Microsoft Word - MS737.rtf
Manuscript submitted as a Letter to the Editor. 
Title:   
Symmetries by base substitutions in the genetic code predict 2’ or 3’ aminoacylation of tRNAs. 
Authors: Jean-Luc Jestina, Christophe Souléb 
Addresses: 
aUnité de Chimie Organique, URA 2128 CNRS 
Département de Biologie Structurale et Chimie, Institut Pasteur 
25 rue du Dr. Roux, 75724 Paris 15, France 
email: jjestin@pasteur.fr (corresponding author) 
tel +33 1 4438 9496; fax +33 1 4568 8404 
bInstitut des Hautes Etudes Scientifiques, CNRS 
35 route de Chartres, 91440 Bures-sur-Yvette, France 
email: soule@ihes.fr 
Key words :  
Mutation; degeneracy; aminoacyl-tRNA synthetase; codon; symmetry breaking. 
Understanding why the genetic code is the way it is, has been the subject of numerous 
models and still remains largely a challenge (Freeland et al., 2000; Sella and Ardell, 
2006). Associations between codons and amino acids were suggested to rely on RNA-
amino acid interactions (Raszka and Mandel, 1972; Yarus, 1998). Closely related 
codons were put in correspondence with closely related amino acids within their 
biosynthetic pathways (Wong, 2005). Codons have also been grouped into systems 
characterized by interlocked thermodynamic cycles (Klump, 2006). Evolutionary 
models that minimise the number of the most frequent mutations provide a rationale for 
the fact that transitions at the third base of codons are mostly neutral mutations 
(Goldberg and Wittes, 1966). Similarly, minimization of the deleterious effects of 
sequence-dependent single-base deletions catalyzed by DNA polymerases provides a 
rationale for the assignment of stop signals to codons (Jestin and Kempf, 1997). While 
in-frame stop codons are strictly selected against, out-of-frame stop codons minimize 
the costs of ribosomal slippages (Seligmann and Pollock, 2004). In this context, the 
frequencies of codons were found to be highly dependent on the reading frame and 
highlighted a symmetrical codon pattern (Koch and Lehmann, 1997). As the genetic 
code is quasi-universal among living organisms, models do not need to be time-
dependent, even though time-dependent models have been suggested (Bahi and Michel, 
2004; Rodin and Rodin, 2006; Sella and Ardell, 2006). Symmetries in the genetic code 
are of special interest as they may highlight underlying organization principles of the 
code. A supersymmetric model for the evolution of the genetic code was proposed: 
successive breaking of these symmetries would provide an evolutive scenario for the 
decomposition into sets of synonymous codons (Hornos and Hornos, 1993; Bashford et 
al., 1997). When the amino acids are mapped to the vertices of a 28-gon, three two-fold 
symmetries were identified for three subsets of the cognate aminoacyl-tRNA 
synthetases (Yang, 2004). 
This letter reports complete sets of two-fold symmetries between partitions of the 
universal genetic code. By substituting bases at each position of the codons according 
to a fixed rule, it happens that properties of the degeneracy pattern or of tRNA 
aminoacylation specificity are exchanged. 
First the set of sixty-four codons of the genetic code was partitionned in two groups of 
thirty-two codons depending on whether the third base of triplets is necessary or not to 
define unambiguously an amino acid or a stop signal (property 1). Rumer reported a 
symmetry by base substitutions that alters property 1 (Rumer, 1966) . The substitutions 
exchanging T and G as well as A and C are applied to all three codon bases and are 
called Rumer’s transformation. If the third base is necessary to define an amino acid, 
then the symmetrical codon by Rumer’s transformation does not require the third base 
of codons to be defined so as to define unambiguously the amino acid. Conversely, if 
the third base does not have to be defined so as to define unambiguously an amino acid, 
then the symmetrical codon by Rumer’s transformation requires the third base to be 
given so as to define unambiguously the amino acid. More recently, one of the authors 
reported a symmetry that leaves unchanged property 1 (Jestin, 2006): this symmetry 
consists in applying to the first base of codons the substitutions exchanging G and C as 
well as T and A. For example, GCN codons coding for alanine are exchanged into CCN 
codons coding for proline; for GCN and CCN codons, the third base does not have to 
be defined so as to define unambiguously the amino acid. 
Here we report a third symmetry that alters property 1 (Fig.1). This symmetry is 
obtained by applying successively the two symmetries described above. It consists in 
applying the substitution exchanging A and G as well as C and T (a transition) to the 
first base in the codon, the substitution exchanging A and C as well as G and T (a 
transversion) to the second base in the codon, and the substitution exchanging A and C 
as well as G and T (a transversion) in the third base of the codon.  
We show further that the only other symmetries exchanging both groups into each other 
are obtained by combining the previous ones with a symmetry acting only on the third 
base of the codons (here we do not include the substitution on the second base which 
exchanges A and C when fixing G and T). This can be seen by counting the number of 
occurrences of A, C, G, and T as first, second or third base in a codon of each group. 
The result is given in Table 1. 
These symmetries are valid for the standard genetic code and for other genetic codes 
such as the vertebrate mitochondrial genetic code which has a higher degree of 
symmetry of its degeneracy pattern as noted earlier (Lehmann, 2000; Jestin, 2006). 
In addition to the existence of Rumer’s transformation, Shcherbak discussed the 
following Rumer’s rule (Shcherbak, 1989), which can be read off Table 1: the ratio R = 
C+G/T+A of the number of occurrences of C and G by the number of occurrences of T 
and A in positions 1, 2 and 3 is equal to 3, 3 and 1 respectively in codons of the first 
group (and hence it is 1/3, 1/3 and 1 for codons of the second group). Similarly, the 
ratio P = T+C/A+G is 1, 3 and 1 in positions 1, 2 and 3 of the first group of codons. 
Secondly, we considered another grouping of codons of the genetic code depending on 
whether the amino acids are acylated by amino acyl-tRNA synthetases at the 2’ or at 
the 3’ hydroxyl group of the tRNA’s last ribose (property 2) (Sprinzl and Cramer, 1975; 
Arnez and Moras, 1994). This classification of amino acyl-tRNA synthetases is very 
similar to the one based on sequence homology and on structural considerations (Eriani 
et al., 1990; Cusack, 1997). Class I synthetases contain HIGH and KMSKS consensus 
sequences, which are absent from class II amino acyl tRNA synthetases. At the 
structural level, class I synthetases also contain a Rossman fold, a domain that binds 
nucleotides, unlike class II synthetases. Class I enzymes catalyse acylation at the 2’ 
hydroxyl group of the tRNA while class II enzymes generally catalyse acylation at the 
3’ hydroxyl group of the tRNA. PheRS as a class II enzyme that catalyses acylation at 
the tRNA’s 2’ hydroxyl group is therefore an exception.  
The case of cysteinyl-tRNACys synthetase (CysRS) is ambiguous and was investigated 
recently. CysRS is a class I synthetase, but establishes contacts with the major groove 
of the acceptor stem of the tRNACys as commonly found for class II enzymes. The 
enzyme from Escherichia coli is able to catalyse the acylation reaction at both 2’ and 3’ 
hydroxyl groups of the tRNACys. The 2’ acylation is about one order of magnitude 
faster than the 3’ acylation when catalysed by E. coli cysteinyl-tRNA synthetase in 
vitro (Shitivelband and Hou, 2005).  
The following classification was then used for 2’ acylated amino acids (Ile, Leu, Met, 
Val, Trp, Tyr, Arg, Gln, Glu, Phe) and for 3’ acylated amino acids (His, Pro, Ser, Thr, 
Asn, Asp, Lys, Ala, Gly).  To the class of 2’ acylated amino acids we also added the 
stop signals, a choice partially justified by the fact that two stop codons of the 
mitochondrial code of vertebrates code for the 2’ acylated amino acid Arg in the 
universal code. Note that if cysteine were not in the class 3’, or if a stop signal was not 
in the class 2’, symmetries could not be identified. If cysteine is assigned to the class 2’ 
as suggested by the previous paragraph, the symmetries are broken. Loss of the 
symmetries might have occurred during the evolution of aminoacyl-tRNA synthetases 
and might be associated to the late appearance of this amino acid in the genetic code 
(Brooks and Fresco, 2002).  
When considering molecular properties such as polarity, volume and hydrophobicity, 
no statistical differences were noted between class 2’ and class I on one hand, class 3’ 
and class II on the other hand (Table 3). 
There exist two symmetries by base substitutions that exchange the class 2’ with the 
class 3’ of the corresponding codon groups (cf. Fig.2). They consist in applying the 
substitution exchanging A and C as well as G and T (a transversion) to the first base of 
the codon, the substitution exchanging A and G as well as C and T (a transition) to the 
second base of the codon, and the substitution exchanging A and C as well as G and T 
or A and T as well as C and G (a transversion) to the third base of the codon. These two 
symmetries differ by the substitution exchanging A and G as well as C and T in the 
third position. They are not related to those depicted in Figures 4 and 5 (Yang, 2004) as 
Yang’s three symmetries act only on three subsets of amino acids whereas the 
symmetries described herein are valid for the whole codon table. 
There are no other symmetries by base substitutions between the two classes 2’ and 3’, 
as can be seen by counting the occurrences of A, C, G and T in each class and each 
position (Table 2). Note also the following analog of the Rumer’s rule: both the ratio R 
= C+G / T+A  and the ratio Q = A+C / G+T  are equal to 1, 1/3, 1 in positions 1,  2,  3 
respectively in the class 2’ (and 1, 3, 1 in the class 3’).  
In this letter we have described new symmetries by base substitutions in the genetic 
code for partitions concerning the codon degeneracy level or the tRNA-aminoacylation 
class. Several evolutionary models have been proposed concerning tRNAs and their 
aminoacyl-tRNA synthetases (Martinez Gimenez and Tabares Seisdedos, 2002; 
Klipcan and Safro, 2004; Chechetkin, 2006; Di Giulio, 2006). Newly introduced amino 
acids may well have been selected to minimize the deleterious effects of 
mistranslations, and possibly according to their molecular volumes (Torabi et al., 
2006). A unique serie of binary divisions of the codon table was recently noted: when 
the same differentiation rule was applied at each division, the class I / class II pattern 
arose consistently (Delarue, 2007). Aminoacyl-tRNA synthetases are likely to have 
evolved by gene duplication and mutation of primordial synthetases within each class, 
as evidenced by sequence homology (Woese et al., 2000). Consistently, the symmetries 
highlighted in this manuscript require three base substitutions per codon, which are 
unlikely to happen, thereby shedding some light on the duplication and divergence 
mechanism of evolution among the two classes of aminoacyl-tRNA synthetases. 
Acknowledgements  :  
We thank H. Epstein, E. Yeramian, D. Moras, B. Prum and J. Perona for their help. 
References : 
Arnez, J. G., Moras, D. 1994. Aminoacyl-tRNA synthetase tRNA recognition. Oxford, 
IRL Press 61-81. 
Bahi, J. M., Michel, C. J. 2004. A stochastic gene evolution model with time dependent 
mutations. Bull. Math. Biol. 66, 763-778. 
Bashford, J. D., Tsohantjis, I., Jarvis, P. D. 1997. Codon and nucleotide assignments in a 
supersymmetric model of the genetic code. Phys. Lett. A 233, 481-488. 
Brooks, D. J., Fresco, J. R. 2002. Increased frequency of cysteine, tyrosine, and 
phenylalanine residues since the last universal ancestor. Mol. Cell. Proteomics 1, 
125-131. 
Chechetkin, V. R. 2006. Genetic code from tRNA point of view. J. Theor. Biol. 242, 922-
934. 
Cusack, S. 1997. Aminoacyl-tRNA synthetases. Curr. Opin. Struct. Biol. 7, 881-889. 
Delarue, M. 2007. An asymmetric underlying rule in the assignment of codons. RNA 13, 
161-169. 
Di Giulio, M. 2006. The non-monophyletic origin of the tRNA molecule and the origin of 
genes only after the evolutionary stage of the last universal common ancestor. J. 
Theor. Biol. 240, 343-352. 
Di Giulio, M., Capobianco, M. R., Medugno, M. 1994. On the optimization of the 
physicochemical distances between amino acids in the evolution of the genetic code. 
J. Theor. Biol. 168, 43-51. 
Eriani, G., Delarue, M., Poch, O., Gangloff, J., Moras, D. 1990. Partition of tRNA 
synthetases into two classes based on mutually exclusive sets of sequence motifs. 
Nature 347, 203-206. 
Freeland, S. J., Knight, R. D., Landweber, L. F., Hurst, L. D. 2000. Early fixation of an 
optimal genetic code. Mol. Biol. Evol. 17, 511-518. 
Goldberg, A. L., Wittes, R. E. 1966. Genetic code: aspects of organization. Science 153, 
420-424. 
Hornos, J. E. M., Hornos, Y. M. M. 1993. Algebraic model for the evolution of the 
genetic code. Phys. Rev. Lett. 71, 4401-4404. 
Jestin, J. L. 2006. Degeneracy in the genetic code and its symmetries by base 
substitutions. C. R. Biol. 329, 168-171. 
Jestin, J. L., Kempf, A. 1997. Chain-termination codons and polymerase-induced 
frameshift mutations. FEBS Letters 419, 153-156. 
Klipcan, L., Safro, M. 2004. Amino acid biogenesis, evolution of the genetic code and 
aminoacyl-tRNA synthetases. J. Theor. Biol. 228, 389-396. 
Klump, H. H. 2006. Exploring the energy landscape of the genetic code. Arch. Biochem. 
Biophys. 453, 87-92. 
Koch, A. J., Lehmann, J. 1997. About a symmetry of the genetic code. J. Theor. Biol. 189, 
171-174. 
Kyte, J., Doolittle, R. F. 1982. A simple method for displaying the hydropathic character 
of a protein. J. Mol. Biol. 157, 105-132. 
Lehmann, J. 2000. Physico-chemical constraints connected with the coding properties of 
the genetic system. J. Theor. Biol. 202, 129-144. 
Martinez Gimenez, J. A., Tabares Seisdedos, R. 2002. On the dimerization of the 
primitive tRNAs: implications in the origin of genetic code. J. Theor. Biol. 217, 493-
498. 
Raszka, M., Mandel, M. 1972. Is there a physical chemical basis for the present genetic 
code? J. Mol. Evol. 2, 38-43. 
Rodin, S. N., Rodin, A. S. 2006. Origin of the genetic code: first aminoacyl-tRNA 
synthetases could replace isofunctional ribozymes when only the second base of 
codons was established. DNA Cell Biol. 25, 365-375. 
Rumer, Y. B. 1966. About the codon's systematization in the genetic code. Proc. Acad. 
Sci. USSR 167, 1393-1394. 
Seligmann, H., Pollock, D. D. 2004. The ambush hypothesis: hidden stop codons prevent 
off-frame gene reading. DNA Cell Biol. 23, 701-705. 
Sella, G., Ardell, D. H. 2006. The coevolution of genes and genetic codes: Crick's frozen 
accident revisited. J. Mol. Evol. 63, 297-313. 
Shcherbak, V. I. 1989. Rumer's rule and transformation in the context of the co-operative 
symmetry of the genetic code. J. Theor. Biol. 139, 271-276. 
Shitivelband, S., Hou, Y. M. 2005. Breaking the stereo barrier of amino acid attachment to 
tRNA by a single nucleotide. J. Mol. Biol. 348, 513-521. 
Sprinzl, M., Cramer, F. 1975. Site of aminoacylation of tRNAs from Escherichia coli with 
respect to the 2'- or 3'-hydroxyl group of the terminal adenosine. Proc. Natl. Acad. 
Sci. USA 72, 3049-3053. 
Torabi, N., Goodarzi, H., Najafabadi, H. S. 2006. The case of an error minimizing set of 
coding amino acids. J. Theor. Biol. in press. 
Woese, C. R., Olsen, G. J., Ibba, M., Soll, D. 2000. Aminoacyl-tRNA synthetases, the 
genetic code, and the evolutionary process. Microbiol. Mol. Biol. Rev. 64, 202-236. 
Wong, J. T. 2005. Coevolution theory of the genetic code at age thirty. Bioessays 27, 416-
Yang, C. M. 2004. On the 28-gon symmetry inherent in the genetic code intertwined with 
aminoacyl-tRNA synthetases--the Lucas series. Bull. Math. Biol. 66, 1241-1257. 
Yarus, M. 1998. Amino acids as RNA ligands: a direct-RNA-template theory for the 
code's origin. J. Mol. Evol. 47, 109-117. 
Figure Legends : 
Figure 1 
Exchange of Group I (codons for which the third base does not have to be defined to 
specify the amino acid) into Group II (codons for which the third base must be 
defined to specify unambiguously the amino acid or the stop signal) by the 
transformation (AG/CT for the first base, GT/AC for the second and third bases). 
N=A,T,G or C; H=A,T or C; Y=T or C; R=A or G. 
Figure 2 
Exchange of the classes 2’ and 3’ by the transformation (AC/GT on the first base, 
AG/CT on the second base, AC/GT on the third base). The special case of cysteine is 
labelled by an asterisk and discussed in the text. 
Table I 
Number of occurences of the bases A, C, G and T at each position within the 
codon in each group. 
Table II 
Number of occurences of the bases A, C, G and T at each position within the 
codon in each class. 
Table III 
Statistical t-values computed from the data on hydrophobicity (Kyte and 
Doolittle, 1982), molecular volume and polarity (Di Giulio et al., 1994) 
comparing the class 2’ with class I, and the class 3’ with class II. These values are 
below the threshold of significance given in the Student’s table. 
                        A        C        G       T 
           
Base 1   Group I              4               12              12             4          
             Group II            12                4               4             12 
   ____________________________ 
Base 2   Group I               0               16              8              8    
              
              Group II            16                 0              8              8 
   ____________________________ 
Base 3   Group I               8                8               8              8 
                  
              Group II              8                8               8              8  
                                                     Table 1   
                   
                        A        C        G       T 
 Base 1    Class 2’            6               10           6           10 
                                   
               Class 3’            10               6             10            6 
_____________________________ 
Base 2    Class 2’             8                 0              8            16 
                        
               Class 3’             8                16             8              0 
_____________________________ 
Base 3     Class 2’           10                6             10             6 
                  
                Class 3’             6               10             6            10  
                                                    Table 2 
                                                                                                                
   Class 2’ / Class I  Class 3’ / Class II 
Hydrophobicity  0.07     0.11 
Polarity   0.017    0.019 
Volume   0.57     0.45 
                                                    Table 3

  This letter reports complete sets of two-fold symmetries between partitions
of the universal genetic code. By substituting bases at each position of the
codons according to a fixed rule, it happens that properties of the degeneracy
pattern or of tRNA aminoacylation specificity are exchanged.

Microsoft Word - MS737.rtf
Manuscript submitted as a Letter to the Editor. 
Title:   
Symmetries by base substitutions in the genetic code predict 2’ or 3’ aminoacylation of tRNAs. 
Authors: Jean-Luc Jestina, Christophe Souléb 
Addresses: 
aUnité de Chimie Organique, URA 2128 CNRS 
Département de Biologie Structurale et Chimie, Institut Pasteur 
25 rue du Dr. Roux, 75724 Paris 15, France 
email: jjestin@pasteur.fr (corresponding author) 
tel +33 1 4438 9496; fax +33 1 4568 8404 
bInstitut des Hautes Etudes Scientifiques, CNRS 
35 route de Chartres, 91440 Bures-sur-Yvette, France 
email: soule@ihes.fr 
Key words :  
Mutation; degeneracy; aminoacyl-tRNA synthetase; codon; symmetry breaking. 
Understanding why the genetic code is the way it is, has been the subject of numerous 
models and still remains largely a challenge (Freeland et al., 2000; Sella and Ardell, 
2006). Associations between codons and amino acids were suggested to rely on RNA-
amino acid interactions (Raszka and Mandel, 1972; Yarus, 1998). Closely related 
codons were put in correspondence with closely related amino acids within their 
biosynthetic pathways (Wong, 2005). Codons have also been grouped into systems 
characterized by interlocked thermodynamic cycles (Klump, 2006). Evolutionary 
models that minimise the number of the most frequent mutations provide a rationale for 
the fact that transitions at the third base of codons are mostly neutral mutations 
(Goldberg and Wittes, 1966). Similarly, minimization of the deleterious effects of 
sequence-dependent single-base deletions catalyzed by DNA polymerases provides a 
rationale for the assignment of stop signals to codons (Jestin and Kempf, 1997). While 
in-frame stop codons are strictly selected against, out-of-frame stop codons minimize 
the costs of ribosomal slippages (Seligmann and Pollock, 2004). In this context, the 
frequencies of codons were found to be highly dependent on the reading frame and 
highlighted a symmetrical codon pattern (Koch and Lehmann, 1997). As the genetic 
code is quasi-universal among living organisms, models do not need to be time-
dependent, even though time-dependent models have been suggested (Bahi and Michel, 
2004; Rodin and Rodin, 2006; Sella and Ardell, 2006). Symmetries in the genetic code 
are of special interest as they may highlight underlying organization principles of the 
code. A supersymmetric model for the evolution of the genetic code was proposed: 
successive breaking of these symmetries would provide an evolutive scenario for the 
decomposition into sets of synonymous codons (Hornos and Hornos, 1993; Bashford et 
al., 1997). When the amino acids are mapped to the vertices of a 28-gon, three two-fold 
symmetries were identified for three subsets of the cognate aminoacyl-tRNA 
synthetases (Yang, 2004). 
This letter reports complete sets of two-fold symmetries between partitions of the 
universal genetic code. By substituting bases at each position of the codons according 
to a fixed rule, it happens that properties of the degeneracy pattern or of tRNA 
aminoacylation specificity are exchanged. 
First the set of sixty-four codons of the genetic code was partitionned in two groups of 
thirty-two codons depending on whether the third base of triplets is necessary or not to 
define unambiguously an amino acid or a stop signal (property 1). Rumer reported a 
symmetry by base substitutions that alters property 1 (Rumer, 1966) . The substitutions 
exchanging T and G as well as A and C are applied to all three codon bases and are 
called Rumer’s transformation. If the third base is necessary to define an amino acid, 
then the symmetrical codon by Rumer’s transformation does not require the third base 
of codons to be defined so as to define unambiguously the amino acid. Conversely, if 
the third base does not have to be defined so as to define unambiguously an amino acid, 
then the symmetrical codon by Rumer’s transformation requires the third base to be 
given so as to define unambiguously the amino acid. More recently, one of the authors 
reported a symmetry that leaves unchanged property 1 (Jestin, 2006): this symmetry 
consists in applying to the first base of codons the substitutions exchanging G and C as 
well as T and A. For example, GCN codons coding for alanine are exchanged into CCN 
codons coding for proline; for GCN and CCN codons, the third base does not have to 
be defined so as to define unambiguously the amino acid. 
Here we report a third symmetry that alters property 1 (Fig.1). This symmetry is 
obtained by applying successively the two symmetries described above. It consists in 
applying the substitution exchanging A and G as well as C and T (a transition) to the 
first base in the codon, the substitution exchanging A and C as well as G and T (a 
transversion) to the second base in the codon, and the substitution exchanging A and C 
as well as G and T (a transversion) in the third base of the codon.  
We show further that the only other symmetries exchanging both groups into each other 
are obtained by combining the previous ones with a symmetry acting only on the third 
base of the codons (here we do not include the substitution on the second base which 
exchanges A and C when fixing G and T). This can be seen by counting the number of 
occurrences of A, C, G, and T as first, second or third base in a codon of each group. 
The result is given in Table 1. 
These symmetries are valid for the standard genetic code and for other genetic codes 
such as the vertebrate mitochondrial genetic code which has a higher degree of 
symmetry of its degeneracy pattern as noted earlier (Lehmann, 2000; Jestin, 2006). 
In addition to the existence of Rumer’s transformation, Shcherbak discussed the 
following Rumer’s rule (Shcherbak, 1989), which can be read off Table 1: the ratio R = 
C+G/T+A of the number of occurrences of C and G by the number of occurrences of T 
and A in positions 1, 2 and 3 is equal to 3, 3 and 1 respectively in codons of the first 
group (and hence it is 1/3, 1/3 and 1 for codons of the second group). Similarly, the 
ratio P = T+C/A+G is 1, 3 and 1 in positions 1, 2 and 3 of the first group of codons. 
Secondly, we considered another grouping of codons of the genetic code depending on 
whether the amino acids are acylated by amino acyl-tRNA synthetases at the 2’ or at 
the 3’ hydroxyl group of the tRNA’s last ribose (property 2) (Sprinzl and Cramer, 1975; 
Arnez and Moras, 1994). This classification of amino acyl-tRNA synthetases is very 
similar to the one based on sequence homology and on structural considerations (Eriani 
et al., 1990; Cusack, 1997). Class I synthetases contain HIGH and KMSKS consensus 
sequences, which are absent from class II amino acyl tRNA synthetases. At the 
structural level, class I synthetases also contain a Rossman fold, a domain that binds 
nucleotides, unlike class II synthetases. Class I enzymes catalyse acylation at the 2’ 
hydroxyl group of the tRNA while class II enzymes generally catalyse acylation at the 
3’ hydroxyl group of the tRNA. PheRS as a class II enzyme that catalyses acylation at 
the tRNA’s 2’ hydroxyl group is therefore an exception.  
The case of cysteinyl-tRNACys synthetase (CysRS) is ambiguous and was investigated 
recently. CysRS is a class I synthetase, but establishes contacts with the major groove 
of the acceptor stem of the tRNACys as commonly found for class II enzymes. The 
enzyme from Escherichia coli is able to catalyse the acylation reaction at both 2’ and 3’ 
hydroxyl groups of the tRNACys. The 2’ acylation is about one order of magnitude 
faster than the 3’ acylation when catalysed by E. coli cysteinyl-tRNA synthetase in 
vitro (Shitivelband and Hou, 2005).  
The following classification was then used for 2’ acylated amino acids (Ile, Leu, Met, 
Val, Trp, Tyr, Arg, Gln, Glu, Phe) and for 3’ acylated amino acids (His, Pro, Ser, Thr, 
Asn, Asp, Lys, Ala, Gly).  To the class of 2’ acylated amino acids we also added the 
stop signals, a choice partially justified by the fact that two stop codons of the 
mitochondrial code of vertebrates code for the 2’ acylated amino acid Arg in the 
universal code. Note that if cysteine were not in the class 3’, or if a stop signal was not 
in the class 2’, symmetries could not be identified. If cysteine is assigned to the class 2’ 
as suggested by the previous paragraph, the symmetries are broken. Loss of the 
symmetries might have occurred during the evolution of aminoacyl-tRNA synthetases 
and might be associated to the late appearance of this amino acid in the genetic code 
(Brooks and Fresco, 2002).  
When considering molecular properties such as polarity, volume and hydrophobicity, 
no statistical differences were noted between class 2’ and class I on one hand, class 3’ 
and class II on the other hand (Table 3). 
There exist two symmetries by base substitutions that exchange the class 2’ with the 
class 3’ of the corresponding codon groups (cf. Fig.2). They consist in applying the 
substitution exchanging A and C as well as G and T (a transversion) to the first base of 
the codon, the substitution exchanging A and G as well as C and T (a transition) to the 
second base of the codon, and the substitution exchanging A and C as well as G and T 
or A and T as well as C and G (a transversion) to the third base of the codon. These two 
symmetries differ by the substitution exchanging A and G as well as C and T in the 
third position. They are not related to those depicted in Figures 4 and 5 (Yang, 2004) as 
Yang’s three symmetries act only on three subsets of amino acids whereas the 
symmetries described herein are valid for the whole codon table. 
There are no other symmetries by base substitutions between the two classes 2’ and 3’, 
as can be seen by counting the occurrences of A, C, G and T in each class and each 
position (Table 2). Note also the following analog of the Rumer’s rule: both the ratio R 
= C+G / T+A  and the ratio Q = A+C / G+T  are equal to 1, 1/3, 1 in positions 1,  2,  3 
respectively in the class 2’ (and 1, 3, 1 in the class 3’).  
In this letter we have described new symmetries by base substitutions in the genetic 
code for partitions concerning the codon degeneracy level or the tRNA-aminoacylation 
class. Several evolutionary models have been proposed concerning tRNAs and their 
aminoacyl-tRNA synthetases (Martinez Gimenez and Tabares Seisdedos, 2002; 
Klipcan and Safro, 2004; Chechetkin, 2006; Di Giulio, 2006). Newly introduced amino 
acids may well have been selected to minimize the deleterious effects of 
mistranslations, and possibly according to their molecular volumes (Torabi et al., 
2006). A unique serie of binary divisions of the codon table was recently noted: when 
the same differentiation rule was applied at each division, the class I / class II pattern 
arose consistently (Delarue, 2007). Aminoacyl-tRNA synthetases are likely to have 
evolved by gene duplication and mutation of primordial synthetases within each class, 
as evidenced by sequence homology (Woese et al., 2000). Consistently, the symmetries 
highlighted in this manuscript require three base substitutions per codon, which are 
unlikely to happen, thereby shedding some light on the duplication and divergence 
mechanism of evolution among the two classes of aminoacyl-tRNA synthetases. 
Acknowledgements  :  
We thank H. Epstein, E. Yeramian, D. Moras, B. Prum and J. Perona for their help. 
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Figure Legends : 
Figure 1 
Exchange of Group I (codons for which the third base does not have to be defined to 
specify the amino acid) into Group II (codons for which the third base must be 
defined to specify unambiguously the amino acid or the stop signal) by the 
transformation (AG/CT for the first base, GT/AC for the second and third bases). 
N=A,T,G or C; H=A,T or C; Y=T or C; R=A or G. 
Figure 2 
Exchange of the classes 2’ and 3’ by the transformation (AC/GT on the first base, 
AG/CT on the second base, AC/GT on the third base). The special case of cysteine is 
labelled by an asterisk and discussed in the text. 
Table I 
Number of occurences of the bases A, C, G and T at each position within the 
codon in each group. 
Table II 
Number of occurences of the bases A, C, G and T at each position within the 
codon in each class. 
Table III 
Statistical t-values computed from the data on hydrophobicity (Kyte and 
Doolittle, 1982), molecular volume and polarity (Di Giulio et al., 1994) 
comparing the class 2’ with class I, and the class 3’ with class II. These values are 
below the threshold of significance given in the Student’s table. 
                        A        C        G       T 
           
Base 1   Group I              4               12              12             4          
             Group II            12                4               4             12 
   ____________________________ 
Base 2   Group I               0               16              8              8    
              
              Group II            16                 0              8              8 
   ____________________________ 
Base 3   Group I               8                8               8              8 
                  
              Group II              8                8               8              8  
                                                     Table 1   
                   
                        A        C        G       T 
 Base 1    Class 2’            6               10           6           10 
                                   
               Class 3’            10               6             10            6 
_____________________________ 
Base 2    Class 2’             8                 0              8            16 
                        
               Class 3’             8                16             8              0 
_____________________________ 
Base 3     Class 2’           10                6             10             6 
                  
                Class 3’             6               10             6            10  
                                                    Table 2 
                                                                                                                
   Class 2’ / Class I  Class 3’ / Class II 
Hydrophobicity  0.07     0.11 
Polarity   0.017    0.019 
Volume   0.57     0.45 
                                                    Table 3
