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Journal of Bacteriology, September 1999, p. 5852-5854, Vol. 181, No. 18
Department of Biochemistry, Robert Wood
Johnson Medical School, University of Medicine and Dentistry of New
Jersey, Piscataway, New Jersey 08854
Received 26 January 1999/Accepted 15 July 1999
Cold shock induction of cspB has been shown to be
primarily regulated at the mRNA level. Here, we demonstrate that the
induction of cspB at low temperature also requires the
translational cis-acting element called the downstream box
(DB). Full induction of cspB at low temperature is achieved
in the presence of both the Shine-Dalgarno sequence and DB. We propose
that the DB sequence functions as a translational enhancer for the
biosynthesis of CspB to bypass the inhibitory effect in translation
caused by cold shock.
Previously, we have shown that the
cold shock induction of cspB is regulated at the level of
transcription (2). Herein, we demonstrate that the
translational cis-acting element called the downstream box
(DB) is essential for the induction of cspB after a
temperature downshift. The presence of a DB sequence in the
cspB mRNA was reported earlier by Mitta et al.
(13). The role of the DB in translation initially proposed
by Sprengart and coworkers (16) indicates that the DB
sequence is complementary to a region in the penultimate stem of the
16S rRNA. Thereby, translation initiation is enhanced by an increase in
the affinity of the ribosome for a DB-containing mRNA (16).
It has been demonstrated that shifting Escherichia coli
cells to low temperature results in a global inhibition of cellular protein synthesis (8). However, under these conditions,
maximal levels of cold shock proteins are observed. Therefore, it has been speculated that cold shock mRNAs, unlike the majority of cellular
mRNAs, are capable of bypassing the protein synthesis inhibitory effect
triggered by cold shock (9). In addition, it has been
demonstrated that after a temperature downshift, translation is
inhibited at the initiation step (9). In this regard, Jones and Inouye (9) have proposed that cold shock mRNAs may be
efficiently translated due to a higher affinity of these mRNAs for the
ribosome at low temperature. Recently, we have demonstrated that
artificial DB sequences inserted in the lacZ gene cause a
significant translational enhancement (3). Herein, we
examined the role of the DB in the translational induction of
cspB, a major cold shock gene of E. coli, at low temperature.
Induction of cspB at low temperature requires DB.
E. coli AR137, a pcnB mutant which maintains
pBR322 derivative plasmids at a low copy number (11), was
transformed with a series of cspB-lacZ translational fusions
(Fig. 1). Figure 1A shows the
cspB-DB sequence located 5 codons downstream of the initiation codon and its complementarity to the anti-DB sequence in the
16S rRNA (from base 1467 to 1481). Figure 1B shows a series of
translational fusions in which cspB was fused to the
lacZ gene after codons 3, 13, and 17 to create pB3, pB13 and
pB17, respectively. Figure 2A shows
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
A Sequence Downstream of the Initiation Codon Is
Essential for Cold Shock Induction of cspB of
Escherichia coli
ABSTRACT
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-galactosidase activity at various time points after a temperature
shift from 37 to 15°C. After 2 and 3 h at 15°C, pB13 and pB17
show the highest levels of -galactosidase activity, indicating that
a region from codons 3 and 13 (containing DB) (see Fig. 1A) plays a
major role in high-level expression of cspB at low
temperature. Primer extension analysis (Fig. 2B) shows that the mRNA
levels of pB3 and pB17 between 1 and 2 h at 15°C are almost
identical, while the -galactosidase activity of pB17 is seven times
higher than that of pB3 (Fig. 2A). This indicates that the differences
in the sequence downstream of the initiation codon cause a significant
effect on the efficiency of mRNA translation but not on the amount of
the mRNAs. Based on the amounts of mRNAs estimated using a
phosphorimager and the increments of -galactosidase activity between
1 and 2 h after cold shock induction, the translational capability
of pB17 was calculated to be 6 times higher than that of pB13 and 18 times higher than that of pB3 (Table 1).
This could be explained by the potential for additional base pairing
between the cspB mRNA (pB17) and the 16S rRNA, as shown in
Fig. 3. There are an additional G-C base
pair and four extra base pairs immediately after codon 13. However, if
mismatches in either cspB mRNA and/or 16S rRNA are
incorporated, the number of G-C base pairs could increase up to four
and five together with other base pairs (Fig. 3B through D). These
extra nucleotides of pB17 complementary to the 16S rRNA possibly
increases the affinity of the pB17 mRNA for the ribosome and in turn
enhances its translation as compared with pB13. This suggests that a
longer DB (pB17) could be more effective for translation than a shorter
DB (pB13). In addition, it could be that specific base pairs between
the mRNA and the 16S rRNA and/or specific mRNA conformations may be
important for a higher translational activity.
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FIG. 1.
Construction of cspB-lacZ fusions. (A)
cspB-DB-anti-DB complementarity. The cspB-DB
sequence is boxed and encompasses the region from codons 5 to 9 (13). The AUG codon is circled, the SD sequence is boxed,
and the L-shaped arrows show the positions where the cspB
gene was fused to lacZ. (B) Translational
cspB-lacZ fusion constructs. On the top, the E. coli
cspB gene is depicted from its 5'-end. In pB3, pB13, and pB17,
cspB is fused to lacZ at residue +177 (3rd
codon), +200 (13th codon), and +212 (17th codon), respectively. The
cspB DNA fragments were amplified by PCR using synthetic
oligonucleotide primers containing a BamHI site at the 5'
end. A plasmid, pSJ7 (10) carrying the wild-type
cspB gene was used as a template DNA to create the PCR
fragments B3, B13, and B17. The 5'-end oligonucleotide primer used in
each of the above PCR reactions is 5'-CCGGATCCAGCTTTAATATAGCT-3'.
The 3'-end oligonucleotide primers for the PCR products B3, B13,
and B17 are 5'-CCGGATCCAGATTTGACATTCTACA-3',
5'-CCGGATCCAGGTTAAACCATTTT-3', and
5'-CCGGATCCAGACCTTTATCAGCGTT-3', respectively. A deletion of
the SD sequence in the cspB gene was created by
site-directed mutagenesis using the QuickChange site-directed
mutagenesis kit (Stratagene). The PCR reaction was carried out using
the pSJ7 plasmid as a template and the oligonucleotides Bsd1
(5'-GAAAGGCTCAAGTTACTTCATGTAGAATG-3') and Bsd2
(5'-CATTCTAC ATGAAGTAACTTGAGCCTTTC-3') to create pSJsd.
Then, pSJsd was used as a template to make the PCR fragments B13sd and
B17sd. The 5' and 3'-end oligonucleotide primers used in these PCR
reactions are the same as the one used for the PCR fragments B13 and
B17. All the above PCR products were cloned at the BamHI
site of the pRS414 vector (10, 15) to create the pB3, pB13,
pB13sd, pB17, and pB17sd constructs.
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FIG. 2.
Enhancement of cspB translation by the DB.
(A) -Galactosidase activity of the cspB-lacZ constructs
obtained before (time zero) and 1, 2, and 3 h after temperature
shift from 37 to 15°C. E. coli AR137 cells transformed
with pB3, pB13, pB13sd, pB17, and pB17sd were grown in Luria-Bertani
medium, and at mid-log phase (optical density at 600 nm = 0.4)
cultures were shifted from 37 to 15°C. -Galactosidase activity was
measured as described by Miller (12). (B) mRNA levels of
pB3, pB13, pB13sd, and pB17 after temperature shift from 37 to 15°C.
The cspB-lacZ mRNAs were detected by primer extension as
described previously (13) at the same time points described
above (panel A). (C) Stabilities of pB3, pB13, pB13sd, and pB17 mRNA.
E. coli AR137 cells transformed with pB3, pB13, pB13sd, and
pB17 were grown under the same conditions as those described above. At
mid-log phase the culture was shifted to 15°C, and after 30 min
rifampin was added to a final concentration of 0.2 mg/ml (time zero).
Total RNA was extracted at 5, 10, 20, and 40 min after rifampin
addition. The cspB-lacZ mRNA was detected by primer
extension as described previously (13).
TABLE 1.
Effect of the DB on translation of cspB at
low temperaturea
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FIG. 3.
Additional base pairing between the cspB mRNA
and the 16S rRNA. (A) Additional base pairs between the pB17 mRNA
(within the sequence from codon 13 to 17) and the 16S rRNA with no
mismatches. (B) pB17 mRNA (within the sequence from codon 13 to 17)-16S
rRNA base pairs with two mismatches in the cspB mRNA. (C)
Same as in panel B with three mismatches in the pB17 mRNA and one
mismatch in the 16S rRNA. (D) Additional base pairs between pB17 mRNA
and 16S rRNA with multiple mismatches in both of them allowed.
Requirement of the SD sequence for the DB activity.
In order
to know if the Shine-Dalgarno (SD) sequence is required for the DB
sequence to exert its function as a translational enhancer, the SD
sequence from pB13 and pB17 was mutated from AGGA to CTTC to create
pB13sd and pB17sd, respectively (Fig. 1A). The low levels of
-galactosidase units from the SD deletion constructs pB13sd and
pB17sd demonstrate that the SD sequence is required for DB-enhanced
cspB expression (Fig. 2A). When the SD sequence was deleted
from pB13, the translational efficiency became even lower than that of
pB3 at low temperature; the translational efficiency of pB13sd was
reduced to 25% of that of pB3 (Table 1). In addition, although the
mRNA half-lives of pB3 (12 min) and pB13sd (17 min) are similar, the
-galactosidase activity of pB3 is four times higher than that of
pB13sd. This result clearly indicates that the role of the DB in
translation initiation is dependent on the SD sequence.
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ACKNOWLEDGMENTS |
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The present work was supported by a grant from the National Institute of Health (GM19043).
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FOOTNOTES |
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* Corresponding author. Mailing address: Dept. of Biochemistry, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, Piscataway, NJ 08854. Phone: (732) 235-4115/4540. Fax: (732) 235-4559/4783. E-mail: inouye{at}rwja.umdnj.edu.
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Journal of Bacteriology, September 1999, p. 5852-5854, Vol. 181, No. 18
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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