RNA-världen: forntid i nutid med framtid
Leif A. Kirsebom
Livets ursprung
Virtanen & Kirsebom, NFRs årsbok 1997
Livets ursprung
history of life www.carolguze.com
Vad kom först?
Livets ursprung – förutsättningar?
http://natgeo.se/vetenskap/rymden/jordens-guld-kommer-fran-asteroid
http://www.apg29.nu/index.php?artid=5019
Centrala dogmat - Hur ser det ut idag
Ribonucleotide
reductase!
Virtanen & Kirsebom, NFRs årsbok 1997
Genetiska koden
RNA – genotyp och fenotyp
Vad kom först?
RNA possess both genotype - represented by a sequence
that can be copied by a polymerase - and
phenotype - represented by some functional trait that varies
according to sequence.
RNA-världen (definition)
1. RNA-världens genetiska material var RNA
2. Basparning enl. Watson-Crick-modell
3. Genetiskt nedärvda proteiner deltog inte
som katalysatorer
• Evidence?
"Molecular fossils"
Molekylära fossiler:
En idag förekommande struktur eller funktion
som har ett gammalt ursprung och som ger
oss en ledtråd om de tidigaste formerna av liv
Struktur:
basen adenin
Funktion: reaktionsväg
ribos (RNA) blir deoxyribos (DNA)
ribosomen (RNA gör protein)
"Molecular fossils"
• Coenzyme – e.g. NAD/ NADH; FAD
Redox reactions/ energy
• Metabolites – e.g. thiamin pyrophosphate,
tetrahydrofolate, pyridoxal phosphate - Riboswitches Nature 441, 1167-1171 (2006)
• Histidine – only amino acid where the
synthesis start with a nucleotide
Adapted from Yarus M Cold Spring Harb Perspect Biol 2011;3:a003590
Små RNA molekyler:!
!
hammarskallestrukturen är katalytisk
RNA genetiskt material – RNA (viroids, virosoides, virus)
RNA katalytiskt!
Virtanen & Kirsebom, NFRs årsbok 1997
RNA katalytiskt – Större
RNase P och andra
Virtanen & Kirsebom, NFRs årsbok 1997
RNase P
Domains and Activity
• Splicing
• Telomerase
• Riboswitches
• Ribosomen etc
Reiter et al. Nature 2010
Metal(II) joner!
RNase P - Domains and Activity
RNA och proteinsyntes
Virtanen & Kirsebom, NFRs årsbok 1997
tRNA, the Genetic code, the Adaptor Hypothesis
(1954/55) and the RNA tie club
Phe
phenylalanine
3'
Phe-tRNA
Phe
5'
T-loop
D-loop
• Pre-tRNA – RNase P RNA (katalytisk)
Anticodon
loop
• Protein synthesis
• Genome replication
e.g. plant virus, RNA primer (bakterie
replikation), retrovirus (HIV), telomers etc
• Synthesis of chlorophyll a
(from eight δ-aminolevulinate)
"Molecular Mimicry"
EF-Tu-tRNA
Shape
Electrostatics
EMBO J 13, 3661-68 (1994)
Czworkowski et al.
EMBO J 19, 489-95 (2000)
Nissen, Kjeldgaard, Nyborg
J Mol Biol 303, 593-603 (2000)
Laurberg et al.
EF-G
RNA-världen:
forntid i nutid med framtid
Virtanen & Kirsebom, NFRs årsbok 1997
"Primordiala soppan"
Darwin letter to his friend and supporter Joseph Hooker
..that life may have taken place in a ”warm little pond,
with all sorts of ammonia and phosphoric salts, lights,
heat, electricity, etc. present, so that a proteine
compound was chemically formed ready to undergo
still more complex changes”.
Citation taken from
RNA: Life’s indispensable molecule
J. Darnell
1. Små organiska föreningar
ex.vis. formaldehyd, aminosyror, adenin, socker
2. Oorganiska föreningar, atomer, molekyler
t ex metalljoner, mineraler, lera
miller-urey-experiment blogs.nature.com
Oparin (1928)
Evidence för en RNA-värld
Byggstenar RNA – ett måste
"Pyrimidine ribonucleotides can be
formed in a short sequence that bypasses
free ribose and the nucleobases, and instead
proceeds through arabinose amino-oxazoline
and anhydronucleoside intermediates."
Matthew W. Powner, Béatrice Gerland and John D. Sutherland
Nature 459, 239-42 (2009)
nNTPs
R + P
RPclosed
RPopen
nPPi
RNA
Evidence för en RNA-värld
Evolution in the test tube - SELEX
Joyce. (2002), Nature 418, 214
Virtanen & Kirsebom, NFRs årsbok 1997
Evolution in the test tube - SELEX
Syntetisk DNA pool
1
T7 promoter
5'
Konstant sekvens
Slumpad sekvens
3'
Konstant sekvens
PCR
Aptamer
Klon
ds DNA pool
PCR
Transkription
RNA
RNA
RT
Binding
Selektion
Anrikning
RNA
Evolution in the test tube - SELEX
Syntetisk DNA pool
1
T7 promoter
5'
Konstant sekvens
2
Slumpad sekvens
3'
Konstant sekvens
PCR
Aptamer
Klon
ds DNA pool
PCR
Transkription
RNA
RNA
RT
Binding
Selektion
Anrikning
RNA
Evolution in the test tube - SELEX
Syntetisk DNA pool
1
T7 promoter
5'
Konstant sekvens
2
Slumpad sekvens
3'
Konstant sekvens
PCR
3
Aptamer
Klon
ds DNA pool
PCR
Transkription
RNA
RNA
RT
Binding
Selektion
Anrikning
RNA
Evolution in the test tube - SELEX
Syntetisk DNA pool
1
T7 promoter
5'
Konstant sekvens
2
Slumpad sekvens
3'
Konstant sekvens
PCR
3
Aptamer
Klon
ds DNA pool
PCR
Transkription
4
RNA
RNA
RT
Binding
Selektion
Anrikning
RNA
Evolution in the test tube - SELEX
Syntetisk DNA pool
1
T7 promoter
5'
Konstant sekvens
2
Slumpad sekvens
3'
Konstant sekvens
PCR
3
Aptamer
Klon
ds DNA pool
PCR
RNA
5
Transkription
4
RNA
RT
Binding
Selektion
Anrikning
RNA
Evolution in the test tube - SELEX
Syntetisk DNA pool
1
T7 promoter
5'
Konstant sekvens
2
Slumpad sekvens
3'
Konstant sekvens
PCR
3
6
Aptamer
Klon
ds DNA pool
PCR
RNA
5
Transkription
4
RNA
RT
Binding
Selektion
Anrikning
RNA
Evolution in the test tube - SELEX
Syntetisk DNA pool
1
T7 promoter
5'
Konstant sekvens
2
Slumpad sekvens
3'
Konstant sekvens
PCR
3
6
Aptamer
Klon
ds DNA pool
PCR
RNA
5
7
4
Transkription
RNA
RT
Binding
Selektion
Anrikning
RNA
Evolution in the test tube - SELEX
• Searching for sequencing space for new functional RNA
sequences.
• Altering the ratio active/inactive sequences.
• Complete coverage N = number of nucleotides
randomized ≈25!
• Compared to for example N = 220, which equals
10117 variants!!!!! Impossible therefore…..
• ….when longer randomized sequences are used it is
a question of probability to score!!
Evolution in the test tube - SELEX
• RNA replicase/ transcriptase
• Binding of various small ligands – e.g. ATP
• RNA catalyzed synthesis of nucleotide
• RNA catalyzed synthesis of nucleotide
• RNA cleavage RNA
Joyce. (2002), Nature 418, 214
Copy RNA - RNA polymerases
RNA based polymerase
≈40 kDa (120 nts)
Protein based polymerase
≈400 kDa
Active site/ Catalytic centre
nNTPs
R + P
RPclosed
Shechner and Bartel (2011) Nat Struc Mol Biol 18, 1036
RPopen
nPPi
RNA
Vassylyev et al. (2002) Nature 417, 712
RNA och proteinsyntes
Virtanen & Kirsebom, NFRs årsbok 1997
Aminoacylering av RNA med RNA
Substrate
5'
3'
G
C
C
U
3'
C
G
G
+
Produkt
U
G
5'
RNA katalysator
Yarus et al., Proc Natl Acad Sci USA (2010)
≈1 800 000-fold down
RNA mediated cleavage of RNA
• Identification of a complex consisting of seven nucleotides
that results in RNA mediated cleavage in the presence of
Mn2+
Substrate
5'
3'
G
A
A
A
Mn2+
+
3'
U
U
U
5'
Produkt
RNA katalysator
Kazakov and Altman Proc Natl Acad Sci USA 89, 7939-43 (1992)
En RNA molekyl mer än en funktion
Genombrottsmolekylen: "One RNA two folding
hur skapades den?
possibilities"
Virtanen and Kirsebom (1997) NFR årsbok
Hur många
förändringar?
Schultes and Bartel Science 289 (2000)
En RNA molekyl mer än en funktion
Genombrottsmolekylen: "One RNA two folding
hur skapades den?
possibilities"
Virtanen and Kirsebom (1997) NFR årsbok
Få!!!!
Schultes and Bartel Science 289 (2000)
Compartmentalization?
Ribonucleotide
reductase!
Szostak et al. (2001), Nature 409, 387
Till slut så fanns det en RNA-värld!
prebiotisk kemi, pre-RNA värld
Virtanen & Kirsebom, NFRs årsbok 1997
Tidsaspekten!
Framtiden
Other
functions
Diagnostics
Virulence
Drug
targets
Regulators
Host
defence
Tillämpning
"RNA and disease"
"RNA and disease"
Infectious diseases - A global threat
Multi resistant
bacteria – e.g. MRSA
Tuberculosis
Globalization and spread
Climate change
Emerging diseases Buruli ulcer
Leptospirosis
Virus infections HIV, Influenza,
SARS
"RNA and disease"
Protein
syntesen
"RNA and disease"
New Findings –
Antibiotics and RNA
New possibilities and strategies
• target should be unique and
essential for the pathogen
• in bacteria there are many!
Binding of paromomycin and Me2+
overlap!
Mikkelsen et al. (2001) Nature Struc. Biol. 8,
510-516.
RNA-världen:
forntid i nutid med framtid
Thank you!
