Effects of
Escherichia coli
ribosomal proteinS12 mutations
on cell-freeprotein synthesis
Namthip Chumpolkulwong
1
, Chie Hori-Takemoto
1
, Takeshi Hosaka
2
, Takashi Inaoka
2
, Takanori Kigawa
1
,
Mikako Shirouzu
1,3
, Kozo Ochi
2
and Shigeyuki Yokoyama
1,3,4
1
RIKEN Genomic Sciences Center, Tsurumi, Yokohama, Japan;
2
National Food Research Institute, Tsukuba, Ibaraki, Japan;
3
RIKEN Harima Institute at SPring-8, Mikazuki-cho, Sayo, Hyogo, Japan;
4
Department of Biophysics and Biochemistry,
Graduate School of Science, The University of Tokyo, Bunkyo-ku, Tokyo, Japan
We examined the effects ofEscherichiacoliribosomal pro-
tein S12mutationson the efficiency ofcell-freeprotein syn-
thesis. By screening 150 spontaneous streptomycin-resistant
isolates from E. coli BL21, we successfully obtained seven
mutants of the S12 protein, including two streptomycin-
dependent mutants. The mutations occurred at Lys42,
Lys87, Pro90 and Gly91 of the 30S ribosomalprotein S12.
We prepared S30 extracts from mutant cells harvested in the
mid-log phase. Their proteinsynthesis activities were com-
pared by measuring the yields of the active chloramphenicol
acetyltransferase. Higher protein production (1.3-fold) than
the wild-type was observed with the mutant that replaced
Lys42 with Thr (K42T). The K42R, K42N, and K42I strains
showed lower activities, while the other mutant strains with
Lys87, Pro90 and Pro91 did not show any significant dif-
ference from the wild-type. We also assessed the frequency
of Leu misincorporation in poly(U)-dependent poly(Phe)
synthesis. In this assay system, almost all mutants showed
higher accuracy and lower activity than the wild-type.
However, K42T offered higher activity, in addition to high
accuracy. Furthermore, when 14 mouse cDNA sequences
were used as test templates, the protein yields of nine tem-
plates in the K42T system were 1.2–2 times higher than that
of the wild-type.
Keywords: ribosomalprotein S12; streptomycin; point
mutation; cell-freeprotein synthesis.
The antibiotic streptomycin inhibits proteinsynthesis and
causes misreading during translation. Ribosomal protein
mutations in Escherichiacoli have been found to confer
resistance to streptomycin [1,2]. These mutations frequently
exist in the ribosomalprotein S12, encoded by rpsL,and
result in streptomycin resistance [3] or streptomycin
dependence [4]. The phenotypes were attributed to the
mutations in the S12protein by Funatsu et al. [5,6]. The
streptomycin-resistance mutations in the ribosomal proteins
S4 and S5 confer ribosomal ambiguity (ram) phenotypes,
and cause a decrease in the translational accuracy [7,8]. In
the 1980s, mutations conferring streptomycin resistance
were found in the 16S rRNA of bacteria and chloroplasts
(rRNA Mutation Database, located at http://www_fandm.
edu). Many of them were near the 530-loop, which has been
proposed to form a pseudoknot structure [9], and were
stabilized by the S12 protein, as shown in a footprinting
study of the 30S ribosomal subunit [10]. A genetic analysis
of the 16S rRNA mutations and chemical probing for each
16S rRNA mutation in the S12 mutant strains demonstra-
ted that the streptomycin resistance was achieved by a lower
affinity for streptomycin, and all of the mutations gave rise
to conformational changes in the rRNA [11,12]. Studies
of streptomycin resistance and dependence in 23S rRNA
mutations have shed light on the relationship between
accurate decoding and GTP hydrolysis by EF-Tu [13–15].
Although the pseudoknot structure and the S12 ribosomal
protein are clearly responsible for translational accuracy,
the streptomycin did not bind to the S12protein itself [3] or
to the 530-loop in helix 18 (H18) [16]. In the crystal structure
of the 30S ribosomal subunit, four molecules of strepto-
mycin were observed [17]. The streptomycin interacted with
the rRNA and the S12 protein, which was the only protein
that formed direct hydrogen bonds with streptomycin. In
2001, the crystal structure of the 30S ribosomal subunit with
the anticodon stem-loop of tRNA in the A-site revealed a
dynamic conformational change in the 30S decoding center,
which consisted of H18, H27, H44 and the S12protein [18].
The structural information agreed well with the hypothes-
ized mechanism of how streptomycin causes misreading on
the ribosome [19].
Recently, in the genus Streptomyces, rpsL mutations
were reported to compensate for a decrease of antibiotic
production in a relA and relC (rplK) mutant strain [20].
These mutations were obtained by selection with a high
concentration of streptomycin. Moreover, the screening by
streptomycin resistance resulted in better antibiotic produc-
tivity in several bacteria [21]. The mutations conferring
streptomycin resistance corresponded to the ribosomal
protein S12mutationson conserved residues, which have
Correspondence to S. Yokoyama, Department of Biophysics and
Biochemistry, Graduate School of Science, The University of Tokyo,
Bunkyo-ku, Tokyo, Japan.
Fax: + 81 3 5841 8057, Tel.: + 81 3 5841 4392,
E-mail: yokoyama@biochem.s.u-tokyo.ac.jp
Abbreviations: H-18, helix 18; CAT, chloramphenicol
acetyltransferase.
(Received 26 December 2003, accepted 28 January 2004)
Eur. J. Biochem. 271, 1127–1134 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04016.x
been characterized well in E. coli. Mutations in the
ribosomal proteinS12 seemed to cause the preservation of
the translation activity, and to enhance the expression of
enzymes involved in antibiotic production in the late
stationary phase. Thus, ribosomalmutations could influ-
ence and enhance the productivity of particular proteins. In
the present study, seven streptomycin-resistant mutants and
two streptomycin-dependent mutants of E. coli were iso-
lated, and were tested in our cell-free translation system.
We found one S12 mutant, K42T, which possessed better
activity than the wild-type. The present paper describes the
translation properties of the S12 mutants in vitro using
poly(U) and mouse cDNAs as test templates.
Materials and methods
Preparation of
rpsL
mutants
Spontaneous streptomycin-resistant or streptomycin-depen-
dent mutants of E. coli BL21 were obtained from colonies
that grew within 2 days after cells were spread on LB
agar containing various concentrations (50, 100, 300 and
600 lgÆmL
)1
) of streptomycin. The mutants were used for
subsequent studies after single-colony isolation.
Mutation analysis of
rpsL
The rpsL genes of the streptomycin-resistant mutants (150
isolates were tested) were obtained by PCR, using the
genomic DNA as the template and the synthetic oligo-
nucleotide primers 5¢-ATGATGGCGGGATCGTTC-3¢
(forward) and 5¢-TTCCAGTTCAGATTTACC-3¢ (rev-
erse), which were based on the E. coli sequence (DDBJ
accession no. J01688). A thermal cycler (Perkin Elmer Cetus)
was used with the following conditions: 5 min of incubation
at 96 °C; 30 cycles of 95 °C for 30 s, 55 °C for 30 s, 72 °Cfor
1 min; and a final step at 72 °C for 7 min. The PCR products
were sequenced directly by the dideoxynucleotide chain
termination method, using the BigDye Terminator Cycle
sequencing kit (Perkin Elmer).
Bacterial strains and culture conditions
Overnight cultures of the wild-type and S12 mutants were
inoculated into 2· YT medium (16 g of tryptone, 10 g of
yeast extract, and 5 g of NaCl per L). The concentration of
streptomycin used for cultivation of the mutant strains was
100 lgÆmL
)1
. The cells were cultivated in a fermenter with
sufficient aeration and an agitation speed of 400 r.p.m. at
37 °C.
S30 preparation
The S30 extracts for proteinsynthesis were prepared from
E. coli strain BL21 and from the S12 mutants as described
previously [22] with minor modifications. The cells were
harvested in mid-log phase and were washed three times with
buffer A [10 m
M
Tris/acetate buffer (pH 8.2) containing
14 m
M
Mg(OAc)
2
,60m
M
potassium acetate, 1 m
M
dithio-
threitol, and 7 m
M
2-mercaptoethanol, supplemented just
before use].The cells(7.2 g)were suspendedin 9 mL ofbuffer
B (buffer A without 2-mercaptoethanol), and were disrupted
with 22.7 g of glass beads in a multibead shocker (Yasui
Kikai, Japan) operated at 2700 r.p.m. for 90 s. The cell debris
and the glass beads were removed by two centrifugation runs
at 30 000 g for 30 min. The clear 30S extract was incubated
for 80 min with a 0.3-fold volume aliquot of a preincubation
mixture, and contained final concentrations of 0.3
M
Tris/
acetate (pH 8.2), 9 m
M
Mg(OAc)
2
,13m
M
ATP, 84 m
M
phosphoenolpyruvate, 4.5 m
M
dithiothreitol, 40 l
M
each of
20 amino acids, and 18.6 lgÆmL
)1
pyruvate kinase. The
reaction was then centrifuged at 30 000 g for 30 min. After
four rounds of dialysis for 45 min each against buffer B, the
extract was centrifuged at 5000 g for 10 min, and the
supernatant was stored in liquid nitrogen.
Cell-free translation
The reaction mixture (30 lL) consisted of the following
components: 58 m
M
Hepes/KOH (pH 7.5), 1.2 m
M
ATP,
0.8 m
M
each of GTP, CTP and UTP, 1.7 m
M
dithiothreitol,
0.64 m
M
cAMP, 170 lgÆmL
)1
E. coli total tRNA (Boeh-
ringer-Mannheim), 200 m
M
potassium glutamate, 27.5 m
M
NH
4
OAc, 13.4 m
M
Mg(OAc)
2
,35lgÆmL
)1
folinic acid,
4 lgÆmL
)1
of plasmid pK7-CAT [23] used as a template
for chloramphenicol acetyltransferase (CAT) synthesis,
66.6 lgÆmL
)1
T7 RNA polymerase prepared in our labor-
atory, 80 m
M
creatine phosphate (Boehringer-Mannheim),
250 lgÆmL
)1
creatine kinase (Boehringer-Mannheim),
500 l
M
each of 20 amino acids, 4% polyethylene glycol
8000 (Sigma), 25 m
M
phosphoenolpyruvate (Roche) and
0.24 vol. of S30 extract. The concentration of Mg(OAc)
2
was varied, corresponding to the S30 extract. The reaction
mixture was incubated at 37 °C for 1 h. The enzyme activity
of the synthesized CAT was determined by the spectro-
photometric procedures described previously [24]. The
protein concentration of the cell extract was determined
according to the Bradford method [25]. The ribosome
concentration was measured by the absorbance at 260 nm.
His-tagged proteins were synthesized from DNA tem-
plates cloned within pPCR2.1 (Invitrogen), in a batch system
for a one-hour incubation. Each reaction mixture was
loaded onto a Ni-nitrilotriacetic acid column (Qiagen)
equilibrated with a buffer containing 20 m
M
Tris/HCl
(pH 7.5), 500 m
M
NH
4
Cl, and 5 m
M
imidazole. The prod-
uct was eluted with 0.2
M
imidazole buffer. The eluted
fraction was separated by SDS/PAGE and stained with
SYPRO Orange (Molecular Probes). The product was deter-
mined by using a LAS-1000 image analyzer (Fuji Film).
Error frequency assay in the
in vitro
translation
The error frequency assay in vitro was performed as
described by Legault-Demare and Chambliss [26], with
some modifications. The error frequency of an extract from
the mutant strain was studied by the misincorporation of
Leu in the poly(U)-dependent poly(Phe) synthesis system.
The reaction mixture (30 lL) contained almost all of the
components described above, with the exception of the T7
RNA polymerase and the plasmid pK7-CAT. The ribo-
somes and the supernatant (S100) were prepared from the
S30 extract by ultracentrifugation at 90 000 g for 2 h. The
ribosomes were suspended in a buffer containing 20 m
M
Hepes pH 7.8, 20 m
M
MgSO
4
, 100 m
M
NH
4
Cl, and 6 m
M
1128 N. Chumpolkulwong et al. (Eur. J. Biochem. 271) Ó FEBS 2004
2-mercaptoethanol. In order to reduce the amount of
endogenous mRNA, the ribosomes and S100 were incuba-
ted at 37 °C for 10 min prior to use. The final concentra-
tions of ribosomes, S100, and each amino acid (except for
Phe and Leu) were 4 A
260
ÆmL
)1
,0.18mgÆmL
)1
,and0.4m
M
in the reaction mixture. The reaction was started with
the addition of 0.75 mgÆmL
)1
poly(U), 200 l
M
[
14
C]Phe
(0.11 MBq, Amersham) and [
3
H]Leu (3.7 MBq, Amer-
sham), and was incubated at 37 °C for 15 min. The
background value was obtained from the reaction in the
absence of poly(U). After the incubation, a 15 lL aliquot of
the reaction mixture was transferred into 5% (v/v) trichlo-
roacetic acid, heated at 95 °C for 10 min, and applied to a
nitrocellulose membrane (Advantec). The membrane was
washed with 1% (v/v) trichloroacetic acid and dried. Then,
the radioactivity remaining in the membrane was measured
with a liquid scintillation counter. The error frequency was
calculated by the ratio of the incorporation of [
3
H]Leu to
that of [
14
C]Phe.
Results
Growth characteristics ofS12 mutants
We isolated 150 colonies of E. coli BL21, grown on plates
containing various concentrations of streptomycin. The
sequence of the rpsL gene, which encodes the ribosomal
protein S12, was confirmed by the PCR technique, and all
mutations were identified as a single substitution of an
amino acid, as shown in Table 1. The phenotypes of the
E. coli BL21 mutations in this study are the same as those
previously reported [27]. Isolation of the streptomycin-
dependent strains, P90L and G91D, required the addition
of 0.1 mgÆmL
)1
streptomycin and resisted to streptomycin
up to 10 mgÆmL
)1
. Mutationsof Lys42 also conferred a
high level of streptomycin resistance up to 10 mgÆmL
)1
,as
well as the K87R mutation.
The cultivation was carried out with a fermenter, under
the conditions described in the Materials and methods.
Reproducible growth curves of the wild-type and mutant
strains are shown in Fig. 1. Most of the mutant strains
showed the same growth pattern as that of the wild-type,
whereas the growth rate of the K42T mutant strain was
slightly lower than that of the wild-type. Exceptionally, the
doubling times of P90Q, K87E and G91D were 1.5–2 times
slower than that of the wild-type (data not shown).
Cell-free CAT proteinsynthesis with the extract
from each strain
An S30 extract was prepared from the cells harvested in the
mid-log phase, when the D
600
was approximately 3.0. To
estimate the proteinsynthesis activity of each mutant strain,
we used S30 extracts in cell-free CAT protein synthesis
(Fig. 2). The plasmid pK7-CAT was used as a standard
template, and the components in the reaction mixtures were
described in the Materials and methods. The yield of the
CAT protein in the wild-type system was approximately
0.68 mg per 1 mL of reaction mixture, for a 1 h incubation.
The efficiencies of CAT synthesis in the mutant systems,
with alterations at residue 87, 90 or 91, were essentially the
same as that of the wild-type system. The mutations of
Lys42 distinguished themselves into two groups. The K42R,
Table 1. Positions ofmutations in rpsL of E. c oli BL21. Position
numbering originates from the start codon of the open reading frame.
Amino acid numbering starts from the N-terminal amino acid.
Resistance level determined after a 24 h incubation on LB agar.
Strain
Position of
mutation in rpsL
Amino acid
replacement
Resistance level to
streptomycin
(mgÆmL
)1
)
BL21
a
–
b
0.01
KO-365 128 (AfiG) K42R >10
KO-368 129 (AfiC) K42N >10
KO-371 128 (AfiC) K42T >10
KO-374 128 (AfiT) K42I >10
KO-375 263 (AfiG) K87R >10
KO-376
c
272 (CfiT) P90L 10
KO-378 272 (CfiA) P90Q 0.03
KO-430 262 (AfiG) K87E 0.3
KO-431
c
275 (GfiA) G91D 10
a
Genotype: E. coli B, F
–
, dcm, ompT, hsdS(r
B
–
,m
B
–
), ga;
b
Wild-
type rpsL gene;
c
These mutant strains showed a streptomycin-
dependent phenotype.
Fig. 1. Growth curves of the wild-type (d), K42T (s), K87R (m), and
K42R (e)strains.Cultivation was performed with a fermenter under
the conditions described in the Materials and methods.
Fig. 2. Comparison ofcell-free CAT proteinsynthesis in the wild-type
and S12 mutant systems. The pk7-CAT plasmid concentration was
4ngÆlL
)1
and the magnesium acetate concentration in each reaction
was 13.4 m
M
. The reaction was incubated for 1 h at 37 °C. The CAT
enzyme activity was measured as described in the Materials and
methods.
Ó FEBS 2004 RibosomalproteinS12mutations (Eur. J. Biochem. 271) 1129
K42I and K42N systems exhibited lower activities than that
of the wild-type system. On the other hand, the CAT protein
yield in the K42T system was about 0.92 mgÆmL
)1
,which
was 1.3 times better than that in the wild-type system.
We tested the effect of streptomycin on CAT protein
synthesis in each system by the addition of various
concentrations of streptomycin. In the wild-type system,
the productivity was reduced to 50% by the addition of
streptomycin up to 0.1 lgÆmL
)1
, and the protein synthesis
was completely inhibited at 0.8 lgÆmL
)1
. In contrast, the
50% inhibitory concentrations were 1.5 mgÆmL
)1
in the
other streptomycin-resistant mutant systems (data not
shown). No enhancement of the productivity was observed
in any of the systems, unlike with the streptomycin-
dependent mutant strains reported previously.
To examine whether the better productivity of the K42T
system was a consequence of the ribosome content in the
S30 extract, we measured the A
260
values of the wild-type
and mutant extracts. All of them exhibited approximately
210–240 A
260
ÆmL
)1
, and there were no significant differ-
ences among the strains. We also analyzed the CAT
synthesis by cell-free systems made with the extracts of late-
log phase cells (6–7 h cultivation), in which the ribosome
content was reduced to approximately 160–180 A
260
ÆmL
)1
.
The CAT productivities of the late-log phase systems were
about 30% of those of the mid-log phase systems (data not
shown). The ribosome contents of the wild-type and K42T
mutant extracts were practically the same in the mid-log
phase as well as in the late-log phase. Therefore, the
difference in the productivity between the wild-type and
K42T systems is not related to the ribosome concentration
in the extract.
To confirm the amounts of 70S ribosome in the S30
extracts, we analyzed them on 6–38% sucrose density
gradients by ultra-centrifugation (17 000 r.p.m. for 17 h,
using a Beckman Coulter Optima XL-80k ultracentrifuge,
SW28 rotor, Beckman Coulter Inc., Palo Alto, CA, USA).
Under conditions using 20 m
M
Mg
2+
, the 70S ribo-
some was observed as the main fraction in all extracts
(Fig. 3A,C,E). On the other hand, when the Mg
2+
concentration was reduced to 5 m
M
, the main 70S ribosome
fraction was still observed in the wild-type (Fig. 3B) and
K42T (Fig. 3D) extracts, whereas the 30S and 50S
ribosomal subunits were observed in the K42R extract
(Fig. 3F). The instability of the 70S ribosome in the K42R
system seems to correlate with the low productivity of this
system. In these experiments, the amount and the stability of
the 70S ribosome in the K42T extract did not appear to
differ from those of the wild-type extract.
Comparison of the optimum concentration conditions
between the wild-type and K42T systems
We analyzed the dependence ofprotein productivity on the
Mg
2+
and DNA concentrations for the wild-type and
K42T systems, in order to examine if there were different
optimum concentrations between the systems. The results
showed that both the wild-type and K42T systems could
synthesize the CAT protein very well with a Mg
2+
concentration range between 10.7 and 13.4 m
M
(Fig. 4A).
Moreover, the K42T system still synthesized the CAT
protein efficiently, even in 16.1 m
M
Mg
2+
. The optimum
concentration of DNA was 4 ngÆlL
)1
(Fig. 4B) for both
systems, and higher DNA concentrations caused decreased
protein synthesis. We also found that the optimum
concentration of Mg
2+
for the other mutant systems was
13.4 m
M
(data not shown). These results indicated that the
K42T system itself could synthesize the CAT protein more
efficiently than the wild-type system, in the examined ranges
of Mg
2+
and template DNA concentrations.
Cell-free proteinsynthesis with mouse cDNA templates
In addition to CAT protein synthesis, we compared the
productivity of other randomly selected templates in the
Fig. 3. Analysis of ribosome fractions in the wild-type extract (A, B), the
K42T extract (C, D), and the K42R extract (E, F) by 6–38% sucrose
gradient density centrifugation. The concentration of MgSO
4
was
20 m
M
(A,C,E)or5m
M
(B, D, F).
Fig. 4. CAT proteinsynthesis in the wild-type (d) and K42T system (m)
with various concentrations of Mg
2+
(A) and template DNA (B).
1130 N. Chumpolkulwong et al. (Eur. J. Biochem. 271) Ó FEBS 2004
wild-type and K42T systems. The results using the 14
sequences are shown in Fig. 5. With nine of the 14
templates, the protein productivity of the K42T system
was 1.2–2 times better than that of the wild-type system.
Four of them showed almost the same production level in
both the wild-type and K42T systems. In only one case, the
K42T system showed slightly lower productivity than the
wild-type system. On average, the protein productivity in
the K42T system was 1.2 times higher than that in the wild-
type system. In summary, the majority of the tested
sequences in this study showed better productivity in the
K42T system.
Translation properties
The translation properties of the wild-type and S12 mutant
strains were examined by an in vitro poly(U)-dependent
translation assay. To estimate the misincorporation rate, the
nearly cognate substrate Leu and the cognate substrate
Phe were labeled by radioisotopes in the same reaction. As
shown in Table 2, the incorporation of Phe in almost all
of the mutant strains was lower than that in the wild-type
(except for K42T and K87R), while the misincorporation of
Leu instead of Phe in all mutant strains were significantly
lower than in the wild-type. The K42R mutant showed the
highest missense error rate among the S12 mutants. This
result was consistent with the previously published results
[28], which reported that the K42R mutant is a nonrestric-
tive phenotype among the streptomycin resistant mutants.
The K42N and K87R mutants reportedly showed higher
fidelity than the K42R mutant [6]. In our assays, the K42T
and K87R mutants showed the same poly (Phe) synthesis
activity as the wild-type, though the K87R strain showed a
slightly lower activity than the wild-type in CAT synthesis.
In the case of the K42T strain, which is reportedly a
restrictive strain [29], we found that its Leu uptake was eight
times lower than that of the wild-type, which is consistent
with studies of E. coli mutants to date. Therefore, the K42T
mutant retained the accuracy together with the high
productivity in the cell-free system.
Discussion
The mutant strains obtained in this study each had a
mutation in the ribosomalprotein S12, at the conserved
residue 42 or among residues 87–91. We investigated the
growth rate, the in vitro resistance level to streptomycin,
and the activity of CAT proteinsynthesis in cell-free
systems prepared from each strain. We found that the
streptomycin-resistant mutant K42T yielded 1.3 times
more CAT protein than the wild-type. The amount of the
CAT protein synthesized in the K42T system approached
1mgÆmL
)1
in a 1 h reaction. The higher productivity in
the K42T system was not caused by any differences from
the wild-type system, in terms of the ribosome content
and the optimum concentrations of Mg
2+
and template
DNA. The replacements of the other amino acids at
Lys42 showed lower protein yields than the wild-type,
while the substitutions within residues 87–91 did not affect
the protein production. Therefore, Lys42 in the S12
protein is important for the efficiency ofprotein synthesis,
and only the replacement by Thr increased the produc-
tivity of the ribosome.
We used CAT proteinsynthesis as the standard assay
for the proteinsynthesis activity in the cell-free system. To
consider the general applicability of the K42T system, we
examined the efficiency of the 14 mouse cDNAs, as test
templates. The K42T extract could synthesize almost all of
the proteins up to two times better than the wild-type. A few
of the proteins were synthesized at the same level as that of
the wild-type system, and one of them was lower. The
average protein productivity of the K42T extract was
approximately 1.2 times better than that of the wild-type
extract. These data indicate that there are two main
characteristics of the K42T system. First, the K42T system
exhibits better productivity, independent of the mRNA
sequence upstream of the decoding region of a target
protein, because all of the mouse cDNA plasmid vectors
were constructed to add the His-tagged sequence at the
N-terminus, and were different from the pK7-CAT protein.
Second, we could not find any particular secondary
structure or biased usage of rare codons in the mouse
Fig. 5. Cell-freeproteinsynthesis using 14 mouse cDNAs was carried
out with an extract of the wild-type or K42T mutant. The concentration
of plasmids no. 1, 4, 5, 6, 7, 8, 10–14 was 2.3 ngÆlL
)1
,andthatof
plasmids no. 2, 3 and 9 was 1 ngÆlL
)1
. The sequence of each cDNA
can be found at http://fantom2.gsc.riken.go.jp/. The ID numbers of the
cDNAs are as follows: No.1, ri2310047C17; No.2, riB230209J06;
No.3, ri1110035A10; No.4, ri2410011D23; No.5, ri1110008I14; No.6,
ri1810013M05; No.7, ri1110012D12; No.8, ri1810074L23; No.9,
ri2810428M05; No.10, ri4930405J06; No.11, ri9830160H04; No.12,
ri2310046C23; No.13, ri4933409B01 and No.14: ri2810454O07.
Table 2. Translation properties ofS12 mutants in vitro. The ratio of the
Leu misincorporation rates of the wild-type and mutant strains,
obtained by using a poly(U)-direct cell-free translation system, as
described in the Materials and methods.
Strain
Leu
incorporation
(pmol)
Phe
incorporation
(pmol)
In vitro missense
error rate
a
(Leu/Phe) · 10
)3
WT 1.00 14.16 70
K42R 0.14 2.86 48
K42T 0.12 14.80 7.9
K42I 0.05 8.75 5.9
K42N 0.01 3.88 3.4
K87E 0.09 9.34 8.0
K87R 0.03 16.30 1.7
P90Q 0.03 5.26 9.2
P90L 0.02 8.73 6.5
G91D 0.07 8.98 2.6
Ó FEBS 2004 RibosomalproteinS12mutations (Eur. J. Biochem. 271) 1131
cDNAs, so the ribosome activity of the K42T extract would
be useful for general protein synthesis.
The translational accuracy was examined in terms of the
misincorporation rates of Leu in the poly(U)-dependent
poly(Phe) synthesis system. The alleviation of the effect of
streptomycin results from a mutation in the ribosomal
protein S12, which decreases the affinity for streptomycin
and increases the translational accuracy [30]. It also
decreases both the efficiency ofproteinsynthesis and the
growth rate. In this study, all of the mutants exhibited
higher fidelity than the wild-type, and K42R showed the
lowest fidelity among the mutants. These results are
consistent with a previous study, which mentioned that
the streptomycin-resistant K42R mutant has a nonrestric-
tive phenotype in E. coli [28]. In Bacillus subtilis,theK56R
mutant (corresponding to K42R of E. coli) was only
reported as nonrestrictive [31]. Mutations at Lys87, Pro90
and Gly91 conferred higher fidelity than the wild-type,
which also agreed with the previous studies [29,30]. The
K42T mutant was reportedly a restrictive phenotype, which
conferred a low level of nonsense readthrough in vivo
together with slower growth than the wild-type [29]. In this
study, the K42T system exhibited high translation fidelity,
without any reduction in the activity ofcell-free protein
synthesis activity.
Two conformations, with open and closed forms in the
A-site, are suggested by the crystal structure of the 30S
subunit with anticodon stem-loop in the A-site [18]. Closure
of the A-site might lead to GTP hydrolysis by EF-Tu. This
would be followed by the release of EF-Tu and the exposure
of an amino acid, attached to the 3¢-end of the tRNA, to the
peptidyl transferase center. The properties of the strepto-
mycin-resistant mutants are explained well by the open-to-
closed hypothesis. Many of the interactions among the S4,
S5 and 16S rRNAs that maintain the open form are
destroyed in ram mutants, which are inclined toward the
closed form and result in an error-prone phenotype [19].
In the crystal structure of the Thermus thermophilus 30S
subunit, the ribosomalproteinS12 is positioned on the
decoding center [17]. The S12protein interacted with H27 of
the 16S rRNA via Lys45-Lys46 (corresponding to Lys42-
Lys43 of E. coli) and with the 530 loop in H18 via Lys91
(Lys87 in E. coli) in the open form. In the closed form,
Lys45 interacted with the A1492-A1493 residues in H44
[32,33]. The high accuracy of the S12 restrictive mutants
would result from the preference of the open form with the
interaction of the 910–912 residues in H27, in which three
base pairs form the restrictive and ram forms, corresponding
to the open and closed conditions, respectively [19,34]. This
may account for the low translation activity. There is
another possible effect of the S12proteinon the translation
activity. The S12protein may also directly participate in the
GTP hydrolysis by EF-Tu, as suggested by a cryo-electron
microscopic study [35,36]. The aminoacylated tRNA in the
A-site interacted with H69 in the 23S rRNA and the S12
protein. Conformational changes in the S12protein and the
16S rRNA should be required to initiate GTP hydrolysis.
According to the K42T mutant results, shown in Table 2,
this mutation affected the translation properties of
the ribosome in two distinguishable manners: the fidelity
and the efficiency. First, the misincorporation level of Leu in
the K42T system was the same as that in the K42R system,
because K42T and K42R would relatively allow the closed
form in all of mutants used here. It suggested that the Lys42
replacements with Thr and Arg had the same effect on the
interaction with H27. Secondly, the efficiency of poly (Phe)
synthesis by K42T mutant was equal to that of the wild-
type, and there was no loss of the translation activity. The
difference in the activities of K42T and the other Lys42
mutants might come from the involvement of the S12 protein
in GTP hydrolysis by EF-Tu, as described above. Unfortu-
nately, the events involved in the initiation of GTP hydrolysis
are still unclear, because many conformational changes
occur in tRNA, EF-Tu, rRNA and ribosomal proteins. The
substitution of Thr instead of Arg could compensate for the
disadvantage caused by its more restrictive phenotype than
the wild-type, during or after the closure of the A-site. Thus,
K42T system acquired both the high accuracy and the better
productivity in the cell-free system.
The crystal structure of the 30S subunit with antibiotics
revealed that the amino group of Lys45 (Lys42 in E. coli)in
the S12protein hydrogen bonds with the streptomycin, and
forms a salt bridge with the phosphate A913 in the 16S
rRNA [17]. The replacement of Lys with Arg was supposed
to disrupt the direct interaction with streptomycin and to
retain the latter interaction, which could contribute toward
stabilizing the ram status. It reduced the affinity of the 30S
subunit for streptomycin, leading to the resistance of K42R
while decreasing the productivity. However, the Lys to Thr
alteration might change the conformation of the 30S
subunit to preferentially support the translation process,
resulting in the better protein yield by K42T. Recently, the
70S ribosome crystal structures of the wild-type and K42R
mutant were determined at 10 and 9 A
˚
resolutions,
respectively [37]. In the near future, it will become possible
to discuss the structural differences between the wild-type
and mutant ribosomes.
Studies aimed toward improving the productivity of
protein synthesis in vitro have employed several strategies.
The development of a continuous flow in vitro protein
synthesis system successfully maintained the activity over
24 h by the continuous supply of substrates and the removal
of low molecular mass products [38]. This method was
successfully used in experiments with both E. coli and wheat
germ extracts [39–41]. The condensed S30 is highly
productive in cell-freeproteinsynthesis augmented with a
dialysis system [23]. The translation components, the
reaction conditions, and the generation and the consump-
tion of the energy source have been optimized in the cell-free
system [42,43]. For high-throughput protein production,
the designed sequence was added upstream of the expressed
genes in expression vectors [44]. There are no reports
focusing on modification of the ribosome, the apparatus of
translation, in terms of high-yield protein production. Our
development of this ribosomalprotein mutation is one of
the strategies to enhance protein production in E. coli-based
cell-free translation systems.
Acknowledgements
We thank T. Matsuda and N. Matsuda for technical assistance, Dr
T. Terada for useful technical advice, and Dr Y. Hayashizaki for
providing the mouse cDNAs used in this study. This work was
supported by a grant from the Organized Research Combination
1132 N. Chumpolkulwong et al. (Eur. J. Biochem. 271) Ó FEBS 2004
System (ORCS) of the Science and Technology Agency of Japan and
by the RIKEN Structural Genomics/Proteomics Initiative (RSGI), the
National Project onProtein Structural and Functional Analyses,
Ministry of Education, Culture, Sports, Science and Technology of
Japan.
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1134 N. Chumpolkulwong et al. (Eur. J. Biochem. 271) Ó FEBS 2004
. Effects of Escherichia coli ribosomal protein S12 mutations on cell-free protein synthesis Namthip Chumpolkulwong 1 , Chie Hori-Takemoto 1 , Takeshi Hosaka 2 ,. modification of the ribosome, the apparatus of translation, in terms of high-yield protein production. Our development of this ribosomal protein mutation is one of the strategies to enhance protein. University of Tokyo, Bunkyo-ku, Tokyo, Japan We examined the effects of Escherichia coli ribosomal pro- tein S12 mutations on the efficiency of cell-free protein syn- thesis. By screening 150 spontaneous