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FEMS Microbiology Letters 176 (1999) 39^43
Transcription of cspA, the gene for the major cold-shock protein
of Escherichia coli, is negatively regulated at 37³C by the
5P-untranslated region of its mRNA
Li Fang 1 , Bing Xia, Masayori Inouye *
Department of Biochemistry, Robert Wood Johnson Medical School, 675 Hoes Lane, Piscataway, NJ 08854, USA
Received 8 April 1999; received in revised form 27 April 1999; accepted 28 April 1999
Abstract
The gene for CspA, the major cold-shock protein in Escherichia coli, is tightly regulated at both optimal and low
temperatures. While CspA is drastically induced after temperature downshift, it is hardly detectable at 37³C. Here we
demonstrate that the deletion of parts of the 5P-untranslated region (5P-UTR) of the cspA mRNA results in constitutive
expression of CspA at 37³C. By analyzing the amounts and the stabilities of the mRNAs produced from the deletion
constructs, we rule out the possibility that the CspA production is due to the stabilization of the mutant mRNAs. We propose
that significant premature termination or pausing occurs during the transcription of the unusually long 5P-UTR of the cspA
mRNA at 37³C, which represents a new mechanism that contributes to the tight repression of CspA production at higher
temperature. z 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights
reserved.
Keywords : Transcription; cspA; Cold shock; 5P-Untranslated region
1. Introduction
When exponentially growing Escherichia coli cells
are shifted from 37³C to low temperature, cells stop
growing as the synthesis of most cellular proteins is
either shut o¡ or signi¢cantly reduced. This growth
lag period is termed the acclimation phase, during
which cold-shock proteins such as CspA [1], CspB
[2], CspG [3] and CspI [4] are speci¢cally and transiently induced (see [5,6] for review). At the end of
* Corresponding author. Fax: +1 (732) 2354559;
E-mail: [email protected]
1
These authors contributed equally to this work.
the acclimation phase cells become adapted to the
low temperature as the synthesis of non-cold-shock
proteins is reinitiated, resulting in the resumption of
cell growth.
The gene for CspA, the major cold-shock protein
of E. coli, is tightly regulated in a temperature-speci¢c manner ([2,7]). In contrast to its dramatic induction after temperature downshift, CspA is barely
detectable at 37³C. It has been shown that mRNA
stability plays an important role in the regulation of
cspA gene expression [8^10]. Even though the cspA
promoter is constitutively active at 37³C, its transcripts are hardly translated because of the extreme
instability of its mRNA at that temperature.
0378-1097 / 99 / $20.00 ß 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
PII: S 0 3 7 8 - 1 0 9 7 ( 9 9 ) 0 0 2 1 6 - 5
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L. Fang et al. / FEMS Microbiology Letters 176 (1999) 39^43
An interesting feature of the cspA mRNA is its
unusually long (159 bases) 5P-untranslated region
(UTR) [7]. We have shown that overexpression of
the entire UTR or even as short as its ¢rst 25 bases
caused the prolonged production of CspA from the
chromosomal cspA gene at low temperature [11].
Such overexpression of the 5P-UTR is considered to
saturate a putative negative regulator whose binding
to the 5P-UTR blocks the cspA transcription or destabilizes its mRNA.
2. Materials and methods
2.1. Strains and media
E. coli strain CL83 [recA ara(lac-proAB)
rpsL(=strA) x80 lacZM15] ([12]) was used for plasmid puri¢cation. Because the E. coli major outer
membrane lipoprotein migrates very close to CspA
on an SDS-PAGE gel, a lpp3 strain was ¢rst isolated
from CL83 by screening for globomycin-resistant
colonies. Globomycin is known to inhibit the biosynthesis of the lipoprotein, causing the accumulation of
its precursor and killing the cells [13]. A strain thus
isolated was con¢rmed to be lpp3 by SDS-PAGE,
named CL83lpp, and used in all other experiments.
M9-glucose medium supplemented with all the amino acids except methionine was used for pulse-label
experiment. M9-glucose medium supplemented with
0.2% casamino acids was used for RNA extraction.
2.2. Plasmid construction
p2TACR was constructed as follows. First, the
PCR fragment obtained with primers 3549 (5PCGGCATTAAGTAAGCAGTTG-3P) and 3550 (5PTAATTAAGTGTGCCTTTCGG-3P) was cloned
into the SmaI site of pUC19. Then the cspA coding
region together with the Shine-Dalgarno (SD) sequence, ampli¢ed by primers 6923 (5P-CCGGTACCTAAAGGTAATACACT-3P) and 6922 (5P-CCGAATTCACAGGCTGGTTAC-3P), was cloned between
the EcoRI and KpnI sites. Finally, the transcriptional
terminator of cspA was ampli¢ed by PCR using
primers 6290 (5P-CGGAATTCAGCCTGTAATCTCT-3P) and 4860 (5P-CTGTCGACTTACTTACGGCGTTGC-3P) and cloned between the EcoRI and
SspI sites. The ¢nal construct is identical to the
wild-type cspA gene, except for having an extra
KpnI site-containing sequence of GGGTACC upstream of the SD sequence.
p2HTACR, p2JTACR and p6mTACR were constructed in the same way as was p2TACR, except
that the ¢rst PCR was carried out with the following
pairs of primers: for p2HTACR, primers 3150 (5PGTCCTGATGACAGACCG-3P) and 3846 (5P-GAATGTGCACTACG-3P); for p2JTACR, primers
3549 (5P-CGGCATTAAGTAAGCAGTTG-3P) and
4428 (5P-CTGGATCCTTTAATGGTCTGTACGTCAAACCGT-3P); and for p6mTACR, primers
3552 (5P-GACAGGATTAAAATCGAG-3P) and
6196 (5P-AACCGTTGATGTGCA-3P).
Plasmid pJJG02, which contains the full-length
wild-type cspA gene, was used as template for all
the PCR ampli¢cations. All the PCR ampli¢ed sequences were con¢rmed by manual sequencing.
2.3. Pulse-labeling of cells and SDS-PAGE
CL83lpp cells transformed with the above plasmids were grown in M9-glucose medium supplemented with all amino acids except methionine. During the mid-exponential phase 0.5 ml of each culture
was pulse-labeled with 30 WCi [35 S]methionine for
5 min and chased for 3 min by adding 100 Wl 30
mg ml31 non-radioactive methionine. Cells were
washed with 20 mM sodium phosphate bu¡er (pH
7.0) and dissolved in 30 Wl SDS-loading bu¡er. After
incubating in a boiling waterbath for 5 min, 10 Wl of
each lysate was analyzed by SDS-PAGE on a 17.5%
gel.
2.4. RNA extraction and primer extension analysis
RNAs were extracted from 1.5-ml fractions of exponentially growing cells by the hot-phenol method
as described [14]. The amounts of RNAs were determined by both OD260 measurement and 1% agarose
gel electrophoresis. An equal amount (3 Wg) of each
RNA was subjected to primer extension assay as
described [15]. Oligonucleotide 3551 (5P-TTTAGAGCCATCGTCAGGAG-3P), which is complementary to the +65^+84 region of cspA coding sequence, was end-labeled with [Q-32 P]ATP using T4
polynucleotide kinase and used as a primer.
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41
3. Results
3.1. E¡ect of deletion mutations in the 5P-UTR on
CspA production
To study the role of the 5P-UTR in the regulation
of cspA at 37³C, we constructed a series of plasmids
containing the intact cspA promoter and the entire
coding sequence but with di¡erent deletions in the
5P-UTR (Fig. 1A). CL83lpp cells transformed with
the above plasmids were pulse-labeled and cellular
protein content analyzed by SDS-PAGE on a
17.5% gel. The cells harboring p2TACR did not pro-
Fig. 2. mRNAs produced from the UTR deletion plasmids at
37³C. RNAs were extracted from cells carrying the deletion constructs at mid-exponential phase and 3 Wg of each RNA was analyzed by primer extension using 32 P-labeled primer 3551 as a
probe. A: Autoradiograph showing the primer extension products. Di¡erent sizes of the products re£ect various deletions in
the 5P-UTR of the cspA mRNA. B: Histograph showing the
amount of mRNAs as quantitated by densitometry, with the
amount of mRNA produced from p2TACR taken as one. Columns 1, 2, 3 and 4 correspond to p2TACR, p2HTACR,
p2JTACR and p6mTACR, respectively.
duce a discernible amount of CspA, while the cells
with plasmids containing 5P-UTR deletion mutations
produced signi¢cant amounts of CspA (Fig. 1B).
Moreover, the longer a deletion a plasmid had, the
more CspA it produced, as p6mTACR expressed the
highest level of CspA at 37³C and p2TACR an almost undetectable amount.
Fig. 1. Construction of cspA 5P-UTR deletion mutations and the
e¡ect of the mutations on the expression of CspA at 37³C. A:
Schematic representation of the 5P-UTR deletion constructs.
p2TACR contains full-length 5P-UTR ; p2HTACR has a 58-base
deletion from +86 to +143 ; p2HTACR has a 118-base deletion
from +26 to +143; p6mTACR has a 137-base deletion from +7
to +143, almost the entire 5P-UTR. Filled black box represents
the Shine-Dalgarno (SD) sequence. B: Autoradiograph showing
the pattern of protein synthesis of the cells harboring the above
plasmids. CL83lpp cells carrying the deletion constructs were
pulse-labeled with [35 S]methionine at 37³C. Total cell lysates were
analyzed on a 17.5% SDS-PAGE. The position of CspA is indicated.
3.2. Amounts of mRNAs transcribed from the deletion
plasmids
Next we analyzed the amount of the cspA mRNA
produced from these plasmids at 37³C. Total RNAs
were extracted and 3 Wg of each RNA was subjected
to primer extension assay. As the wild-type cspA
gene, p2TACR produced very little amount of transcript at 37³C (Fig. 2A). However, cells with the
plasmids containing UTR deletions generated increasingly greater amounts of the mRNA. The
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L. Fang et al. / FEMS Microbiology Letters 176 (1999) 39^43
amounts of mRNAs were quantitated by densitometry and plotted, with that of p2TACR taken as one.
As shown in Fig. 2B, the amount of transcripts produced from p2HTACR, p2JTACR and p6mTACR
was about 3.4, 18 and 50 times of that from
p2TACR, respectively. The di¡erent sizes of the
primer extension products completely agree with the
lengths expected from individual deletion mutations.
3.3. Stabilities of the mutant mRNAs
Since cspA expression at 37³C is known to be
regulated at the level of mRNA stability, we next
investigated whether mRNA stabilization contributed to the CspA production at 37³C observed
above. RNAs were extracted from mid-exponential
phase cells containing di¡erent plasmids at 0, 1, 2, 4,
and 8 min after the addition of rifampicin. 3 Wg of
each RNA was used for primer extension analysis.
As shown in Fig. 3, none of the mRNAs was signi¢cantly stabilized by the deletion mutations. It is important to note that the panel for p2TACR was exposed separately and for a much longer time than for
others.
4. Discussion
Since the mRNA amount of each construct
roughly parallels the amount of CspA produced at
37³C (Figs. 1 and 2A), it is unlikely that the derepression is mainly caused by increased translation
e¤ciency. In addition, the striking di¡erence in the
amounts of mRNAs cannot be adjusted by mRNA
stabilization (Fig. 3). Thus, the 5P-UTR of the cspA
mRNA appears to be involved in negative regulation
of the cspA gene expression at the level of transcription. We propose that a signi¢cant portion of the
cspA transcription at 37³C is prematurely terminated
within its 5P-UTR.
It seems that cspA has been evolved as a low-temperature-speci¢c gene. This is achieved by two mechanisms: the induction of cspA at low temperature
and its repression at higher temperature. The induction mechanisms include dramatic mRNA stabilization at low temperature and the enhanced translation
by having a downstream box in its coding sequence
[16]. As to the repression mechanisms, the cspA expression is e¤ciently inhibited by the extreme instability of its mRNA and by e¡ective premature termination or pausing of the cspA gene transcription
as suggested in this work. Since the cspA promoter is
highly active at both 37³C and 15³C, CspA appears
to be produced in a most e¡ective and urgent fashion
upon cold shock without depending on the de novo
synthesis of new protein factors [17]. These mechanisms are likely to be shared by other cold-shockspeci¢c genes in the cspA family such as cspB, cspG
and the newly characterized cspI [4], since they all
have 5P-UTRs of comparable length.
Acknowledgments
Fig. 3. Stabilities of the 5P-UTR deletion mRNAs. Total RNAs
were extracted from cells containing the deletion plasmids at 0,
1, 2, 4 and 8 min after adding rifampicin. 3 Wg of each RNA
was analyzed by primer extension using the same primer as in
Fig. 2. Autoradiograph shows the primer extension products.
Lanes 1, 2, 3, 4 and 5 correspond to time points 0, 1, 2, 4 and 8
min after the addition of rifampicin (200 Wg ml31 ¢nal concentration). p2TACR panel was exposed separately and for a longer
time than the others.
We thank Dr. Sangita Phadtare for critical reading
of the manuscript. This work is supported by a NIH
Grant (GM 19043) to M.I.
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