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HYPOTHESIS
State Key Laboratory of Protein and Plant Gene
Research, School of Life Sciences, Peking University,
China
Please cite this article as:
Xinmiao Fu. Potential protein-encoded synthesis of DNA and RNA.
Hypothesis 2014, 12(1): e6, doi:10.5779/hypothesis.v12i1.366
*Correspondence:
[email protected]
1/5
Received: 2014/02/27; Accepted: 2014/05/12;
Posted online: 2014/09/12
© 2014 Xinmiao Fu. This is an Open Access article
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Potential protein-encoded
synthesis of DNA and RNA
Xinmiao Fu*
mono- and/or dinucleotides are assumed
presence of antibiotics such as neomy- HYPOTHESIS Synthesis of DNA encoded
to be arranged along the characteristic
cin . In addition, regarding the origin of
by TALE proteins TALE proteins are secret- organisms9.
amino acids of TALE and PUF, and then
the central dogma, it is widely believed
assembled as oligonucleotides by ligase
that the codon-amino acid stereochemi- cifically recognize host DNA sequences
ed by bacterial plant pathogens and spe- On the other hand, the code of DNA
or condensation agents. This hypothesis
cal pairing occurred during early evo- via their modular DNA-binding domain of
suggests a new protein-based strategy for
lution and played a crucial role in the
synthesizing DNA and RNA molecules.
current ribosome-mediated translation
INTRODUCTION The central dogma
process, which does not involve the direct
3,4
ful DNA targeting tool utilized in different
recognition by RVDs of TALE repeats
also suggests the possibility that the
tandem repeats9. Each repeat comprises
sequence information of the RVDs in a
around 34 conserved amino acids and TALE protein can be transferred residue
targets a specific base pair by using re- by residue to DNA. Although the minimal
interaction between codons and corre- peat variable diresidues (RVDs) at posinumber of repeats in an artificial TALE
10,11
sponding amino acids5,6,7,8.
tions
12
and
13
(Figure
1B).
Structural
1
Francis Crick in 1958 and later formuto significantly activate gene expression
lated in 19702, is based on extensive In this article, I suggest the possibility that studies have revealed that multiple TALE in cells is reported to be 6.510, it is con-
of molecular biology, as proposed by
ABSTRACT As stated by the central dogma
repeats form a superhelical structure to
ceivable that mono- and dinucleotide
protein can be artificially transferred resi- track along the sense strand of the DNA
due by residue directly to DNA and RNA, duplex within its internal layer and that the
duplexes can be arranged specifically
respectively, based on transcription ac- 12th residue in each repeat stabilizes the
tivator-like effectors (TALE) and Pumilio/ RVD loop while the 13th residue makes
(Figure 1B) based on the following report-
of molecular biology, in living organisms
genetic and biochemical studies on liv- the sequence information encoded by
the sequence information of DNA is trans-
ing organisms and is fundamental to life
ferred to RNA and then to protein, but
sciences. It follows that the sequence
such information cannot be transferred
information of DNA can be self-replicated
back from protein to nucleic acids. In this
or transferred residue by residue to RNA
article, it is proposed that the sequence in-
and then to protein (Figure 1A). However, fem-3 mRNA-binding factors (PUF). This
formation encoded by protein can be artificially transferred back to DNA and RNA,
respectively, based on transcription activator-like effectors (TALE) and Pumilio/fem-3
mRNA-binding factors (PUF). Specifically,
Illustration by Eloïse Kremer
along the RVD loops of TALE repeats
ed observations. First, the minimal num-
a nucleotide-specific contact12. Since
ber of repeats found in naturally occurring
the
DNA
specificity
can
be
designed
by
the sequence information encoded by hypothesis, if proved experimentally, sugTALE proteins is as low as 1.513, indicating
customizable
assembly
of
TALE
repeats,
protein cannot be transferred back to gests a new strategy for the synthesis of
that a mononucleotide base pair alone
TALEN (a TALE protein fused with a nucle- is likely to be sufficient to interact with
nucleic acids. In some special cases, short DNA/RNA molecules.
ase protein) is being deployed as a power- a TALE repeat. Second, each base pair
DNA can also serve as a template in
directing in vitro protein synthesis in the
from the DNA duplex12 was reported to
HYPOTHESIS
Vol.12, No.1 | 2014 | hypothesisjournal.com
HYPOTHESIS
Potential protein-encoded synthesis of DNA and RNA
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Fu
DNA
independently interact with the RVD loop
contains an RNA-binding domain that
of one TALE repeat, also strengthening the
comprises multiple PUF repeats and also
possibility of an interaction between a
forms a superhelical structure to bind the
TALE repeat and the mononucleotide
RNA molecule within its inner concave
base pair. Third, mono- or dinucleotide
surface18. Three-dimensional structural
base pairs, although unable to exist
studies19 revealed that each PUF repeat,
stably in solution , were reported to
though not making direct interactions
14
translation
RNA
protein
(PUF)
TALE repeats
Characteristic residues
NG NN HD NI
Mono- or dideoxynucleotide
base pairs
T G C A A T G C A T G C
A C G T T A C G T A C G
NI NG NN HD NI NG NN HD
(ligation or condensation)
Oligodeoxynucleotide
duplex
be stably formed in the hydrophobic
with either the phosphate backbone or
cavities
cages15,16.
the 2' hydroxyl groups of RNA, recog
Fourth, the apparent dissociation con-
nizes a single RNA base through its
stant between optimized TALE pro-
three conserved amino acids, two of
of
self-assembled
teins and target DNA was reported to
which make hydrogen bonds or Van der
be as low as 0.16 nM17. Lastly, TALE
Waals interactions with the edge of an
repeats were reported to be flexible in
RNA base and a third residue that stacks
conformation to cope with the B-form
with the same base and/or the preceding
conformation of the DNA duplex12.
base. The code of RNA-binding speci-
Together, these observations strongly
T G C A A T G C A T G C
A C G T T A C G T A C G
PUF repeats
suggest that mono- or dinucleotide base
pairs can be arranged along the corresponding characteristic amino acids of
TALE proteins through sequence-specific
ficity by PUF repeats has been decoded
and artificially evolved20,21 (also illustrated
in Figure 1C), by which modular PUF
repeats capable of selectively binding
specific RNA sequences can be created.
Characteristic residues
CQ NQ SE SR CQ NQ SE SR NQ NQ SE SR
interactions as described above. As
The bioinformatics analysis results de-
Mono- or diribonucleotides
A U G C A U G C A U GC
such, these independent but arranged
scribed here indicate that, although a
(ligation or condensation)
base pairs would be easily ligated or
majority contain 8 PUF repeats, many
A U G C A U G C A U G C
condensed as an oligodeoxynucleotide
of the naturally occurring PUF proteins
duplex (as illustrated in Figure 1B). In this
only carry 2 or 3 PUF repeats (Table 1). In
Oligodeoxynucleotides
regard, the sequence information of mul-
particular, the PUF repeats in some PUF
tiple RVDs discontinuously encoded in
proteins are not sequentially connected
the TALE protein is transferred to DNA.
but discontinuously present as discrete
synthesis of RNA by RNA ligase or condensation agents from mono- or diribonu-
Synthesis of RNA encoded by PUF pro-
units of one, two or three repeats (as
from protein (e.g., TALE or PUF) to DNA or RNA is illustrated (highlighted in red
cleotides that are arranged in a sequence-specific manner along the characteristic
teins Similarly, the transfer of sequence
or blue). B Schematic illustration of the synthesis of a DNA duplex by DNA ligase
residues of multiple PUF repeats. The amino acid code of RNA base specificity
information from protein to RNA can
or condensation agents from mono- or dideoxyribonucleotide base pairs that
was adopted from earlier studies
potentially be achieved using PUF, which
Figure 1 | Illustration of the modified central dogma from potential
are arranged in a sequence-specific manner along the characteristic residues of
synthesis of DNA and RNA encoded by protein. A Sequence information
TALE repeats in a designed TALE protein. The amino acid code of DNA sequence
transfer (from DNA to RNA to protein) takes place in nature and is summarized by
specificity was adopted from Bogdanove et al.9. C Schematic illustration of the
the central dogma of molecular biology (black). Sequence information transfer
.
20,21
HYPOTHESIS
exemplified in Figure 1). These observations indicate that the minimal number of
PUF repeats for their stable interaction
Vol.12, No.1 | 2014 | hypothesisjournal.com
HYPOTHESIS
Potential protein-encoded synthesis of DNA and RNA
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Fu
Repeat No.
2
3
4
5
6
7
8
9
Total
Protein No.a
7
38
94
91
167
91
532
4
1024
Ratio (%)
0.7
3.7
9.2
8.9
16.3
8.9
52
0.4
100
Table 1 | Varying number of PUF repeats in naturally occurring PUF proteins.
a
A total of 1024 PUF proteins were found in SMART database (http://smart.embl.de/; date of 2013/6/5).
The number of PUF repeats in each protein was counted and a summary of PUF proteins with different
repeats is presented here.
PUF = Pumilio/fem-3 mRNA-binding factors; SMART = Simple Modular Architecture Research Tool.
dideoxyribonucleotide base pairs due to
and the mono- and diribonucleotides at
blocks. Specifically, if the building block
space hindrance, and thus would not be
a concentration of approximately 1 mM.
is mononucleoside 3',5'-biphosphate,
suitable to catalyze the ligation. An alternative approach to overcome this difficulty is to use chemical agents, such as
cyanogen bromide23, cyanamide24 and
imidazole25, which have been reported
to be able to condense mono- and dideoxyribonucleotide base pairs into oligonucleotide duplexes. Since the base
If polynucleotide phosphorylase is used
for ligation
, a specific diribonucleoside
26,27
(with a free 3'-terminal hydroxyl group)
corresponding to the last two PUF
mono- and diribonucleotides into oligori-
added and incubated with the PUF pro-
agents such as cyanogen bromide31 and
tein. Polynucleotide phosphorylase can
montmorillonite32,33. To this end, mixed
even 1. It follows that the mono- and/or
of the TALE protein to each of these
RVDs of the TALE protein, condensation
diribonucleotides arranged along the
deoxynucleotides can be evaluated
by these agents should be carried out ef-
concave surface of a designed PUF pro-
by commercial systems based on iso-
ficiently due to the entropy reduction.
tein could be assembled as an oligoribo-
thermal titration calorimetry or surface
PUF-encoded synthesis of RNA To syn- oligoribonucleotides by the enzyme.
nucleotide (as illustrated in Figure 1C). As
plasma resonance. According to the
thesize specific single-stranded RNA, the
such, the oligoribonucleotide sequence
early reports12,22 on the interaction be-
gene encoding multiple PUF repeats can
be designed and assembled according
the preliminary conditions used here are
to the principles and methods described
protein.
as follows: TALE at a concentration of
previously20,21, and the PUF protein can
approximately 2 μM and the mono- and
be expressed and purified similarly as re-
dideoxyribonucleotides at a concentra-
ported previously19,21. The binding affinity
tion of approximately 1 mM.
of the PUF protein to various types of
multiple TALE repeats plus the N- and
One approach to assembling these
mono- and diribonucleotides can be eval-
C-terminal signals of TALE can be
mono- and dideoxyribonucleotide base
uated by commercial systems based on
designed
de-
pairs is to use DNA ligase plus ATP and
isothermal titration calorimetry or those
scribed previously (as reviewed by
optimal ligation buffer. However, given
based on surface plasma resonance.
Bogdanove et al.9) and the protein can
that the DNA duplex has been reported
The results of this kind of experiment will
be expressed and purified as described
to be absorbed in the inner surface of
determine the approach and conditions
recently
. Mixed 5'-phosphate mono-
the superhelical structure of the TALE
that are suitable for the subsequent liga-
(4 types) and dideoxyribonucleotides
protein12, DNA ligase bearing a molecu-
tion and condensation. The preliminary
(16 types) are then incubated with the
lar weight of 43 kDa may not be able to
conditions used here are as follows: PUF
access those partially buried mono- and
at a concentration of approximately 2 μM
EXPERIMENTAL DESIGN TALE-encoded
synthesis of DNA To synthesize specific
DNA duplexes, the gene encoding
12,22
and
assembled
as
purified TALE protein for a certain length
The third approach to assemble these
bonucleotides is to use condensation
pairs are aligned along the characteristic
tween TALE and short DNA duplexes,
is not required.
nucleoside 3',5'-diphosphates can be
of time. In particular, the binding affinity
is solely determined by the characteristic
it is dinucleoside pyrophosphate, ATP
repeats of the PUF protein and mixed
with RNA bases may be as low as 2, or
amino acids of PUF repeats in the PUF
ATP should be included for ligation; if
be added to the mixture to initiate the
5'-biphosphate mono- and diribonucleo-
addition of mononucleoside 3',5'-diphos-
sides are incubated with the PUF protein
phates to the diribonucleoside, which will
for a certain length of time before add-
be further elongated, base by base, to
ing these condensation agents and other
crucial chemicals (e.g., metal ions).
A substitute for polynucleotide phos-
SIGNIFICANCE AND CONCLUSION In
phorylase is RNA ligase28,29,30, which is
summary, it is proposed that sequence-
known to catalyze the formation of an
specific DNA and RNA molecules can
internucleotide phosphodiester bond be-
possibly be synthesized according to
tween an oligonucleotide acceptor with
the characteristic amino acid sequences
a 3'-terminal hydroxyl (minimal acceptor
of designed TALE and PUF proteins, re-
being trinucleotides) and an oligonucle-
spectively. In terms of thermodynamics,
otide donor molecule with a 5'-terminal
TALE and PUF proteins arrange or fix the
phosphate (minimal donor being trinucle-
free mono- or dinucleotides, leading to
oside diphosphate, dinucleoside pyro-
a reduction in the entropy of the nucleo-
phosphate and mononucleoside 3',5'-bi-
tides and thus facilitating the subsequent
phosphates). For this purpose, a specific
ligation or condensation. This hypothesis
triribonucleoside (with a free 3'-terminal
is clearly experimentally testable. If
hydroxyl group) corresponding to the
proved, it is of interest to further exam-
last three PUF repeats of the PUF protein
ine whether Trp RNA-binding attenuation
serves as the starting primer, and dinu-
proteins34 and pentatricopeptide repeat
cleoside pyrophosphate or mononucleo-
proteins35, both interacting with RNA in
side 3',5'-biphosphates serve as building
a sequence-specific manner, can serve
HYPOTHESIS
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HYPOTHESIS
Potential protein-encoded synthesis of DNA and RNA
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Fu
a similar purpose as PUF. More impor-
It was proposed recently8 that the third
2 Crick F. Central dogma of molecular biology.
8 Yarus M, Widmann JJ, Knight R. RNA-amino
http://dx.doi.org/10.1007/978-1-4612-5190-3
tantly, proving these possibilities by
characteristic residue of the PUF re-
Nature. 1970;227(5258):561-3.
acid binding: a stereochemical era for the genetic
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supported by research grants from the
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Habrian CH, Zykovich A, et al. Quantitative analysis
The molecular basis for the genetic code. Proc Natl
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of TALE-DNA interactions suggests polarity effects.
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Saxinger WC. On the fundamental nature and evo-
12Deng D, Yan C, Pan X, Mahfouz M, Wang J,
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Zhu JK, et al. Structural basis for sequence-spe-
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2012;335(6069):720-3.
19Wang X, McLachlan J, Zamore PD, Hall TM.
PMid:5237212
http://dx.doi.org/10.1126/science.1215670
Modular recognition of RNA by a human pumilio-
7 Hlevnjak M, Polyansky AA, Zagrovic B.
PMid:22223738 PMCid:PMC3586824
homology domain. Cell. 2002;110(4):501-12.
Sequence signatures of direct complementarity be-
13Boch J, Bonas U. Xanthomonas AvrBs3 family-
http://dx.doi.org/10.1016/S0092-8674(02)00873-5
tween mRNAs and cognate proteins on multiple lev-
type III effectors: discovery and function. Annu Rev
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els. Nucleic Acids Res. 2012;40(18):8874-82.
Phytopathol. 2010;48:419-36.
A universal code for RNA recognition by PUF pro-
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14Saenger W. Principles of Nucleic Acid Structure.
PMid:21572425
The
potential
sequence
information
transferring from TALE and PUF to DNA
and RNA, respectively, however, would
not occur in nature, presumably due to
PUF proteins were found to be less than
National Natural Science Foundation of
and 10 (Table 1), respectively). In
China (No. 31100559 and No. 31270804
other words, it is unlikely that in nature
to X.F.) and the National Basic Research
genetic information is stored in proteins
Program of China (973 Program) (No.
like TALE and PUF, which is then trans-
2012CB917300 to X.F.).
30
13
ferred into nucleic acids. In this regard,
the hypothesis does not challenge the
central dogma.
ABOUT THE AUTHOR Dr. Fu is a protein
scientist focusing on the mechanism
of protein biogenesis, folding, assembly
Nevertheless, the specific amino acid-
and degradation, as well as protein evo-
nucleotide interaction between TALE
lution. His long-term goal is to elucidate
and DNA, or between PUF and RNA,
how the genetic information encoded
may provide new insights into the ori-
by DNA is accurately transferred to the
gin of the central dogma. For instance,
three-dimensional structure information
regarding the origin of the ribosome-
of proteins, which is usually facilitated
mediated translation from mRNA to
by molecular chaperones and folding
protein, a direct codon-amino acid
catalysts in cells.
stereochemical pairing was suggested
to occur during evolution5,6, which later
evolved to the current translation form
that does not involve such direct pairing.
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HYPOTHESIS
Vol.12, No.1 | 2014 | hypothesisjournal.com