J. theor. Biol.

(1997) 185, 511521

00225193/97/080511 +11 $25.00/0/jt960310 7 1997 Academic Press Limited
Does Escherichia coli Optimize the Economics of the Translation Process?
Jr:N Soioxovici, Tnirrr. LrsNii :Nb Ci:ibr Rriss*
Centre de Genetique Moleculaire, CNRS, bax t 24, Av de la Terrasse, F91198,
Gif Sur Yvette, France
(Received on 20 June 1996, Accepted in revised form on 6 November 1996)
The codon translation rate is usually assumed to be proportional to the cellular concentration of the
cognate tRNA, but synonymous codons sharing the same cognate tRNA may be translated at rather
dierent rates. To account for the latter observation, we assume that the translation process is optimized
in two respects: (i), the codon demand is optimized with respect to the supply of cognate tRNAs
(composition of the tRNA pool); and (ii), for synonymous codons sharing the same cognate tRNA,
the usage frequency of each codon correlates optimally with the stability of the codon-anticodon
complex. These assumptions allow us to compute the relative rate constants of synonymous codons.
Highly expressed genes, which produce 8090% of the protein mass in the E. coli cell, appear to have
selected codons which make an optimal use of the tRNA pool. Assuming the optimization criteria were
valid, a list of codon translation times (in ms) were derived from available experimental data.
7 1997 Academic Press Limited
Introduction
In Escherichia coli, the codon translation rates
correlate fairly well with the concentrations of the
cognate tRNAs (Ikemura, 1981a; Varenne et al.,
1984; Holm, 1986; Liljenstro m & von Heijne, 1987).
It was generally assumed that, under given growth
conditions, all synonymous codons sharing the same
cognate tRNA are translated at a common rate
(Varenne & Lazdunski, 1986; Liljenstro m & von
Heijne, 1987; Bulmer, 1988). Actually, experimental
evidence (Curran & Yarus, 1989; Soerensen &
Pedersen, 1991) argues against this hypothesis, as for
instance the two Glu codons, GAA and GAG, are
translated in E. coli at rates diering by over a factor
of three though they share the same cognate tRNA
(Soerensen & Pedersen, 1991).
In the absence of complete experimental data, we
attempt to evaluate indirectly the translation rates of
synonymous codons. Here, we consider the hypoth-
esis that the translation process is optimized, in order
to best exploit the cellular resources invested in
translation. Protein production is known to claim
most of the cellular energy. Energy expenditure can be
minimized by carefully adaptating the oer (i.e.,
the resources the cell invests for translation:
ribosomes, ternary complexes . . .) to the demand
(codon usage), and the oer/demand handling
eciency. Given these minimization hypotheses,
codon demand turns out to be related simply both to
the cognate tRNA availability, and to the biophysical
chemistry characteristics of the codon-anticodon
match. For synonymous codons sharing the same
cognate tRNA, these relations set the relative time
constants of their translation kinetics.
Using data available for E. coli, our results suggest
that in this species, the production of some 90% of
the protein mass might indeed be optimized for best
cellular resource management.
The Codon Translation Rate
Early biometric data from E. coli [summarized in
Bremer & Dennis (1987)] suggested that the total
number of ternary complexes is only a few times
higher than the number of ribosomes present in the
*Author to whom correspondence should be addressed.
E-mail:claude.reiss.cgm.cnrs-gif.fr.
tRNAs
Codons
. soioxovici ET AL. 512
cell. Recent experimental results obtained in vivo
(Emilsson & Kurland, 1990; Emilsson et al., 1993)
conrm that in this species, ternary complexes are in
short supply. For the most abundant of the tRNA
species studied, the E. coli cell contains only one
tRNA molecule for three ribosomes, and the ratio
is below 1:100 for the less abundant tRNAs. It is
therefore generally accepted that tRNA availability is
the rate-limiting factor of translation elongation. In
the following, we will assume translation kinetics to be
of pseudo-rst order with respect to tRNA concen-
trations. Kinetic data of the main steps of the trans-
lation elongation cycle, obtained in vitro [summarized
in Yarus & Smith (1995)] support this assumption.
In sequenced genes of E. coli, synonymous codons
appear to be used with signicantly dierent
frequencies. In particular, highly expressed genes tend
to restrict to a subset of synonymous codons. On the
other hand, Pedersen (1984) showed that in vivo, the
translation rate of highly expressed genes is larger
than that of weakly expresed genes. Taken together,
these results suggest that codons used often are
translated faster than less frequent, synonymous
codons sharing the same cognate tRNA. For a few
codons, in vivo measurements provide a direct proof
of this assumption (Soerensen & Pedersen, 1991;
Curran & Yarus, 1989). Hence the pseudo-rst order
rate constants of the elongation kinetics are likely to
be codon-specic, even for isoaccepted codons.
Presumably, this specicity is introduced upon
binding of the ternary complex to the A site of the
ribosome. Binding steps include, successively, initial
binding of the ternary complex, codon recognition by
its anticodon, transfer of the cognate ternary complex
to the pre-A site (anticodon bound to mRNA,
acceptor arm still bound to EF-Tu), GTP hydrolysis,
dissociation of EF-Tu and transfer of the aa-tRNA to
the A site (Rednina et al., 1996).
In vitro, the initial binding stepoccurs at a rate which
does not aect (Bilgin et al., 1988) or aects moder-
ately (Rodnina et al., 1995) the rate of cognate amino
acid incorporation into the nascent peptide. This rate
may therefore be assumed codon-independent. In
contrast, the kinetics of the step in which GTP is
hydrolyzed, is codon-dependent (Rodnina et al.,
1995). Since this step follows codon recognition, the
latter is probably responsible for the codon-dependent
rate of GTP hydrolysis. The rate constant of the
translation of a given codon would then depend on the
stability of the codon-anticodon interaction, hence be
characteristic of each recognized codon. This is
consistent with the nding by Ikemura (1981b) that the
stability of the codon-anticodon match correlates with
the codonusage. Inthis study we restrict tothe simpli-
cationassumingthat at anytime, all tRNAsareengaged
in ternary complexes. This is to provide a clear insight
into the minimization mechanism. A more involved
model relating tRNA and cognate codon translation
rate was developed by Liljenstro m et al. (1985).
Optimizing Translation Time by Mutual Adaptation of
tRNA Supply and Codon Demand
Consider a set of coding sequences of a given
species, in which tRNA (i) is present at a
concentration n
i
such that Sn
i
=1, the sum extending
over the number of tRNA species present in the cell
(41 for E. coli for instance). In the set, N
i
codons are
recognized by tRNA (i) (the N
i
codons are identical
or dierent, depending on whether tRNA (i)
recognizes one or more codons).
According to the hypothesis that tRNA availability
is the rate-limiting step for codon translation, and
neglecting codon context eects, the time taken to
translate one codon recognized by tRNA (i) is k/n
i
.
For the N
i
codons, the translation time is t
i
=kNv
i
/n
i
(with SN
i
=N,v
i
=N
i
/N,Sv
i
=1).
(a) We assume that the time constant k does not
depend on (i). Then the time taken to translate the
total number N of codons in the set is T=kNS(v
i
/n
i
),
the sum extending over all the tRNA species. The
frequencies v
i
=N
i
/N are xed for the set of coding
sequences under consideration, so T is a function of
variables n
i
only, linked by Sn
i
=1
The values of n
i
for which T is optimal are
among those which give extrema to the auxiliary
function: F=S(v
i
/n
i
) +l(Sn
i
1), l =constant, and
are among the solutions of the partial dierential
equation 1F/1n
i
=0 or (zv
i
)/n
i
=Szv
i
, which
corresponds to n
i
=(zv
i
)/Szv
i
and T/kN=(Szv
i
)
2
,
as already shown by Pontier (1970).
(b) We next assume that the rate constant 1/k is not
the same for all codons, but is multiplied by a
tRNA-specic eciency factor f
i
, a part of the rate
constant corresponding to the energy invested in the
codon-anticodon (i) match. Replacing k by k/f
i
in (a)
yields, for n
i
=(z(v
i
/f
i
))/Szv
i
/f
i
), the optimal value
of T/kN=(S
z
(v
i
/f
i
))
2
.
(c) The genetic code is degenerate, as 61 codons
encode 20 amino acids, transferred in E. coli by 41
cognate tRNA species. Among the latter, most can
match two or even three dierent codons.
I soacceptors
Synonymous codons
21 22
2
12 13
1
11
I soacceptors
Synonymous codons
2 3 1
1 2
+
1r:Nsi:1ioN oi1ixiz:1ioN 513
Let us assume that tRNA (i) matches codon (i j) with
eciency f
i j
(in E. coli, j =13 maximum, depending
on the multiplicity of tRNA (i); f
i2
and/or f
i3
may be
zero, in case one and/or two codons are only
recognized by tRNA (i); Si j =61).
Then Tis optimal for n
i
=zS
j
(v
i j
/f
i j
)/S
i
z(S
j
i j
/f
i j
),
and corresponds to t
i
=S
j
(z(Sv
i j
/f
i j
))
2
.
(d) So far we have concentrated on the optimiz-
ation of the volume of the oer (the distribution of
the tRNAs characterized by the values of n
i
) and the
volume of the demand (v
i
or v
i j
) to minimize T.
Further optimization can be achieved by adapting
oer and demand to the handling capacity, i.e. to the
eciencies f
i j
. This can be done by considering
t
i
/kN=(1/n
i
)S
j
(v
i j
/f
i j
) as a function of f
i j
. Since
S
j
f
i j
=f
i
, t
i
is optimal for f
i j
/f
i
=zv
i j
/m
i
, where
m
i
=Szv
i j
. It follows that f
i1
:f
i2
:f
i3
=zv
i1
:zv
i2
:zv
i3
.
Then n
i
=(m
i
/zf
i j
)/S
i
(m
i
/zf
i
) and T/kN=t =
(S
i
(m
i
/zf
i
))
2
.
Under the hypotheses made and given the
eciencies f
i j
, which depend mainly on the physical
characteristics of the codon (i j)-anticodon (i)
interaction, it appears that the cell can optimize the
translation time via two compatible routes: (1) by
setting the frequency with which an isoaccepted
codon is used proportional to the square of the
corresponding eciency (related to the strength of the
codon-anticodon match); and (2) by adjusting the
tRNA pool so that the concentration of any tRNA is
proportional to the sum of the square roots of the
cognate codon frequencies.
The model yields only relative values of the f
ij
s. In
the absence of the experimental value of at least one
f
ij
per tRNA (i), we may assume that the most used
codon [say (i1)] is that which ts best the anticodon
of (i), and that tRNA (i) has evolved so as to match
optimally codon (i1), i.e., f
i1
=1. This amounts to
assume that for all codons, processed either by a
tRNA having only one cognate codon, or used most
frequently among the cognate codons, the translation
rate constant is precisely 1/k. It follows then that
f
i j
=z(v
i j
/v
i1
), f
i
=m
i
/zv
i1
and n
i
=KzS
j
zv
i1
v
i j
,
where 1/K=S
i
zS
j
zv
i1
v
i j
.
The optimized time taken to translate codon (i j)
is t
i j
=k/(n
i
zv
i j
/v
i1
) =k/d
i j
, where the parameter
d
i j
(n
i
) =n
i z
v
i j
/v
i1
characterizes the optimal match
between oer and demand for that codon. Introduc-
ing the expression of n
i
, we may also write
d
i j
( f
i j
) =K f
i j
zm
i
.
(e) Some codons benet from degenerate recog-
nition, as they are matched by two isoacceptor tRNA
species (i) and (m). Let (1), (2), . . ., (q) designate all
the codons accepted by tRNA (i) and/or (m) (in the
following, all sums run over j =1 to q). We consider
that the pooled tRNAs (i) and (m) translate these
codons with eciencies f
im,1
, f
im,2
, . . ., f
im,q
, respectively.
The time taken to translate all codons recognized by
tRNAs (i) and (m) is t
im
such that t
im
/kN=S
j
(v
j
/
(n
i
+n
m
)f
im,j
). Optimizing with respect to f
im,j
yields
f
im,j
=f
im
zv
j
/S
j
zv
j
with f
im
=S
j
f
im,j
, and optimizing
with respect to (n
i
+n
m
) yields (n
i
+n
m
) =KS
j
zv
j
/f
im
.
Let us assume that codons (1) and (q) are optimally
translated by tRNA (i) (f
i1
=1) and (m) (f
mq
=1),
respectively. The time taken to translate all codons (1)
in the set, t
1
is such that t
1
/kN=v
1
/n
i
[translation by
tRNA (i) only] and t
1
/kN=v
1
/((n
i
+n
m
)f
im,1
) [trans-
lation by the pool of tRNA (i) and (m)]. Thus
f
im,1
=n
i
/(n
i
+n
m
). Similarily, f
im,q
=n
m
/(n
i
+n
m
). It
follows that f
im,1
+f
im,q
=1, f
im
=Szv
j
/(zv1 +zv
q
),
f
im,j
=zv
j
/(zv1 +zv
q
).
The optimal translation time of codon j is
t
j
=k/d
im,j
, where d
im,j
=(n
i
+n
m
)f
im,j
.
Testing the Optimization Hypothesis in Selected E. coli
Gene Sets
The optimal demand/oer conditions derived in the
previous section can be checked against experimental
data, for sequenced genes (codon frequency usage) of
species for which the tRNA distribution under given
physiological conditions is known.
In the cell, the amount of proteins produced per
gene varies within large limits. The E. coli cell for
instance, synthesizes tens of thousands copies of each
of the 54 ribosomal proteins, but less than ten copies
of the lacl repressor protein. It must be realized that
at any time, less than a hundred genes produce about
90% of the protein molecules present in the E. coli
cell, the remainder being produced by some three
thousand other genes. The former are therefore
prevailing in the global cellular economy.
. soioxovici ET AL. 514
T:nir 1
GenBank acronymes of the set of 41 ribosomal genes of E. Coli (set RIBO in the text)
ggECOHIMA.RPLT gg ggECORPSV.RPSV gg ggECRPOS10.PE11 gg ggECSPC.PE5 gg
ggECORNPA.RPMH gg ggECOSTR1.RPSL gg ggECRPSB.RPSB gg ggECSPC.PE6 gg
ggECORPLRPM.RPMA gg ggECRPOS10.PE3 gg ggECRPSFRI.PE1 gg ggECSPC.PE7 gg
ggECORPLY.RPLY gg ggECRPOS10.PE4 gg ggECRPSFRI.PE3 gg ggECSPC.PE8 gg
ggECORPMBG.PE1 gg ggECRPOS10.PE5 gg ggECRPSFRI.PE4 gg ggECSPC.PE9 gg
ggECORPMBG.PE2 gg ggECRPOS10.PE6 gg ggECRPSI.PE2 gg ggECSPC.PE10 gg
ggECORPMFA.RPMF gg ggECRPOS10.PE7 gg ggECRPSL.RPSJ gg ggECSPC.PE11 gg
ggECORPOA.RPLQ gg ggECRPOS10.PE8 gg ggECRPSO.PE1 gg ggECSPC.PE12 gg
ggECORPSOP.RPSO gg ggECRPOS10.PE9 gg ggECSPC.PE3 gg ggECTRMD.PE2 gg
ggECORPSRPO.RPSU gg ggECRPOS10.PE10 gg ggECSPC.PE4 gg ggECTRMD.PE4 gg
ggECORPST.RPST gg
The trend to optimize the oer/demand volumes
and handling may therefore be expected to be much
greater in heavily expressed genes. We have therefore
selected four sets of E. coli genes: for two of them, the
amount of protein molecules produced per gene has
been determined (Van Bogelen et al., 1992).
(i) 14 genes of high expression (HIGH set). The
total numberof proteinmoleculesproducedbythisset is
222338, the average number of codons per gene is 390.
(ii) 15 genes which yield the lowest number of
protein molecules, among the genes for which the
expression was quantitated (LOW set). These are
by no means the genes with lowest absolute
expression, but are those which could be quantitated
just above the detection limit of the method used to
quantify the protein production. The total number of
protein molecules produced by the genes of this set is
3052, the average number of codons per gene is 503.
Among the 54 ribosomal genes, the 41 listed in
GenBank form the RIBO set. These genes carry an
average number of codons of 230. The total number
of ribosomal protein molecules per cell is in excess of
10
6
(Neidhardt, 1987).
The last set, used as reference, contains the 2129
genes or putative coding sequences extracted from the
E. coli entrance of the GenBank, release 79
(TOTAL set). This set includes the three previous
sets. The average number of codons per gene in the
TOTAL set is 300.
Results
The list of the genes of the RIBO, HIGH and LOW
sets are given in Tables 13.
Table 4 shows the frequencies of usage (%) of
the 61 codons in the four sets. Whereas the TOTAL
and LOW sets use all codons, one (AGG, the less
used codon in the TOTAL and LOW sets) is
avoided in the RIBO set, but as much as 14 codons
are missing in the HIGH set. Though the HIGH
and LOW sets carry comparable total numbers of
codons, the former avoids close to one codon out
of four, the latter none. Some of the avoided
codons (UUA, CUU) are in fair use in the TOTAL
set.
Codon usages in the TOTAL and LOW sets
T:nir 2
GenBank acronymes of the set of the 14 genes of
E. Coli most heavily expressed (set HIGH in the text)
Casename NR Casename NR
ECOAROF 311 ECOMOTAB 150
ECOCARAB 321 ECOPCKA 312
ECOFTSQA 30 ECORPOH 20
ECOGLTA 71 ECGRPEP 211
ECOGLTA 158 ECPPCGP 350
ECOGLTA 250 ECSPCPE 300
ECOGLTA 243 ECTRMDP 80
ECOLYSU 245
(NR=number of expressed mols cell
1
.)
T:nir 3
GenBank acronymes of the set of the 15 genes of E. Coli expressed at the lowest
measurable level (set LOW in the text)
Casenote NR Casename NR
ggECODNAK gg 9000 ggECOTGTUFB.TUFB gg 35 000
ggECOFABB.PE1 gg 28 300 ggECTIG.TIG gg 8800
ggECOFOLA.FOLA gg 9388 ggECACEB.PE2 gg 10 800
ggECOHATP.PE5 gg 4800 ggECACEX.PE4 gg 5645
ggECOOMPA.OMPA gg 20 300 ggECGROES.PE1 gg 2905
ggECORPOA.RPOA gg 6400 ggECRPSFRI.PE1 gg 10 000
ggECOSTR3.TUFA gg 52 000 ggECRSPA.RPSA gg 19 000
(NR=number of expressed mols cell
1
).
1r:Nsi:1ioN oi1ixiz:1ioN 515
correlate quite well (0.964), the HIGH and RIBO sets
correlate fairly (0.867). The correlations of the RIBO
set with the TOTAL set (0.718) and the LOW set
(0.775) are rather low.
T:nir 5
Amino acid usage (%) in the four sets
Amino acid HIGH RIBO LOW TOTAL
ggLYSgg 5.921 9.648 4.727 4.803
ggASNgg 3.135 3.662 3.898 4.065
ggILEgg 6.621 5.678 5.362 5.836
ggMETgg 2.371 1.707 2.877 2.385
ggARGgg 5.232 9.443 5.940 5.798
ggSERgg 4.141 4.423 6.059 5.936
ggTHRgg 6.356 5.431 5.084 5.532
ggTRPgg 0.682 0.535 1.271 1.371
ggTYRgg 2.409 1.851 2.656 2.948
ggLEUgg 7.449 7.036 10.038 10.158
ggPHEgg 3.263 3.086 3.336 3.774
ggCYSgg 0.743 0.411 0.990 1.097
ggGLUgg 8.566 5.986 6.987 6.082
ggASPgg 6.366 4.361 5.288 5.334
ggVALgg 8.895 9.031 6.897 7.080
ggGLYgg 9.541 8.044 7.658 7.384
ggALAgg 8.900 10.471 9.348 9.447
ggGLNgg 3.014 3.477 4.453 4.354
ggHISgg 2.232 2.427 2.366 2.252
ggPROgg 4.162 3.292 4.767 4.363
T:nir 4
Codon usage (%) in the four sets
Casename HIGH RIBO LOW TOTAL
ggAAAgg 4.651 6.629 3.980 3.520
ggAAUgg 0.314 0.551 0.880 1.770
ggAAGgg 1.239 2.937 0.860 1.270
ggAACgg 2.805 3.080 2.850 2.290
ggAUAgg 0.022 0.020 0.110 0.520
ggAUUgg 1.259 1.489 2.580 2.780
ggAUGgg 2.550 1.693 2.860 2.380
ggAUCgg 5.305 4.120 2.910 2.520
ggAGAgg 0.029 0.061 0.090 0.280
ggAGUgg 0.079 0.143 0.430 0.860
ggAGGgg 0.012 0.000 0.070 0.180
ggAGCgg 0.734 1.285 1.220 1.540
ggACAgg 0.245 0.265 0.350 0.800
ggACUgg 2.418 2.529 1.060 1.030
ggACGgg 0.370 0.306 1.000 1.350
ggACCgg 3.289 2.284 2.830 2.330
ggUAUgg 0.573 0.428 1.250 1.610
ggUACgg 1.823 1.407 1.500 1.330
ggUUAgg 0.132 0.163 0.560 1.220
ggUUUgg 0.558 0.795 1.310 2.020
ggUUGgg 0.229 0.367 0.870 1.220
ggUUCgg 2.688 2.264 2.260 1.740
ggUGUgg 0.264 0.143 0.420 0.480
ggUGGgg 0.679 0.530 1.100 1.370
ggUGCgg 0.475 0.265 0.490 0.610
ggUCAgg 0.090 0.143 0.510 0.770
ggUCUgg 1.742 1.693 1.110 0.990
ggUCGgg 0.116 0.061 0.830 0.830
ggUCCgg 1.357 1.061 1.410 0.930
ggGAAgg 6.711 4.140 5.340 4.090
ggGAUgg 1.978 1.734 3.030 3.190
ggGAGgg 1.809 1.795 2.110 1.970
ggGACgg 4.355 2.590 2.440 2.120
ggGUAgg 2.143 2.427 1.260 1.150
ggGUUgg 5.029 4.446 1.880 1.960
ggGUGgg 1.340 1.163 2.680 2.500
ggGUCgg 0.394 0.918 1.380 1.450
ggGGAgg 0.067 0.041 0.440 0.810
ggGGUgg 4.112 5.058 2.940 2.600
ggGGGgg 0.212 0.163 0.670 1.050
ggGGCgg 5.099 2.713 3.500 2.900
ggGCAgg 2.389 2.835 1.770 2.050
ggGCUgg 3.550 4.813 1.720 1.680
ggGCGgg 2.104 1.876 3.590 3.180
ggGCCgg 0.810 0.857 2.290 2.500
ggCAAgg 0.291 0.857 1.350 1.360
ggCAUgg 0.455 0.632 0.830 1.210
ggCAGgg 2.707 2.590 2.840 2.980
ggCACgg 1.765 1.774 1.290 1.030
ggCUAgg 0.040 0.061 0.130 0.370
ggCUUgg 0.296 0.367 0.480 1.080
ggCUGgg 6.402 5.690 6.640 5.220
ggCUCgg 0.310 0.326 0.950 1.020
ggCGAgg 0.007 0.082 0.200 0.350
ggCGUgg 3.739 6.241 3.250 2.220
ggCGGgg 0.033 0.020 0.170 0.560
ggCGCgg 1.416 2.978 2.540 2.180
ggCCAgg 0.429 0.469 0.820 0.840
ggCCUgg 0.163 0.612 0.440 0.690
ggCCGgg 3.464 2.101 3.140 2.310
ggCCCgg 0.084 0.082 0.180 0.510
Amino acid usage in the four sets is given in
Table 5. The aminoacid usage in the LOW and
TOTAL sets is quite close (correlation 0.99), that of
HIGH with these two sets is fair (correlations 0.89).
In contrast, the RIBO set distinguishes from the three
other sets by lower correlations (0.85 with HIGH,
0.81 with the two others), indicating some bias in the
aminoacid usage in the 41 ribosomal proteins selected
(about twice the average use of Lys, Arg, and half that
of Trp and Cys).
We next consider the tRNA supply (oer), and the
codon demand in the four sets. Table 6 shows the
relative concentrations of the 35 tRNA species (rst
column, %, taken from Ikemura (1981a) and Varenne
et al. (1984). For the four sets, the demand (%) in
mono- or isorecognized (including the degenerate
isoaccepted) codons was computed, according to the
optimization hypotheses. The demand was taken as
zS
i
z(v
i1
v
i j
)
, or as S
i
zv
j
/f
im
, assuming f
i1
=f
mq
=1.
The two last columns give the f
i
=Sf
i j
or
f
im
=S
i
S
j
(f
i j
+f
ml
) values for the RIBO and TOTAL
sets.
Figures 1(a)4(a) display the optimized supply
demand data for the four gene sets. The linear
regression lines (dotted lines) are shown. The
correlations between optimized supply and demand
are highest for the two most expressed gene sets,
RIBO (0.96) and HIGH (0.95). Linear regression
coecients show that in these sets, the percentage of
demand is almost exactly proportional to the
percentage of supply [solid line in Figs 3(a) and
4(a)].
The correlation is high too for the TOTAL and
. soioxovici ET AL. 516
T:nir 6
tRNA supply (%), cognate codon demand (%) (dem columns) for the four sets, and the f
j
or f
jm
factors for RIBO
and TOTAL sets, for the 35 tRNA species of E. Coli (Ikemura 1981a)
a.a. (codon(s)), (anticod.) tRNA % demHIGH demRIBO demLOW demTOTAL fRIBO fTOTAL
LYS(AAA;AAG),UUU 5.830 4.998 6.267 4.234 4.083 1.660 1.600
ASN(AAU;AAC),QUU 3.500 3.711 3.942 3.796 3.608 1.420 1.880
ILE(AUU;AUC),GAU 5.830 5.360 4.838 4.094 4.009 1.600 1.960
ILE(AUA),NAU 0.290 0.287 0.267 0.600 1.243 1.000 1.000
MET(AUG),CAU 1.750 3.049 2.450 3.001 2.653 1.000 1.000
ARG(CGU;CGC;CGA),ICG 5.250 4.786 6.321 4.587 4.083 1.800 2.270
ARG(CGG)** 0.640 0.350 0.267 0.702 1.294 1.000 1.000
ARG(AGA;AGG) 0.700 0.412 0.470 0.695 1.225 1.000 1.820
SER(UCU;UCC;UGA;USG), 3.870 4.361 4.201 5.155 4.638 1.760 1.970
(GGA+UGA)
SER(AGU;AGC),GCU 1.460 1.887 2.466 2.263 2.829 1.330 1.750
THR(ACU;ACC),GGU 4.670 4.736 4.182 4.191 3.392 1.950 1.670
THR(ACA;ACG),UGU 1.400 1.562 1.444 2.274 2.659 1.930 1.770
TRP(UGG),CCA 1.750 1.574 1.371 1.992 2.012 1.000 1.000
TYR(UAU;UAC),QUA 2.880 3.499 2.784 2.896 3.051 1.550 1.910
LEU(CUU;CUC),GAG 1.750 1.487 1.595 2.399 2.520 1.940 1.970
LEU(CUA) 0.580 0.387 0.470 0.664 1.051 1.000 1.000
LEU(CUG),CAG 5.830 4.836 4.494 4.609 3.934 1.000 1.000
LEU(UUA;UUG),AAA 1.460 1.212 1.474 2.313 2.691 1.670 2.000
PHE(UUU;UUC),GAA 2.040 1.724 3.574 3.398 3.404 1.590 1.930
CYS(UGU;UGC),GCA 1.170 1.737 1.270 1.761 1.861 1.740 1.860
GLU(GAA,GAG),UUC 5.250 6.110 4.937 5.061 4.544 1.660 1.690
ASP(GAU;GAC),QUC 4.670 5.186 4.084 4.273 4.142 1.820 1.820
VAL(GUU;GUC;GUA;GUG), 8.550 8.609 8.545 6.910 6.878 1.580 1.770
(GAC+UAC)
GLY(GGU;GGC),GCC 6.530 5.935 5.570 4.510 4.093 1.730 1.950
GLY(GGA),UCC 0.880 0.500 0.381 1.239 1.563 1.000 1.000
GLY(GGG),CCC 0.580 0.875 0.762 1.660 1.781 1.000 1.000
ALA(GCU;GCC;GCA;GCG), 10.260 8.372 8.588 7.052 7.878 1.830 1.810
(GGC+UGC)
GLN(CAA),UUG 1.750 1.037 1.739 2.222 2.012 1.000 1.000
GLN(CAG),CUG 2.330 3.149 3.034 3.013 2.973 1.000 1.000
HIS(CAU;CAC),QUG 2.330 3.111 3.172 2.857 2.629 1.600 1.930
PRO(CCU;CCC) 0.990 1.012 1.720 1.710 1.954 1.360 1.860
PRO(CCA;CCG) 3.320 4.148 3.320 3.870 3.313 1.470 1.610
*anticodon not identied.
LOW sets (0.94). However, the linear regression
coecients indicate that oer and demand are less
optimally balanced [dotted lines in Figs 3(a)
and 4(a)], as the demand tends to exceed the
oer, for tRNA species present below about 4%
of the total RNA, but the reverse holds above
this value.
For comparison, we compute the correlations
between supply and demand without the optimization
criteria, i.e., assuming that all codons share a
common rate constant, as it had been generally
assumed in previous works. The data, displayed in
Figs 1(b) to 4(b), show that all correlations are good
in general (q0.91), but now the TOTAL and LOW
sets correlate better (0.96) and their linear regression
parameters indicate good adequation of oer and
(non-optimized) demand [especially for the LOW set,
see solid lines on Figs 1(b) and 2(b)]. For the RIBO
and HIGH sets, the demand appears to exceed the
oer (slope of linear regression 1.22), specially for
most of the abundant tRNAs (q4%), consistent with
the biases in the aminoacid composition in these sets,
as seen in Table 5.
Clearly the translation of the most heavily
expressed genes (producing 8090% of total protein
mass) appears to make optimal use of the cellular
tRNA ressources, by an adapted constraint of the
codon selection in these genes, but this constraint is
somewhat relaxed in the other genes.
Table 7 gives the codon-specic parameters f
i j
Fic. 14. The data are taken from table 6. Ordinate: relative frequency (%) of codon(s) recognized by a given tRNA
species (demand, DEM); abscissa: relative cellular amount (%) of cognate tRNA (supply, SUPP). [1(a)4(a)]:
optimized demand and supply (see text), for the TOTAL, LOW, HIGH and RIBO sets, respectively. [1(b)4(b)]:
non-optimized demand and supply (see text), for the TOTAL, LOW, HIGH and RIBO sets, respectively. Dotted line:
linear regression line of the data points. Regression parameters and correlation are given in the insert of each gure.
The solid line represents the exact supply-demand balance in each gure.
11
11
1 5
5
9
7
3
1
1 3 7 9
4(a) DEMRI BO
DEMRI BO = 0.38679 + 0.87543 * tRNASUPP
Correl ati on: r = 0.96161
11
11
1 5
5
9
7
3
1
1 3 7 9
3(a) DEMHI GH
DEMHI GH = 0.32110 + 0.89644 * tRNASUPP
Correl ati on: r = 0.96631
11
8
1 5
5
7
3
1
1 3 7 9
2(a) DEMLOW
DEMLOW = 1.1410 + 0.63433 * tRNASUPP
Correl ati on: r = 0.93919
11
9
1 5
5
7
3
1
1 3 7 9
1(a) DEMTOTAL
DEMTOTAL = 1.3211 + 0.57672 * tRNASUPP
Correl ati on: r = 0.94216
11
12
1 5
4
10
8
2
0
1 3 7 9
4(b) DEMRI BO
DEMRI BO = 0.7081 + 1.2266 * tRNASUPP
Correl ati on: r = 0.93899
11
11
1 5
5
9
7
3
1
1 3 7 9
3(b) DEMHI GH
DEMHI GH = 0.6138 + 1.1739 * tRNASUPP
Correl ati on: r = 0.94602
11
11
1 5
5
9
7
3
1
1 3 7 9
2(b) DEMLOW
DEMLOW = 0.06893 + 0.97797 * tRNASUPP
Correl ati on: r = 0.95645
11
11
1 5
5
9
7
3
1
1 3 7 9
1(b) DEMTOTAL
DEMTOTAL = 0.50669 + 0.83792 * tRNASUPP
Correl ati on: r = 0.96088
8
6
4
2
0
0
2
4
6
2
6
1r:Nsi:1ioN oi1ixiz:1ioN 517
Fic. 14.
. soioxovici ET AL. 518
T:nir 7
Codon-specic supply-demand parameters
di j tdem tsup
Codon fi j tRNA % fi j . ni (TOTAL) (ms) (ms)
ggAAAgg 1.000 5.830 5.830 4.080 34 51
ggAAUgg 0.880 3.500 3.080 3.177 64 65
ggAAGgg 0.600 5.830 3.498 2.448 57 85
ggAACgg 1.000 3.500 3.500 3.610 57 58
ggAUAgg 1.000 0.290 0.290 1.240 683 168
ggAUUgg 1.000 5.830 5.830 4.010 34 52
ggAUGgg 1.000 1.750 1.750 2.650 113 78
ggAUCgg 0.960 5.830 5.597 3.850 35 54
ggAGAgg 1.000 0.700 0.700 1.230 283 169
ggAGUgg 0.750 1.460 1.095 2.123 181 98
ggAGGgg 0.820 0.700 0.574 1.009 345 206
ggAGCgg 1.000 1.460 1.460 2.830 136 73
ggACAgg 1.000 1.400 6.070 2.660 33 78
ggACUgg 0.670 4.670 3.129 2.271 63 92
ggACGgg 0.770 1.400 4.674 2.048 42 102
ggACCgg 1.000 4.670 4.670 3.390 42 61
ggUAUgg 1.000 2.880 2.880 3.050 69 68
ggUACgg 0.910 2.880 2.621 2.776 76 75
ggUUAgg 1.000 1.460 1.460 2.690 136 77
ggUUUgg 1.000 2.040 2.040 3.400 97 61
ggUUGgg 1.000 1.460 1.460 2.690 136 77
ggUUCgg 0.930 2.040 1.897 3.162 104 66
ggUGUgg 0.860 1.170 1.006 1.600 197 130
ggUGGgg 1.000 1.750 1.750 2.010 113 103
ggUGCgg 1.000 1.170 1.170 1.860 169 112
ggUCAgg 0.460 3.870 1.780 2.134 111 97
ggUCUgg 0.520 3.870 2.012 2.413 98 86
ggUCGgg 0.480 3.870 1.858 2.227 107 93
ggUCCgg 0.510 3.870 1.974 2.366 100 88
ggGAAgg 1.000 5.250 5.250 4.540 38 46
ggGAUgg 1.000 4.670 4.670 4.140 42 50
ggGAGgg 0.690 5.250 3.622 3.133 55 66
ggGACgg 0.820 4.670 3.829 3.395 52 61
ggGUAgg 0.360 8.550 3.078 2.477 64 84
ggGUUgg 0.470 8.550 4.019 3.234 49 64
ggGUGgg 0.530 8.550 4.532 3.646 44 57
ggGUCgg 0.400 8.550 3.420 2.752 58 76
ggGGAgg 1.000 0.880 0.880 1.560 225 133
ggGGUgg 0.950 6.530 6.204 3.885 32 54
ggGGGgg 1.000 0.580 0.580 1.780 341 117
ggGGCgg 1.000 6.530 6.530 4.090 30 51
ggGCAgg 0.420 10.260 4.309 3.310 46 63
ggGCUgg 0.390 10.260 4.001 3.073 49 68
ggGCGgg 0.530 10.260 5.438 4.176 36 50
ggGCCgg 0.470 10.260 4.822 3.704 41 56
ggCAAgg 1.000 1.750 1.750 2.010 113 103
ggCAUgg 1.000 2.330 2.330 2.630 85 79
ggCAGgg 1.000 2.330 2.330 2.970 85 70
ggCACgg 0.930 2.330 2.167 2.446 91 85
ggCUAgg 1.000 0.580 0.580 1.050 341 198
ggCUUgg 1.000 1.750 1.750 2.520 113 83
ggCUGgg 1.000 5.830 5.830 3.930 34 53
ggCUCgg 0.970 1.750 1.698 2.444 117 85
ggCGAgg 0.380 5.250 1.995 1.550 99 134
ggCGUgg 1.000 5.250 5.250 4.080 38 51
ggCGGgg 1.000 0.640 0.640 1.290 309 161
ggCGCgg 0.990 5.250 5.197 4.039 38 51
ggCCAgg 0.610 3.320 2.025 2.019 98 103
ggCCUgg 1.000 0.990 0.990 1.950 200 107
ggCCGgg 1.000 3.320 3.320 3.310 60 63
ggCCCgg 0.860 0.990 0.851 1.677 233 124
Column (2): values of parameter fij assuming fj1 =1; column (3):
tRNA supply nj , %; column (4): codon-specic supply, fij .nj ,
column (4): codon-specic demand dij for the TOTAL set; column
(5): translation time per codon, according to supply; column (6):
translation time per codon, according to demand.
and d
i j
, the latter being computed as f
i j
n
i
(supply) or
as f
i j
m
i
(demand). It can be seen that, for the genes
in the TOTAL set and under optimal translation
conditions, for codons bearing a pyrimidine as the
rst base, f
i j
m
i
exceeds f
i j
n
i
in most instances
(systematically for U). The reverse holds for codons
beginning with a purine (especially G). Stated
dierently, in an average gene of E. coli, the
translation of codons beginning with a pyrimidine
would in general be slower than optimal, due to
insucient supply of the cognate tRNAs, and most of
those beginning with a purine would experience the
reverse.
Estimation of Optimal Codon Translation Times under
Standard Growth Conditions
The total translation time of the lacZ gene (1025
codons) has been measured in E. coli to be
T=82.0 20.6 s, under standard growth conditions
(So rensen et al., 1989). Assuming that the relative
cellular tRNA concentrations were determined under
similar growth conditions, we may compute the value
of the constant k from the equation T=S
i
S
j
k/d
i j
(n
i
).
We may also compute the value of k/K from
T=S
i
S
j
k/d
i j
(f
i j
) for the TOTAL set (to which the
lacZ gene belongs). We obtain the values of k =198
(units: ms/% tRNA) and k/K=254, which allow to
compute the times (t
sup
) and (t
dem
) taken to translate
the 61 codons, according to the tRNA supply and to
the codon demand, respectively (Table 7); t
sup
depends
on n
i
hence on growth conditions, but not on the gene
set. The reverse holds for t
dem
. Under the assumptions
made, t
sup
and t
dem
should be identical, provided the
total translation time of a gene and the cellular tRNA
concentrations are measured under identical physio-
logical conditions. The data, Table 7, deserve the
same remarks made in the previous section on the
values of f
i j
n
i
and f
i j
m
i
computed for the TOTAL set.
Comparison of Codon Translation Rates Measured
in vivo with those Computed According to the
Optimization Hypothesis
In E. coli, ten codon pairs and one codon triplet of
the 29 YNN codons have each a cognate tRNA.
Curran & Yarus (1989) introduced each YNN codon
at the frameshift point of the RF2 gene. A reporter
gene, fused to the 3'-shifted frame, allows to quantify
the frameshift probability. This probability is
assumed to be an index of the rate of the aa-tRNA
selection at the YNN codon, which could be used for
comparison of the translation rates of isoaccepted
codons. For each pair, the ratio of the measured
1r:Nsi:1ioN oi1ixiz:1ioN 519
translation rate indices was compared with the ratio
of their eciency indices (f
i j
), computed for the RIBO
set. These ratios correlate rather satisfactorily for
codons recognized by the following tRNAs: Phe
(measured ratio/computed ratio =1.41/1.68), Arg1
(1.56/1.45), Tyr1 (1.96/1.81), His (2/1.61), Cys
(1.75/1.36), Pro2 (1.56/2.12), Leu(UUR) (2/1.5) and
Leu2 (1.31/1). The ratios of those recognized by Pro1
(1.14/0.36) and the Ser tRNAs ((1.27/0.8 and
1.29/0.65) show that the measured ratios are in
disagreement with those computed for the RIBO set,
which uses seldom the CCC (Pro) and the UCG (Ser)
codons. The measured ratios agree better with those
computed for the TOTAL set (respectively 1.14/0.86,
1.27/1 and 1.29/1.04).
Soerensen & Pedersen (1991) measured directly the
in vivo translation rates of the Glu codons GAA and
GAG, which share the same cognate tRNA. The ratio
of the measured rates (3.4) is over twice that
computed for the RIBO set (1.52).
Though in most instances where experimental data
are available, the codon translated fastest by the
cognate tRNA can be predicted, the quantitative
agreement between measured and computed trans-
lation rates is poor for many codons.
Discussion
We may surmise that the cell has two, non exclusive
ways to optimize its translation economics. One
optimization path makes the best use of the physical
characteristics of the tRNA, in particular the
anticodon structure, by adapting the synonymous
codon demand to the thermodynamic characteristics
of codon-anticodon complexation. This is achieved
by letting the frequency of occurrence of a
synonymous codon be proportional to the square of
the rate constant specic of the codon-anticodon
complexation. Optimization can also be achieved by
mutually adjusting the tRNA pool and the codon
demand, so that the oer of the isoaccepting tRNA
is proportional to the sum of the square roots of the
cognate codon frequencies.
Close to optimal oer/demand correlation is
observed for the subpopulation of the less than 100
genes most expressed in E. coli. These represent
some 8090% of the protein molecules produced,
hence by far the largest cellular energy expenditure,
whatever the physiological status of the cell.
Furthermore, the corresponding genes make a
massive, but by no means exclusive, use of the most
frequent codons.
This observation raises several questions. Why
is there no systematic use of the most frequent
codons in these genes, and why do the 3000 or
so genes, producing the remaining 1020% proteins
of the cell, use fairly often synonymous, less
frequent codons? More generally, is there a selective
advantage for the cell to use codons which do not
optimally t the anticodon, to produce isoaccepting
tRNAs in dierent relative amounts, and why do
these amounts vary among species (Ikemura, 1982)?
Also, why does the highest oer (tRNA Ala, 10.26%)
copiously exceed the demand (8.5%), and more
generally why is there no perfect oer-demand
correlation?
Work performed in this laboratory by Deana
et al. (1996) has demonstrated the strong incidence
of synonymous codon selection on control and
regulation of gene expression. They showed that
the exchange of a few codons in the ompA gene of
E. coli for instance, for synonymous ones, can aect
major steps in gene expression, like messenger
stability, premature transcription abortion by a
polarity eect, translation frameshifting and trans-
lation abortion. These perturbations appear to result
from a marked change of the ribosome trac on the
messenger. In prokaryotes, a silent codon exchange
may in addition result in a deep alteration of
transcription-translation coupling. The global result
of the silent mutations is a severe alteration of the
protein yield.
Obviously, given the relative concentrations of
isoaccepting tRNAs, synonymous codons have been
carefully selected in the gene, in order to produce the
required control and regulation tasks. Then, if a
heterologous gene is to be expressed optimally in a
given species, its sequence should be rewritten in the
dialect of that species in view of optimizing the
ribosome trac on its messenger. This can be done by
taking into account the optimization constraints
imposed by the isoaccepting tRNA structure and
relative concentrations.
Many reasons could explain the conservation of
infrequent codons. WE evoked but one: at several
places (e.g. Purvis et al., 1987; Crombie et al.,
1992, 1994; Hernan et al., 1992; Kolb et al., 1994),
it has been speculated that the rate of translation
could participate in the protein folding process.
The slow translation of rare codons could allot
enough time to the part of the nascent protein leaving
or having left the ribosome, for thorough folding
(Reiss, 1995). Rare codons, which often appear in
clusters, could be auxiliaries helping the nascent
protein to nd readily and quickly its folding
pathway. Experiments are in progress to test this
hypothesis.
. soioxovici ET AL. 520
Although oer and demand correlate quite well,
they are far from being perfectly adapted (Figs 14). It
must rst be stressed that the measurements of the
tRNA concentrations (Ikemura, 1981a) are of limited
precision and that for several tRNAs, the relative
concentrations have been obtained by extrapolation
(Varenne et al., 1984). Updating these data would
certainly be welcome (Emilsson et al., 1993).
Furthermore, tRNAs may full some other functions
in addition to translation, such as N-terminal amino
acid transfer (Andacht et al., 1989), which could
explain, for abundant tRNAs in particular, the excess
of oer over demand.
The answer to the question in the title seems to be
yes for the most expressed genes, which represent
perhaps 3% of the genes in the E. coli cell, and no
for the remaining 97%. The translation factory would
thus use two strategies, one for mass production
(8090% of total proteins), which is optimized, and
another for producing the remaining 1020% of the
proteins, for which the demand seems simply
proportional to the oer. Why two strategies, and in
particular how could the non-optimized strategy be
maintained during evolution? We conjecture that
the latter is used to enable constitutive control and
regulation over the expression, of many genes in
the TOTAL set, as mentioned earlier in this discussion
(Deana et al., 1996). Actually, for these genes, the loss
of optimization in translation demand/oer may be
more than compensated for by the economy in
regulatory pathways whichshouldhave beenbuilt, had
there been no such constitutive regulation. If this
picture is correct, it would mean that the composition
of the tRNA pool in E. coli results from the
compromise to keep the energy invested as low as
possible in the translation of highly expressed genes,
and to provide simple and inexpensive constitutive
control and regulation of less expressed genes.
Finally, for species for which the frequencies of
codon usage are known and in which the tRNAspecies
and their cognate codons have been identied, the
composition of the tRNA pool can be deduced,
provided the optimization hypotheses apply.
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