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 mutations on cell-free protein 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 of Escherichia coli ribosomal pro- tein S12 mutations on the efficiency of cell-free protein 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 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 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 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 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) 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 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 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 protein synthesis 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 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. 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 . 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 D 600 was 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. 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 of cell-free CAT protein synthesis 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 Ribosomal protein S12 mutations (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 of protein 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 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 M (A,C,E)or5m M (B, D, F). Fig. 4. CAT protein synthesis 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 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 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 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 ,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 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 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 Ribosomal protein S12 mutations (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 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,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 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, 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 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, 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 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]. 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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.NatlAcad.Sci.USA99, 14652–14657. 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