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INTRODUCTION |
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
subunits of RNA polymerase to determine how
polyamines influence the functional specificity of transcription. Seven
different types of
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
subunit is an efficient way to alter the global pattern of
gene expression (19). We found that synthesis of the
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.
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EXPERIMENTAL PROCEDURES |
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
subunits
(
70,
54,
38,
32,
28,
24, and
18) were purified, and antisera against the
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)-
-gal mRNA and Cya (AUG)-
-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, [
-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-
-Galactosidase Synthesis by an
Immunoprecipitation Method--
E. coli MA261
lacZ
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-
-galactosidase synthesized was
determined using 1,000,000 cpm of [35S]methionine-labeled
protein and antiserum against
-galactosidase (Sigma) according to
the method of Philipson et al. (46). Radioactivity of
labeled Cya-
-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 (
G) for the formation of the
secondary structure was calculated on the basis of the data of Turner
and Sugimoto (49).
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RESULTS |
Effects of Polyamines on Synthesis of RNA Polymerase
Subunits--
The synthesis of mRNA is catalyzed by the RNA
polymerase core enzyme (
2
' subunits), but its
functional specificity is modified by binding one of the seven
different types of
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
subunits using a polyamine-requiring mutant, MA261, which lacks
the genes for biosynthetic enzymes of putrescine (ornithine
decarboxylase and agmatinase) (20). The levels of
subunits measured
by Western blotting were tentatively defined as synthesis. We studied
the levels of various
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
28 was
stimulated 4.0-fold, and that of
38 was stimulated
2.3-fold by the addition of putrescine to the medium. The synthesis of
four other
subunits (
70,
54,
32, and
24) was not affected by
polyamines. One of the
subunits,
18
(
FecI), could not be detected (data not shown),
indicating that
18 exists in a low amount under these
experimental conditions.

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Fig. 1.
Effect of polyamines on synthesis of
various subunits of RNA polymerase in
E. coli MA261. Western blotting of subunits
was performed using 20 µg protein of cell lysate for
54, 38, 32,
28, and 24 or 10 µg of cell lysate
protein for 70. Cell lysate was prepared from cells
cultured with or without 100 µg/ml putrescine (PUT) and
harvested at A540 = 0.2.
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The relative amounts of seven
subunits shown in Fig. 1 were
apparently different from those of E. coli W3110 (50). It is
known that the amount of
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
subunits may also change by the difference of cell types.
Effect of Polyamines on Gene Expression of Regulatory Factors for
28 Synthesis and on That of Factors Regulated by
28--
As shown in Fig.
2B, the level of
28 (rpoF) mRNA was also markedly increased by
polyamines. The level of
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
28 synthesis. It is known that the flagellar master
operon (flhDC) positively regulates
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 28 synthesis
and that of factors controlled by
28. A, genes that
regulate 28 synthesis and those controlled by
28 were shown. (boldface
arrow), strong stimulation; (lightface
arrow), weak stimulation; , 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
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.
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Because polyamines increase the level of
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
28 (Fig. 2A). As shown in Fig. 2B,
the expression of flgM (which encodes
anti-
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
28 subunit. The syntheses of both
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
28 synthesis in E. coli MA261. As shown in
Fig. 4A,
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
28 synthesis. We
carried out experiments to determine whether cAMP, by itself, could
increase the synthesis of the
28 subunit. As shown in
Fig. 4B, addition of 1 mM cAMP to the medium in
the absence of putrescine caused the synthesis of
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
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
28 subunit, and that polyamines stimulate
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
28 subunit was synthesized in
the absence of putrescine and addition of putrescine to the medium did
not influence the synthesis of
28 subunit significantly
(data not shown).

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Fig. 4.
Effect of polyamines and cAMP on
28 synthesis. Western blotting of
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.
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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.
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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- -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 ( 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- -gal fusion
protein was measured as described under "Experimental Procedures."
PUT, putrescine.
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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-
-galactosidase (Cya-
-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)-
-gal mRNA and Cya
(AUG)-
-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+. , no spermidine (SPD) added; , 2 mM spermidine. Each value is the average of duplicate
determinations.
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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 ( G) for the formation of the secondary
structure were performed as described under "Experimental
Procedures."
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 |
DISCUSSION |
In this study we initially determined whether the synthesis of
seven different species of
subunits was regulated by polyamines because the functional specificity of RNA polymerase is modified by the
subunit. Among seven
subunits, the synthesis of
28 was most influenced by polyamines. We defined the
amount of
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
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-
-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
28, synthesis of
38
subunit was also stimulated by polyamines. However, stimulation of
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
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.
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