Document

Report
MOLECULAR COLONIES:
A plausible form
of compartmentalization
in the RNA world
Alexander Chetverin
Institute of Protein Research
of the Russian Academy of Sciences
Pushchino, Moscow Region
alexch@vega.protres.ru
Oparin was the first who realized that no evolution in the
prebiotic world was possible without a competition and hence
compartmentalization, some form of segregation of
biomolecules from the environment. His 'coagulates' (bits of a
gel) and then 'coacervates' (colloid vesicles) were the first
models of primitive cells.
“The moment when the gel was precipitated or the first
coagulum formed, marked an extremely important stage in the
process of the spontaneous generation of life. At this moment…
the transformation of organic compounds into an organic body
took place. Not only this, but at the same time the body became
an individual… and set itself apart from the environment
surrounding it”.
“Only the most complicated and efficient could grow and
develop, all the rest either ceased to develop or perished”.
Oparin AI (1924) Origins of Life. Moscow, Moskovhii Rabochii. English translation by Ann Synge.
Compartmentalization in liposomes
Growing and division (self-replication) of liposomes
can be coupled to RNA replication
Szostak JW, Bartel DP, Luisi PL (2001) Nature 409, 387-390.
Oberholzer T, Wick R, Luisi PL, Biebricher CK (1995) Biochem. Biophys. Res. Commun. 207, 250-257.
However, replication of liposomes and
self-replication of ribozymes
are hardly compatible
Many ribozymes have optimal activity in the presence of
high concentrations of divalent metal ions. In such
conditions, vesicles composed of acidic phospholipids would
aggregate, possibly interfering with growth and division.
Szostak JW, Bartel DP, Luisi PL (2001) Nature 409, 387-390.
Lipid membrane is a strong barrier for
nucleotide exchange with the surrounding solution
Remained in the liposomes, %
100
ATP (±Mg2+), AMP, ADP
75
ADP + Mg2+
50
AMP + Mg2+
25
2 C13COOH
1 C13COO-Glycerol
50
25
75
ImpA (adenosine-5’phosphorimidazolide)
50
25
2 C13COOH
1 C13COO-Glycerol
100
75
100
75
AMP, dAMP
100
ImpA
3'-NH2-ImpA
2 C13COOH
1 C13COO-Glycerol
50
4 C9COOH
25 1 C9COH
1 C9COO-Glycerol
A
AMP, dAMP
ImpA
3'-NH2-ImpA
Time, hours
Mansy SS, Schrum JP, Krishnamurthy M, Tobé S, Treco DA, Szostak JW (2008) Nature 454,122-125.
Nonenzymic poly(С)-directed synthesis of poly(G)
inside and outside liposomes
Substrate (5 mM 3'-NH2-ImpG) was added to the surrounding solution
Time, hours
30-nt product
15-nt primer
Inside
Outside
Mansy SS, Schrum JP, Krishnamurthy M, Tobé S, Treco DA, Szostak JW (2008) Nature 454,122-125.
Compartmentalization in molecular colonies
Qβ-replicase amplifies RNAs exponentially
Haruna I, Spiegelman S (1965) Science 150, 884–886.
In only 10 min, it produces up to 1010 copies of a single RQ RNA molecule
(a cognate Qβ replicase template)
Qβ replicase synthesizes a variety of RQ RNAs
(for Replicable by Qβ replicase) without the addition
of a template (“spontaneously”)
• Mixing of Qβ replicase with ATP, GTP, CTP and UTP.
• Incubation for 1–3 hours at 37°C.
• Product analysis by gel electrophoresis.
The gel is stained with ethidium bromide
(stains polynucleotides, but not mononucleotides).
223 nt
120 nt
Eigen: RQ RNAs are generated de
novo (self-originate)?
Sumper M, Luce R (1975) Proc. Natl. Acad. Sci. USA 72, 162–166.
Biebricher CK, Eigen M, Luce R (1981) J. Mol. Biol. 148, 369–390.
Biebricher CK, Eigen M, Luce R (1986) Nature 321, 89–91.
Louis Pasteur, 1860:
No life could arise in a boiled meat broth unless solid particles
heavier than air were allowed to enter
Pasteur’s “swan” flask
Detection of airborne RQ RNAs
•
•
•
Qβ replicase- and NTP-containing agarose is poured into 2 dishes
The dishes are incubated for 1 hour: one dish is closed, the other is open
The agarose is stained with ethidium bromide
Closed dish
Open dish
Chetverin A, Chetverina H, Munishkin A (1991) J. Mol. Biol. 222, 3-9
Molecular colonies (nanocolonies, “polonies") provide
for compartmentalization of biochemical reactions in
the absence of membranes
Molecular colonies form when template nanomolecules (DNA or RNA) are amplified in an
immobilized medium whose polymer matrix possesses pores in the nanometer range
Each colony is made up of clustered copies of one parental molecule (a molecular clone)
Chetverin A, Chetverina H, Munishkin A (1991) J. Mol. Biol. 222, 3-9
Selection of RNA molecules for the ability to replicate
Membrane
with NTPs
Nonreplicable RQ RNA fragments
5' fragment
3' fragment
Reacting
sequences
Agarose with Qβ replicase
Recombination
Replicating RNA
One
fragment
Two
fragments
Chetverin A, Chetverina H, Demidenko A, Ugarov V (1997) Cell 88, 503-513.
Growing DNA colonies by carrying out
Polymerase Chain Reaction (PCR) in a
polyacrylamide gel
Reaction well with a
dry polyacrylamide gel
14 mm
Gel swelled
under cover slip
Gel secured the with
adhesive foil
Gene amplification in molecular colonies
109
Obelin gene
(756 bp)
108
107
108
GFP gene
(1570 pb)
107
106
0
30
100
Number of DNA molecules seeded
Number of DNA
molecules in a spot
• Nearly all seeded molecules produce DNA colonies
• Each colony contains up to 108 copies of a gene
Samatov TR, Chetverina HV, Chetverin AB (2005) Nucleic Acids Res. 33, e145.
Gene expression in molecular colonies: Transcription
Drying the PCR gel
Soaking in a transcription cocktail
Incubation
(2 hours at 37°C)
Signal intensity increases as a
results of transcription
After PCR
After transcription
Samatov TR, Chetverina HV, Chetverin AB (2005) Nucleic Acids Res. 33, e145.
Gene expression in molecular colonies:
Transcription + Translation
Number of seeded
DNA molecules
Protein synthesis was monitored by
GFP (green fluorescent protein) fluorescence
30
100
1
2
Reaction time, hours
Since genes are co-localized with their expression product,
they can be selected according to properties of the synthesized proteins
Samatov TR, Chetverina HV, Chetverin AB (2005) Nucleic Acids Res. 33, e145.
Relevance to the problem of the origin of life
Molecular colonies can perform all essential functions of a living cell,
including replication of the genetic material and its expression
(transcription and translation), and can undergo evolution.
Unlike liposomes, compartmentalization is achieved because of a
relatively low rate of diffusion of macromolecules in a porous matrix as
compared with low-molecular substances.
RNA colonies similar to those generated in Qβ replicase-containing
agarose might have served as a pre-cellular form of
compartmentalization in the RNA world.
Instead of agarose, RNA colonies might be formed in a wet
clay or another porous minerals existed on the Earth.
Could life originate in a clay?
1940s: Clay minerals might play an important role
in the early chemical processes leading up to
the origin of life
Minerals could concentrate and catalytically transform
organic molecules toward biofunctionality and confer on
them structural properties such as chirality.
Goldschmidt VM (1947) Geochemical Aspects of the Origin of Complex Organic
Molecules on the Earth, as Precursors to Organic Life. Published: New Biology 1952,
12, 97-105.
Bernal JD (1949) The physical basis of life. Proc. Phys. Soc. 62, 537-558.
Properties of layered clays
Clay mineral
Repeating layers
Density,
g/cm3
Specific surface
area, m2/g
Ion exchange
capacity, meq/g
Kaolinite
Kaolinite type:
1 tetrahedral
1 octahedral
2.60-2.68
8-20
0.03-0.15
Montmorillonite
Smectite type:
2 tetrahedral
1 octahedral
2.35-2.70
600-800
0.8-1.5
Yeremin NI (2004) Non-metallic minerals, 2nd ed. Publishing House of Moscow University.
Montmorillonite structure
Si
O
H
Al
Layer spacing 1-2 nm;
increases several fold
upon hydration
Montmorillonite is a safe haven for organic molecules
Not only it prevents decomposition of methanol under “black smokers”
conditions (300 °C, 100 MPa ), but also promotes synthesis of diverse
organic compounds that may be precursors to biomolecules.
Williams LB, Canfield B, Voglesonger KM, Holloway JR (2005) Geology 33, 913–916.
Provides a 3-fold protection of the adsorbed RNA from the UV light
(judging by retention of a ribozyme activity).
Biondi E, Branciamore S, Maurel MC, Gallori E (2007) BMC Evol. Biol. 7, S2.
Has been found in meteorites and on Mars.
Poulet F et al. (2005) Nature 438, 623–627.
Served as a “primordial womb” for the life?
Montmorillonite can concentrate nucleotides
from even very dilute solutions
• Within 24 hours at 25°C, рН 7, 50 mg (ca. 20 μL) of dispersed
clay absorbs 10% (in the presence of Na+) to 90% (in the
presence of Mg2+) of AMP dissolved at a 15 μM concentration
in 1 L (a 50,000-fold greater volume) of water.
• Hence, the concentration of the nucleotide in the clay
becomes up to 50,000-fold higher, i.e., ca. 750 mM.
Ferris JP, Ertem G, Agarwal VK (1989) Orig. Life Evol. Biosphere 19, 153−164.
Na+
Ca2+
Mg2+
Montmorillonite
strongly binds
RNA or DNA,
- doubleespecially
stranded RNA
single stranded
and in the
presence of
divalent cations
Franchi M, Ferris P, Gallori E (2003) Orig. Life Evol. Biosph. 33, 1–16.
Montmorillonite catalyzes synthesis of >40 nt-long oligo(A)
Reaction time, days (at25°C)
2
4 6 8 14
• In the absence of montmorillonite, the
primer is mostly elongated by 2
nucleotides (maximally 10) due to a rapid
hydrolysis of the activated substrate.
• In the presence of montmorillonite,
condensation is 100-1000 times faster
than hydrolysis, whereas only 10 times
faster in its absence.
Substrate:
15 mM adenosine-5’phosphorimidazolide, added daily
Primer:
[32P]dA(pdA)8pA, adsorbed on
montmorillonite
Ferris JP, Hill AR Jr, Liu R, Orgel LE (1996) Nature 381, 59-61.
With 1-methyl adenine as activating group, 40 nt-long
oligo(A) is formed on montmorillonite within 8 hours,
without any primer or additional substrate portions
72
48
24
8
2
The correct 3'-5' inter-nucleotide
linkages are preferentially formed
Reaction time, hours
Huang W, Ferris JP (2006) J. Amer. Chem. Soc. 128, 8914-8919.
Increase in optical density at 400 nm
Montmorillonite particles increase 100-fold
the rate of liposome formation…
+ Montmorillonite
Time, min
Hanczyc MM, Fujikawa SM, Szostak JW (2003) Science, 302, 618-622
…and become incorporated inside the liposomes,
along with the adsorbed RNA
Fluorescently labeled RNA
Hanczyc MM, Fujikawa SM, Szostak JW (2003) Science 302, 618-622.
Thus, montmorillonite is able to:
● concentrate from the surrounding solution and stabilize activated ribonucleotides;
● catalyze polymerization of the ribonucleotides;
● provide for a certain chiral homogeneity of the synthesized polyribonucleotides
(RNA);
● immobilize polyribonucleotides (RNA), both templates and their copies;
● bind single-stranded RNA more strongly than double stranded one (like a single
strand-binding protein), which stabilizes the state in which replication is possible;
● compartmentalize RNA in the absence of lipid membranes (provide for the
formation of RNA colonies);
● keep different RNA species together, thereby providing for the creation of mixed
colonies and for their joint inheritance (by virtue of being adsorbed on the same
clay microparticle);
● form liposomes around RNA colonies (a new role at a later stage of evolution).
Lipid membranes originated subsequent to peptides?
Hydrophilic surface
Hydrophobic surface
Antimicrobial peptides are short (20 to 40 amino acid residues)
amphipathic α-helical peptides able to form pores across lipid bilayers
Conclusions
Molecular colonies could become an experimental model for de novo creation of
living cells.
They are functional equivalents of cells, provide for assembling cellular
components and checking which variations of the assembly ensure the full
expression of the genome:
• provide for compartmentalization of biological macromolecules,
• are able to perform all biochemical reactions that make up the gene expression,
• allow reaction components to be added or removed by simply soaking the gel
(due to the absence of a membrane),
• link the genotype and phenotype as necessary for natural selection,
• are capable of evolution, i.e., the formation of new genetic material which can
then be expressed.
Molecular colonies growing in clay might have been a form of
compartmentalization at an early step of evolution in the RNA world and during
development of the protein world.
Principal collaborators:
H.V. Chetverina
T.R. Samatov
Financial support:
• Program “Molecular and Cell Biology”
• Russian Foundation for Basic Research

similar documents