Chemical Catalysis by RNA

1
Chemical Catalysis by RNA
1. Discovery of catalytic RNA group I
self-splicing introns
2. Other classic cellular RNA enzymes RNase P
and group II introns
3. Self-cleaving RNAs from viruses and cells
4. Exploring the catalytic potential of RNA by in
vitro selection
2
In Vitro Transcription of 26S rRNA Gene Yields
Excised Intron
Transcription in vitro of intron-containing RNA
gave smaller products (Cech and colleagues, 1982)
Inclusion of polyamine prevented
co-transcriptional splicing, and splicing could
be initiated later. Thus, RNA polymerase is not
required.
Fig. 14.44
3
RNA-accelerated Reaction Gives Ligation of Exons
Transcript had exons of defined size, allowing
detection of ligated exons
Splicing required guanosine or GTP
Fig. 14.46
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Guanosine Nucleotide Is Added to the 5-end of
the Intron
Gel shows that linear intron becomes labeled
from radioactive GTP
RNase digestion also demonstrates G is
incorporated at 5-end
Fig. 14.47
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Two Steps of Group I Intron Self-Splicing
1st step is mediated by guanosine
2nd step is the same (chemically) as for mRNAs
attack by 5 exon upon 3-splice site
RNA forms active site and can perform reaction
in vitro. For many group I introns, proteins
participate in vivo as cofactors or chaperones.
Fig. 14.48
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The Two Reaction Steps Are the Reverse of Each
Other Chemically
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Distribution of Group I Introns
N Nuclear P Plastid M Mitochondrial B
Bacterial
Mobile genetic elements
No apparent function
gt3000 identified
Widely and sporadically dispersed, suggesting
horizontal transfer
Haugen et al, Trends Genet. 21 (2005), 111-119
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Mobility of Group I Introns
Haugen et al, Trends Genet. 21 (2005), 111-119
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The Tetrahymena Group I Ribozyme
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The Tetrahymena Group I Ribozyme
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The Tetrahymena Group I Ribozyme
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The Tetrahymena Group I Ribozyme
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The Tetrahymena Group I Ribozyme
Powerful system for studies of RNA catalysis,
structure and folding
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Group I Introns Are Metalloenzymes
High resolution crystal structure revealed
active site details
Two Mg2 ions poised for catalysis
Stahley and Strobel, Science (2005) 309,1587-90.
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Group I Introns Are Metalloenzymes
Role of metals, and even their contacts,
elucidated previously from in-depth kinetics
studies
Role of third metal revealed in these studies
remains unclear
Shan and Herschlag, PNAS (1999) 96, 12299-304
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RNA Folding Studies Folding, One Molecule At a
Time
Native
Misfolded
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DEAD-box Proteins As RNA Chaperones
Group I introns (and structured RNAs in
general) are prone to misfolding
DEAD-box proteins can use ATP to disrupt
structure non-specifically, allowing a second
chance to fold correctly
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Chemical Catalysis by RNA
1. Discovery of catalytic RNA group I
self-splicing introns
2. Other classic cellular RNA enzymes RNase P
and group II introns
3. Self-cleaving RNAs from viruses and cells
4. Exploring the catalytic potential of RNA by in
vitro selection
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tRNA Processing by RNase P
Present in all organisms includes RNA and
protein components
Shown by Altman and Pace labs that RNA
component retains activity at high Mg2
concentration
True ribozyme enzyme is unchanged by
reaction and can participate in multiple turnovers
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Group II Intron Same Chemical Mechanism as the
Spliceosome
Completely different architecture from group I
Structural analogies between group II introns
and spliceosomal RNAs
Figure from Marlene Belforts groups web page
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Chemical Catalysis by RNA
1. Discovery of catalytic RNA group I
self-splicing introns
2. Other classic cellular RNA enzymes RNase P
and group II introns
3. Self-cleaving RNAs from viruses and cells
4. Exploring the catalytic potential of RNA by in
vitro selection
22
Viral Self-cleaving RNAs Cleavage Gives
Unit-length Genome Sequences
Ribozyme sequence
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Self-cleaving Viral RNAs
Hammerhead ribozyme plant virus
Hairpin ribozyme plant virus
VS ribozyme Neurospora (fungus) satellite DNA
HDV ribozyme Human virus
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Structure of the HDV Ribozyme
Ferre-DAmare et al, Nature (1998)395, 567-74.
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Reaction of the HDV Ribozyme
Like all known self-cleaving viral RNAs, the
reaction is attack by 2-OH on adjacent
phosphodiester linkage
First RNA shown to use general acid-base
catalysis
Nakano and Bevilacqua, Science (2000) 287,
1493-97.
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A Human Self-cleaving RNA Related to the HDV
Ribozyme
Isolated in human genome-wide search for small
catalytic RNAs
Present in an intron of an mRNA sequence
Function unclear
Three other catalytic RNAs isolated in the same
search. They remain uncharacterized.
Salehi-Ashtiani et al, Science (2006) 313,
1788-1792
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New Catalytic RNAs Are Still Being Identified
GlmS riboswitch (bacterial) shown to cleave
upon binding its ligand
Ligand, glucosamine-6-phosphate, apparently
participates directly in chemical mechanism.
Another group of catalytic RNAs some mRNAs,
including mammalian ?-globin gene, self-cleave as
part of transcription termination
Cochrane et al, Chem. Biol. (2007) 14, 97-105
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Chemical Catalysis by RNA
1. Discovery of catalytic RNA group I
self-splicing introns
2. Other classic cellular RNA enzymes RNase P
and group II introns
3. Self-cleaving RNAs from viruses and cells
4. Exploring the catalytic potential of RNA by in
vitro selection
29
A Central Role For RNA
RNA
DNA
Protein
RNA
Ubiquitous roles of RNA in Central Dogma
Was there a time when RNA served primary roles
in information storage and catalysis?
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RNA Has Catalytic Potential Beyond That Found In
Nature
Lends credence to models in which a late stage
RNA world had diverse metabolism and pathways
Chen, Li, and Ellington, Chem. Biodivers. 4
(2007), 633-655
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Key Points
1. Group I introns carry out their own splicing,
although proteins generally assist in vivo. The
group I splicing reaction consists of two steps,
in which the second is chemically the reverse of
the first.
2. There are numerous small RNA motifs that
cleave themselves. More are still being
discovered.
3. The discovery that RNA has substantial
catalytic potential has expanded our view of
biological catalysts and suggests that RNA may
have had even more diverse functions in an
ancient world than it appears to have now.
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