Department of Biology MSc Programme

1
Department of Biology MSc Programme
The RNA World
BL.0103
Fall semester 2007/08
Dr Alessandro Puoti Dept. of Biology Ch. du Musée
10, 0.316 alessandro.puoti_at_unifr.ch
Lecture support http//moodle.unifr.ch/course/vie
w.php?id1606
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I. The Origin of Life
Clock analogy for Earths evolutionary history
Earliest known fossil 3.5 billion
years Atmospheric oxygen 2.7 billion
years Oldest animals fossils 600 mio
years Extinction of dinosaurs 60 mio
years Human lineage 5 mio years ago
origin of life prebiotic world
Campbell 26.2
3
The four-stage hypothesis for the origin of
Life(also chemical evolution)
1) synthesis of small organic molecules (amino
acids, nucleotides) hypothesis of Oparin
and Haldane (1920) abiotic synthesis.
experiment by Miller and Urey (1953) 2) joining
of small molecules into polymers (polypeptides,
RNA, DNA) 3) origin of self-replicating molecules
(inheritance) the chicken and egg
question which came first, DNA or protein? 4)
packaging of organic molecules into protobionts
(vesicles) chemical reactions can occur in
an environment that is different from the
surroundings. natural selection of
protobionts that contain molecules that sustain
better proliferation.
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The Miller-Urey experiment
The atmosphere consisted of H20, H2, CH4,
NH3. Sparks mimic lightnings
After 1 week, organic compounds including amino
acids were found.
The conditions on Earth were quite different from
today The synthesis of such components was only
possible in a reducing atmosphere (no
oxygen). Also, there must be no predators that
would destroy the newly formed molecules.
Voet. 1-37
5
Organic compounds produced by the Miller-Urey
Experiment
amino acid


Under certain conditions, nucleotides could also
be formed condensation of HCN adenine
(HCN)5 polymerization of formaldehyde (HCHO)
sugars (CH2O)n


Organic compounds might have been formed on clays
that contain metal ions to catalyze the reaction.
Voet. Table 1-4
6
Molecular Composition of E. Coli.
(of which approx. 1 is mRNA)
Voet. Table 1-1
7
The central dogma of molecular biology
Genetic information transfers that occur in
normal cells Special transfers of genetic
information (RNA viruses) DNA directly specifying
a protein is not known, but could be
possible Note proteins never specify DNA, RNA
or proteins proteins can only receive genetic
information
Voet. 5-21
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Which came first, the chicken or the egg?
Once amino acids have appeared, how could life
then be sustained? Proteins and amino acids
have no homology to nucleic acids How could
life have arisen if the DNA could multiply and be
transcribed and translated only in the presence
of proteins? Which came first, genes or
enzymes? Hypothesis neither the gene nor the
enzyme came first, but molecules that at the
same time - contained the genetic
information - were able to replicate by
themselves - had a catalytic activity
The RNA World hypothesis
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Abiotic replication of RNA
RNA nucleotides abiotic synthesis of 5-10mers
(copied from the template RNA strand) RNA
nucleotides Zinc abiotic synthesis of
5-40mers (with less than 1 error)
This replication of RNA involves a high degree of
errors and hence a larger diversification. Good
RNAs will be selected.
Campbell 26.11
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Structures of small RNAs
RNA molecules have a genotype (sequence) and a
phenotype (the conformation). The structure is
given by the complementarity leading to
double-stranded regions and single-stranded
loops. Many different structures are possible
when more than three stem loops are present.
The secondary and tertiary structures of an
RNA molecule are crucial for its activity
RNA World 1-4
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How can RNA replicate?
RNA World 1-5
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Protobionts favour natural selection of good
molecules
In this case, there is an interaction between
two molecules (RNA polypeptide).
competition between different RNAs
If protected by a membrane, the active molecules
will be more efficient, since they will react
only with good partners
Campbell 26.13
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II. The RNA World
II.1 RNA can catalyze chemical reactions
One example the splicing of pre-mRNAs is
achieved by RNAs and proteins. The RNAs are the
active players in such reactions.
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Splicing in Eucaryotes
The sequence of transesterification reactions
that splice together the exons of eukaryotic
pre-mRNAs.
Voet. 31-49
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Splicing of pre-mRNAs by the spliceosome
Requires snRNAs (U1-U6) and many proteins. (U3
snRNA does not exist)
RNA World 11-4
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Altman and Cech RNAs can act as catalysts
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II. The RNA World
II.2 Group I introns in Tetrahymena
Tetrahymena thermophila ciliated protozoan 1
macronucleus and 1 micronucleus eukaryote
contains spliced RNAs
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RNA Splicing and Tetrahymena
1977 P. Sharp and others discover that coding
sequences are interrupted by large stretches of
non coding DNA - coding exons - non coding
introns Splicing occurs only in eukaryotes
Splicing provides an important form of regulation
of gene expression 1980 T. Cech studies the
rRNA genes in Tetrahymena - 4 ribosomal RNAs -
2 primary transcripts. One is processed into 3
separate rRNAs A few seconds after
transcription, a 413nt intron is removed from the
6400nt primary transcript Splicing assay
unspliced RNA nuclear protein extracts
salts nucleotides Result Mg2, GTP and the
unspliced RNA were sufficient! The linear
Tetrahymena intron can circularize in presence of
Mg2, without enzymes.
Voet. 31-51
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The Tetrahymena pre-rRNA
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Folded structure of the IVS RNA from Tetrahymena
thermophila.
orange double stranded segments yellow
loops blue involved in long range
interactions gray ends of two exons size 0.4
kb
Scientific American (1986)
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Transcription and splicing in vitro
excised IVS (intron)
Isolated nuclei from Tetrahymena Radiolabeled
RNA was extracted and separated on a
denaturing gel.
Lanes 1-4 incubation time (5-60 min) Lane 5
26S rRNA marker
Cech, Nobel lecture, 1989
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Autoexcision of the IVS in Tetrahymena
In vitro transcription
Deproteination of the RNA
- 413 nt
Removal of free nucleotides
RNA was incubated under splicing
conditions (a32P-GTP, Mg2, salt)
Lanes 1-4 successive washes after
chromatography Lane 5 marker uniformly-labeled
linear IVS RNA
Removal of free GTP by chromatography
Electrophoresis and autoradiography
Cell 31, p.151
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IVS excision is resistant to protease treatment
Not treated
Pronase
Proteinase
MgCl2
circular intron
linear intron
Cech, Nobel lecture, 1989
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How to prove the absence of enzyme?
clone the DNA for the Tetrahymena pre-rRNA
grow in bacteria, purify the DNA
in vitro transcription with pure E. coli enzymes
(no splicing enzymes)
removal of the enzymes
splicing assay the splicing still took place
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The sequence of reactions in the self-splicing of
Tetrahymena group I intron.
3-OH group of a guanine attacks the
introns 5terminal phosphate (The guanine comes
from a distinct molecule)
release of the 5exon and phosphodiester bond
with the free guanine
the newly formed 3-OH group of the
5exon attacks the 5terminal phosphate of the
3exon
the two exons are joined and the intron is
released
the 3-OH group of the intron attacks the
phosphate of the nucleotide (A) that is 15
residues away from the 5 end of the intron
cyclized intron and release of the 5terminal
fragment.
The folding (and therefore the secondary
structure) of the RNA is crucial for these events
Voet. 31-50
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Types of splicing
RNA World 11-2
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II.3 The catalytic portion of Ribonuclease P is
an RNA
RNAse P cuts off the 5end of tRNA
Maturation of tRNA catalyzed by RNAse P. The
5leader sequence is removed and shown as a
dashed ribbon.
RNA World 11-3
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RNAse P RNAs from different species
- Watson-Crick base pairing non-Watson-Crick
base pairing
Min 1 RNA synthetic RNA molecule that still has
the catalytic activity
RNA World 4-2
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RNAse P is a ribozyme
in prokaryotes and organelles of eukaryotes
size in E. coli 377-nucleotide RNA component
(125 kD) 119-amino acid polypeptide (14
kD) The RNA portion is essential for the
cleaving activity of the tRNA precursor
(pTyr), but the protein is necessary to cleave
at least another precursor RNA (p4.5).
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Cell 35. 849-857 (1983)
Nomenclature RNAse P (E. coli) C5
protein M1 RNA RNAse P (B. subtilis) P
protein P-RNA
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RNAse P activity in 10 mM Mg
1. complete RNAse P (E. coli) C5 prot. M1
RNA 2. C5 protein only 3. M1 RNA only 4.
complete RNAse P (B. subtilis) P-prot.
P-RNA 5. complete RNAse P (B. subtilis) 6. P-RNA
only 7. C5 protein P-RNA 8. P-protein M1
RNA 9. no addition (negative control) 10. crude
RNAse P from E. coli ( control)

pTyr precursor to E. coli tRNATyr p4.5
precursor to E. coli 4.5S RNA
-
-


-


-


Splicing activity
precursors
cleavage products
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RNAse P activity in 60 mM Mg
1. complete RNAse P (B. subtilis) P-prot.
P-RNA) 2. C5 prot. P-RNA 3. P-RNA only 4. M1
RNA P-Prot. 5. P-protein only 6. complete RNAse
P (E.coli) C5-prot. M1-RNA) 7. M1 RNA
only 8. C5 protein only 9. no addition (negative
control) 10. crude RNAse P from E. coli (
control)




-


-
-

Splicing activity

precursors
cleavage products
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Mg ion dependance of the M1 RNA reaction
Conditions M1 RNA radiolabelled pTyr
radiolabelled p4.5. 15 ar 37C. Denaturing gel
electrophoresis. Autoradiography.

precursors

cleavage products
1.5 10 20 20 30 30 40 40 50 50 60 60 mM
Mg2
100 50 50 100 50 100 50 100 50 100 50 100 mM
Ammonium chloride
- M1 RNA

crude E. coli RNAse P
M1 RNA is active from 20 mM Mg2 on. Maximum
activity is reached at 50 mM Mg2 M1 RNA does not
cleave p4.5 under these conditions, but the whole
RNAse P does (lane 12)
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Is the RNA the only active portion of RNAse P?
No protein contaminants were found with the
RNA. However traces of protein, that are not
detectable can still be associated with the RNA.
This problem has been solved later by testing in
vitro- transcribed precursor RNAs as
substrates. The protein contaminant must have
survived phenol-SDS extraction. The protein
portion alone has no processing
activity. However The native RNAse P (Protein
M1 RNA) is two times more efficient than the
M1 RNA alone. The M1 RNA does not cleave the
p4.5 RNA while the complete RNAse does. ?his
suggests that the RNAse P is fully functional
only if both the M1 RNA and the protein are
present.