Báo cáo khóa học: Effects of Escherichia coli ribosomal protein S12 mutations on cell-free protein synthesis doc

Effects of Escherichia coli ribosomal protein S12 mutationson cell-free protein synthesis Namthip Chumpolkulwong1, Chie Hori-Takemoto1, Takeshi Hosaka2, Takashi Inaoka2, Takanori Kigawa1

Effects of Escherichia coli ribosomal protein S12 mutations

on cell-free protein synthesis

Namthip Chumpolkulwong1, Chie Hori-Takemoto1, Takeshi Hosaka2, Takashi Inaoka2, Takanori Kigawa1, Mikako Shirouzu1,3, Kozo Ochi2and Shigeyuki Yokoyama1,3,4

1

RIKEN Genomic Sciences Center, Tsurumi, Yokohama, Japan;2National Food Research Institute, Tsukuba, Ibaraki, Japan;

3

RIKEN Harima Institute at SPring-8, Mikazuki-cho, Sayo, Hyogo, Japan;4Department of Biophysics and Biochemistry, Graduate School of Science, The 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 propro-tein

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 ribosomal protein S12

We prepared S30 extracts from mutant cells harvested in the

mid-log phase Their protein synthesis 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: ribosomal protein S12; streptomycin; point mutation; cell-free protein synthesis

The antibiotic streptomycin inhibits protein synthesis and

causes misreading during translation Ribosomal protein

mutations in Escherichia coli have been found to confer

resistance to streptomycin [1,2] These mutations frequently

exist in the ribosomal protein S12, encoded by rpsL, and

result in streptomycin resistance [3] or streptomycin

dependence [4] The phenotypes were attributed to the

mutations in the S12 protein by Funatsu et al [5,6] The

streptomycin-resistance mutations in the ribosomal proteins

S4 and S5confer 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 S12 protein 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 streptostrepto-mycin 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 S12 protein [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 S12 mutations on 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)

been characterized well in E coli Mutations in the

ribosomal protein S12 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, ribosomal mutations 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 ofrpsL 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 ofrpsL

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: 5min of incubation

at 96C; 30 cycles of 95 C for 30 s, 55 C for 30 s, 72 C for

1 min; and a final step at 72C 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 5g 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

37C

S30 preparation

The S30 extracts for protein synthesis were prepared from

E colistrain 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 mM Tris/acetate buffer (pH 8.2) containing

14 mMMg(OAc)2, 60 mMpotassium acetate, 1 mM

dithio-threitol, and 7 mM 2-mercaptoethanol, supplemented just

before use] The cells (7.2 g) were suspended in 9 mL of buffer

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 0.3MTris/ acetate (pH 8.2), 9 mM Mg(OAc)2, 13 mM ATP, 84 mM phosphoenolpyruvate, 4.5mMdithiothreitol, 40 lMeach 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 45min 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 mMHepes/KOH (pH 7.5), 1.2 mM ATP, 0.8 mMeach of GTP, CTP and UTP, 1.7 mMdithiothreitol, 0.64 mM cAMP, 170 lgÆmL)1E coli total tRNA (Boeh-ringer-Mannheim), 200 mMpotassium glutamate, 27.5mM

NH4OAc, 13.4 mM Mg(OAc)2, 35 lgÆmL)1 folinic acid,

4 lgÆmL)1 of plasmid pK7-CAT [23] used as a template for chloramphenicol acetyltransferase (CAT) synthesis, 66.6 lgÆmL)1T7 RNA polymerase prepared in our labor-atory, 80 mMcreatine phosphate (Boehringer-Mannheim),

250 lgÆmL)1 creatine kinase (Boehringer-Mannheim),

500 lM each of 20 amino acids, 4% polyethylene glycol

8000 (Sigma), 25mM 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 37C 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 mM Tris/HCl (pH 7.5), 500 mMNH4Cl, and 5mMimidazole 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 thein 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 mM Hepes pH 7.8, 20 mMMgSO, 100 mMNHCl, and 6 mM

2-mercaptoethanol In order to reduce the amount of

endogenous mRNA, the ribosomes and S100 were

incuba-ted at 37C for 10 min prior to use The final

concentra-tions of ribosomes, S100, and each amino acid (except for

Phe and Leu) were 4 A260ÆmL)1, 0.18 mgÆmL)1, and 0.4 mM

in the reaction mixture The reaction was started with

the addition of 0.75mgÆmL)1 poly(U), 200 lM [14C]Phe

(0.11 MBq, Amersham) and [3H]Leu (3.7 MBq,

Amer-sham), and was incubated at 37C for 15min 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 95C 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 [3H]Leu to

that of [14C]Phe

Results

Growth characteristics of S12 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 coliBL21 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)1streptomycin and resisted to streptomycin

up to 10 mgÆmL)1 Mutations of 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 protein synthesis with the extract from each strain

An S30 extract was prepared from the cells harvested in the mid-log phase, when the D600was approximately 3.0 To estimate the protein synthesis 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 of mutations in rpsL of E coli 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)

KO-365128 (AfiG) K42R >10

KO-368 129 (AfiC) K42N >10

KO-371 128 (AfiC) K42T >10

KO-374 128 (AfiT) K42I >10

KO-375263 (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

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 of cell-free CAT protein synthesis in the wild-type and S12 mutant systems The pk7-CAT plasmid concentration was

4 ngÆlL)1and 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.

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 A260values of the wild-type

and mutant extracts All of them exhibited approximately

210–240 A260ÆmL)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 A260ÆmL)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 mM Mg2+, the 70S

ribo-some was observed as the main fraction in all extracts

(Fig 3A,C,E) On the other hand, when the Mg2+

concentration was reduced to 5mM, 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 of protein productivity on the

Mg2+ 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 Mg2+ concentration range between 10.7 and 13.4 mM(Fig 4A) Moreover, the K42T system still synthesized the CAT protein efficiently, even in 16.1 mM Mg2+ 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 Mg2+for the other mutant systems was 13.4 mM(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 Mg2+and template DNA concentrations

Cell-free protein synthesis 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 (A, C, E) or 5 m (B, D, F).

Fig 4 CAT protein synthesis in the wild-type (d) and K42T system (m) with various concentrations of Mg2+(A) and template DNA (B).

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 ribosomal protein 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 protein synthesis 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

1 mgÆmL)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 Mg2+ 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 of protein synthesis, and only the replacement by Thr increased the produc-tivity of the ribosome

We used CAT protein synthesis as the standard assay for the protein synthesis 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-free protein synthesis 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, and that of

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 of S12 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

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 of protein synthesis 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, the K5 6R

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 of cell-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,

S5and 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 ribosomal protein S12 is positioned on the

decoding center [17] The S12 protein 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,

Lys45interacted 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 S12 protein on the translation

activity The S12 protein 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 S12 protein 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 S12 protein 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-free protein synthesis 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 ribosomal protein 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

System (ORCS) of the Science and Technology Agency of Japan and

by the RIKEN Structural Genomics/Proteomics Initiative (RSGI), the

National Project on Protein Structural and Functional Analyses,

Ministry of Education, Culture, Sports, Science and Technology of

Japan.

References

1 Old, D & Gorini, L (1965) Amino acid changes provoked by

streptomycin in a polypeptide synthesized in vitro Science 150,

1290–1292.

2 Anderson, W.F., Gorini, L & Breckenridge, L (1965) Role of

ribosomes in streptomycin-activated suppression Proc Natl Acad.

Sci USA 54, 1076–1083.

3 Ozaki, M., Mizushima, S & Nomura, M (1969) Identification

and functional characterization of the protein controlled by the

streptomycin-resistant locus in E coli Nature 222, 333–339.

4 Birge, E.A & Kurland, C.G (1969) Altered ribosomal protein

in streptomycin-dependent Escherichia coli Science 166, 1282–

1284.

5 Rosset, R & Gorini, L (1969) A ribosomal ambiguity mutation.

J Mol Biol 39, 95–112.

6 Funatsu, G., Nierhaus, K & Wittmann, H.G (1972) Ribosomal

proteins XXXVII Determination of allele types and amino acid

exchanges in protein S12 of three streptomycin-resistant mutants

of Escherichia coli Biochem Biophys Acta 287, 282–291.

7 Funatsu, G & Wittmann, H.G (1972) Ribosomal proteins

XXXIII Location of amino acid replacements in protein S12

isolated from Escherichia coli mutants resistant to streptomycin.

J Mol Biol 68, 547–550.

8 Zimmermann, R.A., Garvin, R.T & Gorini, L (1971) Alteration

of a 30S ribosomal protein accompanying the ram mutation in

Escherichia coli Proc Natl Acad Sci USA 68, 2263–2267.

9 Woese, C.R & Gutell, R.R (1989) Evidence for several higher

order structural elements in ribosomal RNA Proc Natl Acad Sci.

USA 86, 3119–3122.

10 Stern, S., Powers, T., Changchlen, L & Noller, H.F (1989) RNA–

protein interactions in 30S ribosomal subunit: folding and

func-tion of 16S rRNA Science 244, 783–790.

11 Allen, P.K & Noller, H.F (1989) Mutations in ribosomal proteins

S4 and S12 influence the higher order structure of 16S ribosomal

RNA J Mol Biol 208, 457–468.

12 Powers, T & Noller, H.F (1991) A functional pseudoknot in 16S

ribosomal RNA EMBO J 10, 2203–2214.

13 Tapprich, W.E & Dahlberg, A.E (1990) A single base mutation

at position 2661 in E coli 23S ribosomal RNA affects the binding

of ternary complex to the ribosome EMBO J 9, 2649–2655.

14 Bilgin, N & Ehrenberg, M (1994) Mutations in 23 S ribosomal

RNA perturb transfer RNA selection and can lead to

strepto-mycin dependence J Mol Biol 235, 813–824.

15 Zuurmond, A.M., Zeef, L.A & Kraal, B (1998) A

kirromycin-resistant EF-Tu species reverses streptomycin dependence of

Escherichia coli strains mutated in ribosomal protein S12

Micro-biology 144, 3309–3316.

16 Moazed, D & Noller, H.F (1987) Interaction of antibiotics with

functional sites in 16S ribosomal RNA Nature 327, 389–394.

17 Carter, A.P., Clemons, W.M., Brodersen, D.E., Morgan-Warren,

R.J., Wimberly, B.T & Ramakrishnan, V (2000) Functional

insights from the structure of the 30S ribosomal subunit and its

interaction with antibiotics Nature 407, 340–348.

18 Ogle, J.M., Brodersen, D.E., Clemons, W.M Jr, Tarry, M.J.,

Carter, A.P & Ramakrishnan, V (2001) Recognition of cognate

transfer RNA by the 30S ribosomal subunit Science 292, 897–902.

19 Ogle, J.M., Carter, A.P & Ramakrishnan, V (2003) Insights into

the decoding mechanism from recent ribosome structures Trends

Biochem Sci 28, 259–266.

20 Shima, J., Hesketh, A., Okamoto, S., Kawamoto, S & Ochi, K (1996) Induction of actinorhodin production by rpsL (encoding ribosomal protein S12) mutations that confer streptomycin resistance in Streptomyces lividans and Streptomyces coelicolor A3 (2) J Bacteriol 178, 7276–7284.

21 Hosoya, Y., Okamoto, S., Muramatsu, H & Ochi, K (1998) Acquisition of certain streptomycin-resistant (str) mutations enhances antibiotic production in bacteria Antimicrob Agents Chemother 42, 2041–2047.

22 Pratt, J.M (1984) Coupled transcription-translation in prokary-otic cell-free systems In Transcription and Translation (Hames, B.D & Higgins, S.J., eds), pp 179–209 IRL Press, New York.

23 Kim, D.M., Kigawa, T., Choi, C.Y & Yokoyama, S (1996) A highly efficient cell-free protein synthesis system from Escherichia coli Eur J Biochem 239, 881–886.

24 Shaw, W.V (1975) Chloramphenicol acetyltransferase from chlo-ramphenicol-resistant bacteria Methods Enzymol 43, 737–755.

25 Bradford, M.M (1976) A rapid and sensitive method for the quantitation of protein utilizing the principle of protein-dye binding Anal Biochem 72, 248–254.

26 Legault-Demare, L & Chambliss, G.H (1974) Natural messanger ribonucleic acid-directed cell-free protein synthesizing system of Bacillus subtilis J Bacteriol 120, 1300–1307.

27 Timms, A.R., Steingrimsdottir, H., Lehmann, A.R & Bridges, B.A (1992) Mutant sequences in the rpsL gene of Escherichia coli B/r: mechanistic implications for spontaneous and ultraviolet light mutagenesis Mol Gen Genet 232, 89–96.

28 Kurland, C.G., Hughes, D & Ehrenberg, M (1996) Limitations

of translational accuracy In E coli and Salmonella: Cellular and Molecular Biology (Neidhardt, F.C et al eds), 2nd Edn,

pp 979–1003 ASM, Washington, DC.

29 Ruusala, T., Andersson, D., Ehrenberg, M & Kurland, C.G (1984) Hyper-accurate ribosomes inhibit growth EMBO J 3, 2575–2580.

30 Bohman, T., Ruusala, T., Jelenc, P.C & Kurland, C.G (1984) Kinetic impairment of restrictive streptomycin-resistant ribo-somes Mol Gen Genet 198, 90–99.

31 Inaoka, T., Kasai, K & Ochi, K (2001) Construction of an in vivo nonsense readthrough assay system and functional analysis of ribosomal proteins S12, S4, and S5in Bacillus subtilis J Bacteriol.

183, 4958–4963.

32 Ogle, J.M., Murphy, F.V., Tarry, M.J., & Ramakrishnan, V (2002) Selection of tRNA by the ribosome requires a transition from an open to a closed form Cell 111, 721–732.

33 Brodersen, D.E., Clemons, W.M Jr, Carter, A.P., Wimberly, B.T.

& Ramakrishnan, V (2002) Crystal structure of the 30 S ribosomal subunit from Thermus thermophilus: structure of the proteins and their interactions with 16 S RNA J Mol Biol 316, 725–768.

34 Lodmell, J.S & Dahlberg, A.E (1997) A conformational switch in Escherichia coli 16S ribosomal RNA during decoding of messen-ger RNA Science 277, 1262–1267.

35 Stark, H., Rodonina, M.V., Wieden, H.J., Zemlin, F., Winter-meyer, W & van Heel, M (2002) Ribosome interactions of aminoacyl-tRNA and elongation factor Tu in the codon-recognition complex Nat Struct Biol 9, 849–854.

36 Valle, M., Sengupta, J., Swami, N.K., Grassucci, R.A., Burk-hardt, N., Nierhaus, K.H., Agrawal, R.K & Frank, J (2002) Cryo-EM reveals an active role for aminoacyl-tRNA in the accommondation process EMBO J 21, 3557–3567.

37 Vila-Sanjurjo, A., Ridgeway, W.K., Seymaner, V., Zhang, W & Santoso, S., Yu, K & Cate, J.H (2003) X-ray crystal structures of the WT and a hyper-accurate ribosome from Escherichia coli Proc Natl Acad Sci USA 100, 8682–8687.

38 Spirin, A.S., Baranov, V.I., Ryabova, L.A., Ovodov, S.Y & Alakhov, Y.B (1988) A continuous in vitro translation system

capable of producing polypeptides in high yield Science 242,

1162–1164.

39 Spirin, A.S (1991) Cell-free protein synthesis bioreactor In

Fron-tiers in Bioprocessing II (Todd, P., Sikdar, S.K & Beer, M., eds),

pp 31–43 American Chemical Society, Washington, DC.

40 Baranov, V.I & Spirin, A.S (1993) Gene expression in cell-free

systems on preparative scale Methods Enzymol 217, 123–142.

41 Endo, Y., Otsuzuki, S., Ito, K & Miura, K (1992) Production of

an enzymatic active protein using a continuous flow cell-free

translation system J Biotechnol 25, 221–230.

42 Kigawa, T., Yabuki, T., Yoshida, Y., Tsutsui, M., Ito, Y., Shibata,

T & Yokoyama, S (1999) Cell-free production and stable-isotope labeling of milligram quantities of proteins FEBS Lett.

442, 15–19.

43 Kim, D.M & Swartz, J.R (2001) Regeneration of adenosine tri-phosphate from glycolytic intermediates for cell-free protein synthesis Biotechn Bioeng 74, 309–316.

44 Sawasaki, T., Ogasawara, T., Morishita, R & Endo, Y (2002) A cell-free protein synthesis system for high-throughput proteomics Proc Natl Acad Sci USA 99, 14652–14657.