Polyamine Enhancement of the Synthesis of Adenylate Cyclase at the Translational Level and the Consequential Stimulation of the Synthesis of the RNA Polymerase sigma 28 Subunit*

Madoka YoshidaDagger , Keiko KashiwagiDagger , Gota Kawai§, Akira Ishihama, and Kazuei IgarashiDagger ||

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

Received for publication, December 8, 2000, and in revised form, January 25, 2001


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The effects of polyamines on the synthesis of various sigma  subunits of RNA polymerase were studied using Western blot analysis. Synthesis of sigma 28 was stimulated 4.0-fold and that of sigma 38 was stimulated 2.3-fold by polyamines, whereas synthesis of other sigma  subunits was not influenced by polyamines. Stimulation of sigma 28 synthesis was due to an increase in the level of cAMP, which occurred through polyamine stimulation of the synthesis of adenylate cyclase at the level of translation. Polyamines were found to increase the translation of adenylate cyclase mRNA by facilitating the UUG codon-dependent initiation. Analysis of RNA secondary structure suggests that exposure of the Shine-Dalgarno sequence of mRNA is a prerequisite for polyamine stimulation of the UUG codon-dependent initiation.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Polyamines (putrescine, spermidine, and spermine) have important physiological roles and are essential for normal cell growth (1, 2). Some of their proliferative effects appear to be due to regulation of nucleic acid and protein synthesis. As for protein synthesis, it has been reported that polyamines can stimulate some kinds of protein synthesis in both prokaryotic and eukaryotic cell-free systems (3-5) and in vivo (6, 7). Furthermore, polyamines stimulate the in vivo assembly of 30 S ribosomal subunits (8-10) and increase the fidelity of protein synthesis (11-13). We also estimated that most polyamines exist as a polyamine-RNA complex in cells (14, 15), supporting the idea that polyamines regulate protein synthesis at several different steps.

In Escherichia coli, the synthesis of OppA protein, which is a periplasmic substrate binding protein of the oligopeptide uptake system, is strongly stimulated by the addition of putrescine to a polyamine-requiring mutant MA261 (7). We found that stimulation of OppA synthesis occurs at the level of translation and that the position and secondary structure of the Shine-Dalgarno (SD)1 sequence (16) of OppA mRNA are probably important for stimulation by polyamines (17). Furthermore, we found that polyamines cause a structural change of the SD sequence and the initiation codon AUG of OppA mRNA, facilitating formation of the initiation complex (18).

In the present work, we studied the effects of polyamines on the synthesis of various sigma  subunits of RNA polymerase to determine how polyamines influence the functional specificity of transcription. Seven different types of sigma  subunit have been identified in E. coli, each recognizing a specific set of promoters (19). Modulation of the promoter selectivity of RNA polymerase by replacement of the sigma  subunit is an efficient way to alter the global pattern of gene expression (19). We found that synthesis of the sigma 28 subunit is greatly enhanced by polyamines because of an increase in cAMP level, which is due to stimulation of the synthesis of adenylate cyclase at the level of translation initiation.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Bacterial Strains and Culture Conditions-- A polyamine-requiring mutant, E. coli MA261 (speB speC gly leu thr thi), was kindly provided by Dr. W. K. Maas (New York University School of Medicine, New York, NY) (20). Adenylate cyclase- and cAMP receptor protein-deficient mutants E. coli HT28 (W3110 cya::Km) and IT1409 (W3110 crp::Tet) were kind gifts from Dr. H. Aiba (Nagoya University, Nagoya, Japan) (21). E. coli MA261 lacZ::EM was prepared as described previously (22). E. coli MA261 cya::Km, MA261 crp::Tet, MA261 cya::Km crp::Tet, and MA261 lacZ::EM cya::Km were isolated by transduction with phage P1 (23) using E. coli HT28 and IT1409 as the donor. These cells, E. coli Q13 (rna pnp) and E. coli W3110 were grown at 37 °C in medium A supplemented with 5 amino acids (100 µg/ml each Gly, Leu, Met, Ser, and Thr) in either the presence (100 µg/ml) or absence of putrescine (7). Where indicated, 0.4% glycerol was used as carbon source instead of 0.4% glucose. Methionine content in medium A was decreased from 100 to 3 µg/ml in order to label proteins with [35S]methionine; this modification did not influence their growth rate. Another polyamine-requiring mutant DR112 (speA speB) was kindly provided by Dr. D. R. Morris (University of Washington, Seattle, WA), and was grown according to the method of Linderoth and Morris (24). Antibiotics used were 100 µg/ml ampicillin, 50 µg/ml kanamycin, 200 µg/ml erythromycin, and 15 µg/ml tetracycline. Cell growth was monitored by measuring the absorbance at 540 nm.

Plasmids-- Total chromosomal DNA from E. coli W3110 was prepared according to the method of Ausubel et al. (25). To make the cya (TTG)-lacZ fusion gene, polymerase chain reaction (PCR) was performed using total chromosomal DNA as template, and 5'-AGTGAATTCTGACAGGCGTTTCAC-3' (P1) and 5'-AGATCTAGAGCCGGATAAGCCTCG-3' (P2) as primers. pMW Cya (TTG) containing the promoter region and the first 258 nucleotides of open reading frame was constructed by inserting the 0.54-kbp EcoRI-BamHI fragment of the PCR product into the same restriction sites of pMW119 (Nippon Gene, Tokyo). The 3.1-kbp BamHI fragment containing the lacZ gene was obtained from the pMC1871 fusion vector (26) and then inserted into the same restriction site of pMW Cya (TTG). The plasmid thus obtained was named pMW Cya (TTG)-lacZ.

Site-directed mutagenesis by overlap extension using PCR (27) was performed to prepare pMW Cya (ATG)-lacZ. The template used for first PCR was chromosomal DNA from E. coli W3110. Primers used for the first PCR were P1 and 5'-CTGTTTCAGAGTCTCAATATAGAGGTACAT-3' (underlined base for TTG substitution with ATG), and 5'-TAGCAAATCAGGCGATACGTCATGTACCTC-3' (underlined base for TTG substitution with ATG) and P2. Then the second PCR was performed using the first PCR products as templates and P1 and P2 as primers. pMW Cya (ATG), in which TTG initiation codon was replaced by ATG, was constructed by inserting the 0.54-kbp EcoRI-BamHI fragment of the second PCR product into the same restriction sites of pMW119. The pMW Cya (ATG)-lacZ was constructed in a similar manner as for pMW Cya (TTG)-lacZ. The nucleotide sequence of the plasmid DNA was confirmed by the Gene Rapid System (Amersham Pharmacia Biotech).

Western Blot Analysis-- Seven kinds of sigma  subunits (sigma 70, sigma 54, sigma 38, sigma 32, sigma 28, sigma 24, and sigma 18) were purified, and antisera against the sigma  subunits were prepared as described previously (28-30). Antisera against cAMP receptor protein (CRP), H-NS, and SpoT were kindly provided by Dr. H. Aiba, Dr. T. Mizuno, Dr. C. O. Gualerzi, and Dr. M. Cashel (31-34). Rabbit antibody for adenylate cyclase was prepared according to the method of Posnett et al. (35) using the multiple antigenic peptide, DESNRVEVYHHCEGSKEE, which corresponds to amino acids 761-778 of adenylate cyclase (36). Western blot analysis was performed by the method of Nielsen et al. (37). Protein was detected with a Proto Blot Western blot AP system (Promega).

Northern Blot Analysis-- Total RNA was prepared from various E. coli strains according to the method of Emory and Belasco (38). Northern blot analysis was performed using 20 µg of total RNA and the 32P-labeled probes as described previously (39). The probes used were prepared by PCR, and the sizes of probes for rpoF, rpoD, flhDC, fliC, flgM, hns (H-NS), cya, crp, and cpdA (cAMP PDE) were 720 (40), 1842 (41), 941 (41), 1497 (41), 294 (41), 414 (42), 2547 (43), 633 (44), and 828 (41) bp, respectively, in which the 5' end was the ATG or TTG initiation codon of each open reading frame. Primers used for PCR are available from the authors upon request. Probe used for measurement of Cya (UUG)-beta -gal mRNA and Cya (AUG)-beta -gal mRNA was the 1.1-kbp SacI-BamHI fragment of pMW Cya (ATG)-lacZ.

Measurement of cAMP Content and Assay for Adenylate Cyclase-- Cyclic AMP was extracted from cells with 5% trichloroacetic acid. After trichloroacetic acid was removed by ether, cAMP was measured using cAMP enzyme immunoassay system (Amersham Pharmacia Biotech) according to the accompanying manual. The reaction mixture (0.1 ml), for the assay of adenylate cyclase, contained 25 mM Bicine·Na (pH 8.5), 1 mM ATP·2Na, 10 mM Mg2+, 1 mM dithiothreitol, 20 mM creatine phosphate, 10 units/ml creatine kinase, 1 mM cAMP, [alpha -32P]ATP (specific activity: 20-50 cpm/pmol), and 100 µg of cell lysate protein. After incubation at 30 °C for the designated time, [32P]cAMP formed was measured according to the method of Liberman et al. (45).

Measurement of Fusion Cya-beta -Galactosidase Synthesis by an Immunoprecipitation Method-- E. coli MA261 Delta lacZ Delta cya/pMW Cya (TTG or ATG)-lacZ was grown in polyamine-deficient medium. When A540 reached 0.05, the cells were divided into 5-ml aliquots and grown in the presence (100 µg/ml) or absence of putrescine for 10 min. Then, [35S]methionine (1 MBq) was added to each 5-ml aliquot, and the cells were allowed to grow for 20 min. They were harvested after the addition of methionine at a final concentration of 20 mM and disrupted by a French pressure cell at 20,000 p.s.i. containing 1 ml of buffer A (10 mM sodium phosphate, pH 7.4, 100 mM NaCl, 1% Triton X-100, and 0.1% SDS). The amount of Cya-beta -galactosidase synthesized was determined using 1,000,000 cpm of [35S]methionine-labeled protein and antiserum against beta -galactosidase (Sigma) according to the method of Philipson et al. (46). Radioactivity of labeled Cya-beta -galactosidase was quantified using a Fujix Bas 2000II imaging analyzer.

Assays for fMet-tRNAi Binding to Ribosomes and cAMP Phosphodiesterase (cAMP PDE)-- Triplets UUG and AUG were synthesized by DNA/RNA synthesizer Expedite 9809 (PerkinElmer Life Sciences) according to the manufacturer's instructions. Salt (0.25 M NH4Cl)-washed ribosomes, (NH4)2SO4-fractionated crude initiation factors, and f[3H]Met-tRNAi were prepared from E. coli Q13 essentially as described previously (5, 9). UUG- and AUG-dependent fMet-tRNAi binding to ribosomes was measured as described previously (18). cAMP PDE activity was measured by the method of Nielsen and Rickenberg (47).

Prediction of the Secondary Structure of RNA-- Optimal computer folding of mRNAs was performed by the method of Zucker and Stiegler (48). Free energy (Delta G) for the formation of the secondary structure was calculated on the basis of the data of Turner and Sugimoto (49).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Effects of Polyamines on Synthesis of RNA Polymerase sigma  Subunits-- The synthesis of mRNA is catalyzed by the RNA polymerase core enzyme (alpha 2beta beta ' subunits), but its functional specificity is modified by binding one of the seven different types of sigma  subunit (19). To examine possible influence of polyamines on the functional specificity of the transcription apparatus, we first examined the effect of polyamines on the synthesis of sigma  subunits using a polyamine-requiring mutant, MA261, which lacks the genes for biosynthetic enzymes of putrescine (ornithine decarboxylase and agmatinase) (20). The levels of sigma  subunits measured by Western blotting were tentatively defined as synthesis. We studied the levels of various sigma  subunits in MA261 cells after treatment with putrescine (100 µg/ml). In this strain, putrescine can be converted to spermidine. Under these conditions, cell growth was stimulated approximately by 3-5-fold by polyamines (see below). As shown in Fig. 1, synthesis of sigma 28 was stimulated 4.0-fold, and that of sigma 38 was stimulated 2.3-fold by the addition of putrescine to the medium. The synthesis of four other sigma  subunits (sigma 70, sigma 54, sigma 32, and sigma 24) was not affected by polyamines. One of the sigma  subunits, sigma 18 (sigma FecI), could not be detected (data not shown), indicating that sigma 18 exists in a low amount under these experimental conditions.


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Fig. 1.   Effect of polyamines on synthesis of various sigma  subunits of RNA polymerase in E. coli MA261. Western blotting of sigma  subunits was performed using 20 µg protein of cell lysate for sigma 54, sigma 38, sigma 32, sigma 28, and sigma 24 or 10 µg of cell lysate protein for sigma 70. Cell lysate was prepared from cells cultured with or without 100 µg/ml putrescine (PUT) and harvested at A540 = 0.2.

The relative amounts of seven sigma  subunits shown in Fig. 1 were apparently different from those of E. coli W3110 (50). It is known that the amount of sigma  subunits changes with the growth phase and various growth conditions (29, 50), and that the density of bands in Western blotting are not always correlated with protein concentration. The amount of sigma  subunits may also change by the difference of cell types.

Effect of Polyamines on Gene Expression of Regulatory Factors for sigma 28 Synthesis and on That of Factors Regulated by sigma 28-- As shown in Fig. 2B, the level of sigma 28 (rpoF) mRNA was also markedly increased by polyamines. The level of sigma 70 (rpoD) mRNA was measured as a control, and it was not increased by polyamines. Since our hypothesis is that the effects of polyamines on macromolecular syntheses mainly occur at the level of translation, we examined the effects of polyamines on gene expression of regulatory factors for sigma 28 synthesis. It is known that the flagellar master operon (flhDC) positively regulates sigma 28 synthesis (Fig. 2A) (51). Thus, the effect of polyamines on expression of flhDC was examined, and it was clearly stimulated by polyamines (Fig. 2B). The expression of flhDC is regulated by both cAMP/CRP complex and H-NS (Fig. 2A) (52, 53). We also studied the effects of polyamines on expression of the cya, crp, and hns genes, which encode adenylate cyclase, CRP, and H-NS, respectively. The levels of these mRNAs were not influenced by polyamines (Fig. 3A), suggesting that polyamines do not affect transcription of the cya, crp, and hns genes. Accordingly, if polyamines do affect the levels of adenylate cyclase, CRP, or H-NS, they presumably do so at a post-transcriptional level.


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Fig. 2.   Effect of polyamines on gene expression of regulatory factors of sigma 28 synthesis and that of factors controlled by sigma 28. A, genes that regulate sigma 28 synthesis and those controlled by sigma 28 were shown. right-arrow (boldface arrow), strong stimulation; right-arrow (lightface arrow), weak stimulation; perp , inhibition; gene X, unidentified gene. B, Northern blotting of various mRNAs was performed using 20 µg of total RNA prepared from cells cultured with or without 100 µg/ml putrescine (PUT) and harvested at A540 = 0.2. For Northern blotting of rpoD mRNA, 10 µg of total RNA was used instead of 20 µg.


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Fig. 3.   Effect of polyamines on synthesis of Cya, CRP, and H-NS. A, Northern blotting of Cya, CRP, and H-NS mRNAs was performed as described in the legend of Fig. 2. B, protein of cell lysate used for Western blotting of sigma 28 subunit, Cya, CRP, and H-NS was 20, 20, 5, and 20 µg, respectively. Cells were cultured in the presence and absence of 100 µg/ml putrescine (PUT), and harvested at A540 = 0.05, 0.1, or 0.2.

Because polyamines increase the level of sigma 28 protein (Fig. 1) and the corresponding rpoF mRNA (Fig. 2B), we analyzed possible effects of polyamines on the downstream genes (flgM and fliC) whose expression is regulated by sigma 28 (Fig. 2A). As shown in Fig. 2B, the expression of flgM (which encodes anti-sigma 28) and fliC (which encodes flagellin) was enhanced by polyamines. Stimulation of flagellin synthesis by polyamines was confirmed by Western blot analysis (data not shown).

In control experiments, the effects of polyamines on gene expression were examined using E. coli W3110, which can synthesize putrescine and spermidine. As shown in Fig. 2B, addition of putrescine to the medium did not influence the expression of flhDC, rpoF, flgM, and fliC, indicating that a sufficient amount of polyamines can be synthesized in wild type cells.

Polyamines Stimulate Synthesis of Adenylate Cyclase-- The effects of polyamines on the synthesis of adenylate cyclase (Cya), CRP, and H-NS were examined by Western blot analysis (Fig. 3B). As a positive control, we also measured synthesis of the sigma 28 subunit. The syntheses of both sigma 28 subunit and Cya were stimulated by polyamines. On the other hand, polyamines did not affect the synthesis of CRP or H-NS (Fig. 3B). These results suggest that polyamines may stimulate synthesis of adenylate cyclase at the translational level.

Experiments were carried out to determine whether cAMP regulates sigma 28 synthesis in E. coli MA261. As shown in Fig. 4A, sigma 28 synthesis, measured by Western blot analysis, was not observed in mutants with disrupted cya and/or crp genes, indicating that cAMP is involved in sigma 28 synthesis. We carried out experiments to determine whether cAMP, by itself, could increase the synthesis of the sigma 28 subunit. As shown in Fig. 4B, addition of 1 mM cAMP to the medium in the absence of putrescine caused the synthesis of sigma 28 subunit. Under these conditions, significant amounts of flhDC and rpoF mRNAs were synthesized in the absence of putrescine (data not shown). Thus, the degree of stimulation by polyamines of the synthesis of flhDC and rpoF mRNAs and the subsequent synthesis of sigma 28 subunit became small. This effect of cAMP was observed in a cya gene-disrupted mutant, but not in a crp gene-disrupted mutant (Fig. 4C). These results indicate that CRP is necessary together with cAMP for synthesis of sigma 28 subunit, and that polyamines stimulate sigma 28 synthesis through increase in the level of cAMP. It has been reported that the intracellular cAMP level was increased when cells were cultured in the presence of glycerol instead of glucose (54). When E. coli MA261 was cultured in the presence of glycerol, a high level of sigma 28 subunit was synthesized in the absence of putrescine and addition of putrescine to the medium did not influence the synthesis of sigma 28 subunit significantly (data not shown).


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Fig. 4.   Effect of polyamines and cAMP on sigma 28 synthesis. Western blotting of sigma 28 was performed using 20 µg of of cell lysate protein from various mutants cultured in the presence or absence of 100 µg/ml putrescine (PUT) and/or 0.1 or 1 mM cAMP, and harvested at A540 = 0.2.

The level of cAMP and the activity of adenylate cyclase were measured in cells cultured in the absence and presence of putrescine. After addition of putrescine into the medium, there was a stimulation of cell growth together with an increase in intracellular cAMP (Fig. 5, A and C). Activity of adenylate cyclase in cells cultured with putrescine was about 3-fold higher than that in cells cultured without putrescine (Fig. 5B). The increase in activity of adenylate cyclase in cells cultured with putrescine is consistent with the increased level of enzyme observed by Western blot analysis in those cells (Fig. 3B). When E. coli W3110 was used instead of E. coli MA261, addition of putrescine into the medium did not influence the level of intracellular cAMP (Fig. 5C). The level of cAMP in E. coli W3110 was slightly higher than that of cAMP in E. coli MA261 cultured with putrescine.


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Fig. 5.   Effect of putrescine on cell growth (A), adenylate cyclase activity (B), and level of cAMP (C). A, cell growth was monitored by measuring the absorbance at 540 nm. B, the activity of adenylate cyclase was measured using 100 µg of cell lysate protein prepared from cells cultured in the presence and absence of 100 µg/ml putrescine (PUT) and harvested at A540 = 0.2. C, intracellular cAMP content was measured using cells harvested at A540 = 0.2. E. coli MA261 and W3110 were cultured in the presence and absence of 100 µg/ml putrescine. Each value is the average of duplicate determinations.

Polyamines Affect the UUG Codon-dependent Initiation in the Synthesis of Adenylate Cyclase-- Experiments were carried out to study the mechanism underlying polyamine stimulation of adenylate cyclase at the translational level. It is known that transcription of the cya gene is negatively regulated by binding of the cAMP-CRP complex (43). This is due to a repressor effect of CRP together with cAMP at a site in the promoter region of the cya gene (Fig. 6A). Therefore, polyamine regulation was studied using a cya gene-disrupted mutant of E. coli MA261. Thus, in this mutant, there is no negative regulation of the cya gene by the cAMP-CRP complex, which could otherwise confound studies of the effects of polyamines on translational regulation.


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Fig. 6.   Effect of polyamines on synthesis of Cya-beta -gal fusion protein derived from UUG and AUG initiation codon. A, structure of Cya (TTG)-lacZ and Cya (ATG)-lacZ fusion genes. B, possible secondary structure of the initiation codon region of Cya mRNA. Optimal computer folding of the region from -25 to +45 of the mRNA and calculation of free energy (Delta G) for the formation of the secondary structure were performed as described under "Experimental Procedures." C, Northern blot analysis was performed using 30 µg of total RNA. D, synthesis of Cya-beta -gal fusion protein was measured as described under "Experimental Procedures." PUT, putrescine.

The nucleotide sequence of the initiation region of Cya mRNA and its possible secondary structure were analyzed (Fig. 6B). It is known that the initiation of protein synthesis is mainly dependent on the SD sequence and initiation codon (17, 18). The SD sequence of Cya mRNA was positioned normally and relatively exposed, but the initiation codon was UUG instead of AUG (55). Since the SD sequence of Cya mRNA probably interacts with 16 S rRNA efficiently, we hypothesized that polyamines may facilitate the UUG codon-dependent initiation. Thus, fusion genes were constructed that contained portions of cya and a lacZ reporter gene in which the initiation codon was either UUG or AUG (Fig. 6A). The effects of polyamines on synthesis of the fusion protein were examined using these constructs. With the construct containing the AUG initiation codon, the basal synthesis of Cya-beta -galactosidase (Cya-beta -gal) increased by 4.5-fold compared with that synthesized from the UUG initiation codon (Fig. 6D). However, the degree of polyamine stimulation decreased from 2.5-fold to 1.2-fold. The levels of Cya (UUG)-beta -gal mRNA and Cya (AUG)-beta -gal mRNA were nearly equal and were not influenced by polyamines (Fig. 6C). These results indicate that polyamines stimulate the synthesis of adenylate cyclase through the stimulation of the UUG codon-dependent initiation.

The effect of spermidine on UUG- and AUG-dependent fMet-tRNAi binding to ribosomes was then examined (Fig. 7). Although the amount of fMet-tRNAi binding to ribosomes was much greater with AUG than with UUG, spermidine stimulated only UUG-dependent fMet-tRNAi binding to ribosomes significantly. The results support an idea that polyamines stimulate the synthesis of adenylate cyclase through the stimulation of the UUG codon-dependent initiation.


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Fig. 7.   Effect of spermidine on UUG-dependent (A and C) and AUG-dependent (B and D) fMet-tRNAi binding to ribosomes. The assay was performed under standard conditions, in which the molar ratio of UUG or AUG/ribosomes was 90 (18). In A and C, fMet-tRNAi binding to ribosomes was measured by incubating the reaction mixture at 30 °C for 8 min. In B and D, time course of fMet-tRNAi binding to ribosomes was measured in the presence of 8 mM Mg2+. open circle , no spermidine (SPD) added; , 2 mM spermidine. Each value is the average of duplicate determinations.

The UUG codon is used as the initiation codon for 34 identified genes in E. coli (41). We therefore examined the effect of polyamines on two other UUG codon-dependent initiations. As shown in Fig. 8 (A and C), the synthesis of cAMP PDE was slightly inhibited, and that of SpoT, ppGpp metabolizing enzyme (56), was not influenced significantly by polyamines. The amount of cAMP PDE mRNA obtained from cells cultured with or without putrescine was nearly equal (data not shown), indicating that the effect of polyamines is at the level of translation. Secondary structure of the initiation region of these mRNAs is also shown in Fig. 8 (B and D). The SD sequences of cAMP PDE and SpoT mRNAs were mainly located in the stem region of mRNAs. The results suggest that the secondary structure of the initiation region of mRNA is important for stimulation by polyamines of the UUG codon-dependent initiation. Since polyamines preferentially bind to double-stranded RNA (14), polyamines may stabilize the stem structure of the SD sequence of cAMP PDE and SpoT mRNAs. Analysis of RNA secondary structure suggests that exposure of the SD sequence of mRNA is a prerequisite for polyamine stimulation of the UUG codon-dependent initiation.


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Fig. 8.   Effect of polyamines on the activity of cAMP phosphodiesterase (A) and the amount of SpoT protein (C). Cell lysate was prepared from cells cultured in the presence and absence of 100 µg/ml putrescine and harvested at A540 = 0.2. The activity of cAMP PDE was measured as described under "Experimental Procedures." 100% activity: 24.5 pmol of adenosine recovered/min/mg of protein. The amount of SpoT protein was measured by Western blotting. Each value is the average of duplicate determinations. Nucleotide sequence of cAMP PDE (B) and SpoT (D) mRNAs was sited from Refs. 62 and 55, respectively. Optimal computer folding of the nucleotide region from -25 to +45 of the mRNAs and calculation of free energy (Delta G) for the formation of the secondary structure were performed as described under "Experimental Procedures."


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In this study we initially determined whether the synthesis of seven different species of sigma  subunits was regulated by polyamines because the functional specificity of RNA polymerase is modified by the sigma  subunit. Among seven sigma  subunits, the synthesis of sigma 28 was most influenced by polyamines. We defined the amount of sigma 28 measured by Western blotting as "synthesis," because the degree of stimulation by polyamines at the mRNA level was nearly parallel with that at the protein level. Most of our experiments were carried out using a polyamine-requiring mutant, MA261 (20), but similar results were obtained with another polyamine-requiring mutant, DR112 (24). Furthermore, when E. coli MA261 was grown in medium A supplemented with 20 amino acids instead of 5 amino acids, the rate of cell growth increased and similar results on polyamine effects were obtained (data not shown).

Our hypothesis is that polyamines regulate macromolecular synthesis mainly at the translational level since most polyamines exist as a polyamine-RNA complex in cells (14, 15). Therefore, we searched for a target protein, related to the polyamine stimulation of sigma 28 synthesis, whose translation is altered by polyamines. We found that the translation of adenylate cyclase was stimulated by polyamines. It is known that the efficiency of translation initiation depends on the stability of mRNA secondary structure, location, and length of SD sequence and on the nature of the initiation codon (17, 57, 58). Therefore, the possible secondary structure of the initiation region of Cya mRNA was analyzed. We found that both the SD sequence and the initiation codon were exposed on the mRNA, and that the position of the SD sequence was normal, occurring 5 nucleotides upstream from the initiation codon. However, the initiation codon was UUG (see Fig. 6). If the UUG codon is replaced by the initiation codon AUG, the cells are nonviable (55). The results strongly suggest that the UUG initiation codon of Cya mRNA limits cya gene expression at the translational level. If the AUG initiation codon was used instead of UUG in Cya-beta -gal fusion mRNA, protein synthetic activity increased by 4.5-fold, but the polyamine effect was almost abolished. Given that such an increase in adenylate cyclase activity causes cell death (55), the level of cAMP in cells must normally be tightly regulated. In constructs containing the normal UUG initiation codon, protein synthesis was enhanced 2.5-fold by polyamines. Thus, polyamines may contribute to maintenance of an optimal cAMP level by facilitating the UUG codon-dependent initiation.

The UUG codon is used as the initiation codon for 34 identified genes in E. coli (41). If polyamines stimulate the interaction between the initiation codon UUG and the anticodon fMet-tRNAi, CAU (59), protein synthesis from all mRNAs having UUG as the initiation codon would be stimulated by polyamines. However, this was not the case, as shown in Fig. 8. The secondary structure of the initiation region of mRNA is probably important for causing the stimulation of the UUG codon-dependent initiation by polyamines. Analysis of RNA secondary structure suggests that exposure of the SD sequence of the mRNA is a prerequisite for polyamine stimulation of the UUG codon-dependent initiation. The structural change of Cya, cAMP PDE, and SpoT mRNAs by polyamines is under investigation.

Three initiation factors (IF1, -2, and -3) are involved in the initiation of protein synthesis. Among the three initiation factors, IF3 functions as a discrimination factor for the non-canonical initiation codons AUU, AUC, AUA, and CUG (60). However, UUG was not discriminated by IF3 (61), and the amount of IF3 was nearly equal in E. coli MA261 cells cultured with or without putrescine (data not shown). Therefore, the possibility that IF3 is involved in the polyamine stimulation of the UUG codon-dependent initiation is probably low.

In addition to sigma 28, synthesis of sigma 38 subunit was also stimulated by polyamines. However, stimulation of sigma 38 synthesis by polyamines was not influenced by the cAMP-CRP under our experimental conditions (data not shown). Experiments are now in progress to clarify the mechanism of polyamine stimulation of sigma 38 synthesis.

The results obtained thus far show that polyamines stimulate protein synthesis by changing the structure of the SD sequence as in the case of OppA mRNA (17, 18) as well as the efficiency of UUG codon-dependent initiation as in the case of Cya mRNA (this paper). In the latter case, exposure of the SD sequence of the mRNA is probably a prerequisite for the stimulation. Models of polyamine modulation of the formation of OppA mRNA (A)- and Cya mRNA (B)-dependent initiation complex are shown in Fig. 9.


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Fig. 9.   Models of polyamine modulation of the formation of OppA mRNA-dependent (A) and Cya mRNA-dependent (B) initiation complex. 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 the interaction between the initiation codon UUG of Cya mRNA and the anticodon fMet-tRNAi, CAU. In this case, exposure of the SD sequence of the mRNA is probably a prerequisite for the stimulation.


    ACKNOWLEDGEMENTS

We thank Drs. K. Williams and A. J. Michael for their help in preparing the manuscript. We also thank Drs. W. K. Maas, H. Aiba, D. R. Morris, T. Mizuno, C. O. Gualerzi, and M. Cashel for their generous contributions of E. coli strains and antibodies.

    FOOTNOTES

* This work was supported by a grant-in-aid for scientific research from the Ministry of Education, Science, Sports and Culture, 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, January 30, 2001, DOI 10.1074/jbc.M011059200

    ABBREVIATIONS

The abbreviations used are: SD, Shine-Dalgarno; PCR, polymerase chain reaction; kbp, kilobase pair(s); PDE, phosphodiesterase; IF, initiation factor; CRP, cAMP receptor protein; H-NS, histone-like protein; Cya, adenylate cyclase; beta -gal, beta -galactosidase; Bicine, N,N-bis(2-hydroxyethyl)glycine; SpoT, (p)ppGpp 3'-pyrophosphohydrolase.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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