Gene Therapy and Molecular Biology Vol 10, page 1
1
Gene Ther Mol Biol Vol 10, 1-12, 2006
Low-usage codons and rare codons of Escherichia
coli
Mini Review
Dequan Chen* and Donald E. Texada
Department of Ophthalmology, Louisiana State University Health Sciences Center, Shreveport, LA 71130
__________________________________________________________________________________
*Correspondence: Dequan Chen, Ph.D., Institute for Retina Research, 8210 Walnut Hill Lane, PBI, Suite 010, Dallas, TX 75231, USA;
Phone: (214) 345-6801; email: dequan.chen@irrdallas.org.
Key words: low-usage codon, rare codon, common codon, major codon, codon usage, infrequent codon, rare tRNA, codon optimization,
and rare tRNA supplementation
Abbreviations: Escherichia coli, (E. coli); rare codon, (RC); rare codon cluster, (RCC)
Received: 4 September 2005; Accepted: 28 December 2005; electronically published: January 2006
Summary
In Escherichia coli (E. coli), a low-usage codon is defined as a codon that is used rarely or infrequently in the
genome with usage frequency lower than the smallest value (or frequency cut-off) among the usage frequencies of
non-degenerate codons (Met codon AUG and Trp codon UGG) and the optimal codons for amino acids Leu, Ile,
Val, Ser, Pro, Thr, Ala, Arg, Gly and Gln that have 2 or more degenerate codons with each having specific
corresponding cognate tRNA for the optimal codon. A rare codon (RC), an infrequent codon or a minor codon is
equivalently defined as a synonymous codon or a stop codon that is not only used rarely or infrequently in a genome
but also decoded by a low-abundant tRNA (rare tRNA) or other factor(s) in an organism. The translational rate for
a sense RC is much lower than that for a common (major) codon due to tRNA availability. A low-usage codon is not
necessarily a RC, e.g., Cys codons UGU and UGC, Thr codons ACU and ACG, or His codons CAC and CAU are
not rare codons of E. coli. However, a RC is definitely a low-usage codon. In E. coli, there are about 30 low-usage
synonymous sense codons but only 20 of them are determined to be the bacterial RCs including 7 (AGG, AGA,
CGA, CUA, AUA, CCC and CGG) used at a frequency of < 0.5% (Group I) and 13 (ACA, CCU, UCA, GGA, AGU,
UCG, CCA, UCC, GGG, CUC, CUU, UCU and UUA) used at a frequency of > 0.5% (Group II). Studies have
demonstrated that all the RCs in Group I and the first 6 RCs in Group 2 can cause translational problems in E. coli.
I. Introduction
Many proteins including those that can be used in
treatment of certain disease (e.g., insulin), can rarely be
obtained in large quantities from their natural sources.
Besides, their purification or isolation is often not easy,
and the cost is often pretty high. Recombinant DNA
techniques have been successfully used in the past to
express and purify these kinds of proteins. The bacterium
E. coli has been and will continue to be the main, popular
and first-choice expression host because it facilitates
recombinant protein expression by its relative simplicity,
its inexpensive and fast high-density cultivation, its wellknown
genetics and the availability of a large number of
compatible tools including mutant strains, recombinant
fusion partners and plasmids (Gold, 1990; Hodgson, 1993;
Olins and Lee, 1993; Kane, 1995; Makrides, 1996;
Jonasson et al, 2002; Sorensen and Mortensen, 2005a;
Sorensen and Mortensen, 2005b). However, not every
foreign gene can be efficiently expressed in E. coli,
probably due to the unique and subtle structure of the
target gene, the mRNA low stability and slow translational
efficiency, the uneasy protein folding, the target protein
degradation by E. coli proteases, the different codon usage
between the organism of the foreign gene and native E.
coli, or the toxicity of the expressed target protein (Olins
et al, 1993; Makrides, 1996; Jonasson et al, 2002).
A number of studies have revealed that RCs and rare
codon clusters (RCC) are capable of qualitatively and
quantitatively causing expression problems in E. coli or
other organisms (Kane, 1995; Makrides, 1996; Gurvich et
al, 2005), and these problems mainly occur on translation
level rather than on transcription level or other levels. The
main translational problems caused by RCs or RCCs
include (a) that rare codons reduce the translation rate of
the target gene, (b) the expressed target protein is low or
undetectable, (c) amino acids are misincorporated into the
target protein, (d) truncated or amino acids-deleted
peptides or proteins are synthesized, and (e) frame-shifted
peptides or proteins are synthesized (Pedersen, 1984;
Pohlner et al, 1986; Sorensen et al, 1989; Gurskii et al,
Chen and Texada: E. coli low-usage codons and rare codons
2
1992b; Kane et al, 1992; Gursky and Beabealashvilli,
1994; Vilbois et al, 1994; Kane, 1995; Calderone et al,
1996; Kleber-Janke and Becker, 2000; Kapust et al, 2002;
McNulty et al, 2003; Flick et al, 2004; Shu et al, 2004;
Choi et al, 2004; Chen et al, 2004; Gurvich et al, 2005).
However, different groups often arbitrarily used different
sets of codons as their rare or low-usage codons, or
equivalently used low-usage codons and rare-tRNA
associated codons. This may at least result in the following
problems: (a) some codons are rare codons or low-usage
codons to some groups but not to others, and vice versa;
and (b) over- or under-estimation of the effects of rare
codons on the expression of a gene even in the same
system just because of the difference of low-usage or rare
codons being defined or studied. To overcome these
problems, universal meanings for low-usage codon and/or
rare codon should be defined and the list of low-usage
codons or rare codons in an organism should be
determined. Therefore, the objectives of this review are
mainly to unify and differentiate the meanings of a lowusage
codon and a rare-tRNA associated codon (RC in
short) as well as to determine the lists of the low-usage
codons and rare-tRNA associated codons in E. coli.
II. Codon usage in E. coli
Codon usage was defined by Zhang et al in 1999 as
the number of times (frequency) a codon is translated per
unit time in the cell of an organism. This is a definition for
real-time codon usage. But it is hard to be measured in
vivo. Zhang et al, used 3 different methods to estimate the
codon usage in E. coli and other organisms in their studies
including measuring the average frequencies of codons in
the sequenced protein-coding genes in an organism. All
their methods gave approximately the same results as
regards the hierarchy for “most used’ and “least used”
codons within each synonymous codon family (Zhang et
al, 1991). Therefore, it is reasonable to use averaged
codon frequency of the sequenced protein-coding reading
frames of an organism to roughly represent the real-time
codon usage although this may over-estimate the usages of
infrequently or rarely used codons and underestimate those
of frequently used codons because different reading
frames are used and translated for different number of
times in the organism at a given time (Zhang et al, 1991).
Besides, this is what codon usage generally means to
many scientists in the past and at present.
Before the 1980s and after the discovery of genetic
code redundancy or degeneracy (an amino acid except Met
and Trp is encoded by 2 to 6 codons), it was often thought
that degenerate codons for the same amino acid were used
randomly in a genome. This is based on the simplest
assumption that all genomes have uniform codon usage
meaning that synonymous codons (degenerate codons for
same amino acid) are used with equal frequency. As more
and more sequence data (especially the gene sequences of
bacterium E. coli and yeast Saccharomyces cerevisiae)
appeared in the late 1970s and early 1980s, it came to light
that (a) synonymous codon usage is consistently similar
for all genes within each type of genome or organism
(Grantham et al, 1980a,b, 1981), and (b) synonymous
codon usage was not random, i.e., synonymous codons are
not used with equal frequency in a genome (Ikemura,
1985; Sharp et al, 1988; Zhang et al, 1991; Sorensen et al,
2005a). This is also true for non-synonymous codon usage
(non-random usage of different codons for different amino
acids). Therefore, the codon usage among degenerate
codons in each organism is biased, with some codons
more preferred (at higher usage frequency or used more
frequently) than the other(s). Further, studies also found
that codon usage bias is greater in highly expressed genes
than poorly expressed genes (Gouy and Gautier, 1982;
Sharp and Li, 1986; Makrides, 1996). That is to say,
highly expressed genes in an organism mostly use
preferred codons (especially the most preferred or optimal
codons) and avoid non-preferred codons while poorly
expressed genes use fewer preferred or optimal codons but
more non-preferred codons (Ikemura, 1985). Meanwhile,
codon pair usage was even found not to be random
(Nussinov, 1981; Lipman and Wilbur, 1983; Gutman and
Hatfield, 1989; Irwin et al, 1995).
The codon usage frequencies for the 64 codons (3
stop codons, and 61 sense codons - codons that encode
amino acids) of E. coli, calculated from the GenBank
genetic sequence data (Releases # 63, 69 and 147), are
shown in Table 1. The data in the table demonstrate that
as the total number of the codons or protein-coding genes
included in each GenBank release increases (especially
from #69 to #147, which is about 8 times increase), the
calculated frequency for a given sense codon changes as
follows:
(a) The frequencies of low-usage codons (highlighted
by red, purpurple and blue) has a tendency to increase
(those of CUC, GUA, UCG, CCA, CAU, UGU, CGA,
UCU and ACU increase very little while those of others a
lot) except those of CAC, UGC and UCC (the last two
usage frequencies decrease very little);
(b) The frequencies of some high-usage codons
change very little (those of GUA, ACU, GCC, GCA and
GAU increase while those of AUG, GUU, GUC, GCU,
AAA, GAG and AGC decrease),
(c) The frequencies of some high-usage codons have
a tendency to increase (for those of UUU, AUU, UAU,
CAA, AAU and UGG) while the frequencies of other
high-usage codons, on the contrary, have a tendency to
decrease (for those of UUC, CUG, AUC, GUG, CCG,
ACC, GCG, UAC, CAG, AAC, GAC, GAA, CGU, CGC
GGU and GGC).
The above results may imply the following:
(1) Some codons, whether at high-usage (see above
b) or at low-usage (see above a), are used at about the
same frequency in the old sequenced proteins (e.g., the
proteins included in GenBank release #69) as in the new
sequenced proteins (e.g., the proteins included in GenBank
release #147 but not in #69). Therefore, their usagefrequencies
change very little between the GenBank
releases, and the usage frequency calculated from
GenBank release #69 or 147 should all well represent their
real-time codon usage frequencies (Table 1).
(2) Most low-usage codons (see above a) and some
high-usage codons (see above c) are not well used by the
old sequenced proteins, and the new sequenced proteins
(e.g., the proteins included in GenBank release #147 but
Gene Therapy and Molecular Biology Vol 10, page 3
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not in #69) have used more of them. Therefore, their usage
frequencies increase over the total number of protein
genes included in the GenBank releases. Two factors
should contribute to the phenomenon: one factor is that
these codons especially low-usage codons are more
frequently used in the new sequenced protein genes (most
of them are poorly expressed), which results in the
increase of their calculated usage frequencies; the other
factor, on the contrary, is that poorly expressed genes have
low expression rates at a give time of the bacterial life, and
averaging over the entire genome without weighting the
number of times different reading frames are being
translated leads to over-estimation of their codon usage
frequencies (Zhang et al, 1991). Therefore, the real-time
codon usage frequencies for these codons should be much
lower than the data calculated from GenBank release #147
but somewhere around the data from GenBank release #69
(Table 1).
(3) Some high-usage codons (see above c) are well
used by the old sequenced proteins, and the new
sequenced proteins (e.g., the proteins included in GenBank
release #147 but not in #69) have used less of them.
Therefore, their usage frequencies decrease over the total
number of protein genes included in the GenBank releases.
The above two factors should also contribute to this but in
a reverse direction: the first factor is that these codons are
less frequently used in the new sequenced protein genes
(most of them are poorly expressed), which results in the
true decrease of their calculated usage frequencies; the
second is that poorly expressed genes have low expression
rates at a give time of the bacterial life, and averaging over
the entire genome without weighting the number of times
different reading frames are being translated leads to
under-estimation of the codon usage frequencies for these
codons (Zhang et al, 1991). Therefore, the real-time codon
usage frequencies for these codons should be much higher
than the data from GenBank release #147 but somewhere
around the data from GenBank release #69 or even #63
(Table 1).
The above analysis suggests that the frequency
values listed in the II columns (calculated from GenBank
release #69) of Table 1 most likely and approximately
better represent the real-time codon usage frequencies in
E. coli.
Dong et al, measured E. coli codon usage frequencies
at different bacterial growth rates (0.4-2.5 doublings per
hour), which were calculated from the coding frames of
140 protein mRNAs (Dong et al, 1996). The results has
been adapted and presented to Table 2.
Table 1. Codon frequencies used by protein-coding reading frames of E. colia
I b
II c
III d
I b
II c
III d
I b
II c
III d
I b
II c
III d
UUU 18.85 19.2 22.46 UCU 10.47 10.4 10.94 UAU 15.09 15.4 18.34 UGU 4.80 4.7 5.35
UUC 18.07 18.2 15.62 UCC 9.43 9.4 9.29 UAC 13.29 13.4 12.01 UGC 6.07 6.1 5.99
UUA 10.52 10.9 14.98 UCA 6.52 6.8 9.94 UAA 1.99 2.0 1.99 UGA 0.80 0.8 1.04
UUG 11.33 11.5 12.86 UCG 7.89 8.0 8.52 UAG 0.20 0.2 0.29 UGG 12.90 12.8 13.78
CUU 9.92 10.2 12.49 CCU 6.57 6.6 7.90 CAU 11.35 11.6 12.47 CGU 24.70 24.1 18.92
CUC 9.70 9.9 10.08 CCC 4.19 4.3 5.63 CAC 10.74 10.7 8.82 CGC 21.50 22.1 18.38
CUA 2.97 3.2 4.47 CCA 8.12 8.2 8.63 CAA 13.07 13.2 14.38 CGA 3.06 3.1 4.03
CUG 54.10 54.6 46.04 CCG 23.91 23.8 19.35 CAG 29.68 30.1 28.12 CGG 4.62 4.6 6.49
AUU 27.27 27.2 29.67 ACU 10.83 10.2 11.02 AAU 16.30 16.3 22.83 AGU 7.37 7.2 10.73
AUC 26.97 26.5 22.69 ACC 24.37 24.3 21.39 AAC 24.35 23.9 21.20 AGC 14.95 15.2 15.00
AUA 3.94 4.1 8.22 ACA 6.53 6.5 10.70 AAA 37.47 36.5 35.60 AGA 2.14 2.1 4.47
AUG 26.33 26.5 25.95 ACG 12.54 12.7 13.78 AAG 11.94 12.0 13.05 AGG 1.32 1.4 2.56
GUU 20.79 20.1 20.04 GCU 17.86 17.4 17.36 GAU 32.14 32.3 32.88 GGU 28.48 27.6 24.93
GUC 14.09 14.2 14.04 GCC 23.18 23.5 23.87 GAC 22.03 21.8 18.83 GGC 30.41 30.2 25.66
GUA 12.06 11.6 11.90 GCA 20.92 20.8 21.60 GAA 43.75 43.4 38.02 GGA 6.95 7.0 10.61
GUG 24.68 25.3 23.47 GCG 32.94 33.1 27.99 GAG 19.03 19.2 18.80 GGG 9.63 9.7 11.58
a. The usage of each codon is expressed as the frequency per 1000 codons, which is calculated by division of the absolute number of the
indicated codon by the total number of codons used in all the sequenced E. coli protein-coding sequences or reading frames.
b. Taken from Zhang et al (1991). Codon usage frequency was calculated from 323059 codons of 968 protein coding reading frames
(CDS) (GenBank Version 63.0, 15 March 1990).
c. Taken from Wada et al (1991). Codon usage frequency was calculated from 524410 codons of 1562 protein coding reading frames
(CDS) (GenBank Version 69.0, September 1991).
d. Taken and adapted from http://www.kazusa.or.jp/codon (Nakamura et al, 2000). Codon usage frequency was calculated from 4182749
codons of 13778 protein coding reading frames (CDS) (GenBank Version 147.0, 1 June 2005).
Chen and Texada: E. coli low-usage codons and rare codons
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Table 2. Real-time codon frequencies used by protein-coding reading frames of E. colia
Growth Rate b
Growth Rate b
Growth Rate b
Growth Rate b
0.4 1.07 2.5 0.4 1.07 2.5 0.4 1.07 2.5 0.4 1.07 2.5
UUU 12.55 10.30 7.92 UCU 13.12 14.14 16.33 UAU 10.68 8.90 6.72 UGU 4.23 3.64 2.76
UUC 22.68 22.44 23.25 UCC 11.15 12.09 11.68 UAC 16.20 16.71 16.52 UGC 5.29 4.77 3.81
UUA 6.13 4.64 2.73 UCA 3.89 3.09 1.98 UAA 2.77 3.38 4.18 UGA 0.31 0.23 0.19
UUG 6.63 5.72 4.27 UCG 6.05 4.58 2.51 UAG 0.00 0.00 0.00 UGG 9.76 8.69 7.03
CUU 5.70 4.64 3.86 CCU 4.99 4.79 4.38 CAU 9.23 8.11 6.78 CGU 31.12 36.61 43.82
CUC 6.19 5.52 4.09 CCC 3.32 2.10 1.09 CAC 13.90 13.91 14.21 CGC 22.25 22.39 20.59
CUA 2.15 1.53 0.82 CCA 6.52 6.40 5.18 CAA 10.91 8.98 7.01 CGA 1.32 0.99 0.67
CUG 60.13 61.29 60.75 CCG 29.51 28.88 28.82 CAG 29.24 28.33 27.28 CGG 1.75 1.23 0.62
AUU 21.38 19.26 15.79 ACU 13.88 16.76 20.64 AAU 9.79 7.79 5.61 AGU 3.99 3.01 2.19
AUC 36.68 39.15 43.86 ACC 26.51 27.10 26.70 AAC 27.95 28.64 29.21 AGC 11.97 10.69 9.31
AUA 0.93 0.75 0.52 ACA 3.48 2.99 2.61 AAA 44.43 49.07 55.01 AGA 1.12 0.84 0.63
AUGc
25.32 25.82 25.90 ACG 7.53 6.21 4.17 AAG 12.08 13.74 17.22 AGG 0.09 0.05 0.03
GUU 31.31 35.63 43.18 GCU 28.85 32.14 39.49 GAU 24.25 22.40 19.27 GGU 38.29 40.49 45.55
GUC 11.25 9.71 7.67 GCC 19.80 16.81 11.81 GAC 28.72 30.93 33.74 GGC 35.62 35.54 34.17
GUA 15.87 18.65 22.31 GCA 22.13 22.38 24.87 GAA 53.10 55.10 57.86 GGA 2.71 2.21 1.26
GUG 21.40 18.93 14.98 GCG 30.33 28.45 24.11 GAG 16.57 17.04 16.97 GGG 4.81 3.57 2.36
a. Taken from Dong et al (1996). The usage of each codon is expressed as the frequency per 1000 codons. The codon frequencies were
the averages from 140 proteins and calculated on the basis of the relative weight fraction of each protein and on the assumption that the
amount of a protein accumulated in the cell during the steady growth is proportional to the amount of its corresponding mRNA in the
bacteria.
b. Growth rate is expressed as doublings per hour. Different growth rates were obtained by varying the nutrient contents of the culturing
media (Dong et al, 1996).
c. The data for AUG usage frequency are the sum of the frequencies for Metf1, Metf2, and Metm.
Although the number of protein coding frames used is
very small, the frequency values were obtained by
weighting every protein amount at each growth rate of E.
coli according to the data reported by Pedersen et al
(Pedersen et al, 1978). Therefore, the codon usage
frequencies in Table 2 are real-time codon usage values.
The data in Table 2 demonstrate that (a) E. coli codon
usage is biased at all studied bacterial growth rate, (b) the
frequencies of low-usage sense codons (marked by red and
purple) decrease with increasing growth rate, and (c) the
frequencies of some high-usage sense codons (UUC,
AUC, GUU, GUA, UCU, ACU, GCU, GCA, CAC, AAC,
AAA, GAC, GAA, CGU and GGU) increase while those
of others decrease over the increase of growth rate. In
addition, most codon usage frequencies in Table 2 are in
good agreement with those in Table 1.
III. tRNA abundance in E. coli
Codon usage bias in an organism may have been
formed during evolution by the combinatory effects of
various factors such as the adaptation of gene expressivity
to various growth conditions (Gouy et al, 1982), the
adaptation of codons to tRNA availability (Ikemura, 1980,
1981a,b, 1985), the adaptation of codon-anticodon paring
or interaction to have optimal or intermediate energy
strength (Grosjean et al, 1978; Grosjean and Fiers, 1982),
the adaptation of codon mutations to form specific mRNA
secondary structure(s), etc. But codon adaptation to tRNA
availability are attributed to have played a key role in the
formation of biased codon usage because organismspecific
codon usage patterns were demonstrated to
correlate with the abundance spectra of organism-specific
populations of isoaccepting or cognate tRNAs (Ikemura,
1980, 1981a,b, 1985).
The relative contents of tRNAs for normally growing
E. coli, which were measured by Ikemura (1981a, 1981b,
1985), are listed in Table 3. The data (relative contents for
38 or 40 tRNAs) in the table demonstrate that: (a) the
abundance of tRNAGly3
is the highest (relative amount is
1.1) among all the E. coli tRNAs and it can
recognize/decode two codons (GGU and GGC),
immediately followed by tRNAVal1
, tRNAAla(GCY)
and
Gene Therapy and Molecular Biology Vol 10, page 5
5
tRNAIle1
(relative amounts are 1.05, 1.04 and 1.0
respectively) in succession with the first recognize 3
codons (GUA, GUG and GUU) while the latter 2
recognizing 2 codons (GCC and GCU, AUU and AUC
respectively); (b) tRNALeu1
is a tRNA that recognizes only
one single codon (CUG) and at the same time has the
highest abundance (relative amount is 1.0); (c) some
tRNAs including the cognate tRNAs for CUA, AUA,
CGG, AGA and AGG, ACA and ACG, CCC, or UGU and
UGC, have very low abundances while the abundances for
other tRNAs are different with relative amounts ranging
from 0.1 to 0.9; and (d) UCU (for Ser), GUU (for Val),
GCU (for Ala), and GGG (for Gly) are recognized by 2
isoacceptor-tRNAs. In addition, the relative contents of
tRNAs (43 or 45 tRNAs) for E. coli growing at different
rates (0.4, 0.7, 1.07, 1.6 and 2.5 doublings/hour), measured
by Dong et al (Dong et al, 1996), are listed in Table 4.The
data in Table 4 suggest that tRNA abundance in E. coli
varies with bacterial growth rate - increases over the
increase of growth rate (the increase amplitude varies with
different tRNAs). Most tRNA relative contents in Table 4
are in agreement with those in Table 3. The data of
Tables 1 and 2, with those of Tables 3 and 4, altogether
support the concept that the usage frequency of
synonymous codon often reflects or correlates with the
abundance of its cognate tRNA in E. coli (Garel, 1974;
Garel et al, 1981; Ikemura, 1985; Bulmer, 1987; Emilsson
and Kurland, 1990; Emilsson et al, 1993; Kane, 1995;
Makrides, 1996; Dong et al, 1996).
IV. Definition of low-usage codon and
rare codon
Low-usage codon is often called RC, minor codon,
and infrequent codon because all of them imply that the
usage of such a codon in a genome or an organism is low
or very low, in other words, the codon is used rarely or
infrequently in a genome or an organism. All the above
terms have been equivalently used in the past. But
different groups defined different sets of codons as their
low-usage codons (although most groups included the
several least usage codons in their low-usage codon sets)
due to (a) the different numbers of the available proteincoding
gene sequences for calculating the codon usage
frequencies, and (b) the arbitrary frequency cut-offs which
were used by different people (e.g. 0.5%, 1.0%, or 1.1%)
to define the boundary between low-usage codons and
common codons. The above may result in the following
problems: (a) some codons, are low-usage codons to some
people but not to others, and vice versa; e.g., GUC and
GCC were considered as RC by Pedersen (1984) but not
us; (b) different results or conclusions regarding the
effects of low-usage codons on the expression of a gene(s)
(often over- or under-estimation occurs) may be obtained
for the same system just because of the difference of lowusage
codons being defined or studied; (c) the results from
different groups, for the same gene, are often hard to be
compared with each other. Therefore, universal
definition(s) for the above terms, or universal terms with
fixed meanings is required.
The correlation of the usage frequency of a
synonymous codon with its cognate tRNA abundance
(such as high-usage codons with high-abundant tRNAs
and low-usage codons with low-abundant tRNAs),
together with the so far reported expression problems
derived from low-usage codons and/or their cognate tRNA
availability, suggest that just one term to cover all the
above meanings is not enough. To satisfy the above
requirements, a RC, an infrequent codon or a minor codon
is equivalently defined as a synonymous codon that is not
Table 3. Relative contents of tRNAs in E. colia
tRNA Recognized codon Content b
Leu: 1 CUG 1.00
2 CUU, CUC 0.30
UUR UUA, UUG 0.25
CUA CUA minor
Val: 1 GUA, GUG, GUU* 1.05
2 GUC, GUU* 0.40
Gly: 1 GGG* 0.10
2 GGA,GGG* 0.15
3 GGU, GGC 1.10
Ala: 1 GCA, GCG, GCU* 0.85
GCY GCC, GCU* 1.04
Arg: 1, 2 CGU, CGC, CGA 0.90
CGG CGG minor
AGR AGA, AGG minor
Ile: 1 AUU, AUC 1.00
2 AUA 0.05
Lys AAA, AAG 1.00
Glu 2 (1) GAA, GAG 0.90
Asp 1 GAU, GAC 0.80
Thr: 1+3 ACU, ACC 0.80
4 ACA, ACG minor
Asn AAU, AAC 0.60
Gln: 1 CAA 0.30
2 CAG 0.40
Tyr: 1+2 UAU, UAC 0.50
Ser: 1 UCU*, UCA, UCG 0.25
3 AGU, AGC 0.25
UCY UCC, UCU*
His CAC, CAU 0.40
Trp UGG 0.30
Pro: 1 CCG major
2 CCC minor
3 CCU, CCA, CCG major
Phe UUU, UUC 0.35
Cys UGU, UGC minor
Met: m AUG 0.30
f1 AUG 0.40
f2 AUG 0.10
a. Taken and adapted from Ikemura (1981a,b, 1985)
b. The content is the relative amount to that of tRNALeu1(CUG)
that
is normalized to 1.0 and approximately on the order of 104
molecules per cell for normally growing E. coli.
*. A single codon is recognized by 2 tRNAs.
Chen and Texada: E. coli low-usage codons and rare codons
6
Table 4. Relative contents of tRNAs in E. coli at different growth rates
tRNA Recognized codon(s) Growth Rate (doublings per hour)
0.4 0.7 1.07 1.6 2.5
Leu: 1 CUG 1.00 1.06 1.19 1.51 1.57
2 CUC, CUU 0.21 0.25 0.29 0.33 0.42
3 CUA, CUG 0.15 0.18 0.19 0.23 0.22
4 UUG 0.43 0.45 0.49 0.68 0.66
5 UUA,UUG 0.25 0.25 0.29 0.26 0.27
Val: 1 GUA, GUG, GUU 0.86 0.86 0.78 1.35 1.45
2A GUC, GUU 0.14 0.14 0.17 0.19 0.20
2B GUC, GUU 0.14 0.17 0.19 0.26 0.31
Gly: 1+2 (GGG) / (GGA,GGG) 0.48 0.51 0.55 0.78 0.79
3 GGC, GGU 0.98 1.08 1.19 1.41 1.77
Ala: 1B GCU, GCA, GCG 0.73 0.83 1.00 1.24 1.49
2 GCC 0.14 0.15 0.17 0.23 0.25
Arg: 2 CGU, CGC, CGA 1.06 1.03 1.10 1.68 1.81
3 CGG 0.14 0.18 0.10 0.16 0.16
4 AGA 0.19 0.17 0.19 0.23 0.25
5 AGG 0.09 0.11 0.11 0.17 0.16
Ile: 1+2 (AUC, AUU) / AUA 0.78 0.84 0.94 1.34 1.75
Lys AAA, AAG 0.43 0.48 0.52 0.62 0.74
Glu 2 GAA, GAG 1.05 1.10 1.18 1.71 2.08
Asp 1 GAC, GAU , 0.54 0.58 0.60 0.85 1.10
Thr: 1 ACC, ACU 0.02 0.03 0.04 0.04 0.05
2 ACG 0.12 0.14 0.15 0.19 0.22
3 ACC, ACU 0.25 0.26 0.27 0.34 0.39
4 ACA, ACU, ACG 0.20 0.22 0.23 0.35 0.49
Asn AAC, AAU 0.27 0.27 0.31 0.43 0.52
Gln: 1 CAA 0.17 0.19 0.26 0.22 0.31
2 CAG 0.20 0.22 0.25 0.36 0.44
Tyr: 1 UAC, UAU 0.17 0.17 0.19 0.33 0.30
2 UAC, UAU 0.28 0.27 0.27 0.37 0.36
Ser: 1 UCA, UCU, UCG 0.29 0.39 0.39 0.49 0.52
2 UCG 0.08 0.07 0.08 0.10 0.10
3 AGC, AGU 0.31 0.31 0.32 0.38 0.40
5 UCC, UCU 0.17 0.18 0.20 0.26 0.29
His CAC, CAU 0.14 0.16 0.19 0.24 0.31
Trp UGG 0.21 0.20 0.24 0.29 0.36
Pro: 1 CCG 0.20 0.17 0.25 0.19 0.19
2 CCC, CCU 0.16 0.18 0.16 0.28 0.27
3 CCA, CCU, CCG 0.13 0.13 0.16 0.18 0.18
Phe UUC, UUU 0.23 0.26 0.30 0.33 0.36
Cys UGC, UGU , 0.36 0.35 0.37 0.50 0.50
Met: m AUG 0.16 0.18 0.21 0.29 0.31
f1 AUG 0.27 0.34 0.43 0.45 0.72
f2 AUG 0.16 0.16 0.17 0.24 0.27
only used rarely or infrequently in a genome but also
decoded by a low-abundant tRNA (rare tRNA) or other
factor(s) such as less-efficient translation releasing
factor(s) in an organism. Therefore, a RC encoding an
amino acid is a rare-tRNA associated codon while a RC
for translation termination is a stop codon with lowest
usage frequency in a genome. Meanwhile, a low-usage
codon is defined as a codon (whether synonymous or not)
that is used rarely or infrequently in a genome, and its
usage frequency should be: (a) lower than the usage
frequencies of the non-degenerate codons (that is, AUG
for Met, and UGG for Trp); (b) lower than the usage
frequencies of the optimal codons for amino acids (Leu,
Ile, Val, Ser, Pro, Thr, Ala, Arg, Gly and Gln) with 2 or
more degenerate codons because these amino acids have 2
or more tRNA carriers with at least one to specify the
corresponding optimal codon of each amino acid; (c)
lower than the smallest value (cut-off frequency) among
Gene Therapy and Molecular Biology Vol 10, page 7
7
the usage frequencies listed in (a) and (b). Therefore, cutoff
frequency is an objective value rather than an arbitrary
one for defining the boundary between low-usage codons
and common codons in each organism. Data in Tables 1
and 2 suggest that the usage frequency of Trp codon UGG
is the very cut-off frequency value of E. coli.
V. Determination of E. coli low-usage
codons and rare codons
Based on the above definitions, a low-usage codon is
not necessarily a RC but a RC is definitely a low-usage
codon. All the 3 stop codons (UAA, UAG and UGA) of E.
coli (Tables 1 and 2) are the least usage codons of the
bacterium. According to the above definitions, they should
be low-usage codons. However, UAA cannot be regarded
as the rare stop codon but a major stop codon of E. coli
because it has the highest usage among the 3 stop codons.
The low-usage sense codons of E. coli, based on the
1.28% cut-off of usage frequency calculated from
GenBank release #69 (Table 1, columns of “II”) and on
the 0.869% cut-off of real-time usage frequency calculated
from 140 proteins of E. coli growing at the rate of 1.07
doublings/hour (Table 2), are all listed in Table 5. The
relative tRNA contents measured by Ikemura (1980,
1981a,b, 1982, 1985) and Dong et al, 1996 are also
included in the table. The 3 stop codons are all E. coli lowusage
codons, but not listed in Table 5. Based on the
above RC definition, 10 low-usage sense codons out of the
30 listed in Table 5 are excluded from the list of E. coli
rare sense codons because of the following reasons:
(a) UGU and UGC. They are the only synonymous
codons of Cys, and both are decoded by a single tRNACys
which has a relative amount of > 0.36 (Dong et al, 1996).
(b) ACU and ACG. They are 2 synonymous codons
of Thr (the total is 4), but they recognized by more than 2
tRNAs. ACU is recognized by tRNAThr1
, tRNAThr3
and
tRNAThr4
, and the sum of the relative contents of these 3
tRNAs is > 0.45 (Dong et al, 1996) or 0.8 (Ikemura,
1985). ACG is recognized by tRNAThr2
and tRNAThr4
, and
the sum of the relative contents of these 2 tRNAs is > 0.32
(Dong et al, 1996) or minor (Ikemura, 1985).
(c) CAC and CAU. They are the only synonymous
codons of His, and both are decoded by a single tRNAHis
which has a relative amount of 0.4 (Ikemura, 1985) or >
0.14 (Dong et al, 1996).
(d) UUG. UUG is one of 6 synonymous codons of
Leu, and is decoded by 2 tRNAs-tRNALeu4(UUG)
and
tRNALeu5(UUA,UUG)
. The relative amount of tRNALeu4(UUG)
is
0.43 (Dong et al, 1996) while tRNALeu5(UUA,UUG)
has a the
relative amount of 0.25 (Ikemura, 1985) or > 0.25 (Dong
et al, 1996).
(e) GUA. GUA is one out of the 4 synonymous
codons of Val. The single tRNA that can decode this
codon is very high abundant and the relative amount is
1.05 (Ikemura, 1985) or > 0.78 (Dong et al, 1996).
(f) AAG. Lys has only 2 synonymous codons AAA
and AAG, and the 2 codons are decoded by a single
tRNALys
which has a relative amount of 1.0 (Ikemura,
1985) or > 0.43 (Dong et al, 1996).
(g) AAU. Asn has only 2 synonymous codons AAU
and AAC, and the 2 codons are decoded by a single
tRNAAsn
which has a relative amount of 0.6 (Ikemura,
1985) or > 0.27 (Dong et al,1996). Besides, the usage
frequencies of AAU calculated from the 3 GenBank
releases are much higher than those (14.95, 15.2 and 15.0
per thousand) of AGC (the optimal codon of Ser).
Moreover, UGU and UGC for Cys, CAC and CAU
for His, AAG for Lys, and AAU for Asn are not regarded
as rare codon in Table 5 because these amino acids all
have 2 synonymous codons that are respectively decoded
by a single tRNA (Crick’s “wobble hypothesis” can
explain why a single tRNA can recognize multiple
degenerate codons (Crick, 1966). In addition, whether the
above 10 low-usage sense codons that have been excluded
from E. coli rare codon list can cause significant
expression problems, has never been reported.
There are 9 amino acids (Phe, Tyr, His, Gln, Asn,
Lys, Asp, Glu and Cys) that have only 2 synonymous
codons. Tables 3 and 4 demonstrate that (a) the 2
synonymous codons of Phe, His, Asn, Lys, Cys, Asp or
Glu are specified by a single tRNA; (b) the 2 codons
(UAU and UAC) of Tyr are non-differentially recognized
by the 2 tyrosinyl tRNAs (tRNATyr1
and tRNATyr2
); and (c)
Gln has 2 tRNAs and each recognize a Gln codon
(tRNAGln1
for CAA and tRNAGln2
for CAG). Although
there exists usage difference in the 2 synonymous codons
for each of the 8 amino acids Phe, Tyr, His, Asn, Lys,
Asp, Glu and Cys, it is not due to their tRNA availability
(Grosjean et al, 1978) but mainly to gene expressivity
(Gouy et al, 1982; Ikemura, 1985). The usage differences
in the 2 synonymous codons of the above 8 amino acids
may be explained by the “rules” proposed for the choice or
usage preference of the synonymous codons that are
decoded by a single tRNA (Gouy et al, 1982; Ikemura,
1985). Therefore, any codon for the above amino acids
(except Gln) cannot be regarded as a rare codon even
though it may be a low-usage codon according to the
usage cut-off determined as described above.
In this paper, the following codons are considered to
be the rare sense codons of E. coli and are classified into 2
groups:
(a) Group I: AGG, AGA, CGA, CUA, AUA, CCC
and CGG (arranged from least usage to high usage based
on the average usage frequency). The usage frequency of
each codon calculated from GenBank release #69 is <
0.5%.
(b) Group II: ACA, CCU, UCA, GGA, AGU, UCG, CCA,
UCC, GGG, CUC, CUU, UCU and UUA (arranged from
least usage to high usage based on the average usage
frequency). The usage frequency of each codon calculated
from GenBank release #69 is > 0.5% but < 1.1%.
Some rare sense codons in Group II (namely the first
highlighted 6 in the above list) have been reported to be
involved in translational problems (Konigsberg and
Godson, 1983; Chen and Inouye, 1990a; Ma et al, 2003;
Zhou et al, 2004). According to this, Group II can further
be classified into 2 subgroups:
Group IIa: ACA, ACU, UCA, GGA, AGU and UCG;
Group IIb: CCA, UCC, GGG, CUC, CUU, UCU and
UUA.
At present, whether rare sense codons in Group IIb
can causeexpression problems have not been reported, and
Chen and Texada: E. coli low-usage codons and rare codons
8
Table 5. Low-usage sense codons of E. colia
Codon frequency (per thousand)b
Cognate tRNA relative amount and other codons c
GenBank release # Growth Rate Growth Rate f
Amino
Acid Codon 63 69 147 0.4 1.07 2.5
Amount d
Other Codons
e
0.40 1.07 2.50
Other Codons
e
Arg AGG 1.3 1.4 2.6 0.09 0.05 0.03 minor AGA 0.09 0.11 0.16 /
Arg AGA 2.1 2.1 4.5 1.32 0.99 0.67 minor AGG 0.19 0.19 0.25 /
Arg CGA 3.1 3.1 4 1.32 0.99 0.67 minor CGU,CGC 1.06 1.1 1.81 CGU,CGC
Leu CUA 3 3.2 4.5 2.15 1.53 0.82 minor / 0.15 0.19 0.22 CUG
Ile AUA 3.9 4.1 8.2 0.93 0.75 0.52 minor / 0.78 0.94 1.75 AUC,AUU g
Pro CCC 4.2 4.3 5.6 3.32 2.1 1.09 minor / 0.16 0.16 0.27 CCU
Arg CGG 4.6 4.6 6.4 1.75 1.23 0.62 minor / 0.14 0.1 0.16 /
*Cys UGU 4.8 4.7 5.3 4.23 3.64 2.76 minor UGC 0.36 0.37 0.5 UGC
*Cys UGC 6.1 6.1 6 5.29 4.77 3.81 minor UGU 0.36 0.37 0.5 UGU
Thr ACA 6.5 6.5 10.7 3.48 2.99 2.61 minor ACG 0.2 0.23 0.49 ACG,ACU
Pro CCU 6.6 6.6 7.9 4.99 4.79 4.38 major CCG,CCA 0.16 0.16 0.27 CCC
CCU 0.13 0.16 0.18 CCA,CCG
Ser UCA 6.6 6.8 9.9 3.89 3.09 1.98 0.25 UCU,UCG 0.29 0.39 0.52 UCU,UCG
Gly GGA 7 7 10.6 2.71 2.21 1.26 0.15 GGG 0.48 0.55 0.79 GGG g
Ser AGU 7.4 7.2 10.7 3.99 3.01 2.19 0.25 AGC 0.31 0.32 0.4 AGC
Ser UCG 7.9 8 8.5 6.05 4.58 2.51 minor UCU,UCA 0.29 0.39 0.52 UCU,UCA
UCG 0.08 0.08 0.1 /
Pro CCA 8.1 8.2 8.6 6.52 6.4 5.18 major CCG,CCU 0.13 0.16 0.18 CCG,CCU
Ser UCC 9.4 9.4 9.3 11.2 12.1 11.7 / UCU 0.17 0.2 0.29 UCU
Gly GGG 9.6 9.7 11.6 4.81 3.57 2.36 0.15 GGA 0.48 0.55 0.79 GGA g
GGG 0.1 / /
Leu CUC 9.7 9.9 10.1 6.19 5.52 4.09 0.3 CUU 0.21 0.29 0.42 CUU
Leu CUU 9.9 10.2 12.5 5.7 4.64 3.86 0.3 CUC 0.21 0.29 0.42 CUC
§Thr ACU 10.8 10.2 11 13.9 16.8 20.6 0.8 ACC 0.02 0.04 0.05 ACC
ACU 0.25 0.27 0.39 ACC
ACU 0.2 0.23 0.49 ACA,ACG
Ser UCU 10.5 10.4 10.9 13.1 11.1 16.3 / UCC 0.17 0.2 0.29 UCC
*His CAC 10.8 10.7 8.8 13.9 13.9 14.2 0.4 CAU 0.14 0.19 0.31 CAU
Leu UUA 10.5 10.9 15 6.13 4.64 2.73 0.25 UUG 0.25 0.29 0.27 UUG
§Leu UUG 11.3 11.5 12.9 6.63 5.72 4.27 0.25 UUA 0.43 0.49 0.66 /
UUG 0.25 0.29 0.27 UUA
*His CAU 11.3 11.6 12.5 9.23 8.11 6.78 0.4 CAC 0.14 0.19 0.31 CAC
#Val GUA 12.1 11.6 11.9 15.9 18.7 22.3 1.05 GUU.GUG 0.86 0.78 1.45 GUU,GUG
*#Lys AAG 11.9 12 13.1 12.1 13.7 17.2 1.0 AAA 0.43 0.52 0.74 AAA
§Thr ACG 12.5 12.7 13.8 7.53 6.21 4.17 minor ACA 0.12 0.15 0.22 /
ACG 0.2 0.23 0.49 ACA,ACU
*#Asn AAU 16.3 16.3 22.8 9.79 7.79 5.61 0.6 AAC 0.27 0.31 0.52 AAC
a. Low-usage sense codons of E. coli were selected and include: all the codons at a cut-off of < 1. 28% frequency calculated from GenBank
release #69, and all the codons at a cut off < 0.869% real-time frequency when E. coli was at the growth rate of 1.07 doublings/hour. The
cut-offs are the lowest frequency values among those of non-degenerate codons (Met and Trp) and the optimal codons for amino acids with
more than 2 degenerate codons (Leu, Ile, Val, Ser, Pro, Thr, Ala, Arg and Gly).
b. The data are codon usage frequencies calculated from the sequence data of GenBank release #63, 69 or 147 (refer to Table 1) or
calculated by Dong et al, 1996 from 140 proteins coding frames when E. coli was at different growth rate of 0.4 doublings/hour (refer to
Table 2).
c. The relative amount is the amount relative to that of tRNALeu1(CUG)
that is normalized to 1.0.
a. Taken and adapted from Ikemura Ikemura (1981a,b, 1985) (refer to Table 3).
b. The other synonymous codons that are also recognized by the same tRNA.
c. Taken and adapted from Dong et al, 1996 (refer to Table 4).
Gene Therapy and Molecular Biology Vol 10, page 9
9
d. The tRNAILe2
co-migrated with tRNAILe1
on 2-D PAGE. The latter recognizes AUU and AUC while the former recognizes AUA, and the
relative content for different growth rate is the sum of both tRNAs. The tRNAGly1(GGG)
also co-migrated with tRNAGly2 (GGA,GGG)
on 2-D
PAGE, and the relative content for different growth rate is similarly the sum of both tRNAs.
* Cys, His, Lys and Asn all have 2 synonymous codons that are recognized by one single tRNA.
§ Codons ACU and ACG of Thr are decoded by 3 and 2 tRNAs, respectively. Codon UUG of Leu are also decoded by 2 tRNAs.
# The relative amounts for the single tRNAs which decodes GUA (Val), AAG (Lys) and AAU (Asn) are high (> 0.6 according to
Ikemura (1985) or > = 0.27 according to Dong et al, 1996).
needs further studies. However, the rare sense codons in
Group I, especially Arg rare codons AGG and AGA, have
been extensively studied, and most effects of RCs and
RCCs as well as their underlying mechanisms are obtained
from studies of this group of rare sense codons (Hackett
and Reeves, 1983; Pedersen, 1984; Misra and Reeves,
1985; Fang et al, 1986; Garcia et al, 1986; Pohlner et al,
1986; Harms and Umbarger, 1987; Chen et al, 1990a,b,
1991; Gurskii et al, 1992a, b; Ivanov et al, 1992; Kane et
al, 1992; Gursky et al, 1994; Hua et al, 1994, 1996;
Vilbois et al, 1994; Curran, 1995; Del, Jr. et al, 1995;
Kane, 1995; Bouquin et al, 1996; Calderone et al, 1996;
Major et al, 1996; Saraffova et al, 1996; Zahn and Landy,
1996; Zahn, 1996; Babic et al, 1997; Ivanov et al, 1997;
Schwartz and Curran, 1997; Tsai and Curran, 1998;
Wakagi et al, 1998; Jiang et al, 1999; Imamura et al, 1999;
Roche and Sauer, 1999; Sauer and Nygaard, 1999; KleberJanke
et al, 2000; Zdanovsky and Zdanovskaia, 2000;
Acosta-Rivero et al, 2002; Hayes et al, 2002; Kapust et al,
2002; Laine et al, 2002; Park et al, 2002; McNulty et al,
2003; Olivares-Trejo et al, 2003; Tan et al, 2003; Chen et
al, 2004; Sakamoto et al, 2004; Shu et al, 2004; Gurvich et
al, 2005).
Codon optimization (a kind of nucleotide substitution
which replaces the rare codons in a gene by synonymous
optimal or other major codons) and rare tRNA
supplementation (co-expression of rare-tRNA genes) are
the 2 strategies to overcome the expression problems
caused by rare sense codons or study the underlying
mechanisms. In order to highly express a foreign gene in
bacterium E. coli, either one or both of the 2 strategies
may be adopted. Because mutant strains such as Rosetta
2(DE3) of E. coli (Chen et al, 2004) are commercially
available for rare tRNA supplementation, it is
recommended to first try this strategy when a foreign gene
cannot be expressed to satisfaction in regular expression
host such as BL21(DE3). Further, if rare tRNA
supplementation cannot correct the expression problem(s),
codon optimization to replace some or all of the E. coli
RCs or their RCCs in a foreign gene is likely to be a must.
The E. coli RCs that should be considered in codon
optimization, based on the so far reports, at least include
all the above 7 Group I RCs and probably the 6 Group IIa
RCs.
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