(Received for publication, September 16, 1996, and in revised form, November 5, 1996)
From the Faculty of Pharmaceutical Sciences, Chiba University, 1-33 Yayoi-cho, Inage-ku, Chiba 263, Japan
Polyamine stimulation of the synthesis of
oligopeptide-binding protein (OppA) was shown to occur mainly at the
level of translation by measuring OppA synthesis and its mRNA
level. Several artificial oppA genes were constructed by
site-directed mutagenesis. These synthesize different kinds of OppA
mRNAs: mRNAs differing in the size of 5-untranslated region;
mRNAs having the Shine-Dalgarno (SD) sequence in a different
position; mRNAs having different secondary structure in the region
of the SD sequence; and fusion mRNAs consisting of the
5
-untranslated region of OppA mRNA and the open reading frame of
-galactosidase. By measuring the synthesis of OppA or
-galactosidase from these mRNAs, we found that the 171-nucleotide 5
-untranslated region and 145 nucleotides of the ORF of
OppA mRNA are involved in the polyamine stimulation of OppA
synthesis. When the secondary structure of the above region of OppA
mRNA was analyzed by optimal computer folding, it was shown that
the degree of polyamine stimulation of OppA protein synthesis was
dependent on the structure of the SD sequence in addition to its
position. Loose base pairing of the SD sequence with other regions of
the mRNA caused strong polyamine stimulation, while intense base
pairing of the SD sequence with other regions of the mRNA resulted
in insignificant or weak polyamine stimulation.
Polyamines, aliphatic cations present in almost all living organisms, are known to be necessary for normal cell growth (1). Their proliferative effects are probably caused by the stimulation of nucleic acid and protein synthesis. We previously reported that polyamines can stimulate some kinds of protein synthesis in both prokaryotic and eukaryotic cell-free systems (2, 3) and in vivo (4, 5). Furthermore, it has been reported that assembly of 30 S ribosomal subunits is stimulated by polyamines (6, 7) and the fidelity of protein synthesis is increased by polyamines (8, 9). We also found that most polyamines exist as a polyamine-RNA complex in cells, and that the amount of polyamines (spermidine plus spermine) bound to RNA in rat liver is about 2 mol/100 mol of phosphate of RNA (10). Under the condition of spermine-stimulated globin synthesis in a rabbit reticulocyte cell-free system (11), the amount of polyamine bound to RNA was very close to the estimated value in cells.
In Escherichia coli, synthesis of a protein (polyamine-induced protein) was strongly stimulated by the addition of putrescine to growing cells of a polyamine-requiring mutant MA261 (12). The protein was identified, by cloning the corresponding gene, as OppA1 and is a periplasmic substrate-binding protein of the oligopeptide uptake system (13). In the present work, we have shown that stimulation of OppA synthesis by polyamines occurs mainly at the level of translation, and the position and secondary structure of the Shine-Dalgarno (SD) sequence (14) are probably involved in the stimulation of protein synthesis by polyamines.
E. coli MA261 (speB speC gly leu thr thi) was kindly provided by Dr. W. K. Maas, New York University School of Medicine. E. coli MA261 oppA::Km and lacZ::Em were prepared as described previously (15, 16). These cells were grown at 37 °C in medium A in either the presence (100 µg/ml) or absence of putrescine (13). 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, HT283, was kindly provided by Dr. H. Tabor, National Institutes of Health, and was grown according to the method of Hafner et al. (17).
PlasmidspACYCoppA, equivalent to pPI5.1, was
prepared as described previously (13). The 4.3-kb
HindIII-SalI fragment of pPI57 (13) was ligated
into the same restriction sites of pUC119. Subsequently, the 3.2-kb
oppA gene-containing SmaI fragment of the plasmid
was ligated into the same restriction site of pMW119 purchased from Nippon Gene (pMW975). pMW211 was constructed by inserting the 3.4-kb
NruI fragment of pACYCoppA into the
SmaI site of pMW119. A PCR product was obtained using the
HindIII-digested pMW975 as template, and
5-CAAATAGGTTACCTGGT-3
and 5
-GGGGAATTCCAATCTCTATTTGATTGA-3
as
primers. pMW412 was constructed by inserting the 1.1-kb
EcoRI-BstEII fragment of the PCR product into the
same restriction sites of pMW211.
Site-directed mutagenesis by overlap extension using PCR (18) was
performed to prepare pMWSD and pMWSD*. The template used
for first PCR was EcoRI digested pMW975. Primers used for
first PCR were 5
-GGGGAATTCCGGGCAATAAGGGCGC-3
(P1, sequence for
958
to 942 of oppA gene),
5
-TTTTTGGACTC
TCATTATAATT-3
(complementary sequence for
27 to
4 of oppA gene except two underlined bases),
5
-AATTATAATGA
GAGTCCAAAAA-3
(sequence for
27 to
4 of
the oppA gene except two underlined bases) and
5
-TGGCTCCAGCCAAACCATTCT-3
(P2, complementary sequence for 1068-1088
of the oppA gene) to construct pMWSD
, and were
P1, 5
-TCATTGTTTTT
ACTCCCTCATT-3
(complementary sequence
for
21 to 4 of oppA gene except three underlined bases), 5
-AATGAGGGAGT
AAAAACAATGA-3
(sequence for
21 to 4 of
oppA gene except three underlined bases) and P2 to construct
pMWSD*, respectively. Then, a second PCR was performed using initial
PCR products as templates and P1 and P2 as primers. Two PCR products thus obtained were digested with EcoRI and
BstEII, and inserted into the same restriction sites of
pMW211. The same method was applied to prepare pMWSD1 to pMWSD5 with
appropriate primers.
To make pMW9-lacZ and pMW45-lacZ, PCR was performed using
EcoRI digested pMW975 as template. Primers used were
5-GGGGGATCCGTCGCGAAAGAATAATGT-3
(P3) and
5
-AACGCCAGCTGCAGCTAAACTTCTCT-3
to construct pMW9-lacZ, and P3 and
5
-ACTGAACTTCTGCAGCATTGTTACGTA-3
to construct pMW45-lacZ. After the
products were digested with BamHI and PstI, they
were inserted into the same restriction sites of pUC119. The 3.1-kb PstI fragment containing the lacZ gene was
obtained from pMC1871 fusion vector (19) and was then inserted into the
same restriction site of the above plasmids (pUC9-lacZ and pUC45-lacZ).
Then, the 3.4- and 3.5-kb KpnI-HindIII fragments
of pUC9-lacZ and pUC45-lacZ were inserted into the same restriction
sites of pMW119.
The strains and plasmids used in this study are listed in Table I.
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The
nucleotide sequence of the upstream region of the oppA gene
was determined by the dideoxy method of Sanger et al. (20) using the M13 phage system. S1 nuclease mapping was performed according
to the method of Ausubel et al. (21). A 5-terminal 32P-labeled 600-nucleotide fragment of single-stranded DNA
(5 × 104 cpm), which is complementary to the upstream
region of OppA mRNA (from
579 to 21), was used as a probe. The
probe was hybridized with 60 µg of total RNA from E. coli
MA261 prepared by the method of Emory and Belasco (22) and digested
with S1 nuclease. The length of the remaining DNA fragment was
determined by electrophoresis on a 5% polyacrylamide sequencing gel.
OppA mRNA was measured by dot blot analysis (23) using a
32P-labeled 319-nucleotide fragment of DNA (from 996 to
1314) as a probe. The DNA fragment was labeled with
[
-32P]dCTP using TaKaRa
BcaBESTTM labeling kit.
OppA was purified from the 100,000 × g supernatant of E. coli MA261 as described previously (12). Immunoglobulin for OppA was prepared as described previously (24) and partially purified from the antiserum by precipitation with 40% saturated (NH4)2SO4.
Measurement of OppA Synthesis by an Immunoprecipitation MethodE. coli MA261 was grown in polyamine-deficient medium. When A540 reached 0.15, 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 OppA protein synthesized was determined using 1,000,000 cpm of [35S]methionine-labeled protein according to the method of Philipson et al. (25) with some modifications (26). Radioactivity of labeled OppA protein was quantified using a Fujix Bas 2000II imaging analyzer.
Measurement of Polyamine ContentsPolyamine levels in E. coli were determined by high pressure liquid chromatography as described previously (27).
Prediction of the Secondary Structure of RNAOptimal
computer folding of the 65 to 65-nucleotide region of OppA mRNA
or lacZ mRNA was performed according to the method of Zuker and
Stiegler (28). Free energy (
G) for the formation of the
secondary structure was calculated on the basis of the data of Turner
et al. (29).
As shown in Fig. 1, we confirmed polyamine
stimulation of OppA synthesis in polyamine-requiring mutant MA261 cells
by immunoprecipitation of [35S]methionine-labeled OppA
protein. If the oppA gene was disrupted, there was no OppA
synthesis. When cells were transformed with pACYCoppA, a
relatively high copy number plasmid, large amounts of OppA were
synthesized, and polyamines did not stimulate OppA synthesis
significantly. In contrast, when cells were transformed with pMW975, a
low copy number plasmid, OppA synthesis was greatly stimulated by
polyamines. These results indicate that polyamine stimulation of OppA
synthesis is observed only when the copy number of the oppA
gene is low. Under these conditions, putrescine and spermidine contents
in cells grown in the presence and absence of 100 µg/ml putrescine
were 43.5 and <0.1 nmol/mg protein for putrescine and 5.45 and 0.45 nmol/mg protein for spermidine. Thus, it is clear that significant
amounts of putrescine were converted into spermidine. The function of
polyamines was not replaced by Mg2+ or Ca2+
(data not shown).
Determination of the Transcription Initiation Sites of the oppA Gene
First, we determined the nucleotide sequence upstream of the
oppA gene. As shown in Fig. 2, insertion
sequence 2 (IS2) (30) was found in the upstream region.
Although IS2 was observed in another polyamine-requiring mutant HT283
(EWH319), it was not in E. coli W3110, the parental strain
of MA261 and HT283. The upstream region of the gene also included the
leucine responsive element observed in the upstream region of the
oppA gene of Salmonella typhimurium (31). We
confirmed leucine stimulation of OppA synthesis in E. coli
HT283, but polyamine stimulation of OppA synthesis was not influenced
by leucine (data not shown). The effect of leucine on OppA synthesis in
E. coli MA261 could not be determined since leucine is
necessary for cell growth in this strain.
Transcription initiation sites of the oppA gene were
determined by S1 nuclease mapping (Fig. 3). In E. coli MA261, there were three initiation sites (P1,
P2, and P3). However, initiation mainly occurred
from P1, suggesting that IS2 has a strong promoter activity. When OppA
mRNA was synthesized from pMW975, pMW412, and pMW211, the major
initiation site of transcription was P2, P2, and P3, respectively. It
remains to be clarified why P1 is not the initiation site in
pMW975.
Polyamine Stimulation of OppA Synthesis at the Translational Level
To determine the level of polyamine stimulation of OppA
synthesis, the amount of OppA mRNA and OppA synthesis were measured by dot-blotting of RNA and immunoprecipitation of
[35S]methionine-labeled OppA protein (Fig.
4). When OppA mRNA was transcribed from P1,
polyamines significantly stimulated OppA mRNA synthesis (3.2-fold).
When OppA mRNA was transcribed from P2 and P3, polyamines only
slightly stimulated the OppA mRNA synthesis (1.2-1.4-fold). In the
region upstream of P2 and P3, there were no typical 35 and
10
promoter regions, but transcriptional activity was stronger from P2
than from P3.
OppA synthesis measured by immunoprecipitation of
[35S]methionine-labeled OppA protein was stimulated by
polyamines with all versions of the OppA mRNAs (Fig. 4). The degree
of polyamine stimulation relative to the transcription initiation site
was in the order P1 > P2 P3. This result indicates that
polyamine stimulation of OppA synthesis occurs mainly at the level of
translation, and that transcription from P1 was also stimulated by
polyamines. It also suggests that the 171-nucleotide 5
-UTR is enough
to cause polyamine stimulation of OppA synthesis.
We previously
reported that polyamines stimulate protein synthesis mainly at the
level of translational initiation (2). For initiation of protein
synthesis, the most important elements in the mRNA are the
initiation codon AUG and SD sequence. When the SD sequence was removed
from the OppA mRNA, there was no significant OppA synthesis (Fig.
5). These results confirmed the importance of the SD
sequence in protein synthesis (32-34). The SD sequence of the OppA
mRNA was relatively distant (12 nucleotides) from the AUG compared
with other mRNAs, in which the typical position of SD sequence was
7 nucleotides upstream from the AUG. Therefore a new SD sequence was
inserted 7 nucleotides upstream from the AUG. When OppA was synthesized
from the mRNA with the new SD sequence, polyamine stimulation was
observed, but to a lesser degree (pMWSD*, 2.3-fold) (Fig. 5). However,
OppA synthesis from the mRNA with the new SD sequence in the
absence of putrescine was greater than that from the normal OppA
mRNA. The results suggest that the position of the SD sequence may
influence polyamine stimulation of OppA synthesis.
We next examined whether the 5-UTR of the OppA mRNA is sufficient
for polyamine stimulation of OppA synthesis. As shown in Fig.
6, synthesis of
-galactosidase from the chromosomal
lacZ gene was not stimulated by polyamines. When the 5
-UTR
and 27 nucleotides encoding first 9 amino acids for OppA protein was fused to
-galactosidase (9-lacZ mRNA), the degree of polyamine stimulation of
-galactosidase synthesis was 1.4-fold. The length of
OppA mRNA used for the construction of a fused mRNA was then increased to include the 5
-UTR and 135 nucleotides encoding first 45 amino acids (45-lacZ mRNA). As a result, the degree of polyamine stimulation increased to 4.2-fold. Possible secondary structure of the
initiation codon surrounding region (
65 to 65) of OppA mRNA and
9-lacZ mRNA was then compared (Fig. 7). Stability of the mRNAs was nearly equal (
46.6 and
47.8 kcal/mol). The SD sequence of the 9-lacZ mRNA was tightly base paired. However, the
SD sequence of OppA mRNA, which is equivalent to 45-lacZ mRNA, was loosely base paired. This suggests that the secondary structure of
the SD sequence may play an important role in polyamine stimulation of
OppA synthesis.
The secondary structure of the SD sequence was then changed by
site-directed mutagenesis at positions of the oppA gene
corresponding to the 5-UTR of OppA mRNA (Table I). Since
polyamines bind to double-stranded RNA, especially GC-rich RNA rather
than to single-stranded RNA (10, 35), the GC-rich double-stranded
region of the 5
-UTR of OppA mRNA (stems I and
II of Fig. 7A) was mutated. As shown in Table
II, the change of the secondary structure of the SD
sequence was observed by the mutation on stem I (pMWSD1 and 2) or stems I and II (pMWSD5), but not by the mutation on stem II (pMWSD3 and 4).
When the SD sequence was loosely base-paired as in mRNAs synthesized from pMW975, pMWSD4, pMWSD3, pMWSD5, and pMWSD1, polyamines significantly stimulated OppA synthesis (Table II). The degree of
polyamine stimulation was 3.7-5.1-fold. The amounts of OppA mRNA
from the cells cultured with putrescine were nearly equal to those from
the cells cultured without putrescine. When the SD sequence was tightly
base paired as in mRNAs synthesized from pMW9-lacZ and pMWSD2,
polyamines did not influence protein synthesis significantly. The
secondary structure of the AUG region was not correlated with the
polyamine stimulation of OppA synthesis (Fig. 7).2
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Polyamines can stimulate synthesis of proteins such as OppA protein (1, 2) and ribosomal proteins (26), which are important for cell growth. In this communication, the molecular mechanism of polyamine stimulation of OppA synthesis has been studied. It was found that polyamine stimulation occurs mainly at the translational level.
In E. coli cells, IS2 was inserted at some stage of
evolution. IS2 enhanced transcriptional efficiency of oppA
gene and the IS2-dependent transcription was stimulated by
polyamines. This also contributed to the polyamine stimulation of OppA
synthesis. In E. coli W3110 lacking IS2 at the upstream
region of oppA gene, the transcription started mainly from
P2 and the efficiency was low because only weak 35 and
10 regions
exist upstream of P2 (Fig. 2). Thus, IS2 was inserted at some stage of
evolution by chance so that OppA protein was synthesized more
effectively in E. coli MA261 and HT283. A plasmid containing
461 nucleotides of the 3
-end of IS2 (pMW975) led to transcriptional
initiation starting from P2 (Fig. 3). This was unexpected, and it
remains to be clarified why P1 is not the initiation site in pMW975.
The whole sequence of IS2 may be necessary as a signal for
transcriptional initiation.
When transcription started from P2 or P3, polyamines did not influence
transcriptional efficiency significantly. Thus, we could study the
polyamine effect on OppA synthesis at the level of translation using
E. coli MA261oppA::Km/pMW975 or pMW211.
When pMW975 and pMW211 were used as the template for OppA mRNA
synthesis, the size of the 5-UTR was 266 and 171 nucleotides,
respectively. Although the polyamine effect on OppA protein synthesis
was slightly greater with pMW975 than with pMW211, essentially the same
results were obtained with both plasmids. The results with pMW211 were only shown with fused mRNAs containing the 5
-UTR of OppA mRNA and the open reading frame of lacZ mRNA.
For initiation of protein synthesis in E. coli, the most
important elements in the mRNA are the initiation codon AUG and SD sequence. The latter can base pair with the 3-end of 16 S rRNA so that
translational efficiency increases. Thus, translational efficiency
decreases if the SD sequence undergoes intrastrand base pairing. The
polyamine effect on OppA synthesis was examined by changing the
secondary structure of the SD sequence and the AUG region through
site-directed mutagenesis at positions of oppA gene corresponding to
the 5
-UTR of OppA mRNA. Although the secondary structure of the
AUG region did not influence the polyamine stimulation of OppA protein
synthesis, that of SD sequence did. When the SD sequence was loosely
base paired with another region of OppA mRNA, polyamines
significantly stimulated protein synthesis. When the SD sequence was
strongly base paired, polyamines did not influence protein synthesis.
Since polyamines bind to double-stranded RNA more strongly than to
single-stranded RNA (10, 35), it is to be expected that a strongly base
paired SD sequence would become further stabilized by spermidine
binding. It is also noted that the disappearance of the GC-rich stem I
in OppA mRNA synthesized from pMWSD2 caused no polyamine
stimulation of OppA synthesis. Our results suggest that polyamines may
contribute to unwinding weak secondary structure of the SD sequence
through their binding to a region(s) such as stem I on the OppA
mRNA close to SD sequence. However, an alternative explanation may
also be possible. Experiments are in progress to determine how
polyamines change the secondary structure of OppA mRNA.
In contrast, polyamines are known to inhibit some kinds of protein
synthesis like ribosome modulation factor and OmpC protein (36). It is
of interest to know the secondary structure of the SD sequence of these
mRNAs. In case of eukaryotic protein synthesis, polyamines can
regulate the initiation complex formation of
Met-tRNAi·mRNA·40 S ribosomal subunits positively and
initiation factor-dependent RNA helicase activity
negatively (37). Thus, polyamine regulation of protein synthesis is
dependent on the size and base composition of the 5-UTR of the
mRNA. There is a tendency that polyamines regulate protein
synthesis directed by the mRNAs having long 5
-UTR in both
prokaryotes and eukaryotes. We consider that these studies will help to
establish the physiological importance of polyamines.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) D83137[GenBank].
We thank Dr. A. J. Michael for his help in preparing this manuscript.