REASSIGNMENT OF CODONS FROM ARG TO ALA BY MULTIPLE TRNAALA
VARIANTS
Kazuaki Amikuraa and Daisuke Kigaa,baDepartment of Computational Intelligence and Systems Science, Interdisciplinary Graduate School of
Science and Engineering, Tokyo Institute of Technology, Midori-ku, Yokohama-shi, Kanagawa
226-8503, Japan: E-mail: kiga@sb.dis.titech.ac.jpbEarth-Life Science Institute, Tokyo Institute of Technology, 35 Meguro-ku, Tokyo 152-8551, Japan(Received: April 4, 2013; Accepted: June 17, 2013)
Abstract
We have recently developed “simplified genetic
codes” in which only 19 amino acids are assigned to
one sense codon each. In one simplified code, forexample, the UGG codon for tryptophan isreassigned to alanine (Ala) by a tRNAAla variant. We
have previously generated such codes each of which
was constructed using only one kind of tRNA variant.
In this study, we describe a novel simplified genetic
code in which the codons of Arg were reassigned to
Ala using 3 kinds of tRNAAla variants. The usage ofmultiple tRNA variants will be important to constructmore simplified genetic codes containing less than 19
amino acids. A Simplified genetic code will provide
not only new insights into primordial genetic codes,
but also an engineering tool for the assessment ofprotein evolution.

amino acid. This tRNA-dependent conversion, in thecase of synthesizing asparagine, is the sole pathway
for asparagine biosynthesis in Deinococcus
radiodurans and Thermus thermophilus [6, 7].
When all proteins were synthesized from aprimordial genetic code encoding fewer than 20
amino acids, was an organism able to survive using
only these proteins? In other words, were these
proteins able to construct a genetic code and toachieve all essential biological functions? The
characterization of so-called “simplified proteins”
supports the notion that not all the 20 canonicalamino acids are required in order to construct a
protein with biological function and structure.
We recently proposed that “simplified genetic
codes” that encode less than 20 amino acids will bean effective tool to prepare a simplified protein [8].
Though addition of a single tRNA variant wasenough to creation of a simplified genetic code with
19 amino acids, addition of multiple tRNA variants isrequired for creation of a more simplified genetic
code encoding fewer than 19 amino acids requires..
In this work, we show that multiple tRNAAla variants
reassign codons to amino acids to construct a
simplified genetic code lacking Arg.

(Keywords)
simplified genetic code, amino acids, engineering,
evolution, tRNA, aminoacyl-tRNA synthetase,anticodon, translation, cell-free, rewiring
Introduction
Transfer RNA achieves the correspondence
between 20 amino acids and 61 codons in a genetic
code in almost all known organisms[1]. Each aminoacid is bonded to its cognate tRNA(s) by a distinct
aminoacyl-tRNA
synthetase(aaRS).
These
assignments are also achieved by functional
interaction of aaRS, tRNAs, 20 amino acids, andother components of the translational apparatus. EachtRNA is bonded to its cognate amino acid by adistinct aaRS.
It is generally accepted that the number ofamino acids in the genetic code has increased throughevolution from fewer than 20 amino acids throughevolution [1, 2]. Phylogenetic analysis ofaminoacyl-tRNA synthetases suggests a way to add
amino acid into the genetic code [3]. All known
eukaryotes and a small number of bacteria have 20
canonical aaRSs to directly synthesize cognateaminoacyl-tRNAs. On the other hand, all known
archaea and most bacteria do not have at least one of
the 20 canonical aaRSs [4, 5]. Therefore, only 19
amino acids are directly connected to cognate tRNAs
by cognate aaRS. Instead of the direct
aminoacylation, the amino acid except the 19 aminoacids is indirectly connected to cognate tRNA. In thiscase, first, the amino acid is connected to a
non-cognate tRNA by a non-cognate aaRS. Second,
the non-cognate amino acid moiety on themischarged tRNA is transformed into the cognate

Viva Origino 41 (2013) 20 - 23
© 2013 by SSOEL Japan

Material and Method
DNA constructs
For the expression of His-tagged ras, we used
the pK7 plasmid containing the gene [9]. An
expression
plasmidforchloramphenicolacetyltransferase with an N-terminal His tag wasconstructed in our previous study [8]. Genes
encoding tRNA variants were cloned into thepUC119 plasmid (Takara, Shiga, Japan). Each tRNAvariant is created by site-directed mutagenesis from aplasmid encoding tRNAAlaCCA [8]. In the mutagenesis
experiments, polymerase chain reaction (PCR)
amplification was performed with KOD-plus(Toyobo,
Tokyo, Japan) using the following PCR conditions: 2
min at 94°C for 1 cycle, followed by 20 cycle of 0.25
min at 94°C, 0.5 min at 65°C and 3.2 min at 68°C.
Preparation of tRNA transcript
The tRNA variants were prepared by run-off
transcription using T7 RNA polymerase and a
PCR-amplified linear DNA template. The variants
are purified using 8% urea polyacrylamide gelelectrophoresis in a gel composed of TBE(Tris-EDTA-Borate Buffer; 5×) 8 ml, urea (19 g),
and 40% acrylamide gel (29:1) 8ml. After denaturing
PAGE, the band located by UV shadowing was cut
out and was allowed to rotate for 12 hours with 1 ml

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of 0.3 M NaCl.. Finally, the overnight product issterilized by Millipore filtration (0.45 µM pore size).
Cell-free protein expression
The Escherichia coli S30 cell-free protein
synthesis method was used in this study. The
composition of the cell-free protein synthesis reaction
has been previously described [10], except for theomission of a specific amino acid, and the addition of
the tRNA variant and 5.0 µM a.a.-SA(5'-O-[N-(L-a.a.)sulfamoyl]adenosine
aminoacyl
adenylate analogs, Integrated DNA Technologies,
Coralville, IA). The S30 extract was prepared fromthe E.coli BL21 (DE3) strain. The batch mode was
employed, with 20-µl reaction volumes and areaction time of 1h.
Detecting the radiolabeled products
Translation of CAT and Ras was performed
using the 20 µl scale batch mode of synthesis at 37°C
for 1 h with the components described above, except
for the addition of [14C] Leu. The non-purified
products were analyzed on 12% Bis–Tris gels with
MES running buffer (50 mM MES, 50 mM Tris–base,
3.47 mM SDS, 1.0 mM EDTA, pH 7.3). Scanningwas performed using an image analyzer, FLA-5000(Fujifilm, Tokyo, Japan), and an imaging plate,
BAS-IP MS 2040 (Fujifilm, Tokyo, Japan), tomeasure the radioactivity of the products.

Fig.1. Strategy for constructing a simplified genetic code.
(A) The anticodon stem loop sequence of the tRNAAla
variant that was designed to translate the CGC codons for
Arg on mRNA. (B) Schematic view of the simplified
genetic code in the E. coli S30 cell-free translation
mixture. The tRNAAla variant, with its anticodon
corresponding to the CGC codon of Arg, assigns Ala tothe codon. Arg is removed from the translation mixture.
Arginyl-tRNA synthetase (ArgRS) is inhibited by
Arg-SA.
Table 1. Oligonucleotides used to construct tRNAvariants.

Results
Construction of Arg-lacking simplified genetic codeby a combination of tRNAAla variants.
Using multiple tRNAAla variants, we expanded
our previous strategy for constructing a simplified
genetic code. We designed 3 tRNAAla variants withthe anticodon loop corresponding to Arg[8](Fig.1A).
In the universal genetic code, 6 codons, with thesequences CGN and AGR, are assigned to Arg. We
first unassigned those 6 codons by removing Arg
from the E. coli S30 cell-free translation mixture andadding Arg-SA, a strong inhibitor of arginyl-tRNA
synthetase (ArgRS) We then constructed 3 tRNAAla
variants, which had the anticodons GCG, UCG, andUCU, respectively. We named these tRNA variants
AR_GCG, AR_UCG and AR_UCU respectively. Ala
is considered to be attached to these tRNAAla variantsby alanyl-tRNA synthetase (AlaRS), because AlaRS
does not recognize the anticodon loop [11-13].
Therefore, the tRNAAla variants can assign Ala to Arg
codons (Fig.1B).
The tRNA variants is used in constructing asimplified genetic code are prepared by traditional
biochemical methods. From a tRNAAlaCCA-expressing
construct, each tRNA variant-expressing construct
was generated by site-directed mutagenesis. The
oligonucleotides necessary to create the plasmid bythese methods are shown in Table1. The tRNAvariants were prepared by run-off transcription using
T7 RNA polymerase and a PCR-amplified linear
DNA template. The transcription products were
purified by urea polyacrylamide gel electrophoresis(see Materials and Methods). The AR_GCG (3.2nmol), AR_UCG (2.9 nmol), and AR_UCU(2.9nmol)tRNA variants were produced from 150-µl reaction
solution. These results showed that the efficiency oftranscription by T7 RNA polymerase was not
significantly affected by the anticodon sequence.
In order to produce the 6 unassigned CGN and AGR
codons that encode Arg in the universal code, weremoved Arg from the mixture and added Arg-SA to
the mixture. Arg-SA inhibits the activity of ArgRS
that is required for protein synthesis. We confirmedthat protein synthesis was reduced depending on theconcentration of Arg-SA, with a range of 0.05 µM to
5 µM in the translation mixture (Fig.2A, lane 3-5).
Finally, to reassign Ala to the unassigned codons, we
added the 3 tRNAAla variants to the cell-freetranslation mixture with the 6 unassigned codons.
Protein synthesis was increased depending on the
addition of the tRNA variant mixture (1 µM each;Fig. 2B, lane 4). Based on this, we concluded that an
Arg-lacking simplified genetic code was created by
the 3 tRNAAla variants. Additionally, our results showthat the Arg-lacking simplified genetic code canproduce protein efficiently, on par with the universal
genetic code (Fig. 2B, lane 1 and 4).

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between the a.a.-SA of the amino acid and its cognate
aaRS. If the inhibition constant of a.a.-SA is higher
than the Michaelis constant of a.a.-AMP, we will
have to add more of the a.a.-SA corresponding to theamino acid.
Our results with respect to constructing asimplified genetic code using multiple tRNA variants
pave a new path to create a more simplified genetic
code with less than 19 amino acids. By removal of
multiple amino acids from the genetic code, we candesign various simplified genetic codes eachcontaining sets of amino acids which are considered
as elements of a hypothetical primordial code. For
example, 10 amino acids obtained from prebiotic
biosynthesis were considered to be encoded in theprimordial genetic code proposed in phase 1 of thecoevolution hypothesis [15, 16]. From otherviewpoints,thephylogenetic
analysisofaminoacyl-tRNA synthetases suggested the kinds ofamino acids that may be present in the primordial
genetic code [17, 18]. Since there are many otherhypothesis about the kinds of amino acids in theprimordial genetic code in each stage of evolution
before commonotes, we refer to the reports by
Trifonov, which summarizes various studies ofsimplified genetic codes [19].
We suggest that the creation of a simplified
protein, as well as the construction of a simplified
genetic code, provides new insights to assess earlylife forms. Although we do not know whether these
simplified proteins are able to achieve all essential
biological functions, it is generally accepted thatearly life forms used only simplified proteins thathave produced by a simplified genetic code [19, 20].
Previous studies showed a functional protein
composed of 9 kinds of amino acids [21]. In otherstudies, many simplified proteins with greaternumbers of amino acids have been created [22, 23].
However, these previous experiments are not enoughto show that an organism can live with a primordial
genetic code that employs less than 20 amino acids.
Thus, we consider it is necessary to create sets ofsimplified proteins that can realize an essential
biological function such as energy metabolism andassignments by aaRSs.
The simplified genetic code with the removalof multiple amino acids will be a more effective tool
to create a simplified protein. Previously, the creationof a simplified protein by selection from a randommutagenesis library using the universal genetic code
has been frustrated by the reappearance of codons ofan amino acid that is required to be removed from thelibrary. When removing many amino acids from aprotein, the probability of such reappearance
becomes higher. In contrast, with the use of asimplified genetic code, every protein sequencetranslated from any mRNA in the library does notinclude the eliminated amino acids. The tRNAcomposition versatility that we have demonstrated by
the usage of multiple tRNA variants in this study willbe crucial for the creation of a more simplified
protein that encodes fewer than 19 kinds of aminoacids.

Fig.2. (A) Validation of the activity of Arg-SA. Ras wastranslated under the conditions noted at the top of eachlane. (B) Reassignment of unassigned codons by 3tRNAAla variants. CAT was translated under the conditions
noted at the top of each lane. An autoradiogram of apolyacrylamide gel, with the products labeled with [14C]
Leu, is shown.

Discussion and Conclusion
We describe an Arg-lacking simplified genetic
code in which multiple tRNAAla variants are used.
This represents our strategy of constructing asimplified genetic code to have the versatility toreassign amino acids. Using a set of tRNA variants
that correspond to anticodons of multiple amino acids,
we will be able to construct a more simplified genetic
code which encodes fewer than 19 kinds of aminoacids. Furthermore, we can construct various
simplified genetic codes using a combination oftRNAAla and tRNASer. Our previous work showed thattRNASer variants could be used to construct a
simplified genetic code [8].
In addition to this work, previous work on a
Trp-lacking code and a Cys-lacking code (personalcommunication) produced unassigned codons by theaddition of a.a.-SA, in the range of 0.5 to 5 µM. We
will be able to produce unassigned codons for otheramino acids by the addition of 5 µM a.a.-SA
corresponding to the amino acid that we require to
remove from the genetic code. However, for various
reasons, we might have to change the concentration
depending on the kind of the amino acid. For
example, the proportion of each amino acid removed
from the translation mixture by dialysis might bedifferent. A few amino acids are known to be
produced in the metabolism of S30 cell-free extracts[14]. When the amino acid is only partly removed,with a significant quantity remaining in thetranslation mixture, more a.a.-SA corresponding to
the amino acid is needed. Additionally, the affinity
between the a.a.-AMP of a particular amino acid andits cognate aaRS could be different from the affinity

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(2002).

23. C. E. Schafmeister, S. L. LaPorte, L. J. Miercke, R. M. Stroud,

Acknowledgments
We acknowledge funding from the KAKENHI
programs [19680016, 21680026, 23119005, and23650155 to D.K.]; Japan Society for the Promotionof Science (JSPS) and the Ministry of Education,
Culture, Sports, Science and Technology (MEXT).
We also thank Takanori Kigawa and Shigeyuki
Yokoyama for providing the Ras constructs.

A designed four helix bundle protein with native-like structure,
Nat Struct Biol. 4, 1039-1046 (1997).

References
1. G. R. Moura, J. A. Paredes, M. A. S. Santos, Development of
the genetic code: Insights from a fungal codon reassignment,
FEBS Letters. 584, 334-341 (2010).
2. S. Osawa, Evolution of the genetic code. (Oxford University
Press, New York, 1995).
3. Nureki et al., Structure of an archaeal non-discriminating
glutamyl-tRNA synthetase: a missing link in the evolution of
Gln-tRNAGln formation, Nucleic Acids Res. 38, 7286-7297(2010).
4. D. L. Tumbula, H. D. Becker, W. Z. Chang, D. Soll,
Domain-specific recruitment of amide amino acids for protein
synthesis, Nature. 407, 106-110 (2000).
5. K. Sheppard et al., From one amino acid to another:tRNA-dependent amino acid biosynthesis, Nucleic Acids Res.
36, 1813-1825 (2008).
6. B. Min, J. T. Pelaschier, D. E. Graham, D. Tumbula-Hansen,
D. Soll, Transfer RNA-dependent amino acid biosynthesis: anessential route to asparagine formation, Proc Natl Acad Sci U
S A. 99, 2678-2683 (2002).
7. H. D. Becker, D. Kern, Thermus thermophilus: a link inevolution of the tRNA-dependent amino acid amidation
pathways, Proc Natl Acad Sci U S A. 95, 12832-12837 (1998).
8. Kawahara-Kobayashi et al., Simplification of the genetic
code: restricted diversity of genetically encoded amino acids,
Nucleic Acids Res. 40, 10576-10584 (2012).
9. T. Kigawa, Y. Muto, S. Yokoyama, Cell-free synthesis andamino acid-selective stable isotope labeling of proteins for
NMR analysis, J Biomol NMR. 6, 129-134 (1995).
10. T. Kigawa et al., Preparation of Escherichia coli cell extract
for highly productive cell-free protein expression, J Struct
Funct Genomics. 5, 63-68 (2004).
11. Y. M. Hou, P. Schimmel, A simple structural feature is amajor determinant of the identity of a transfer RNA, Nature.
333, 140-145 (1988).
12. C. Francklyn, P. Schimmel, Aminoacylation of RNA
minihelices with alanine, Nature. 337, 478-481 (1989).
13. M. Guo et al., The C-Ala domain brings together editing andaminoacylation functions on one tRNA, Science. 325, 744-747(2009).
14. T. Kigawa et al., Cell-free production and stable-isotope
labeling of milligram quantities of proteins, FEBS Lett. 442,
15-19 (1999).
15. J. T. Wong, Coevolution theory of the genetic code at age
thirty, BioEssays. 27, 416-425 (2005).
16. J. T. Wong, Coevolution of Genetic-Code and Amino-Acid
Biosynthesis, Trends in Biochemical Sciences. 6, 33-36(1981).
17. L. Ribas de Pouplana, P. Schimmel, Aminoacyl-tRNA
synthetases: potential markers of genetic code development,
Trends Biochem Sci. 26, 591-596 (2001).
18. L. Ribas de Pouplana, R. J. Turner, B. A. Steer, P. Schimmel,
Genetic code origins: tRNAs older than their synthetases?,
Proc Natl Acad Sci U S A. 95, 11295-11300 (1998).
19. E. N. Trifonov, Consensus temporal order of amino acids andevolution of the triplet code, Gene. 261, 139-151 (2000).
20. K. Miura et al., Synthesis and expression of a synthetic genefor the activated human c-Ha-ras protein, Jpn J Cancer Res. 77,
45-51 (1986).
21. K. U. Walter, K. Vamvaca, D. Hilvert, An active enzyme
constructed from a 9-amino acid alphabet, J Biol Chem. 280,
37742-37746 (2005).
22. S. Akanuma, T. Kigawa, S. Yokoyama, Combinatorial
mutagenesis to restrict amino acid usage in an enzyme to a
reduced set, Proc Natl Acad Sci U S A. 99, 13549-13553

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