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J. Biol. Chem., Vol. 277, Issue 40, 37139-37146, October 4, 2002

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Polyamines Enhance Synthesis of the RNA Polymerase ³⁸ Subunit by Suppression of an Amber Termination Codon in the Open Reading Frame^*

Madoka Yoshida, Keiko Kashiwagi, Gota Kawai^§, Akira Ishihama^¶, and Kazuei Igarashi

From the  Graduate School of Pharmaceutical Sciences, Chiba University, 1-33 Yayoi-cho, Inage-ku, Chiba 263-8522, Japan, the ^§ Department of Industrial Chemistry, Faculty of Engineering, Chiba Institute of Technology, 2-17-1 Tsudanuma, Narashino-shi, Chiba 275-8588, Japan, and the ^¶ Department of Molecular Genetics, National Institute of Genetics, Mishima, Shizuoka 411-0801, Japan

Received for publication, July 4, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The mechanisms by which polyamines stimulate synthesis of the RNA polymerase ³⁸ subunit in Escherichia coli were studied. Polyamine stimulation was observed only in strains in which the 33rd codon of RpoS mRNA is a UAG termination codon instead of a CAG codon for glutamine in wild-type E. coli. Readthrough of the termination codon by Gln-tRNA^supE was stimulated by polyamines. This stimulation was found to be caused by an increase in both the level of suppressor tRNA^supE and the binding affinity of Gln-tRNA^supE for ribosomes. The stimulatory effect was observed with a UAG termination codon but not with UGA and UAA codons. Readthrough of the UAG termination codon at the 270th amino acid position of RpoS mRNA was also stimulated by polyamines, indicating that polyamines stimulate readthrough of a UAG codon regardless of its location within the RpoS mRNA. When cell viability of an E. coli strain having a termination codon in the 33rd position of RpoS mRNA was compared using cells cultured with or without putrescine, it was higher in cells cultured with putrescine than in cells cultured without putrescine. The level of ³⁸ subunit in the cells cultured with putrescine was higher than that in cells cultured without putrescine on days 2, 4, and 8, but the level of ⁷⁰ subunit was almost the same in cells cultured with or without putrescine. These results confirm that elevated expression of the rpoS gene is important for cell viability at late stationary phase.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Polyamines, aliphatic cations present in almost all living organisms, are necessary for normal cell growth (1, 2). Because polyamines interact with nucleic acids and mostly exist as polyamine-RNA complexes in cells (3, 4), their proliferative effects are presumed to be caused by stimulation of nucleic acid and protein synthesis. In fact, it has been reported that polyamines stimulate the synthesis of some protein species in vitro (5-7) and in vivo (8, 9), induce the in vivo assembly of 30 S ribosomal subunits (10-12), and increase the fidelity of protein synthesis (13-15), altogether suggesting that polyamines regulate protein synthesis at several different steps.

In Escherichia coli, the synthesis of OppA protein, a periplasmic substrate-binding protein of the oligopeptide uptake system, is strongly stimulated by the addition of putrescine to a polyamine-requiring mutant, MA261 (9). We found that (i) the stimulation of OppA synthesis takes place at the level of translation; (ii) the position and secondary structure of the Shine-Dalgarno (SD)¹ sequence (16) on OppA mRNA are important for this stimulation (17); and (iii) polyamines cause a structural change of the SD sequence and the initiation codon AUG of OppA mRNA, facilitating formation of the initiation complex (18). We also found that polyamines increase the translation of adenylate cyclase (Cya) mRNA by facilitating UUG codon-dependent initiation (19). Analysis of RNA secondary structure suggests that exposure of the SD sequence of mRNA is a prerequisite for polyamine stimulation of UUG codon-dependent initiation (19).

In the present study we found a novel mechanism of the polyamine stimulation, which is involved in the enhanced synthesis of RNA polymerase ³⁸ subunit by polyamines (19). This type of the polyamine stimulation was observed only in E. coli strains in which the 33rd codon of RpoS mRNA is a UAG amber termination codon instead of a CAG codon for glutamine in wild-type E. coli strains. Readthrough of the termination codon by Gln-tRNA^supE was stimulated by polyamines.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Bacterial Strains and Culture Conditions-- Polyamine-requiring mutants, E. coli MA261 (speB speC gly leu thr thi) (20), HT283 (speA speB speC speED thr pro thi) (21), and DR112 (speA speB) (22) were kindly provided by Dr. W. K. Maas (New York University School of Medicine), Dr. H. Tabor (National Institute of Health), and Dr. D. R. Morris (University of Washington), respectively. A ³⁸-deficient mutant, E. coli KT1100 rpoS::tet (23), was kindly supplied by Dr. K. Tanaka (University of Tokyo). E. coli MA261 rpoS::tet, HT283 rpoS::tet, and DR112 rpoS::tet were isolated by transduction with P₁ phage (24) using E. coli KT1100 as the donor. E. coli W3110 type A, B, and D used were characterized previously (25), and another W3110 strain used was a new (type F) derived from type A. E. coli C600 (supE44 hsdR thi-1 thr-1 leuB6 lacY1 tonA21) was from our laboratory stock.

E. coli MA261 and MA261 rpoS::tet were grown at 37 °C in medium A supplemented with 5 amino acids (100 µg/ml each of Gly, Leu, Met, Ser, and Thr) in the presence (100 µg/ml) or absence of putrescine (9). HT283 and DR112 strains were cultured according to the method of Hafner et al. (21) and Linderoth and Morris (22), respectively, in the presence (100 µg/ml) or absence of putrescine. Antibiotics used were 100 µg/ml ampicillin, 50 µg/ml kanamycin, 30 µg/ml chloramphenicol, and 15 µg/ml tetracycline. Cell growth was monitored by measuring the absorbance at 540 nm.

Plasmids-- Plasmids pMRTCG (=pMW33TCG) and pMRTAG (=pMW33TAG) (26) were kind gifts from Dr. Y. Kamio (Tohoku University). Total chromosomal DNA from E. coli was prepared according to the method of Ausubel et al. (27). For construction of pMWrpoS(33CAG) (=pMW33CAG) or pMWrpoS(270TAG) (=pMW270TAG), PCR was performed using 5'-CAACGAATTCCGTGACCTTGCTCAGCGCA-3' (P1) and 5'-TGCAAGCTTGTATGGGCGGTAATTTGACC-3' (P2) as primers and total chromosomal DNA of W3110 types A and B, respectively, as templates. After cutting with EcoRI and HindIII, the 1.9-kb fragment was inserted into the same restriction sites of pMW119 (Nippon Gene, Japan). Site-directed mutagenesis by overlap extension using PCR (28) was performed to prepare pMWrpoS(33TGA) (=pMW33TGA) and pMWrpoS(33TAA) (=pMW33TAA). The template used for the first PCR was chromosomal DNA from E. coli W3110 type A. To make pMWrpoS(33TGA) primers used for the first PCR were P1 and 5'-TTCCTCTTCGGCCAAATCGTTATCACTGGGTTCTCATTCTACTAA-3' (underlined base for CAG substitution with TGA) and 5'-GCCTTAGTAGAATGAGAACCCAGTGATAACGATTTGGCCGAAGAGGAACTGTTATCGCAG-3' and P2. The second PCR was performed using the first PCR products as templates and P1 and P2 as primers. To make pMWrpoS(33TAA) primers used for the first PCR were P1 and 5'-TTCCTCTTCGGCCAAATCGTTATCACTGGGTTCTTATTCTACTAA-3' (underlined base for CAG substitution with TAA) and 5'-GCCTTAGTAGAATAAGAACCCAGTGATAACGATTTGGCCGAAGAGGAACTGTTATCGCAG-3' and P2. The second PCR was performed using the first PCR products as templates and P1 and P2 as primers. After cutting with EcoRI and HindIII, the 1.9-kb fragment was inserted into the same restriction sites of pMW119.

PCR was performed to make pUCsupE and pACYCsupE, using chromosomal DNA of MA261 as templates and 5'-ACGCTGTTCGGATCCTAACCAAACAGTCAC-3' (P3) and 5'-GAAGGATCCGACGTGTCAACATCGCATTCG-3' (P4) as primers. After cutting with BamHI, the 0.9-kb fragment was inserted into the same site of pUC119 (Takara Bio Inc., Japan) and pACYC184 (PerkinElmer Life Sciences).

For preparation of pSTrpoS(33TAG)-His₆, PCR was performed using MA261 chromosomal DNA as templates and 5'-AGGGAATTCGGGTAGGAGCCACCTTATG-3' and 5'-AGCCAAGCTTGAGATTAGTGGTGGTGGTGGTGGTGCTCGAGCTCGCGGAACAGCGCTTCG-3' as primers. The 1.1-kb PCR product was inserted into the EcoRV site of pSTBlue-1 using pSTBlue-1 Perfectly Blunt cloning kit (Novagen) according to the manufacturer's instructions. A plasmid in which the rpoS(33TAG)-His₆ gene is under the control of T7 promoter was selected.

PCR and DNA Sequencing of rpoS Gene and metT Operon-- PCR for rpoS gene or metT operon was performed using chromosomal DNA of various E. coli strains as templates and the primer pair of 5'-GCGAATTCCATAGTCAAGGGATCACG-3' (P5) and 5'-GCGGGATCCCTCGAGTTACTCGCGGAACAGCGCTTC-3' (P6) and of 5'-GTCACAGGTTCGAATCCCGTC-3' and 5'-GACGTGTCAACATCGCATTCG-3', respectively. The nucleotide sequence was determined by the Gene Rapid System (Amersham Biosciences).

Western Blot Analysis-- Antisera against ³⁸ and ⁷⁰ subunits were prepared as described previously (29). Western blot analysis was performed by the method of Nielsen et al. (30) using Proto Blot Western blot AP system (Promega).

Dot Blot and Northern Blot Analysis of RNA-- Total RNA was prepared from various E. coli strains according to the method of Emory and Belasco (31). Dot blot analysis of RpoS mRNA was performed according to the method of Sambrook et al. (32). The 1.1-kb PCR product prepared as described above was labeled with [-³²P]dCTP using BcaBEST labeling kit (Takara Bio Inc.) and used as a probe. Northern blot analysis of tRNAs encoded by the metT operon was performed using the 0.9-kb PCR product prepared similarly to the probe described above (19).

Measurement of Aminoacylation Level of tRNA^Gln and Gln-tRNA Binding to Ribosomes-- Total RNA was prepared from E. coli cells according to the method of Chomczynski and Sacchi (33) using TRIzol reagent (Invitrogen). Polyacrylamide gel electrophoresis, blotting, and detection of Gln-tRNA^Gln and tRNA^Gln were performed according to the methods of Varshney et al. (34) and Kowal et al. (35) using the 5'-end ³²P-labeled primer 5'-CCTCGGAATGCCGGAATTAGAATCC-3' as a probe for hybridization. Assay of Gln-tRNA formation was performed as described previously (36) except that tRNA obtained from E. coli MA261/pUCsupE (2 A₂₆₀ units) and crude aminoacyl-tRNA synthetases (110 µg of protein) (37) were used. [³H]Gln-tRNA binding to ribosomes was measured as previously described (37) except that the reaction mixture (0.05 ml) contained 4 A₂₆₀ units and 0.3 M NH₄Cl-washed ribosomes, and mRNA was omitted. [³H]Gln-tRNA was prepared using tRNA of E. coli MA261/pUCsupE and [³H]Gln (1.06 GBq/mmol, ARC) by crude aminoacyl-tRNA synthetases (37). Hexaribonucleotide AUGUAG was synthesized by DNA/RNA synthesizer Expedite 8909 (PerkinElmer Life Sciences).

Measurement of Cell Viability-- Cell viability was determined by counting colony numbers in aliquots of the culture grown on LB-containing 1.5% agar plate at 37 °C. Thus, the definition of viable cells is that the cells are able to grow on agar plate.

Preparation of mRNA and in Vitro Translation-- pSTrpoS(33TAG)-His₆ was linearized by HindIII, and RpoS(33UAG)-His₆ mRNA was synthesized by T7 RNA polymerase using AmpliScribe T7 High Yield transcription kit (Epicentre Technologies). The 30,000 × g supernatant (S-30) of E. coli C600/pUCsupE was prepared as described previously (37). For in vitro translation, a reaction mixture (0.1 ml) containing 50 mM Tris-HCl, pH 7.5, 60 mM NH₄Cl, 6 mM 2-mercaptoethanol, 2 mM ATP, 0.5 mM GTP, 4 mM phosphoenolpyruvate, 2.5 µg of pyruvate kinase, 2 µg of folinic acid, 50 kBq of [³⁵S]methionine (37 TBq/mmol), 0.2 mM each of 19 amino acids without methionine, 10 µg of RpoS(33UAG)-His₆ mRNA, 17 A₂₆₀ units S-30, and magnesium acetete and spermidine at the specified concentration was incubated at 30 °C for 1 h. A 45-µl aliquot of each reaction mixture was placed on a 3MM paper disc (Whatman, 24 mm in diameter) and radioactivity insoluble in hot 5% trichloroacetic acid (TCA) was measured with a liquid scintillation spectrometer. A 10-µl aliquot of nickel-nitrilotriacetic acid agarose suspension (50% V/V in water, Qiagen) was added to the residual 50-µl aliquot, and the mixture was incubated for 30 min on ice. After removal of supernatant, the pellet was mixed with 30 µl of sample buffer for SDS-polyacrylamide electrophoresis (38) and boiled for 2 min. A 20-µl aliquot was used for 12% SDS-polyacrylamide electrophoresis and fluorography was performed according to the method of Laskey and Mills (39). Radioactivity of labeled ³⁸-His₆ was quantified using a Fuji Bas-2000II imaging analyzer.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Polyamine Stimulation of the Synthesis of RNA Polymerase ³⁸ Subunit Based on the Readthrough of an Amber Codon in the Open Reading Frame (ORF) of RpoS mRNA-- We reported previously that synthesis of the RNA polymerase ³⁸ subunit was stimulated by polyamines using one of the polyamine-requiring mutants, MA261 (19). To confirm this observation, the effect of polyamine addition on the synthesis of ³⁸ subunit was examined using three independent isolates of polyamine-requiring E. coli mutants, MA261, HT283, and DR112 (20-22). As shown in Fig. 1A, the increased synthesis of ³⁸ subunit was observed for MA261 and HT283 but not for DR112. The level of ⁷⁰ subunit was, however, nearly equal for all three mutants and was not affected by the addition of polyamines (Fig. 1A). The level of RpoS mRNA in E. coli MA261, as measured by dot blotting, was nearly equal in the presence and absence of polyamine addition (Fig. 1B), indicating that the stimulation of ³⁸ synthesis by polyamines takes place at a post-transcriptional step(s).

View larger version (23K): [in this window] [in a new window]   Fig. 1.   Effect of polyamines on the synthesis of 38 subunit of RNA polymerase in polyamine-requiring mutants of E. coli. A, Western blotting of  subunits was performed using 10 µg of cell lysate protein for 38 or 5 µg of cell lysate protein for 70. Cell lysates were prepared from cells cultured with or without 100 µg/ml putrescine (PUT) and harvested at A540 = 0.2. B, dot blotting of RpoS (38) mRNA was performed using 1, 3, 10, and 30 µg of total RNA. Total RNA was prepared as described under "Experimental Procedures" from E. coli MA261 cells cultured with or without 100 µg/ml putrescine and harvested at A540 = 0.2.

To understand why the polyamine stimulation takes place in only two strains (MA261 and HT283) among three polyamine-requiring mutants, the nucleotide sequence of the rpoS gene was determined. As shown in Fig. 2, a TAG termination codon (instead of CAG codon for glutamine) was present at the 33rd position of the ORF of the rpoS gene in MA261 and HT283. We also determined the nucleotide sequence of the rpoS gene in two kinds of E. coli W3110 from our laboratory stock. One (type D) has a CAG codon for glutamine, but the other (type F, new type) has the termination codon TAG (Fig. 2). In the E. coli strain WC196, the 33rd amber codon of the rpoS gene is suppressed by the supD gene, which translates the UAG codon into serine (26). Together these results suggest that a high incidence of mutations are accumulated at the 33rd codon of the rpoS gene.

View larger version (36K): [in this window] [in a new window]   Fig. 2.   Nucleotide sequence of rpoS and its encoded amino acid residues in various E. coli strains. A, nucleotide sequence of rpoS and its encoded amino acid residues in various E. coli strains are shown. B, nucleotide sequences for 31-35 amino acid residues of the ORF of rpoS gene are shown.

The three E. coli strains, MA261, HT283, W3110 (type F), carry the TAG amber codon at the 33rd position of the rpoS gene, suggesting that these strains carry a suppressor for UAG and that the efficiency of suppression is enhanced by polyamines. To test the UAG suppression of the mutant rpoS gene in MA261 (one of the three mutants) we constructed an MA261 derivative with rpoS disrupted by insertion of the tet gene, and we tested the expression of a plasmid-encoded ³⁸ after transfection of various plasmids containing the rpoS gene with various mutations at the 33rd codon. In the presence of polyamine addition, the level of ³⁸ subunit increased only for the rpoS mutant with a UAG amber termination codon at the 33rd position of RpoS mRNA (Fig. 3B, 33TAG), whereas the ³⁸ level remained unaltered for the rpoS mutant with a UGA codon at the same position (Fig. 3B, 33TGA). The ³⁸ subunit was not detected for the mutant rpoS gene with the UAA termination codon (Fig. 3B, 33TAA), suggesting that the MA261 strain does not carry the UAA suppressor.

View larger version (18K): [in this window] [in a new window]   Fig. 3.   Effect of polyamines on the synthesis of 38 subunit derived from RpoS mRNA containing sense codon or nonsense codon at the 33rd position. A, structures of pMW33CAG, pMW33TCG, pMW33TAG, pMW33TGA, and pMW33TAA are shown. B, Western blotting of 38 subunit was performed using 10 µg of cell lysate protein. Cell lysates were prepared from cells cultured with or without 100 µg/ml putrescine (PUT) and harvested at A540 = 0.2.

The expression level of ³⁸ was essentially the same in the presence and absence of polyamines for the rpoS gene with CAG (Gln) or UCG (Ser) codons at the 33rd position (Fig. 3B, 33CAG and 33TCG). Taken together we conclude that neither CAG-dependent Gln-tRNA and UCG-dependent Ser-tRNA binding to ribosomes nor readthrough of the UGA termination codon were influenced by polyamines.

One of the W3110 strains (type B) carries a UAG termination codon at the 270th position of the RpoS mRNA (25). We checked whether polyamines enhance the level of suppression at this position. As shown in Fig. 4, polyamines enhanced the synthesis of full-length ³⁸ subunit from the mutant RpoS mRNA about 2-fold in MA261 rpoS::tet carrying pMW270TAG, indicating that polyamines stimulate the suppression of the UAG codon on the rpoS gene regardless of the position of UAG codon. The percentage of readthrough at the 270th position, i.e. the ratio of full-length ³⁸ to the COOH-terminal truncated ³⁸, in the presence and absence of polyamines was ~18 and 11%, respectively. The stimulation of the amber mutant rpoS expression by polyamines was, however, not observed when the same plasmid was expressed in the DR112 rpoS::tet strain, and the level of full-length ³⁸ subunit in DR112 was very low (Fig. 4C, DR112), suggesting that the level or activity of suppressor tRNA for UAG is low or that the UAG suppressor tRNA does not exist in the strain DR112.

View larger version (25K): [in this window] [in a new window]   Fig. 4.   Effect of polyamines on the synthesis of 38 subunit derived from RpoS mRNA containing amber codon at the 270th position. A, the nucleotide sequence of rpoS and its encoded amino acid residues in E. coli W3110 type A and B are shown. B, structures of pMW33TAG and pMW270TAG are shown. C, Western blotting of 38 subunit was performed using 10 µg of cell lysate protein. Cell lysates were prepared from cells cultured with or without 100 µg/ml putrescine (PUT) and harvested at A540 = 0.2.

Four different genes encoding tRNA^Gln, glnU, glnW, glnV, and glnX, exist in the metT operon (40). A commonly occurring suppressor tRNA for the amber termination codon in E. coli is encoded by the supE gene, which can be generated after mutation in either glnV or glnX of the metT operon. To identify the nature of the amber suppressor in each of the three polyamine-requiring mutant strains, we determined the nucleotide sequences of the tRNA^Gln genes for all three mutants. The nucleotide sequences of the tRNA^GlnU, tRNA^GlnW, and tRNA^GlnV genes were the same among the three polyamine-requiring mutants (data not shown). However, the tRNA^GlnX gene was changed to the tRNA^supE gene in both MA261 and HT283 but not in DR112 (Fig. 5). These results indicate that tRNA^supE is involved in the stimulation of ³⁸ subunit synthesis by polyamines.

View larger version (34K): [in this window] [in a new window]   Fig. 5.   Nucleotide sequence of glnX in polyamine-requiring mutants of E. coli. A, nucleotide sequences of glnX in polyamine-requiring mutants of E. coli strains are shown. The gene for glnX of MA261 and HT283 was changed to the gene for supE. B, possible secondary structures of tRNAGlnX and tRNAsupE are shown (48).

Mechanism of Polyamine Stimulation of Readthrough of the Amber Termination Codon in RpoS mRNA-- One possible mechanism of the enhancement of amber suppression by polyamines is an increase in the level of suppressor tRNA. To test this possibility, we first measured the level of tRNA^supE in E. coli MA261. Total cellular RNA was subjected to Northern blot hybridization using a probe that hybridizes to both tRNA^supE and tRNA^GlnV. As shown in Fig. 6A, the combined level of tRNA^supE and tRNA^GlnV in E. coli MA261 was higher for the culture with polyamines than that without polyamines. This was confirmed by Northern blot analysis of the combined levels of all seven species of tRNA (see Fig. 5A) encoded by the metT operon (Fig. 6B). The level was found to be 1.8× higher in the presence of polyamines than that found in its absence. Most of the tRNA^supE and tRNA^GlnV was aminoacylated in E. coli MA261 cultured with or without the addition of polyamines (Fig. 6A). These results suggest that polyamines stimulate transcription of the metT operon or stabilize the tRNAs.

View larger version (28K): [in this window] [in a new window]   Fig. 6.   Effect of polyamines on the level of tRNAGln encoded by supE and glnV (A) and of tRNAs encoded by metT operon (B) and effect of suppressor tRNAGln on polyamine stimulation of the synthesis of 38 subunit in polyamine-requiring mutants of E. coli (C and D). A, total RNA was prepared from E. coli MA261 cells cultured with or without 100 µg/ml putrescine (PUT) and harvested at A540 = 0.2. Polyacrylamide gel electrophoresis was performed using 8 µg of total RNA. OH, RNA was incubated at 37 °C for 2 h at pH 9.5. B, Northern blotting of metT operon tRNAs of E. coli MA261 was performed using 5 µg of total RNA. Hybridized RNA is processed tRNAs. C, E. coli MA261 containing pUC119 or pUCsupE was cultured with or without 100 µg/ml putrescine (PUT) and harvested at A540 = 0.2 and 0.6. Western blotting of 38 subunit was performed using 10 µg of cell lysate protein. D, E. coli DR112, DR112rpoS::tet/pMW33TAG containing pACYCsupE or DR112rpoS::tet/pMW33TAG containing pUCsupE was cultured with or without 100 µg/ml PUT and harvested at A540 = 0.2. Western blotting of 38 subunit was performed using 10 µg of cell lysate protein.

The effects of high-level tRNA^supE on the polyamine stimulation of the synthesis of ³⁸ subunit were then examined. As shown in Fig. 6C, the degree of stimulation of the synthesis of ³⁸ subunit by polyamines became smaller in both early and middle logarithmic phases by transforming a high-copy number plasmid (pUC119) containing the gene for tRNA^supE. This was caused by the increase in the level of ³⁸ subunit in cells cultured without polyamines. When E. coli DR112 rpoS::tet was transformed with the gene for tRNA^supE in middle-copy number plasmid (pACYC184) but not in high-copy number plasmid (pUC119), it was found that polyamines stimulated the synthesis of ³⁸ subunit (Fig. 6D). As for the cells transformed with pUCsupE, the decrease in polyamine stimulation of the synthesis of ³⁸ subunit was also caused by the increase in the level of ³⁸ subunit in cells cultured without polyamines. The results indicate the necessity of limiting amounts of tRNA^supE for polyamine stimulation of the synthesis of ³⁸ subunit.

Next we analyzed possible effects of polyamines on Gln-tRNA formation and RpoS mRNA-dependent protein synthesis in a cell-free system. Detailed analysis was carried out using spermidine because it is more effective than putrescine (at least for protein synthesis in vitro) (5, 13). The tRNA preparation used for aminoacylation in vitro was rich in tRNA^supE because tRNA was isolated from E. coli MA261 containing pUCsupE. As shown in Fig. 7A, Gln-tRNA formation was not influenced by the addition of 1 mM spermidine. Similar results were obtained in the presence of 2.5 mM spermidine (data not shown).

View larger version (29K): [in this window] [in a new window]   Fig. 7.   Effect of spermidine on Gln-tRNAsupE formation (A), on in vitro synthesis of 38 subunit directed by RpoS-His6 mRNA containing amber codon at the 33rd position (B and C), and on Gln-tRNAsupE binding to ribosomes (D). A, assay for Gln-tRNA formation was performed under standard conditions in the presence of various concentrations of Mg2+. , no spermidine (SPD); , 1 mM SPD. B, in vitro synthesis of 38 subunit was performed as described under "Experimental Procedures." , no SPD; , 1 mM SPD. C, SDS-polyacrylamide gel electrophoresis and fluorography of [35S]methionine-labeled 38-His6 were performed as described under "Experimental Procedures." D, the binding of Gln-tRNAsupE was measured in the presence () and absence () of 1 mM spermidine as described under "Experimental Procedures." Values in A, B, and D are the means of duplicate determinations.

Possible effects of polyamines on mutant RpoS(33UAG) mRNA-dependent synthesis in vitro of the ³⁸ subunit were then analyzed by measuring the incorporation of [³⁵S]methionine into the TCA-insoluble fraction. As shown in Fig. 7B, 1 mM spermidine significantly stimulated the overall activity of protein synthesis. Because the protein synthesis activity in the in vitro translation system employed depends on the addition of externally added mRNA (data not shown), the result indicates the stimulation of ³⁸ subunit synthesis by spermidine. To confirm this interpretation the protein products were analyzed by SDS-PAGE, and the gels were subjected to fluorography. Fig. 7C shows one of the fluorograms, which indicates that the band intensity of ³⁸ subunit increased, albeit at low levels, in the presence of spermidine.

Stimulation of poly(U)-dependent polyphenylalanine synthesis by polyamines is attributable to the increase in aminoacyl-tRNA binding to ribosomes but not at the levels of peptide bond formation and translocation (5). Thus, we first examined the effect of polyamines on mRNA-independent binding of Gln-tRNA^supE to ribosomes. Although the binding activity was low, it was clearly stimulated by 1 mM spermidine (Fig. 7D), indicating that the affinity of Gln-tRNA^supE to ribosomes is increased by polyamines. Then, the effect of polyamines on AUGUAG-dependent binding of Gln-tRNA^supE was examined. Polyamines also enhanced Gln-tRNA^supE binding to ribosomes (data not shown). These results, taken together, suggest that polyamines stimulate the synthesis of ³⁸ subunit through at least two steps: (i) the increased level of tRNA^supE and (ii) the increased binding of Gln-tRNA^supE to ribosomes.

Physiological Significance of Polyamine Stimulation of Suppression of Amber Mutation in RpoS mRNA-- The rpoS gene is essential for cell viability in the stationary phase (41). Thus, we compared cell viability between MA261 cells cultured with or without putrescine. The rate of cell growth was enhanced by putrescine as reported (17). When rpoS was disrupted, cell growth slowed slightly, but polyamine enhanced cell growth greatly (Fig. 8A). As for cell viability, determined by colony formation on a rich plate, it increased greatly when the cells were cultured in the presence of putrescine. However, cell viability of MA261 cultured in the absence of putrescine was higher than that of MA261 in which rpoS was disrupted (Fig. 8B). Cell viability was parallel with the level of ³⁸ subunit (Fig. 8C). The results suggest that elevated expression of rpoS gene is important for cell viability.

View larger version (40K): [in this window] [in a new window]   Fig. 8.   Effect of polyamines on cell growth of E. coli MA261 (A), cell viability (B), and on the level of 38 and 70 subunits of RNA polymerase (C). A, E. coli MA261 ( and ) and MA261rpoS::tet ( and ) cells were cultured in medium A supplemented with five amino acids in the presence (closed symbols) and absence (open symbols) of 100 µg/ml putrescine, and cell growth was followed by measuring A540. B, viable cells were counted at designated times as described under "Experimental Procedures." Each value is the average of duplicate determinations. C, Western blotting of  subunits was performed using 10 µg of cell lysate protein for 38 or 5 µg of cell lysate protein for 70.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Polyamines stimulate the synthesis of a set of proteins such as oligopeptide-binding protein (OppA) (17), adenylate cyclase (Cya) (19), and ³⁸ subunit (RpoS) (this paper). Up to now two different mechanisms have been identified: a structural change of OppA mRNA, leading to enhanced template activity for translation (17), and stimulation of initiation codon UUG-dependent fMet-tRNA binding to Cya mRNA-ribosomes (19). The results herewith described show a novel mode of the polyamine stimulation of protein synthesis. Polyamines stimulate readthrough of the amber codon by enhancing the binding of amber codon UAG-dependent Gln-tRNA^supE on ribosome-associated RpoS mRNA (see Fig. 9). Thus polyamines modulate protein synthesis not only at the level of initiation but also at the level of elongation of translation. We propose that genes whose expression is modulated by polyamines at the level of translation are referred to as "polyamine modulon." Experiments are in progress to find other members of the polyamine modulon.

View larger version (22K): [in this window] [in a new window]   Fig. 9.   Polyamine effects on three kinds of protein syntheses directed by OppA mRNA, Cya mRNA, and RpoS mRNA. A, polyamines cause a structural change of the SD sequence and the initiation codon AUG of OppA mRNA, facilitating formation of the initiation complex. B, polyamines stimulate interaction between the initiation codon UUG of Cya mRNA and the anticodon CAU of fMet-tRNAi. In this case, exposure of the SD sequence of the mRNA is probably a prerequisite for the stimulation. C, polyamines stimulate the readthrough of amber codon of RpoS mRNA probably through its stimulation of Gln-tRNAsupE binding to ribosomes. RF1, release factor 1.

The increase in readthrough of the amber codon by polyamines has been found for translation of the mutant gene 1 protein mRNA from T7 phage (42). Stimulation of readthrough of both UAG amber and UGA opal codons by polyamines also has been reported in E. coli and eukaryotic cell-free systems (43-45). In these cases, however, the mechanism was not studied in detail. As for polyamine stimulation of the synthesis of ³⁸ subunit, the translation suppression was found to be caused by stimulation of Gln-tRNA^supE binding to ribosomes via two processes, i.e. an increase in the level of tRNA^supE and an increase in the binding affinity of Gln-tRNA^supE to ribosomes. As to the increase in tRNA^supE level, it is of interest to know whether polyamines stimulate transcription of the metT operon or stabilize tRNA^supE. If the latter is the case, the increase in both intracellular level and intrinsic function of tRNA^supE can be explained by a structural change of tRNA^supE by polyamines.

Readthrough in vivo of the UGA opal codon at the 33rd codon of RpoS mRNA was not stimulated by polyamines in cells under our experimental conditions (see Fig. 3). The frequency of the use of UAG, UGA, and UAA as the termination codon in E. coli is 7.6, 29.3, and 63.1%, respectively (46). Furthermore, 316 among 4288 genes in E. coli K12 use tandem termination codons (47). Because not so many genes use UAG as the real termination codon at the end of full-length reading frames, the gene expression as a whole may not be influenced strongly by polyamines even if polyamines stimulate the readthrough of UAG at the natural termination sites.

We found that a mutation at the 33rd position of the ORF of RpoS mRNA occurs frequently. In such strains, polyamines enhance cell viability. The results confirmed that the ³⁸ subunit is important for cell viability (41) and indicate that polyamines play an important role in cell viability of E. coli cells having an amber codon at the 33rd position of the ORF of RpoS mRNA.

	ACKNOWLEDGEMENTS

We thank Drs. A. J. Michael and K. Williams for help in preparing the manuscript. We also thank Drs. W. K. Maas, H. Tabor, D. R. Morris, K. Tanaka, and Y. Kamio for generous contributions of E. coli strains and plasmids, and we thank Ms. H. Maeda for technical assistance.

	FOOTNOTES

^* This work was supported by a grant-in-aid for scientific research from the Ministry of Education, Culture, Sports, Science, and Technology, Japan, and by Research for the Future Program Grant JSPS-RFTF 97L00503 from the Japan Society for the Promotion of Science.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 81-43-290-2897; Fax: 81-43-290-2900; E-mail: iga16077@p.chiba-u.ac.jp.

Published, JBC Papers in Press, July 29, 2002, DOI 10.1074/jbc.M206668200

	ABBREVIATIONS

The abbreviations used are: SD, Shine-Dalgarno; Cya, adenylate cyclase; ORF, open reading frame.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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