAs
2. RNPzymes
3. RNA handles
4. Guide RNAs
Complexity/Importance of protein component
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Non-coding RNAs, Category 1
Category 1

• Ribozymes
• Riboswitches
• RNA forms a specific structure by itself
• Proteins not essential
• Test RNA crystallizes well (if not easily)

Azoarcus (IC3-Strobel) 3.1Å-Nature, 2004
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A Natural Riboswitch Recognizes Guanine and
Hypoxanthine (HX)
Batey et al. Nature 432 411 (2004)
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Non-coding RNAs, Category 2RNPzymes
Category 2

• RNA active site ribosome, snRNPs
• Protein active site telomerase snoRNPs

a b
Test RNA by itself floppy, not crystallizable
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Non-coding RNAs, Category 3
Category 3
RNPs in which RNA provides handles for proteins

• Signal Recognition Particle
• IRESs (esp. poliovirus)
• Xist RNA
• Adeno VA,
• HIV TAR RRE

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Non-coding RNAs, Category 4
Category 4
Protein function is guided by RNA

• gRNA in trypanosome RNA editing
• Micro RNAs in mRNA silencing and
  heterochromatization

PIWI-like
piRNAs rasiRNAs
Ago-like
siRNAs miRNAs
via Greg Hannon
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Drosha
identification of precursors
pri-miRNA
Pasha
export
dsRBD
Dicer
pre-miRNA dsRNAs
asymmetry detection loading
Argonaute
miRNA siRNA
Silencing
via Greg Hannon
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Drosha
identification of precursors
pri-miRNA
Pasha
export
dsRBD
Dicer
pre-miRNA dsRNA
asymmetry detection loading
Argonaute
miRNA siRNA
via Greg Hannon
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TERT
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Est1p
Yeast Telomerase RNA Flexible Scaffold for
Protein Binding
Est2p
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Zappulla, Goodrich Cech, Nature SMB (2005)
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Telomerase RNA Exemplifies Whole Spectrum
Importance of RNA component Complexity of RNA
1. Ribozymes, other functional RNAs
2. RNPzymes
3. RNA handles
4. Guide RNAs
Complexity/Importance of protein component
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Est1p
Est2p
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Yeast Telomerase RNA
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Zappulla, Goodrich Cech, Nature SMB (2005)
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Evolution from an RNA World
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University of Colorado, Boulder Cech Lab