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Journal of Bacteriology, October 1999, p. 6284-6291, Vol. 181, No. 20
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Mutation Analysis of the 5' Untranslated Region of
the Cold Shock cspA mRNA of Escherichia
coli
Kunitoshi
Yamanaka,
Masanori
Mitta,
and
Masayori
Inouye*
Department of Biochemistry, Robert Wood
Johnson Medical School, Piscataway, New Jersey 08854
Received 7 April 1999/Accepted 6 August 1999
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ABSTRACT |
The mRNA for CspA, a major cold shock protein in Escherichia
coli, contains an unusually long (159 bases) 5' untranslated region (5'-UTR), and its stability has been shown to play a major role
in cold shock induction of CspA. The 5'-UTR of the cspA
mRNA has a negative effect on its expression at 37°C but has a
positive effect upon cold shock. In this report, a series of
cspA-lacZ fusions having a 26- to 32-base deletion in the
5'-UTR were constructed to examine the roles of specific regions within
the 5'-UTR in cspA expression. It was found that none of
the deletion mutations had significant effects on the stability of
mRNA at both 37 and 15°C. However, two mutations (
56-86 and
86-117) caused a substantial increase of 
-galactosidase activity
at 37°C, indicating that the deleted regions contain a negative
cis element(s) for translation. A mutation (2-27)
deleting the highly conserved cold box sequence had little effect on
cold shock induction of -galactosidase. Interestingly, three
mutations (28-55, 86-117, and 118-143) caused poor cold shock
induction of -galactosidase. In particular, the 118-143 mutation
reduced the translation efficiency of the cspA mRNA to
less than 10% of that of the wild-type construct. The deleted region
contains a 13-base sequence named upstream box (bases 123 to 135),
which is highly conserved in cspA, cspB, cspG, and cspI, and is located 11 bases
upstream of the Shine-Dalgarno (SD) sequence. The upstream box might be
another cis element involved in translation efficiency
of the cspA mRNA in addition to the SD sequence and the
downstream box sequence. The relationship between the mRNA
secondary structure and translation efficiency is discussed.
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INTRODUCTION |
When a culture of Escherichia
coli is shifted from 37 to 15 or 10°C, a number of proteins,
called cold shock proteins, are transiently induced during its growth
lag period (17, 32, 34). CspA, consisting of 70 amino acid
residues, has been identified as a major cold shock protein
(12), and its three-dimensional structure has been
determined by both X-ray crystallography (28) and nuclear
magnetic resonance spectroscopy (9, 24) to consist of a
five-antiparallel -stranded structure. CspA can bind to single-stranded DNA and RNA without high sequence specificity and has
been proposed to function as an RNA chaperone at low temperature (16).
In E. coli, nine genes encoding CspA-like proteins,
cspA to cspI, have been identified
(34). Among them, cspA (12),
cspB (18), cspG (23), and
cspI (33) are cold shock inducible, and
interestingly, cspD is induced during stationary phase and upon nutritional starvation (35). It was proposed that the
large CspA family of E. coli may have a function in response
to different environmental stresses (34).
Among these cold shock-inducible genes, cspA has been quite
extensively investigated for the mechanism of its cold shock induction (34). The cspA promoter is highly active at
37°C, although CspA is hardly detectable at this temperature (8,
21). Even if the cspA promoter is replaced with the
lpp promoter, a constitutive promoter for a major outer
membrane protein, cspA expression is still cold shock
inducible (8), indicating that the cspA induction upon cold shock occurs mainly at the levels of mRNA stability and
translation. However, it should be mentioned that the cspA promoter contains an AT-rich upstream element (25)
immediately upstream of the 
35 region, which is considered to play an
important role in efficient transcription initiation at low temperature (8, 11, 21). It has been demonstrated that the
cspA mRNA is extremely unstable at 37°C but becomes
stable upon cold shock, indicating that the stability of mRNA plays
a crucial role in cold shock induction of cspA (3, 8,
10). Furthermore, it was shown that the 14-base downstream box
located 12 bases downstream of the translation initiation codon of the
cspA mRNA, which is partially complementary to a region,
called anti-downstream box, of 16S rRNA (30), plays an
important role in efficient translation at low temperature (6,
21). Thus, cspA expression is regulated in a complex
manner at the levels of transcription, mRNA stability, and translation.
An important and unique feature of the cspA mRNA is its
unusually long 5' untranslated region (5'-UTR) consisting of 159 bases (31). This feature is also shared with several cold shock
genes, which are dramatically induced after temperature downshift, such as cspB (7), cspG (23), and
cspI (33). The 5'-UTR is considered to play a
crucial role in the cold shock induction of cspA (1, 3,
8, 10, 11, 15, 21). Here, we constructed a series of deletion
mutations in the 5'-UTR of cspA and analyzed their effects
on cspA expression by examining the amount, stability, and
translation efficiency of mRNA. It was found that besides mRNA
stability, the 5'-UTR plays an important role in translation efficiency
of the cspA mRNA.
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MATERIALS AND METHODS |
Bacterial strain and media.
E. coli AR137
[MC4100 (malT-ompB) pcnB80] (13)
was used and grown in M9-Casamino Acids medium supplemented with
D-biotin (50 µg/ml). When necessary, ampicillin was added
to a final concentration of 25 µg/ml.
General techniques.
All DNA cloning was carried out
according to the method of Sambrook et al. (26). PCR was
carried out according to the manufacturer's instructions (Boehringer)
with 25 cycles of amplification steps of 0.5 min at 94°C, 2 min at
50°C, and 1 min at 72°C.
Plasmid construction.
Each 5'-UTR deletion was prepared by
two-step PCR. A plasmid, pJJG02 (15), which contains the
wild-type cspA gene, was used as a template for PCR. For the
first step, two independent PCRs were carried out for each mutation.
One reaction was done with a combination of primer 67F,
5'-ccttgctagCCGATTAATCATAAATATG-3' (nucleotides 67 to 49 of cspA), and mutation
primer R, which contains a desired deletion mutation and is
complementary to the sense cspA sequence, and another
reaction was done with primer 4311, 5'-ccggatccagGTTGAACCATTTT-3' (complementary to
nucleotides +186 to +198), and mutation primer F, which also contains
the same desired mutation as primer R. In these regions, 5' tails are
shown in lowercase where NheI and BamHI sites are
underlined. For the construction of pMM022, pMM023, pMM024, pMM025,
pMM026, and pKNJ37, primer D1R, 5'-ACTACACT/TTGATGTGCATTAGC-3'
(complementary to 15 to +1/+28 to +35), and primer D1F,
5'-GCACATCAA/AGTGTAGTAAGGCAA-3' (8 to +1/+28 to +42);
primer D2R, 5'-CAACGATAA/GCTTTAATGGTCTGT-3' (complementary to +13 to +27/+56 to +64), and primer D2F,
5'-TAAAGC/TTATCGTTGATACCC-3' (+22 to +27/+56 to +70); primer
D3R, 5'-TAAAGG/CTCTTGAAGGGACTT-3' (complementary to +41 to
+55/+86 to +91), and primer D3F, 5'-TCAAGAG/CCTTTAACGCTTCAAAA-3' (+49 to +55/+86 to +102); primer D4R,
5'-CGGCGATAT/AATGTGCACTACGAGGG-3' (complementary to +69 to
+85/+118 to +126), and primer D4F, 5'-GCACATT/ATATCGCCGAAAGGC-3' (+79 to +85/+118 to +132); primer D5R,
5'-TACCTTTAA/GGCGTGCTTTACAGATT-3' (complementary
to +101 to +117/+144 to +152), and primer D5F, 5'-AAAGCACGCC/TTAAAGGTAATACACT-3' (+108 to +117/+144 to
+159); and primer 8064, 5'-TAATTAAG/GATATGGCGTGCTTT-3'
(complementary to +108 to +122/+136 to +143), and primer 8063, 5'-GCCATATC/CTTAATTATTAAAGG-3' (+115 to +122/+136 to +150),
were used, respectively, where the position of each deletion is
indicated by a slash. Each set of the first PCR products was mixed,
heat denatured, annealed, and extended with Taq DNA
polymerase. The resulting products were then amplified again by PCR
with primer 67F and primer 4311. The final PCR fragments were digested
with NheI and BamHI and inserted into the
XbaI-BamHI site of pKM005 (14). For
pKNJ37, the PCR fragment was cloned into the
XbaI-BamHI site of pRS414X, a pRS414 derivative
(29) in which the unique SmaI site has been
changed to an XbaI site.
For the construction of pMM007, PCR was carried out with primer 67F and
primer 4311 as primers and pJJG02 as a template. The PCR fragment
was digested with NheI and BamHI and
inserted into the XbaI-BamHI site of pRS414X.
pKNJ38 was constructed as follows: oligonucleotide 8509, 5'-CTAGCCGAA AGGCACAAATTAAGAGGGTATTAATAATGAAAGGGGGAATTCCA- 3',
and
oligonucleotide 8510, 5'-AGCTTGGAATTCCCCCTTTCATTATTAATACCCTCTTAATTTGTGCCTTTCGG-3',
were first annealed and then cloned into pKM67
(
21) digested
with
XbaI and
HindIII.
The DNA sequences of all the constructs were confirmed by DNA
sequencing by the chain-termination method (
27).
-Galactosidase assay.
E. coli AR137 harboring
different plasmids was grown at 37°C to mid-log phase in M9-Casamino
Acids medium and then transferred to 15°C. The -galactosidase
assay was carried out according to the protocol of Miller
(20). The assay was done in duplicate at each time point.
Isolation of RNA and primer extension.
E. coli AR137
harboring different plasmids was grown under the same conditions used
for the -galactosidase assay described above. RNA extraction and
primer extension methods were described previously (21).
Primer M13-47, 5'-CGCCAGGGTTTTCCCAGTCACGAC-3', which
is complementary to a coding sequence of lacZ, was used. The
products were analyzed on a denatured polyacrylamide gel and quantified
by using a PhosphorImager (Bio-Rad).
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RESULTS |
Deletion analysis of the cspA 5'-UTR.
Earlier, we
constructed two cspA-lacZ fusions, in which the
lacZ gene was transcriptionally fused to
cspA at +26 (pKM67) (21) or at +143 (pJJG78)
(15) of the cspA mRNA. The -galactosidase activity of the cells harboring pJJG78 was very low at 37°C and increased about 10-fold at 2 h after temperature downshift to 15°C, whereas the -galactosidase activity of the cells harboring pKM67 was very high even at 37°C (21), indicating that the
region from +26 to +143 in the 5'-UTR of the cspA mRNA
plays a crucial role in cspA expression.
In order to further investigate which part of the 5'-UTR is responsible
for the positive or negative regulation of
cspA expression,
we attempted deletion analysis of the 5'-UTR and examined the
effects
of various deletions on cold shock induction of
cspA.
For
this purpose, a series of 5'-UTR deletion mutants, in which
a 26- to
32-base deletion was created at about every 30 bases,
were constructed,
and the resultant 5'-UTRs were translationally
fused to
lacZ
at the 13th amino acid residue of CspA as described
in Materials and
Methods. The resultant plasmids, pMM022, pMM023,
pMM024, pMM025, and
pMM026, contain deletion mutations in the
5'-UTR from +2 to +27, from
+28 to +55, from +56 to +86, from
+86 to +117, and from +118 to +143,
respectively (Fig.
1A). In
the case of
pMM024, a deletion from +56 to +85 was originally
designed, but all the
transformants that we analyzed contained
an extra base deletion at
position +86. Plasmid pMM67 (
21),
which is the wild-type
cspA-lacZ translational fusion construct,
was used as a
control.
E. coli AR137, a
pcnB mutant, which is
known to maintain pBR322 derivatives in a low copy number
(
19),
was used in order to minimize multicopy effects of the
constructed
gene on their expression.
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FIG. 1.
Cold shock induction of -galactosidase. (A)
Construction of cspA-lacZ fusions. The wild-type
cspA is shown on the top. The cspA-lacZ fusion in
each expression plasmid is shown from the 5' end of the cspA
promoter upstream region to lacZ. Nucleotide numbers are
given starting from the transcription initiation site as +1, as
determined by Tanabe et al. (31). The crosshatched, open,
dotted, and diagonally striped bars represent the cspA
promoter, its 5'-UTR, the cspA coding region, and the
lacZ coding region, respectively. The solid boxes indicate
the SD sequence. The positions of deleted regions are shown with
nucleotide numbers. (B) Induction patterns of various deletion
constructs. At mid-log phase, cultures of E. coli AR137
harboring various plasmids were shifted from 37 to 15°C. Samples were
taken at 0, 1, 2, 3, 5, 7, and 10 h after the shift, and
-galactosidase activity was measured. The cspA-lacZ
fusions were pMM67 ( ), pMM022 ( ), pMM023 ( ), pMM024 ( ),
pMM025 ( ), and pMM026 ( ).
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Transformed cells were grown in M9-Casamino Acids medium at 37°C, and
-galactosidase activities were measured after temperature
downshift
from 37 to 15°C. At 37°C (zero time point in Fig.
1B),
-galactosidase activities were 10-fold higher in cells harboring
pMM024 (
56-86) and pMM025 (
86-117) than in cells harboring the
wild-type pMM67, while other deletion mutants [pMM022 (
2-27),
pMM023 (
28-55), and pMM026 (
118-143)] showed very low
-galactosidase
activities. These results suggest that the 5'-UTR
from base +56
to +117 is involved in the repression of
cspA
expression at 37°C.
Interestingly,
-galactosidase activity
increased almost fivefold
with pMM024 (
56-86) after temperature
downshift, while it increased
only less than twofold with pMM025
(
86-117), suggesting that
the region deleted in pMM025 (
86-117)
plays an important role
in cold shock induction of
cspA.
Similar to pMM025 (
86-117),
-galactosidase activity with pMM023
(
28-55) was poorly induced
at a low temperature. In particular, the
region deleted in pMM026
(
118-143) appears to play a crucial role in
cspA expression at
both high and low temperatures, since
-galactosidase activity
was very low at both 37 and 15°C (Fig.
1B). The deletion of the
region from base +2 to +27 (pMM022), which
contains the cold box
sequence involved in
cspA
autoregulation (
15), has little effect
on the cold shock
induction of
cspA (Fig.
1B).
Analysis of cspA-lacZ mRNA.
As described
above, all deletion mutations, except for pMM022 (2-27), of the
5'-UTR of the cspA mRNA affected cspA-lacZ
expression. The cspA promoter is known to be active even at
37°C (8, 11, 21). Since all the deletion constructs have
the intact cspA promoter (Fig. 1A), transcription
efficiencies of these constructs are likely to be identical. On the
other hand, stability of the cspA mRNA is known to be
significantly different depending on growth temperatures (1, 3, 8,
10, 11, 21). Therefore, the effect of the deletion mutations on
cspA-lacZ expression may be due to different mRNA
stabilities of the constructs. To examine this possibility, primer
extension analysis was carried out to quantitate the amounts of the
cspA-lacZ transcripts for each mutant at different time
points after the addition of rifampin at both 37 and 15°C (Fig.
2A). The amounts of transcripts at each
time point were estimated with a phosphorimager, and the amounts of mRNA remaining (percentage of the amount at the zero time point) were plotted as shown in Fig. 2B. All the transcripts were unstable at
37°C with their half-lives estimated as being between 30 and 45 s. At 15°C, however, they became very stable with half-lives of
between 20 and 40 min. These half-lives are similar to those for the
wild-type construct pMM67 obtained previously (21) as well
as to those for the wild-type chromosomal cspA (8,
11). It is important to note that in contrast to the similar
mRNA half-lives at low temperature, -galactosidase activities
induced at 15°C widely varied among all these constructs as shown in
Fig. 1B.
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FIG. 2.
Analysis of mRNA stability. (A) Primer extension
analysis of the cells harboring the cspA-lacZ fusions. At
mid-log phase, cultures of E. coli AR137 harboring various
plasmids were shifted from 37 to 15°C. For measurement of the
mRNA stability at 37°C, cultures were shifted back to 37°C
after 30 min of incubation at 15°C and rifampin was added to the
cultures to a final concentration of 200 µg/ml. RNAs were extracted
at 0 (lanes 1), 1 (lanes 2), 3 (lanes 3), and 5 (lanes 4) min after the
addition of rifampin. For measurement of the mRNA stability at
15°C, rifampin was added 1 h after the temperature downshift,
and then RNAs were extracted at 0 (lanes 5), 5 (lanes 6), 10 (lanes 7),
and 20 (lanes 8) min after the addition of rifampin. Primer extension
was carried out as described previously (21). (B) Graphic
presentation of the results shown in panel A for 37 and 15°C,
respectively. The radioactivities of transcripts were measured with a
phosphorimager and plotted by using the transcript at the zero time
point as 100%. , pMM022; , pMM023; , pMM024; , pMM025; and
, pMM026.
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These results indicate that
-galactosidase activity of each
cspA-lacZ construct is not correlated with the stability of
mRNA
but rather with the amount of mRNA and/or its translation
efficiency.
Therefore, we next examined the amounts of mRNA for
each construct
at different time points after cold shock by the primer
extension
method. The results are shown in Fig.
3A, and the amounts of transcripts
were
estimated with a phosphorimager. Their relative amounts were
calculated
by using the amount of the transcript of pMM67 at zero
time as 1 (Fig.
3B). At 37°C, the amounts of transcripts for pMM022
(
2-27) and
pMM024 (
56-86) were very similar to that of the wild-type
construct
pMM67 (Fig.
3B, column 1). It should be noted that
-galactosidase
activity of pMM024 (
56-86) at 37°C was more than 10 times higher
than that of pMM022 (
2-27) (Fig.
1B). In the case of pMM023
(
28-55),
pMM025 (
86-117), and pMM026 (
118-143), the
amounts of transcripts
at 37°C are approximately half of that of
pMM67. Again, it should
be noted that
-galactosidase activity of
pMM025 (
86-117) was
10 times higher than those of pMM023 (
28-55)
and pMM026 (
118-143)
(Fig.
1B). These results indicate that there is
no correlation
between the amounts of transcripts and the
-galactosidase activities
at 37°C. This is consistent with the
previous notion that although
cspA mRNA was stabilized
and accumulated at the nonpermissive
temperature in the
temperature-sensitive RNase E mutant, CspA
was not produced under this
condition (
8).
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FIG. 3.
Analysis of mRNA level and translational efficiency.
(A) Primer extension analysis of the cspA-lacZ fusions. At
mid-log phase, cultures of E. coli AR137 harboring various
plasmids were shifted from 37 to 15°C. RNAs were prepared from the
culture at 37°C (0 h; lanes 1) and at 0.5 (lanes 2), 1 (lanes 3), 2 (lanes 4), and 3 (lanes 5) h after the temperature downshift. Primer
extension was carried out as described previously (21). (B)
Graphic presentation of the relative amounts of mRNA. Relative
mRNA amounts (mean values of two experiments) were calculated from
the radioactivities of transcripts shown in panel A with the transcript
of pMM67 at 37°C as 1. The relative amount is shown on the top of
each column. Columns 1, 0 h; columns 2, after 0.5 h; columns
3, after 1 h; columns 4, after 2 h; and columns 5, after
3 h. (C) Relative translational efficiencies of the
cspA-lacZ mRNAs. Translational efficiencies at 15°C
were calculated by dividing the increment of -galactosidase activity
during the first 2 h after cold shock by the amount of mRNA
with the following formula: [(Gal 2 h) × (OD 2 h) (Gal
0 h) × (OD 0 h)]/(ave mRNA), where (Gal 0 h) and
(Gal 2 h) are -galactosidase activities at 0 and 2 h after
temperature downshift, respectively; (OD 0 h) and (OD 2 h)
are the optical densities at 600 nm of the cultures at 0 and 2 h
after temperature downshift, respectively; and (ave mRNA) is the
average of relative mRNA amounts at 0.5, 1, and 2 h after
temperature downshift. Relative translational efficiency of each
mRNA was calculated by using the efficiency of mRNA of pMM67 as
100%.
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After temperature downshift, the amounts of the
cspA-lacZ
mRNAs dramatically increased in all the constructs, and the
induction
patterns are shown in Fig.
3. They showed patterns in
accumulation
of the transcripts very similar to that of the wild-type
pMM67,
such that the maximal induction was observed at 1 h after
temperature
downshift. The patterns of mRNA levels were very
similar between
pMM67 and pMM024 (
56-86), while the others also
showed a similar
induction pattern although the amounts of their
mRNAs were approximately
half of that of the pMM67 mRNA at each
time point. Since the promoter
activities of all the deletion
constructs are considered to be
the same, and in addition their
mRNA stabilities were also very
similar to that of the wild-type
construct (Fig.
2), lower amounts
of mRNAs for all the deletion
constructs except pMM024 (
56-86)
are probably due to their lower
transcription elongation rate
and/or transcription attenuation within
the 5'-UTR as reported
recently (
2). A remarkable finding
was that the amounts of
mRNA for pMM026 (
118-143) accumulated
after cold shock were also
identical to those for pMM022 (
2-27),
pMM023 (
28-55), and pMM025
(
86-117) (Fig.
3B). Nevertheless, cold
shock induction of
-galactosidase
activity was extremely low for
pMM026 (
118-143) throughout cold
shock treatment (Fig.
1B),
indicating that the mRNA for this construct
was very poorly
translated.
Translational regulation by the 5'-UTR.
Relative translation
efficiencies at 15°C were calculated for all the constructs from the
increments of -galactosidase activity during the cold shock and the
amounts of mRNA (see the legend to Fig. 3). As shown in Fig. 3C,
the translation efficiency of pMM026 (118-143) mRNA was
extremely poor and calculated to be 9.5% of that of pMM67 mRNA.
Besides pMM026 (118-143), the translation efficiencies of the
mRNAs of pMM023 (28-55) and pMM025 (86-117) were relatively
low and calculated to be 54 and 36% of that of pMM67 mRNA,
respectively. These results clearly indicate that the translation
efficiency of mRNA plays an important role in the regulation of
cspA expression and that, in particular, the region from
base +118 to +143 of the 5'-UTR plays a major role in translation efficiency.
Nucleotide sequence comparison of the 5'-UTRs of the
cold-shock-inducible genes,
cspA,
cspB,
cspG, and
cspI, reveals that
there is a 13-base
sequence named upstream box (UB) sequence (from
base +123 to +135 of
cspA) very well conserved among these genes
as shown in Fig.
4. Interestingly, these 13-base sequences
are
located immediately upstream of the Shine-Dalgarno (SD) sequence
and contain a palindromic sequence to form a stable secondary
structure
(
G =
9.5 kcal [Fig.
4, see also Fig.
6]). It is
also
complementary to the region from base 1023 to 1035 of 16S rRNA
(Fig.
4). The significance of these facts will be discussed in
the
Discussion. In the pMM026 (
118-143) mRNA, this UB region
has
been deleted, which may be the major cause for the poor translation
efficiency of the mRNA. In order to characterize the role of the
UB
sequence in translation efficiency, two new constructs were
made; in
one construct (pKNJ37), the exact 13-base UB sequence
was deleted from
the wild-type
cspA-lacZ construct (pMM007), and
in another
(pKNJ38) the 13-base sequence was added at the upstream
region of the
SD sequence of pKM67 (
21) as shown in Fig.
5A.
In pKM67, the
lacZ gene is
fused at base +26, and it has been
shown that as a result of the
substantial deletion in the 5'-UTR
-galactosidase became expressed
even at 37°C without any further
induction upon cold shock
(
21). Note that both pMM007 and pMM67
have the exactly
identical insert of
cspA in different vectors,
pRS414 and
pKM005, respectively. Cold shock induction patterns
of
-galactosidase activities of these two plasmids (pMM007 and
pMM67)
are similar (data not shown). Cells were transformed with
pKNJ37,
pMM007, pKNJ38, and pKM67, and cold shock induction of
-galactosidase activity was examined. Deletion of the UB sequence
(pKNJ37) significantly lowered
-galactosidase activity not only
at
37°C but also upon cold shock (Fig.
5B). Consistently, when
the UB
sequence was inserted into pKM67, the constitutive expression
of
-galactosidase at 37°C increased by approximately 20% (Fig.
5B).
This increment was also kept upon cold shock. These results
suggest
that the UB sequence may be associated with efficient
translation of
cspA, although the loss of the UB sequence is unlikely
to be
the sole defect in pMM026 (
118-143).
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FIG. 4.
Sequence similarities of cspA,
cspB, cspG, and cspI mRNAs around
the SD sequence and potential base pairing between cspA
mRNA and 16S rRNA. Nucleotide numbers of cspA
(31), cspB (7), cspG
(23), and cspI (33) mRNA are given
starting from the major transcription initiation site as +1. The
sequence of 16S rRNA is from the work of Brosius et al. (4).
Nucleotides identical in the four csp mRNAs are shown in
boldface. The 13-base homologous sequences in cspA,
cspB, cspG, and cspI are boxed (the
UB). Positions of the SD sequence and the initiation codon are
underlined. Potential base pairings between cspA mRNA
and 16S rRNA are indicated by vertical lines.
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FIG. 5.
Role of the 13-base UB sequence in the cspA
5'-UTR in cspA-lacZ expression. (A) Construction of
cspA-lacZ fusions. The constructs are drawn in the same
manner as shown in Fig. 1A except for a box with wavy lines, which
represents the 13-base UB sequence (5'-GCCGAAAGGCACA-3')
located upstream of the SD sequence. pKNJ37 is identical to
pMM007 except for the deletion of the 13-base sequence. In both pMM007
and pKNJ37, cspA was translationally fused to
lacZ. pKNJ38 is identical to pKM67 (21) except
for the addition of the 13-base sequence by replacing the DNA fragment
between XbaI and HindIII with synthesized
oligonucleotides as described in Materials and Methods. In both pKM67
and pKNJ38, cspA was transcriptionally fused to
lacZ. (B) Cold shock induction of -galactosidase. The
cspA-lacZ fusions were pMM007 (), pKNJ37 (), pKNJ38
(), and pKM67 ().
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DISCUSSION |
In the present paper, we have attempted to further elucidate the
roles of the unusually long 5'-UTR of the cspA mRNA in
cspA expression. We made a series of 26- to 32-base deletion
mutations encompassing the entire 5'-UTR. These mutated 5'-UTRs were
translationally fused to lacZ at the 13th amino acid residue
of CspA after the downstream box sequence. At 37°C, pMM022
(2-27), pMM023 (28-55), and pMM026 (118-143) showed
-galactosidase activities similar to the wild-type pMM67, while
pMM024 (56-86) and pMM025 (86-117) showed higher
-galactosidase activities than pMM67, indicating that
the region from base +56 to +117 is involved in the repression of
cspA expression at 37°C to some extent. As shown in Fig. 2 and 3, this repression is not due to the decrease in the amounts and
the stability of mRNA. Although it is possible that a factor binding this region might be involved in the repression, a precise mechanism for the repression is unclear at present. Note that the most
important fact for the repression of the wild-type cspA expression at 37°C is the extreme instability of the cspA
mRNA (3, 8, 10).
Based on the -galactosidase activities after temperature downshift,
mutants can be classified into three classes: class I [pMM022
(2-27) and pMM024 (56-86)], in which -galactosidase is
induced in a fashion similar to that of pMM67; class II [pMM023 (28-55) and pMM025 (86-117)], in which cold shock
induction of -galactosidase is poor; and class III [pMM026
(118-143)], with very low -galactosidase activities both before
and after cold shock. It is worth mentioning that these differences
were due to neither the amounts nor the stability of mRNA as
evident from Fig. 2 and 3. This supports the notion that the 5'-UTR has another role in translation efficiency in addition to the stability of mRNA.
The relative translation efficiencies after temperature downshift for
different constructs showed surprisingly significant differences (Fig.
3C), which coincided well with the classification of the constructs at
15°C as described above. The translation efficiencies with pMM022
(2-27) and pMM024 (56-86) (class I) are a little better than that
of the wild-type pMM67, those with pMM023 (28-55) and pMM025
(86-117) (class II) are 40 to 50% of that of pMM67, and that with
pMM026 (118-143) (class III) is less than 10% of that of pMM67.
In pMM026 (118-143), the deletion mutation is clearly
affecting the translation efficiency but not the stability of
mRNA. The deleted region was found to contain a 13-base sequence
(bases +123 to +135) well conserved in the mRNAs for all the cold
shock-inducible CspA family genes, cspA, cspB,
cspG, and cspI (Fig. 4). This sequence, designated the UB sequence, may form a distinct secondary
structure in both the wild-type pMM67 and class I constructs [pMM022
(2-27) and pMM024 (56-86)] (Fig.
6). Class I constructs showed a
translation efficiency similar to that of the wild type. In class II
[pMM023 (28-55) and pMM025 (86-117)], which showed 50% of the
translation efficiency of the wild type, this secondary structure
disappears; however, the predicted secondary structures around the SD
sequence in these constructs are still similar to that of the wild-type construct. In contrast, when the UB region is deleted [pMM026 (118-143); class III, which showed a very poor translation
efficiency], the SD region forms a more stable secondary structure.
This likely prevents recognition of the mRNA by ribosomes, causing
a very poor translation efficiency in pMM026 (118-143). Therefore,
the UB sequence may function to punctuate the formation of a stable secondary structure immediately upstream of the SD sequence, allowing it to be highly accessible to ribosomes. Alternatively, as the UB
sequence is complementary to the 16S rRNA sequence from bases 1023 to
1035 (Fig. 4), it is possible that the UB sequence may be another
cis element, which may enhance translation efficiency by
forming a duplex with 16S rRNA in addition to the SD sequence and the
downstream box (21). Since all the constructs use the identical site of cspA to fuse to lacZ, the
observed differences in translation efficiency are considered to be at
the level of translation initiation but not at the level of translation
elongation.
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|
FIG. 6.
Comparison of the secondary structures of the 5'-UTRs
for the deletion constructs. Secondary structures of the 5'-UTR for
each deletion construct were predicted with a nucleotide sequence
analysis program (DNASIS-Mac; Hitachi Software Engineering Co. Ltd.)
based on the method of Zuker and Stieger (36). Nucleotides
are numbered as the position in the cspA mRNA starting
from the transcription initiation site as +1. The position of the
deletion in each mutant is shown by an arrow with the nucleotide
numbers of the deleted region. The highly conserved 13-base sequences
upstream of the SD sequence designated the UBs are boxed. The
initiation codon and the SD sequence are also boxed.
|
|
Consistent with the proposed role of the UB sequence in the
translation, the addition of the UB sequence to pKM67 resulted in the
increase of -galactosidase activity by approximately 20%. On the
other hand, the deletion of the exact 13-base UB sequence [pKNJ37
(123-135) in Fig. 5] resulted in a 50% reduction of
-galactosidase activity at 37°C and a lower level of induction
upon cold shock. Note that the predicted secondary structure
surrounding the SD sequence of pKNJ37 (123-135) is the same as that
of the wild type (Fig. 6).
In summary, stabilization of the cspA mRNA upon cold
shock is prerequisite for CspA production. This stabilization does not require any de novo protein synthesis (5). As presented
here, translation efficiency of cspA mRNA turns out
to play an important role in cspA expression in
addition to the mRNA stabilization. The region from base +118
to +143, containing the UB sequence, of the 5'-UTR was clearly required
for the cold shock induction (Fig. 1). However, it is unlikely that a
de novo-synthesized activator binds to this region to induce
translation upon cold shock, since the cold shock induction of CspA was
observed even in the presence of a translation inhibitor such as
chloramphenicol (7), although it cannot be ruled out that a
preexisting factor might be activated upon cold shock. It is possible
that the secondary structure of the 5'-UTR of cspA mRNA
at low temperatures, in particular surrounding the SD and UB sequences,
might be different from that at 37°C. This structural change might
make cspA mRNAs more accessible to ribosomes. It is thus
possible that the 5'-UTR of the cspA mRNA might act as
an RNA thermometer, as was recently proposed for the rpoH
mRNA (22). To know the more precise molecular mechanism of the regulation of cspA expression, determination of the
secondary structure of the cspA mRNA and the
relationship between the secondary structure and the translation
efficiency remain to be addressed. The point mutation analysis in
addition to the deletion analysis presented here will give us
information on the molecular anatomy of the structure and function of
the 5'-UTR of the cspA mRNA.
| |
ACKNOWLEDGMENTS |
Kunitoshi Yamanaka and Masanori Mitta contributed equally to this work.
We thank W. Bae, J.-P. Etchegaray, and S. Phadtare for comments.
K.Y. was partly supported by the Uehara Memorial Foundation. This work
was supported by a grant from the National Institutes of Health (GM 19043).
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Biochemistry, Robert Wood Johnson Medical School, 675 Hoes Lane,
Piscataway, NJ 08854. Phone: (732) 235-4115. Fax: (732) 235-4559. E-mail: inouye{at}umdnj.edu.
Present address: Takara Shuzo Co., Ltd., Biotechnology
Research Laboratories, Seta 3-4-1, Otsu, Shiga 520-2193, Japan.
| |
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Abstract
Introduction
