Translational Enhancement by an Element Downstream of the
Initiation Codon in Escherichia coli*
Jean-Pierre
Etchegaray and
Masayori
Inouye
From the Department of Biochemistry, University of Medicine and
Dentistry of New Jersey, Robert Wood Johnson Medical School,
Piscataway, New Jersey 08854
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ABSTRACT |
The translation initiation of Escherichia
coli mRNAs is known to be facilitated by a cis
element upstream of the initiation codon, called the Shine-Dalgarno
(SD) sequence. This sequence complementary to the 3' end of 16 S rRNA
enhances the formation of the translation initiation complex of the
30 S ribosomal subunit with mRNAs. It has been debated that a
cis element called the downstream box downstream of the
initiation codon, in addition to the SD sequence, facilitates formation
of the translation initiation complex; however, conclusive evidence
remains elusive. Here, we show evidence that the downstream box plays a
major role in the enhancement of translation initiation in concert with
SD.
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INTRODUCTION |
Sprengart and co-workers (1, 2) have proposed that in genes
0.3 and 10 of bacteriophage T7, a specific region
located downstream of the initiation codon serves as an independent
translational signal. This region of the RNA sequence designated as the
downstream box (DB)1 is
complementary to bases 1469-1483 within the Escherichia
coli 16 S rRNA (anti-DB sequence). It is speculated that
formation of a duplex between the DB and anti-DB of 16 S rRNA is
responsible for translational enhancement (2). The DB sequence has also been implicated in the translation of the
c1 mRNA, an
mRNA that lacks any untranslated region and the SD sequence (3).
Interestingly,
c1 translation was enhanced at 42 °C in
a temperature-sensitive strain in which the amount of ribosomal protein
S2 decreased at 42 °C. It was proposed that the anti-DB sequence in
S2-deficient ribosomes indirectly becomes more accessible to DB,
resulting in enhancement of translation initiation of the
c1
mRNA (3, 4). However, the role of the DB in
c1
translation initiation was disputed by Resch and co-workers (5). These
authors constructed lacZ translational fusions with the
c1 gene to test the DB function. Since a deletion of 6 bases encompassing a portion of the DB sequence did not reduce the
formation of the translation initiation complex, they disputed the
existence of DB. Despite these elusive roles of DB (6), we have shown
that the presence of a DB sequence in cold-shock mRNAs plays an
important role in translation efficiency, and we proposed that the DB
is involved in the formation of a stable initiation complex at low
temperature before the induction of cold ribosomal factors (7).
Furthermore, we pointed out the SELEX enrichment of DB-like sequences
in an mRNA when the 30 S ribosomal subunit was used as a ligand
(8, 9).
It was suggested that the results obtained by Resch et al.
(5) could be explained by recreating a new DB as a result of the
deletion of the original DB in the
c1 mRNA (6).
Indeed, a 6-base deletion eliminating 5 out of 8 matches in the
original DB recreated a new 9-base matching DB sequence including the
initiation codon. Therefore, the results by Resch and collaborators (5) could be explained by the newly created DB. In the present paper, we
performed both biochemical and genetic experiments to reexamine the
role of the DB in translation initiation, and we determined that DB
plays a crucial role in regulation of gene expression in E. coli by enhancing the formation of the translation initiation complex.
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EXPERIMENTAL PROCEDURES |
Plasmid Construction--
The cspA-lacZ fusion
constructs were made by the insertion of annealed oligonucleotides
at the EcoRI site of the pJJG78 (10). Annealed
oligonucleotides DB1 (5'-AATTAATCACAAAGTGGG-3') with DB1'
(5'-AATTCCCACTTTGTGATT-3') or DB2 (5'-AATTATGAATCACAAAGTGGG-3') with
DB2' (5'-AATTCCCACTTTGTGATTCAT-3') were used to create pJJG78DB1 or pJJG78DB2 constructs, respectively.
The pIN-lacZ constructs were made by inserting the
XbaI-SalI fragments from pJJG78 or pJJG78DB2 into
the XbaI-SalI sites of pIN-III (10) to create
pINZ and pINZDB1, respectively. Then, the annealed
oligonucleotides ZDB2
(5'-CTAGCCCTTATTAATAATGAAAGGGGGAATTATGAATCACAAAGTGGG-3') with ZDB2'
(5'-AATTCCCACTTTGTGATTCATAATTCCCCCTTTCATTATTAATAAGGG-3') were inserted
at the XbaI-EcoRI sites of pINZ to create
pINZDB2. Annealed oligonucleotides ZDB3
(5'-CTAGCCCTTATTAATAATGAATCACAAAGTGGG-3') with ZDB3'
(5'-AATTCCCACTTTGTGATTCATTATTAATAAGGG-3') or ZDB4
(5'-CTAGAGGGTATTAATAATGAATCACAAAGTGGG-3') with ZDB4'
(5'-AATTCCCACTTTGTG-ATTCATTATTAATACCCT-3') were inserted at the
XbaI-EcoRI sites of pINZ to construct pINZDB3 and
pINZDB4, respectively.
-Galactosidase Activity--
E. coli AR137
(pcnB
) (11) or JM83
(pcnB+) harboring different plasmids were grown
at 37 °C to mid-log phase in 20 ml of LB medium containing 50 µg/ml ampicillin in a 125-ml flask. The cultures were then
transformed to a 15 °C shaking water bath, or
isopropyl-
-D-thiogalactopyranoside (IPTG, 1 mM) was added to a final concentration of 1 mM.
A 100-µl culture was taken at each time point.
-Galactosidase
activity was measured according to Miller's procedure (12).
Primer Extension--
E. coli AR137
(pcnB
) or JM83 (pcnB+)
carrying different plasmids were grown under the same condition as used
for the
-galactosidase assay described above. To estimate the
mRNA amounts of the lacZ fusion constructs, 1.5 ml of
culture was taken at each time point, and total RNA was extracted by
the hot phenol method (13).
For the mRNA stability experiments at 15 °C, rifampicin (0.2 mg/ml) was added 30 min after the temperature downshift from 37 °C.
The mRNA amounts from the cspA-lacZ fusion constructs
were estimated by primer extension using the 32P-labeled
M13-47 antisense primer complementary to the region of lacZ
between codons 14 and 22 as described previously (7). The reverse
transcription reaction was carried out with AMV-RT according to the
manufacturer's procedure (Boehringer Mannheim), and the cDNA
products were resolved on a 6% Sequencing Gel and quantified by
PhosphorImager (Bio-Rad).
Pulse Labeling--
Cultures of E. coli AR137
(pcnB
) cells carrying pINZ or pINZDB1 were
grown at 37 °C under the same conditions used for the
-galactosidase assay. IPTG (1 mM) was added at mid-log
phase to each culture. At each time point, 1 ml of the culture was
labeled for 5 min with 100 µCi of
trans-[35S]methionine (1, 175 Ci/mmol) (NEN
Life Science Products) as described previously (14). Cell extracts from
each time point were loaded on a 5% SDS-PAGE, and
-galactosidase
synthesis was measured by PhosphorImager.
Ribosome Isolation--
Cultures of E. coli JM83
(pcnB+) cells carrying pINZ or pINZDB1 were
grown in 600 ml of LB medium in a 4-liter flask under the same
condition as described above. At mid-log phase IPTG (1 mM)
was added to each culture. Ribosomal particles were isolated by the
procedure described by Dammel and Noller (15) with some modifications
as follows: a 100-ml aliquot from the original culture was taken at
each time point, and chloramphenicol was added to a final concentration
of 0.1 mg/ml to stop cell growth. Cells were immediately collected by
centrifugation (5,000 × g for 10 min at 4 °C),
resuspended in buffer 1 (20 mM Tris-HCl (pH 7.6), 10 mM MgCl2, 100 mM NH4Cl,
6 mM
-mercaptoethanol and 1 mg/ml lysozyme), and frozen
at
80 °C for few hours. The cells were lysed by the freeze-thaw
method (16). The cell extracts (0.5 ml) were then layered on top of a
5-40% (w/w) sucrose gradient (7.5 ml), and the polysomes and
ribosomal subunits were separated by centrifugation at 151,000 × g for 2.5 h at 4 °C using a Beckman SW-41 rotor. The
polysome profiles were detected by a fast protein liquid chromatography
system, and a total of 15 fractions of 0.5 ml each were collected.
Detection of the lacZ-mRNA--
From each polysome fraction
(0.5 ml) 0.2 ml was spotted on a nitrocellulose membrane using the
Minifold II Slot-Blot System (Schleicher & Schuell). The
lacZ-mRNA was detected by hybridization using the
32P-labeled M13-47 primer (17), and the amount of
lacZ-mRNA was estimated by PhosphorImager.
In Vitro Translation--
Using the E. coli S30
Extract System for Linear Templates Kit (Promega), the
transcription-translation coupled reaction was carried out according to
the manufacturer's protocol as follows. To 20 µl of pre-mix
containing all the amino acids except for methionine, 10 µCi of
trans-[35S]methionine (1,175 Ci/mmol; NEN Life
Science Products) and the E. coli S30 extracts, 160 ng of
pINZ or pINZDB1 (1 µl), were added, and the mixture was incubated at
37 °C for 45 min. The products were precipitated with acetone and
analyzed by 15% SDS-PAGE.
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RESULTS |
The Effect of a Perfectly Matching DB--
It has been previously
shown that DB is essential for the production of CspA at low
temperature (7). However, the wild-type DB of cspA has 10 matches out of 15 possible matches to the anti-DB in 16 S rRNA (7).
Therefore, we added DBs of 12 (pJJG78DB1) or 15 (pJJG78DB2) bases that
are complementary with the anti-DB of 16 S rRNA to the site after the
5th codon of lacZ under the cspA regulatory
system in pJJG78 (see Fig. 1A)
to examine if they enhance lacZ expression at 15 °C.
Mid-log phase cells (pcnB
) grown at 37 °C
were shifted to 15 °C, and
-galactosidase activity was measured
at 1, 2, and 3 h after the shift. Fig. 1B shows that at
1 h at 15 °C the
-galactosidase activity was 3- and 8-fold higher with pJJG78DB1 and pJJG78DB2, respectively, than with pJJG78. After 2 and 3 h at 15 °C, the
-galactosidase activity was
increased 3.5 and 10.5 times with pJJG78DB1 and pJJG78DB2,
respectively, than with pJJG78 (Fig. 1B). Moreover, the
effect of the DB was observed at 37 °C in which the
-galactosidase activity of pJJG78DB1 and pJJG78DB2 was 2- and 4-fold
higher as compared with pJJG78. The amount of the lacZ
mRNA (Fig. 1C) at each time point as well as the
mRNA stability (Fig. 1D) did not vary significantly
between these constructs. The lacZ mRNA half-life from
pJJG78, pJJG78DB1, and pJJG78DB2 was calculated to be 27, 23, and 25 min, respectively. In addition, computer analysis (18) revealed no
significant differences in the mRNA secondary structures among
pJJG78, pJJG78DB1, and pJJG78DB2, suggesting that the insertion of the
perfectly matching DB may not have a particular effect in the mRNA
secondary structures that could account for the difference in their
-galactosidase expression. These results indicate that DB functions
as a translational enhancer and that greater complementarity to the
anti-DB improves translational efficiency and/or that specific base
pairings like the first three nucleotides of the DB from pJJG78DB2 may
play an important role for the DB activity.

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Fig. 1.
Perfectly matching DB enhances the
translation of cspA. A, translational
cspA-lacZ fusion constructs. The cspA
gene structure from its 5' end is shown at the top.
pJJG78DB1 and pJJG78DB2 were constructed from pJJG78 as described under
"Experimental Procedures." The DB sequences of pJJG78DB1 (12 matches) and pJJG78DB2 (15 matches) are shown at the bottom.
B, -Galactosidase activity of the cspA-lacZ
fusion constructs after cold shock at 15 °C. E. coli
AR137 cells transformed with pJJG78, pJJG78DB1, or pJJG78DB2 were grown
in LB medium, and at mid-log phase (A600 = 0.4)
cultures were shifted from 37 to 15 °C. -Galactosidase activity
was measured before (time 0) and 1, 2, and 3 h after
the shift. C, detection of the cspA-lacZ
mRNAs. Total RNA from E. coli AR137 cells carrying
pJJG78, pJJG78DB1, or pJJG78DB2 was extracted at the same time points
indicated above (B) and used as a template for primer
extension as described under "Experimental Procedures."
D, mRNA stability from the cspA-lacZ
constructs. E. coli AR137 cells transformed with pJJG78,
pJJG78DB1, and pJJG78DB2 were grown as described above. At mid-log
phase the cultures were shifted to 15 °C, and after 30 min
rifampicin was added to a final concentration of 0.2 mg/ml (time
0). Total RNA was extracted at 5, 10, and 40 min after rifampicin
addition. The cspA-lacZ mRNAs were detected by primer
extension as described under "Experimental Procedures."
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DB Functions at 37oC--
The experiments described
above were carried out at 15 °C. In order to examine whether DB also
works at 37 °C, the cspA cold-shock regulatory regions
upstream of SD of pJJG78 and pJJG78DB2 were replaced with the
constitutive lpp promoter and the lac
promoter-operator fragment using a pINIII vector (19), yielding pINZ
and pINZDB1, respectively (Fig.
2A). Cells
(pcnB
) transformed with pINZ or pINZDB1 showed
very low
-galactosidase activity in the absence of IPTG, an inducer
of the lac promoter (Fig. 2B, time 0).
Upon the addition of 1 mM IPTG,
-galactosidase activity
was induced in both cells. After 3 h induction,
-galactosidase activity increased 18- and 37-fold for pINZ and pINZDB1, respectively (Fig. 2B). However, the levels of
-galactosidase activity
show a dramatic difference between the two; the activity with DB
(pINZDB1) was 34 times higher than that without DB (pINZ),
demonstrating that DB functions at 37 °C as well. Specific
activities of
-galactosidase produced from vector pINZ and pINZDB1
are almost identical (data not shown), and thus the addition of the
five amino acid residues in the
-galactosidase sequence of pINZDB1
(due to DB) does not affect the enzymatic activity. Furthermore, the
stabilities of
-galactosidase from pINZ and pINZDB1 are also
identical with a half-life of approximately 3 h (data not
shown).

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Fig. 2.
Perfectly matching DB enhances translation at
37 °C. A, pIN-lacZ constructs. The
XbaI-SalI fragment from pJJG78 or pJJG78DB2 was
inserted into the XbaI-SalI sites of pIN-III to
create pINZ and pINZDB1, respectively, which then were used to create
pINZDB2, pINZDB3, and pINZDB4 as described under "Experimental
Procedures." B, -Galactosidase activity of the
pINZ-lacZ constructs. Cultures of E. coli AR137
cells transformed with pINZ, pINZDB1, pINZDB2, pINZDB3, and pINZDB4
were grown at 37 °C under the same conditions described in Fig. 1.
IPTG (1 mM) was added at mid-log phase to each culture.
-Galactosidase activity was measured before (time 0) and
at 0.5, 1, 2, and 3 h after IPTG addition. C, mRNA
sequences of the pIN-lacZ constructs showing the position of
SD, AUG, and DB. The lacZ in pJJG78 has a 10-match DB. The
perfectly matching DB located after the 5th codon has 16 residues
complementary with the anti-DB.
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SD Requirement for DB Function--
Next, we examined whether DB
functions independently from SD, the initial ribosome-binding site. For
this purpose, the SD sequence of pINZDB1, GAGG was changed to GCCC,
yielding pINZDB2 (Fig. 2, A and C). The
-galactosidase level of pINZDB2 induction was reduced to 1/7 of that
of pINZDB1 but still 3-fold higher than that of pINZ at 3 h after
IPTG induction (Fig. 2B). However, since pINZDB2 has the
second AUG codon 6 codons downstream as a result of DB insertion, it
might serve as a secondary initiation codon for the lacZ
gene. Indeed, the N-terminal sequence analysis showed that 100% of the
-galactosidase produced from pINZDB2 is initiated at the second AUG
codon as compared with pINZDB1, which is more than 90% initiated at
the first AUG codon (data not shown). Furthermore, the second AUG codon
is presided by a potential but poor SD sequence (AAGG) at the region
corresponding to the 2nd and 3th codons (underlined; Fig.
2C). Indeed, when this secondary SD was removed by deletion
of the 15-base sequence (codons 1-5; pINZDB3 in Fig.
2A),
-galactosidase activity at all time points was
reduced to the background level (Fig. 2B), indicating that
the secondary SD played a crucial role in the translation of the
pINZDB2 lacZ mRNA. When the SD sequence was recreated by
5-base substitution in pINZDB3 (pINZDB4; Fig. 2C),
-galactosidase activity of this construct was recovered to a comparable level to that of pINZDB1 (Fig. 2B). It is
important to notice that the DB sequence starting from the first AUG
codon was eliminated in pINZDB4 (Fig. 2C). Therefore, the
high expression of
-galactosidase from pINZDB1 and pINZDB4 is due to
the perfectly matching DB sequence (Fig. 2B). These results
indicate that (a) DB functions only in the presence of SD,
and (b) the position of DB is flexible starting from either
codon 1 or 6.
Enhancement of Protein Synthesis by DB--
The
-galactosidase
activity shown in Fig. 2B indicates that DB enhances the
translation of pINZDB1. Therefore, in order to test the effect of the
DB in translation efficiency, the rate of
-galactosidase synthesis
from pINZ and pINZDB1 was analyzed. The rate of
-galactosidase
synthesis was measured by pulse labeling cells for 5 min with
[35S]methionine after the addition of IPTG using cells
harboring pINZ and pINZDB1. After SDS-PAGE, the amounts of radioactive
-galactosidase were estimated using a PhosphorImager (Fig.
3). Prior to the addition of IPTG, the
rate of
-galactosidase synthesis from pINZ and pINZDB1 was
identical. However, upon IPTG induction the rates of
-galactosidase synthesis from pINZDB1 were continuously increasing at each time point,
whereas the rate of
-galactosidase synthesis from pINZ was almost
not affected. After 4 h of IPTG addition the rate of
-galactosidase synthesis from pINZDB1 was 6.5 times higher than that
of pINZ. This result demonstrates that DB enhances the translation efficiency of pINZDB1 as reflected by the increment in the synthesis of
-galactosidase.

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Fig. 3.
Rate of
-galactosidase synthesis of the
pINZ-lacZ constructs. Cultures of E. coli AR137 cells carrying pINZ or pINZDB1 were grown at 37 °C
under the same conditions described above. IPTG (1 mM) was
added at mid-log phase to each culture. Rate of -galactosidase
synthesis was measured before (time 0) and 0.5, 1, 2, 3, and
4 h after IPTG addition. Cells were pulse-labeled with
trans-[35S]methionine as described under
"Experimental Procedures." Cell extracts from each time point were
analyzed by 5% SDS-PAGE, and the -galactosidase synthesis was
measured by PhosphorImager. The rate of -galactosidase synthesis
from pINZ (filled circles) and pINZDB1 (open
circles) is shown at each time point.
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Enhancement of Translation Initiation by DB--
In order to
examine whether DB enhances translation initiation, we next analyze the
ability of lacZ mRNA from pINZ and pINZDB1 to form
polysomes. For this experiment, pcnB+ cells were
used to amplify the effect of DB. Interestingly, cells with pINZDB1
could not form colonies on LB plates in the presence of 1 mM IPTG, whereas cells with pINZ formed colonies. The
lethal effect of IPTG on the cells with pINZDB1 is considered to be due to overexpression of
-galactosidase. After the addition of IPTG, cell growth was stopped by the addition of chloramphenicol (0.1 mg/ml)
at 15, 30, and 60 min, and then polysome profiles were examined as
shown in Fig. 4. From each gradient
fraction (500 µl), 200 µl were spotted on a nitrocellulose
membrane, and the amount of the lacZ mRNA analyzed using
a 24-base antisense oligonucleotide (M13-47 oligonucleotide). The
amounts of the lacZ mRNA were quantified by a
PhosphorImager and are displayed in Fig. 4. Although the polysome
profiles are similar, there are significant differences in the
distribution of the lacZ mRNA; at 15 min the
lacZ mRNA mainly exists in the upper half of the
gradient (fraction 8-14, corresponding to 70 S to 30 S ribosomes)
with pINZ, while with pINZDB1 a major peak (fraction 3 to 8) is formed
in the lower half of the gradient. At 30 min, the lacZ
mRNA from pINZ moved to the position of the 70 S ribosome, whereas
the lacZ mRNA from pINZDB1 maintained a similar pattern
as that at 15 min. At 60 min a major fraction of the lacZ
mRNA from pINZ remained in the upper half of the gradient, whereas
the lacZ mRNA from pINZDB1 was broadly distributed from
higher order polysomes to 70 S ribosome fraction. Therefore, the
reason why cells harboring pINZDB1 could not form colonies on LB plates
containing 1 mM IPTG may be due to a decrease in the
concentration of free ribosomes as a result of the massive expression
of a highly translatable DB-containing mRNA (20). These results
indicate that DB enhances the efficiency of polysome formation probably
due to a translation initiation enhancement.

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Fig. 4.
Ribosomal fractionation of E. coli
JM83 cells transformed with pINZ or pINZDB1. Ribosomal
particles were isolated as described by Dammel and Noller (15).
Cultures of E. coli JM83 cells carrying pINZ or pINZDB1 were
grown at 37 °C in LB medium containing 50 µg/ml ampicillin. At
mid-log phase (A600 = 0.4) 1 mM of
IPTG was added to each culture. Chloramphenicol (0.1 mg/ml) was added
at 15, 30, and 60 min after IPTG addition. The cell extracts prepared
as described under "Experimental Procedures" were then layered on
top of a 5-40% (w/w) sucrose gradient. The polysomes and ribosomal
subunits were separated by centrifugation at 151,000 × g for 2.5 h at 4 °C. The polysome profiles were then
detected by using a fast protein liquid chromatography system. 0.2 ml
from each fraction (0.5 ml) were spotted on a nitrocellulose membrane
using the Minifold II Slot-Blot System (Schleicher & Schuell). The
lacZ mRNA was detected by hybridization using the
32P-labeled M13-47 as described under "Experimental
Procedures." PhosphorImager values from the hybridization are plotted
at the right. The pINZ and pINZDB1 mRNAs are shown in
closed and open squares, respectively.
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In order to estimate the exact effect of DB from the above experiment,
the amount of the lacZ mRNA and the
-galactosidase activity were measured at the same time points taken in the polysome profiles (15, 30, and 60 min after IPTG induction). As shown in Fig.
5A, the amounts of the
lacZ mRNA reached almost the maximal level at 15 min for
both pINZ and pINZDB1. The PhosphorImager analysis of this result
revealed that the amounts of the pINZDB1 mRNA are 1.5, 1.4, and 1.3 times higher than those of the pINZ mRNA at 15, 30, and 60 min,
respectively. The higher mRNA levels for pINZDB1 are probably
attributable to the highly efficient polysome formation of pINZDB1 that
may stabilize the mRNA (21). The induction of
-galactosidase
activity is shown in Fig. 5B. In the case of pINZDB1, the
activity is very high even in the absence of IPTG, and upon the
addition of IPTG, it increased from 18,500 to 64,400 units (3.5-fold)
after 2.5 h incubation. In the case of pINZ, the background
activity prior to IPTG induction was much lower, and it increased from
900 to 2,900 units (3-fold) at the 2.5-h time point. The increment of
the
-galactosidase activity of pINZDB1 between 30 and 60 min is 35 times higher than that of pINZ, and therefore the efficiency of
-galactosidase production for pINZDB1 is calculated to be 26 times
higher than that for pINZ on the basis of the amount of mRNA.
Therefore, the higher levels of
-galactosidase production from
pINZDB1 are due to a high efficiency of polysome formation.

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Fig. 5.
Translational enhancement by a perfectly
matching DB at 37 °C. A, estimation of pINZ and
pINZDB1 mRNAs. Cultures of E. coli JM83 carrying pINZ or
pINZDB1 were grown at 37 °C under the same conditions described in
Fig. 4. Total RNA extracted at 15, 30, and 60 min after IPTG (1 mM) addition was used as a template for primer extensions
according to the procedure described previously (7). B,
-galactosidase activity of pINZ and pINZDB1 in multi-copy expression
system. E. coli JM83 cells transformed with pINZ or pINZDB1
were grown at 37 °C under the same condition described in Fig. 4.
-Galactosidase activity was measured before (time 0) and 0.5, 1, 1.5, 2, and 2.5 h after IPTG (1 mM) addition
(open circles and squares). Closed
circles and squares represent the activities in the
absence of IPTG.
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Next, in order to demonstrate more directly the translation-enhancement
effect of DB, the
-galactosidase synthesis was examined in a
cell-free system using pINZ and pINZDB1. The
[35S]methionine incorporation into
-galactosidase
(band G) with pINZDB1 (2nd lane, Fig.
6A) was 8-fold higher than
that with pINZ (1st lane), whereas the
-lactamase (band L) production was almost identical in both lanes.
Fig. 6B shows a time course of in vitro production of
-galactosidase from pINZ and pINZDB1 performed as
described above. The same reaction was carried out with non-radioactive methionine, spotted on a nitrocellulose membrane, and hybridized with
the M13-47 oligonucleotide as described under "Experimental Procedures." As shown in Fig. 6C, at each time point the
amount of lacZ mRNA from pINZ and pINZDB1 was almost
identical. This result supports the role of the DB as a translational
enhancer from the in vivo data described above.

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Fig. 6.
Cell-free synthesis of
-galactosidase from pINZ and pINZDB1.
A, PINZ or pINZDB1 DNA (160 ng; 1 µl) was added to the
E. coli 30 S extract from Promega, and the
transcription-translation-coupled reaction was carried out as described
under "Experimental Procedures"). 1st lane,
pINZ DNA; 2nd lane, pINZDB1 DNA; and
3rd lane, a control reaction without added DNA.
Samples were precipitated with acetone and analyzed by 15% SDS-PAGE to
detect the production of -galactosidase. band G,
-galactosidase; band L, -lactamase. B, time
course in vitro synthesis of -galactosidase from pINZ and
pINZDB1 was carried out as described above. Samples were taken after
15, 30, 60, and 120 min incubation at 37 °C. C, each
reaction from the time course experiment described above was done in
duplicate with non-radioactive methionine, spotted on nitrocellulose
membrane, and hybridized with 32P-labeled M13-47
oligonucleotide as described under "Experimental Procedures."
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Further Enhancement of DB-dependent Translation by
S2ts--
It has been proposed that in the absence of
ribosomal protein S2, structural changes in 16 S rRNA result in the
release of the anti-DB sequence from the penultimate stem making it
more accessible to base pair with DB (3, 4). We analyzed the
-galactosidase expression of pINZ, and pINZDB1 in E. coli
CS239 that carries an S2 temperature-sensitive mutation (3). Fig. 7A shows that the
-galactosidase activity of pINZDB1 significantly increases upon
shifting the temperature from 30 to 42 °C in the S2ts
strain (CS239) (6.3-fold from 0 to 3.5 h), whereas the activity in
the wild-type strain (CS240) slightly increased (1.1-fold from 0 to
3.5 h). If the initial ratio of the activity of CS239 to that of
CS240 at time 0 is taken as 1, the ratio dramatically increased,
reaching 5.8 at 3.5 h after temperature shift (Fig. 7B). In contrast, the lacZ gene without DB, pINZ,
did not show any significant differences in its expression between
CS240 and CS239, and the ratios of the activity of CS239 to that of
CS240 remained also at the initial level throughout the incubation time (Fig. 7B). A similar experiment was carried out with pINZDB3
(SD
, DB+), and the ratio of the activity in
CS239 to that in CS240 increased 3.4-fold at 3.5 h after the
temperature shift (data not shown). These results clearly demonstrate
that the low levels of S2 protein at 42 °C causes significant
stimulation of the lacZ expression only if the
lacZ gene contains DB, consistent to the proposal of Shean
and Gottesman (3).

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Fig. 7.
Translational enhancement of pINZDB1 in cells
with S2-depleted ribosomes. A, -galactosidase
activity from pINZ and pINZDB1. E. coli CS240 and CS239 (3)
were transformed with pINZ or pINZDB1, and cultures were grown at
30 °C in LB medium. At mid-log phase the cells were shifted to
42 °C in the presence of 1 mM IPTG. -Galactosidase
activity was measured as Miller units before (time 0) and at
0.5, 1, 1.5, 2.5, and 3.5 h after shift to 42 °C. B,
relative induction of the lacZ expression between pINZDB1
and pINZ in cells with S2-depleted ribosomes. Before the shift to
42 °C (time 0), the ratio of the -galactosidase
expression from pINZ and pINZDB1 in CS239 to that of CS240 was
estimated as 1, and the ratios after the shift to 42 °C were
calculated accordingly.
|
|
 |
DISCUSSION |
The present results clearly demonstrate that there is a
cis element downstream of the initiation codon, which in
concert with SD plays an important role in the translation efficiency
of mRNAs in E. coli. Our results provide supporting
evidence for the DB hypothesis (1, 2) that DB forms a complex with the
anti-DB in the 16 S rRNA to enhance translation initiation of
DB-containing mRNA.
The present data reveal the following five features of the DB function.
(a) The
-galactosidase activity of the pJJG78DB2 is
2-3-fold higher than that of the pJJG78DB1, indicating that a better
complementarity between DB and anti-DB yields better translation and/or
that the first 3 residues (AUG) in the DB are required for the better
activity of the DB. (b) The position of DB in the mRNA
is quite flexible as DB can start from codon 1 (pINZDB1) or codon 6 (pINZDB4). (c) DB itself works very poorly in the absence of
SD, suggesting that the formation of translation initiation complex
could be first initiated by the SD-16 S rRNA interaction, which
subsequently leads to the DB-anti-DB interaction. Alternatively, the
SD-16 S rRNA interaction could be stabilized by DB-anti-DB base
pairing. It has been shown that the 30 S ribosomal subunit can bind to
mRNA in the absence of initiator tRNA to form an intermediate
translation initiation complex (22). This intermediate complex could be
stabilized by the interaction of DB with anti-DB. (d) DB
enhances the translation initiation as judged by increased mRNA
translational efficiency both in vivo and in
vitro as observed by the increase of polysome formation and
-galactosidase production in a cell-free system. (e) In
the absence of S2 protein, the DB function is enhanced, consistently
with the notion that anti-DB becomes more accessible to DB (3, 4).
Since that the major conclusions for the behavior of the DB in
translation have been made from artificially created DBs in
overexpression systems as shown above, it would be important to analyze
the effect of DB under more physiological conditions. In this regard,
we have reported earlier that the DB is crucial for the induction of
the cold-shock protein
A (CspA) at low temperature and, furthermore, that major cold-shock genes contain DB sequences (7). Moreover, we have recently
postulated that the DB is essentially required for the induction of
major cold-shock proteins under conditions completely blocking protein
synthesis at low temperature (23). Future experiments testing
artificially created DBs using single copies of DB-containing lacZ mRNAs would greatly support the conclusions about
the role of the DB as a translational enhancer.
It should be noted that there are a number of mRNAs without the
5'-untranslated region such as
c1 (3), mRNA from
other phages (5), and Caulobacter crescentus (24). In these
leaderless mRNAs, conclusive evidence indicates that DB plays an
essential role in the formation of the translation initiation complex
in the absence of SD (3). Such complexes in the absence of SD may be
formed easier without any extra sequence upstream of the initiation
codon (3, 24).
Among a number of cis elements in E. coli
mRNAs known to enhance translation (25), DB next to SD appears to
be found most often (6, 26-28). It is engaging to elucidate which of
the two sequences (DB or SD) binds first to 30 S ribosomes for the
formation of the initiation complex. In this regard, it should be noted that the SELEX method using 30 S ribosomes as a ligand resulted in
enrichment of DB sequences accompanied with SD (8, 9).
 |
ACKNOWLEDGEMENTS |
We thank M. Gottesman for the E. coli CS239 and CS240 strains; S. Inouye, M. Kozak, P. Jones, S. Matsuyama, K. Yamanaka, O. Mirochnitchenco, L. Fang, L. Egger, W. Bae,
and U. Shinde for helpful discussions; T. Kinzy, D. Reinberg and A. Shatkin for critical reading of the manuscript; and M. Mitta (Takara
Shuzo Co., Japan) for the N-terminal sequence of
-galactosidase from pINZDB1 and pINZDB2.
 |
FOOTNOTES |
*
This work was supported by Grant GM19043 from the National
Institutes of Health.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.:
732-235-4115/4540; Fax: 732-235-4559/4783; E-mail:
inouye{at}rwja.umdnj.edu.
 |
ABBREVIATIONS |
The abbreviations used are:
DB, downstream box;
SD, Shine-Dalgarno;
IPTG, isopropyl-
-D-thiogalactopyranoside;
PAGE, polyacrylamide
gel electrophoresis.
 |
REFERENCES |
-
Sprengart, M. L.,
Fatscher, H. P.,
and Fuchs, E.
(1990)
Nucleic Acids Res.
18,
1719-1723[Abstract]
-
Sprengart, M. L.,
Fuchs, E.,
and Porter, A. G.
(1996)
EMBO J.
15,
665-674[Abstract]
-
Shean, C. S.,
and Gottesman, M. E.
(1992)
Cell
70,
513-522[CrossRef][Medline]
[Order article via Infotrieve]
-
Powers, T.,
Stern, S.,
Changchien, L.,
and Noller, H. F.
(1988)
J. Mol. Biol.
201,
697-716[CrossRef][Medline]
[Order article via Infotrieve]
-
Resch, A.,
Tedin, K.,
Grundling, A.,
Mundlein, A.,
and Blasi, U.
(1996)
EMBO J.
15,
4740-4748[Abstract]
-
Sprengart, M. L.,
and Porter, A. G.
(1997)
Mol. Microbiol.
24,
19-28[Medline]
[Order article via Infotrieve]
-
Mitta, M.,
Fang, L.,
and Inouye, M.
(1997)
Mol. Microbiol.
26,
321-335[CrossRef][Medline]
[Order article via Infotrieve]
-
Ringquist, S.,
Jones, T.,
Snyder, E. E.,
Gibson, T.,
Boni, I.,
and Gold, L.
(1995)
Biochemistry
34,
3640-3648[Medline]
[Order article via Infotrieve]
-
Etchegaray, J.-P.,
Xia, B.,
Jiang, W.,
and Inouye, M.
(1998)
Mol. Microbiol.
27,
873-874[CrossRef][Medline]
[Order article via Infotrieve]
-
Jiang, W.,
Fang, L.,
and Inouye, M.
(1996)
J. Bacteriol
178,
4919-4925[Abstract]
-
Harlocker, S. L.,
Rampersaund, A.,
Yang, W.-P.,
and Inouye, M.
(1993)
J. Bacteriol.
175,
1956-1960[Abstract]
-
Miller, J. H.
(1972)
Experiments in Molecular Genetics, pp. 352-355, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
-
Sarmientos, P.,
Sylvester, J. E.,
Contente, S.,
and Cashel, M.
(1983)
Cell
32,
1337-1346[Medline]
[Order article via Infotrieve]
-
Jones, P. G.,
Mitta, M.,
Kim, Y.,
Jiang, W.,
and Inouye, M.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
76-80[Abstract/Free Full Text]
-
Dammel, C. S.,
and Noller, H. F.
(1995)
Genes Dev.
9,
626-637[Abstract]
-
Ron, E. Z.,
Kohler, R. E.,
and Davis, B. D.
(1966)
Science
153,
1119-1120[Medline]
[Order article via Infotrieve]
-
Inouye, S.,
and Inouye, M.
(1991)
in
Directed Mutagenesis: A Practical Approach (McPherson, M. J., ed), p. 202, IRL Press at Oxford University Press, Oxford
-
Zuker, M.,
and Stieger, P.
(1982)
Nucleic Acids Res.
9,
133-148[Abstract]
-
Inouye, M.
(1983)
in
Experimental Manipulation of Gene Expression (Inouye, M., ed), pp. 15-32, Academic Press, New York
-
Vind, J.,
Sorensen, M. A.,
Rasmussen, M. D.,
and Pedersen, S.
(1993)
J. Mol. Biol.
231,
678-688[CrossRef][Medline]
[Order article via Infotrieve]
-
Iost, I.,
and Dreyfus, M.
(1995)
EMBO J.
14,
3252-3261[Abstract]
-
Wu, X.-Q.,
Lyengar, P.,
and RajBhandary, U. L.
(1996)
EMBO J.
15,
4734-4739[Abstract]
-
Etchegaray, J.-P., and Inouye, M. (1999) in press
-
Winzeler, E.,
and Shapiro, L.
(1997)
J. Bacteriol.
179,
3981-3988[Abstract]
-
McCarthy, J. E. G.,
and Brimacombe, R.
(1994)
Trends Genet
10,
402-408[CrossRef][Medline]
[Order article via Infotrieve]
-
Faxen, M.,
Plumbridge, J.,
and Isaksson, L. A.
(1991)
Nucleic Acids Res.
19,
5247-5251[Abstract]
-
Nagai, N.,
Yuzawa, H.,
and Yura, T.
(1991)
Proc. Natl. Acad. Sci. U. S. A.
88,
10515-10519[Abstract]
-
Ito, K.,
Kawakami, K.,
and Nakamura, Y.
(1993)
Proc. Natl. Acad. Sci. U. S. A.
90,
302-306[Abstract]
Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.