Alexander Chetverin
OVERCOMING CONFORMATIONAL PARADOX:
Template circularization
might prevent the formation of double strands
during RNA replication
Institute of Protein Research of the Russian Academy of Sciences
Pushchino, Moscow Region; alexch@vega.protres.ru
Life: a form of propagation of a genetic material
At present, the simplest imaginable way for
accomplishing this goal is provided by the concept
of the RNA world, because RNA is the only type of
molecules that can serve both as templates and
catalysts for their amplification.
Arguments for the feasibility of the RNA world
1. Nucleotides can spontaneously form under conditions that
existed on the early Earth or a similar planet.
2. Activated nucleotides can spontaneously polymerize into
long (≥ 40 nucleotides) strand.
3. RNA molecules can spontaneously recombine to produce
even longer strands.
4. Pools of random oligonucleotides consisting of 1012 – 1015
molecules (0.01 – 10 μg of a 40 nt-long RNA) always contain
molecules from which one can select ribozymes (RNA
enzymes) with virtually any desired catalytic function.
5. Selected ribozymes can catalyze the synthesis of RNA
strands that are complementary to RNA templates.
A problem not yet solved:
The synthesized complementary strand and the template form
a double helix along the entire length.
Thus, the template and the synthesized complementary copy
are locked in the double helix and therefore unavailable as
templates for the synthesis of more RNA copies.
Hence, no propagation of the genetic material
(NO LIFE) is possible.
Paradox:
“Something inconsistent with
common experience or having
contradictory qualities”
Webster’s Dictionary
Complementary (matching) nucleotides
H
H3C
T
O
H
N
N
H
N
N
A
N
N
C 1'
N
1' C
O
H
C
N
H
O
N
H
N
N
N
1' C
N
G
C1'
N
O
H
N
H
For a complementary strand be synthesized according to the Watson-Crick rules
it must base pair with a template, i.e., be a part of the double helix.
However, to enable further replication, the template and the complementary
copy must remain single stranded, i.e., unpaired.
Conformational paradox:
Double helix is needed
for a template-directed RNA synthesis,
but it prevents RNA amplification
Like the artificial ribozymes, hypothetical replicases
of the ancient RNA world must had encountered
with this problem
Gilbert W. & de Souza S.J. (1999) Introns and the RNA world. In The RNA World,
2nd edn., pp. 221–231, CSHL Press, Cold Spring Harbor, NY.
In Polymerase Chain reaction (PCR),
the paradox is overcome by temperature cycling
Primer
Duplex
Strand
annealing
melting
elongation
(50-60°C)
(>90°C)
(72°C)
Double-stranded DNA
Temperature cycling is not a proper solution
of the conformational paradox in the RNA
world, as it generates another (chemical)
paradox:
divalent cations (Mg2+, Ca2+) are needed for the catalysis of
RNA synthesis at low temperatures,
but they catalyze RNA hydrolysis (depolymerization) at the
high temperatures required for melting RNA duplexes
Replication of the Qβ phage RNA:
a double-stranded intermediate?
Step 1
% от всей РНК
Double-stranded RNA
Partially doublestranded RNA
Infectious (+) strand
Step 2
or
Small (RQ) RNAs
Время, мин
Spiegelman S. et al. (1968) The mechanism of RNA replication.
Cold Spring Harbor Symp. Quant. Biol. 33, 101-124.
There are no double-stranded intermediates
in the Qβ RNA replication cycle
Single strand-specific ribonuclease
Double-stranded RNA
Single-stranded RNA
• All double-stranded and partially double stranded
structures are isolation artifacts: they are induced by
any agent that denature the replicase: phenol,
detergents, or protease.
• Like ribozymes, Qβ replicase cannot use the double
helix as a template.
Weissmann С. et al. (1968) In vitro synthesis of phage RNA: The nature of the
intermediates. Cold Spring Harbor Symp. Quant. Biol. 33, 83-100.
How does Qβ replicase overcome
the conformational paradox?
Possible solution No. 1:
The double helix is unwound by Qβ replicase itself acting like a zipper to
separate the template and the complementary nascent strand, which are then
stabilized in the single stranded conformation by the intramolecular secondary
structure.
Weissmann C. et al. (1968) In vitro synthesis of phage RNA: the nature of the
intermediates. Cold Spring Harbor Symp. Quant. Biol. 33, 83–100.
However:
• The double helix formed by complementary RNA strands are
thermodynamically more stable than are the intrastand secondary structures:
If a mixture of complementary is annealed (melted and then slow cooled),
they are completely converted into double helix.
• Within the replicative complex, the template and the nascent strands are
close to one another, which favors their annealing.
• These stands immediately collapse into the double helix under action of
proteases and detergents that cannot affect the stability of the RNA
secondary structure, but destroy or unfold the protein structure.
Possible solution No. 2:
The unzipped strands are kept from annealing by a single strand-binding protein
that coats the strands along the entire length.
Weissmann C. et al. (1968) In vitro synthesis of phage RNA: the nature of the
intermediates. Cold Spring Harbor Symp. Quant. Biol. 33, 83–100.
However:
The replicative complex remains single-stranded even in a purified cell-free
system that contains no proteins but Qβ replicase.
Possible solution No. 3:
The replicase holds the 3‘ end of the template and the 5‘ end of the nascent strand
during the entire replication cycle. This poses topological constraints to winding the
strands into the double helix.
Weissmann C. et al. (1968) In vitro
synthesis of phage RNA: the nature
of the intermediates. Cold Spring
Harbor Symp. Quant. Biol. 33, 83–
100.
3'
5'
5'
3' Матрица
5'
Растущая цепь
However:
Several nascent strands can simultaneously be synthesized on the same
template strand.
Thach S.S. & Thach R.E. (1973) Mechanism of viral replication. I. Structure of replication
complexes of R17 bacteriophage. J. Mol. Biol. 81, 367–380.
Functional circularity:
The ability of a template to present to replicase its 5′ end,
in addition to the 3′ end, at the initiation step
5'
Template activity of the
genomic RNA of phage Qβ
drastically drops upon its
fragmentation into two
halves.
It looked like Qβ replicase
may sense during initiation
if the template strand is
intact.
3'
The Amphora model: The
template strand could form a
circle if it had complementary
termini capable of base-pairing;
the replicase could then
recognize the terminal helix
(“panhandle”).
Haruna I. & Spiegelman S. (1965) Recognition of size and sequence by an RNA
replicase. Proc. Natl. Acad. Sci. USA 54, 884–886.
Replicable RNAs indeed have complementary termini
5' PPPGGG
CCCAOH 3'
However:
• The complementary stretches are too short (3-4 nt) to form a stable circular
structure.
• Inability of the fragmented template to replicate might be a mere
consequence of the fact that the initiator 3’ end of the complementary strand
cannot be synthesized.
5' PPPGGG
3' HOACCC
CCCAOH 3'
X
GGGPPP 5'
Weissmann C., Billeter M.A., Goodman H.M., Hindley J. & Weber H. (1973)
Structure and function of phage RNA. Annu. Rev. Biochem. 42, 303–328.
All known replicale RNAs are capable of formation a hairpin that
involves the 3‘ и 5‘ terminal structures
Mung
bean RNase
RNase V1
RQ135 RNA
115
120
115
120
Munishkin A.V., Voronin L.A., Ugarov V.I., Bondareva L.A., Chetverina H.V. & Chetverin A.B.
(1991) Efficient templates for Qβ replicase are formed by recombination from
heterologous sequences. J. Mol. Biol. 221, 463-472.
Is there any functional linkage
between the 3’ и 5’ ends of replicable RNAs?
RQ135 RNA
3’ fragment
Point mutations
G →A
5 fragment
Ugarov V.I. & Chetverin A.B. (2008) Functional circularity of legitimate Qβ replicase
templates. J. Mol. Biol. 379, 414-427.
Full-sized product, relative units
Damage to the 5’ terminus results in a drop
of the initial rate of RNA synthesis
Reaction time, s
Ugarov V.I. & Chetverin A.B. (2008) Functional circularity of legitimate Qβ replicase
templates. J. Mol. Biol. 379, 414-427.
Point mutations at the 5’ end increase
the requirement of RNA replication
for the concentration of the initiator nucleotide (GTP)
A
A
AA
GTP concentration, μM
Ugarov V.I. & Chetverin A.B. (2008) Functional circularity of legitimate Qβ replicase
templates. J. Mol. Biol. 379, 414-427.
Mutations at the 5’ end of decrease
the rate and yield of initiation
Full-sized product, relative units
Varied time of
initiation (GTP only)
Initiation stop
(+aurintricarboxylic
acid: ATA)
ATP
Elongation +CTP
UTP
Initiation time (before the addition of ATA), min
Ugarov V.I. & Chetverin, A. B. (2008). Functional circularity of legitimate Qβ replicase
templates. J. Mol. Biol. 379, 414-427.
Mutations at the 5’ end of the template destabilize the
post-initiation replicative complex
+ATA
Varied time of
incubation with ATA
ATP
Elongation +CTP
UTP
Full-sized product, relative units
10-min
initiation
(+GTP)
Time of incubation with ATA, min
Ugarov V.I. & Chetverin, A. B. (2008). Functional circularity of legitimate Qβ replicase
templates. J. Mol. Biol. 379, 414-427.
Thus, the 5’ end of the template interacts with
the 3’ end at the initiation step and thereafter.
Nascent
strand
The terminal helix of the
template might, by itself or
with the assistance of a
replicase molecule, fasten the
template in a circular
conformation and thereby help
to keep the replicative complex
single stranded during the
elongation phase.
Replicase
Terminal
helix
Nascent
strand
Template
Nascent
strand
Similarly, the conformation paradox might be overcome at RNA
replication in the ancient RNA world.
There is growing body of evidence that
various viral RNAs and even eukaryotic
mRNAs form circles.
This feature might be a relic inherited by the
contemporary DNA world from the RNA world
in which a circular structure was a prerequisite
for the ability of genetic material to
propagate.