From the Department of Microbiology and Cell Biology,
Indian Institute of Science, Bangalore 560 012, India and the
¶ Department of Biology, University of Rochester,
Rochester, New York 14627
Received for publication, December 29, 2000
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ABSTRACT |
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The momP1 promoter of the
bacteriophage Mu mom operon is an example of a weak
promoter. It contains a 19-base pair suboptimal spacer between
the Optimal activity of bacterial promoters depends on the precise and
controlled interactions between the promoter with regulatory proteins
and RNA polymerase (RNAP).1
In many instances, promoter activity is modulated by protein-induced changes in DNA structure such as DNA distortion, looping, bending, and
unwinding (1-3). DNA structural distortions are known to influence
promoter activity (4, 5). Certain oligo (A/T) tracts exhibit unusual
curvature (6) and play an important role in the regulation of
transcription initiation (7-11). A different role is attributed to the
A tract when it is positioned upstream and in phase with the promoter
elements. A number of Escherichia coli promoters have
A+T-rich tracts (also known as UP elements) upstream to the The regulatory region of the mom operon of bacteriophage Mu,
which controls a unique DNA modification function (see Ref. 16 for a
recent review), exhibits several interesting features. The promoter,
momP1, which directs the transcription of com-mom
dicistronic mRNA, is a typical example of a weak promoter with a
poor The regulation of mom operon expression occurs at both the
transcriptional and translational levels (16). The Mu C protein, a
"middle" gene product, is an obligatory transcriptional activator of the momP1 promoter (19, 20), as well as for the other
three late promoters (21, 22). C protein binding to a site located at
Mutants have been isolated that relieve the dependence of
momP1 on C activation. In one such (partially) C-independent
mutant, tin7, there is a single-base change (T to G at
position Strains, Plasmids, Primers, Enzymes, and Chemicals--
E.
coli DH10B was used for generating the different plasmid
constructs. E. coli LL306 Construction of momP1 and momP2 Mutants--
The mutants used in
this study were generated by site-directed mutagenesis using either
pLW4 (17) or pUW4 (31) as the template DNA. pUW4 was used as template
for the polymerase chain reaction-based mutagenesis methods. The
mutants, pT2G, pT3G, and pT2GT3G (Fig. 1b), were generated
by using the Stratagene QuickChangeTM site-directed
mutagenesis method involving a pair of mutagenic oligonucleotides and
PfuI DNA polymerase. The mutant pT3C was generated by using
a Promega Gene Editor site-directed mutagenesis kit. pT1C, pT5C, pT4C,
pWT-P2, ptin7-P2, pT2GT3G-P2, and pG21C were generated by
using the modified mega primer method. In this method, a mega primer
was first generated using the pUC reverse primer and the mutagenic
oligonucleotide (as described in Ref. 32). The mega primer was then
used in the Stratagene QuickChangeTM site-directed
mutagenesis method. All of the mutants generated in the pUW4 background
were subcloned into pLW4 using EcoRI and BamHI
restriction enzymes to generate the promoter mutants as lacZ
transcriptional fusions. All of the mutants generated were confirmed by
carrying out Sanger's dideoxy method of sequencing (30).
The promoter expression plasmid pLC1 (22) was generously provided by
Dr. M. M. Howe; it contains an
EcoRI-SmaI-BamHI linker upstream of a
promoterless lacZ gene. Plasmid pLO1 was created by cloning
the smaller PstI-BamHI fragment from pLC1 into
pRSGC3 SmaI (a derivative of phagemid pGC1; Ref. 33). A
synthetic duplex containing either momP1 or momP2
was generated by annealing pairs of appropriate synthetic
oligodeoxynucleotides that had appropriately located 5'
EcoRI and 5' BamHI single-strand overhangs.
Plasmids pLO1/P1 and pLO1/P2 were constructed by ligating the synthetic duplexes into the EcoRI and BamHI sites,
respectively, of pLO1 and were used for generating site-directed
mutations in momP1 and momP2, respectively. After
DNA sequencing confirmed the nature of each mutation, the
mom promoter-containing PstI-BamHI
fragment was cloned into the corresponding sites in pLC1 for promoter
expression analyses (additional details of the plasmid and mutant
constructions are available upon request).
Promoter Strength Analysis--
Isolated colonies of E. coli DH10B cells harboring either a promoter mutant plasmid alone
or with plasmid pVN184 were inoculated into LB broth containing 100 µg/ml of ampicillin (for mutant promoter plasmids alone) or
ampicillin and 25 µg/ml chloramphenicol (both plasmids present); the
cultures were incubated at 37 °C for ~16 h with vigorous shaking.
The overnight cultures were diluted 100-fold into 3 ml of fresh medium
in duplicate tubes and incubated at 37 °C till the cultures reached
an A600 of 0.3-0.7. The samples were
then placed on ice. DNase I and (OP)2Cu Cleavage Reactions--
2.0 µg
(~0.36 pmol) of negatively supercoiled DNA was incubated with DNase I
(final concentration, 0.1 ng/µl) in presence of buffer (20 mM Tris-HCl, pH 7.2, 1 mM EDTA, 5 mM MgCl2, and 50 mM NaCl) in a
total reaction volume of 20 µl. After 30 s at 22 °C, the
reaction was terminated by addition of 20 µl of stop buffer (0.1 M Tris-HCl, pH 7.5, 25 mM EDTA, and 0.5% SDS).
The sample volume was made to be 400 µl with water and extracted
successively with phenol/chloroform/isoamyl alcohol (25:24:1) and
chloroform/isoamyl alcohol (24:1) and then precipitated with 2.5 volumes of 100% (v/v) ethanol in the presence of glycogen as a
carrier. Primer extension protocol is adapted from Gralla (35). The
extension reactions were performed with end-labeled mom
forward and reverse primers as previously described (27).
For the (OP)2Cu cleavage reaction, 2.0 µg of negatively
supercoiled DNA was incubated with a 10-µl sample of 4 mM
1,10-phenanthroline, 0.3 mM CuSO4, and 10 µl
of 58 mM 3-mercaptopropionic acid on ice for 1 min.
Reactions were quenched by adding 7.0 µl of 100 mM 2,9-dimethyl-1,10-phenanthroline; the samples were then deproteinized by phenol/chloroform/isoamyl alcohol (25:24:1) and chloroform/isoamyl alcohol (24:1) extractions, and the DNA was precipitated with 2.5 volumes of 100% (v/v) ethanol. The DNA was used for primer extension
with end-labeled mom forward and mom reverse primers.
In Vivo KMnO4 Footprinting Reaction--
In
vivo KMnO4 footprinting reaction was carried out as
described by Sasse-Dwight and Gralla (36). E. coli DH10B
cells harboring a momP1 promoter mutant plasmid alone or
along with pVN184 were grown to A600 0.6 in 4.0 ml of LB broth. The cultures were treated with 200 µg/ml rifampicin
for 20 min. The samples were then incubated with 30 mM
KMnO4 for 2 min. Reactions were stopped by transferring the
cultures to prechilled tubes. The cells were harvested, and plasmid DNA
was isolated. Primer extension reactions were carried out as described above.
Total RNA Isolation and Primer Extension--
Total RNA was
isolated from E. coli DH10B cells harboring the various
promoter mutant plasmids using the hot acid phenol method. Primer
extension was carried out as per the manufacturer's protocol (Life
Technologies, Inc.) using superscript reverse transcriptase and
end-labeled mom forward (for momP2 transcript
detection) and reverse (for momP1 transcript detection)
primers. An end-labeled primer annealing 150 bases downstream of the
ampicillin transcription +1 start site was used to normalize the levels
of transcripts produced in the different mutant promoter constructs.
Scanning of the autoradiographs was carried out using a Bio-Rad GS710
Calibrated Imaging Densitometer. Quantification was done using Quantity
One software.
DNA Structure Analysis of T6 Run Mutants--
The
variation in helical structure of the DNA depends on base sequence.
Specific sequences contribute to alterations in groove width and DNA
curvature (37). In addition to their use in probing DNA-protein
interactions, nucleases are often used to detect distortions in DNA.
Cleavage reaction of orthophenanthroline cuprous complex ((OP)2Cu) depends on the local DNA structure rather than
the base sequences as demonstrated previously by Spassky et
al. (38). We used (OP)2Cu to probe possible structural
or conformational differences between the wild type (WT) and the
tin7 mutant in the region of the T6 tract in
momP1 (Fig. 1). Negatively
supercoiled plasmid DNA harboring the WT or tin7 mutant
momP1 promoter was subjected to in vitro single
hit cleavage, and the sensitivity pattern was assessed by primer
extension analysis (Experimental Procedures). The results of a typical
(OP)2Cu footprinting are shown in Fig.
2 (a and b). The
sensitivity patterns of both the top and the bottom strands in the
region containing the T6 run were different for the two
promoters. Several hypersensitive sites are seen in the WT that are not
reactive in tin7. For example, at
Because the T4G (tin7) mutant showed a difference in DNA
conformation with respect to wild type, the effect of base
substitutions at other position in the T6 run were
examined. To this end, negatively supercoiled DNAs of various mutants
were subjected to in vitro cleavage with DNase I. Mutants
T2G, T3G, T2GT3G, and T4G (tin7) were selected as
representatives for this analysis (Fig. 2c). The mutants
showed hypersensitivity patterns different from one another, as well as
from the WT. For example, the top strand residue Mutations in the T6 Run Result in Increased Promoter
Activity--
The differences in chemical nuclease and enzymatic
cleavage patterns of WT and mutant T6 run promoters
reflect structural differences among these promoters. To determine
whether the changes also influence promoter activity,
promoter-lacZ fusion constructs were generated for all of
the mutants, and promoter strength was assessed indirectly by measuring
T6 run mutants T2G, T2C, T3G, T3C, T2GT3G, and T5C produced
5-26-fold higher levels of enzyme compared with the WT promoter (Table
I); in contrast, T1C and T4C showed
little or no increased expression. However, all of the mutants remained
responsive to transactivation by C protein (Table I), producing enzyme
levels comparable with that of the activated WT momP1
promoter. Thus, all of the mutant promoters are C
protein-dependent for their full activity, indicating that
the C transactivation mechanism was unaltered. As a control, the
mutation G21C was created (shown in Fig. 1b) upstream from
the T6 run yet within the spacer region. As expected, this
mutant showed levels of
The above experiments were carried out using a momP1
promoter directing production of a Com-LacZ translational fusion. We carried out similar experiments with a momP1
promoter-lacZ transcriptional fusion vector. This was
constructed by subcloning momP1 mutations (T4G, T4A, T4C,
T3G, T3A, and T3C, produced in a momP1-containing synthetic
oligonucleotide duplex) into a site 5' to a promoterless, reporter
lacZ gene (see "Experimental Procedures"), and
momP1 promoter activity was assayed by measuring
In view of the different levels of momP1 expression observed
with the three T4X mutations, we compared their in vitro
sensitivity to cleavage by DNase I. As shown in Fig. 2d, the
T4C promoter region showed a cleavage pattern similar to that of the
WT, which is in sharp contrast to all of the other T6 run
mutants. Because the T4C substitution does not appear to alter the WT
momP1 DNA conformation, we suggest that it possesses the
same unfavorable distortion as the WT and, hence, requires protein C
activation for any transcription.
Formation of Open Complexes by T6 Run
Mutants--
Increased T6 Run Mutants Show Increased P1 Transcript
Levels--
Because only mutants with higher (>8-fold the WT basal
level) expression of Mutations Disrupting the momP2
To further examine the effect (if any) of momP2 expression
on momP1 expression, the momP2 We have addressed the importance of the run of six T nucleotides
located in the momP1 promoter (Fig. 1a) in the
regulation of mom operon expression. An intrinsic DNA
distortion caused by the presence of the T6 tract
overlapping the 5' end of the One could argue that the increase in promoter activity observed in the
T6 run mutants could be a consequence of new base-specific contacts made by RNAP in the substituted positions instead of removal
of intrinsic DNA distortion. However, this seems unlikely because
changing different residues ( Of all of the mutants whose promoter strength we analyzed, T4G
(tin7) showed the highest momP1 transcriptional
activity. This mutation, a T It has been shown earlier that it is primarily the length, not the
sequence, of spacer DNA between the two promoter consensus sequences
( Complex regulatory mechanisms have evolved in bacteriophages to
ensure the precise expression of phage genes. Expression of the
bacteriophage Mu mom gene during the late transcription
phase is a good example of one such regulatory scheme. Although
mom gene expression seems to be dispensable for Mu growth or
lysogeny, premature activation or constitutive expression of
mom function is detrimental to the host cells (49, 50).
Hence, it is not surprising that intricate mechanisms have been evolved
for the regulation of mom expression at both the
transcriptional and translational levels (16). Apart from these modes
of regulation, recently, Sun and Hattman (18) have suggested another
possible regulatory control over mom expression. It was
suggested that the leftward transcription at the momP2
promoter (Fig. 1 and Ref. 18) might prevent low level rightward
transcription of momP1 (and, hence, mom
expression) in two possible ways. First, momP2 might compete with momP1 for RNAP binding in the absence of C protein.
Second, leftward transcription produces an antisense transcript that
might prevent gin mRNA elongation into mom.
The present study rules out the first possibility because disruption of
the momP2 Existence of overlapping promoters in many systems add additional
regulatory complexities (51-53). The momP1 and
momP2 promoters (Fig. 1a) are overlapping and
oriented in a divergent fashion. Normally such organization would lead
to competition in the transcription machinery as exemplified in case of
other overlapping/competing promoters (53). However, earlier DNase I
footprinting analysis with the mom promoter revealed that
RNAP is bound to momP2 in the absence of C protein (17).
Partitioning of RNAP between the two promoters was not observed,
although neither promoter appears to be a strong one. Further,
momP2 disruption did not lead to increased momP1
expression (Fig. 7c), underlining the importance of the
T6 run as a cis-acting negative element that prevents RNAP binding to momP1. Thus, the primary role
of the T6 run is to prevent low level rightward
transcription initiation at momP1.
It is noteworthy that the Plys promoter, another
bacteriophage Mu late gene promoter, also has a T6 stretch
in the position corresponding to that in momP1, but it is
absent in the other two late promoters (Pi and
Pp). Substitutions in two of the T bases in the
Plys spacer region show an UP phenotype depending on the
substituted base (22). Because the premature expression of
mom and lys is detrimental to the host cells, the
DNA negative element seems to be a common fail-safe mechanism to keep
these two late genes tightly regulated until the right time for their expression. Thus, the phage Mu seems to have evolved one common strategy to keep two potentially cytotoxic genes under control.
35 (ACCACA) and
10 (TAGAAT) hexamers. Escherichia coli RNA polymerase is unable to bind to momP1 on its
own. DNA distortion caused by the presence of a run of six T
nucleotides overlapping the 5' end of the
10 element might prevent
RNA polymerase from binding to momP1. To investigate the
influence of the T6 run on momP1 expression,
defined substitution mutations were introduced by site-directed
mutagenesis. In vitro probing experiments with copper
phenanthroline ((OP)2Cu) and DNase I revealed distinct differences in cleavage patterns among the various mutants; in addition, compared with the wild type, the mutants showed an increase (variable) in momP1 promoter activity in vivo.
Promoter strength analyses were in agreement with the ability of these
mutants to form open complexes as well as to produce
momP1-specific transcripts. No significant role is
attributed to the overlapping and divergently organized promoter,
momP2, in the expression of momP1 activity, as
determined by promoter disruption analysis. These data support the view
that an intrinsic DNA distortion in the spacer region of
momP1 acts in cis as a negative element in
mom operon transcription. This is a novel mechanism of
regulation of toxic gene expression.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
35
hexamer of
70 promoters (12, 13). UP elements, when
present, are integral components of promoters, because they interact
with the carboxy terminal domain of the RNAP
-subunit (12). In
vitro studies showed that the E. coli RNAP
(
70) holoenzyme alone is sufficient for
transcriptional activity from several such promoters (12, 14,
15).
35 (ACCACA) element and a suboptimal spacing of 19 bp between
the two consensus elements (Fig. 1a). The spacer region of
the promoter contains a run of six T nucleotides from
12 to
17.
RNAP does not bind to momP1 by itself (17). Instead it binds
an overlapping, divergent promoter region, momP2, which
brings about "leftward" transcription (18). The stretch of six A
nucleotides complementary to the T6 run appears to be part
of an UP element for leftward transcription from momP2
(18).
28 to
57 in the momP1 region (23, 24) brings about an asymmetric distortion and unwinding of the DNA (25-27).
14) that disrupts the T6 run in the
momP1 spacer region (17). Few explanations could account for
the increased promoter activity of tin7 mutant: 1) T
tract-mediated intrinsic DNA curvature is lost because of disruption of the T6 run (to
T3GT2), resulting in RNAP binding to
momP1; 2) because P1 and P2 are overlapping divergent
promoters, weakening of the UP element of momP2 (18) may
facilitate the binding and activity of RNAP at momP1; and 3)
the T(
14) to G change converts momP1 to an extended
10
promoter (28). The present study is an attempt to delineate the role of
the T6 run in momP1 promoter activity.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(pro-lac) was from
L. Lindahl (29). Plasmid pVN184 (+), a C protein-producing construct,
has been described earlier (17). The primers used in this study to
generate site-directed mutants or synthetic duplexes are available upon request. Restriction and modifying enzymes were purchased from Stratagene, New England Biolabs, and Roche Molecular Biochemicals and
were used according to the suppliers' recommendations. DNase I was
from Worthington, and E. coli DNA polymerase (PolIk) was from New England Biolabs. Superscript reverse transcriptase was purchased from Life Technologies, Inc. Chemicals and other reagents were purchased from Life Technologies, Inc. and Sigma. Primers were
synthesized by Bangalore Genei (Pvt.) Ltd. (Bangalore, India), Life
Technologies, Inc., and the University of Rochester Core Lab Facility.
[
-32P]ATP (6000 Ci/mmol) was purchased from New
England Nuclear. Most of the standard procedures were carried out as
described by Sambrook et al. (30).
-Galactosidase activity in
SDS-CHCl3-treated cells was determined as described by
Miller (34). In the experiments with plasmid pLC1 constructs,
-galactosidase assays were carried out with exponential cultures
grown from isolated colonies. The values in the tables are the averages
from at least two separate experiments, and replicate assays were done
on each culture. The variation was 10-20% around the mean value.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
14T (top strand) and at
15A,
16A,
17A, and
18C (bottom strand), the WT was cleaved more
often by (OP)2Cu. In contrast, tin7 DNA was
relatively refractory to cleavage by (OP)2Cu at these residues, whereas
10A in the top strand was hypersensitive. We also
probed the promoter structure by using DNase I as a footprinting agent.
DNase I reaction also revealed substantial differences in cleavage
sensitivity patterns (indicated by the asterisks in Fig.
2c, lanes 1 and 2). DNase I cleavage
gave rise to two hypersensitive sites, at
9G and
17T (top strand)
in the WT promoter, compared with hypersensitive sites at
12T and
13T (in the T6 run) of the tin7 promoter.
These results show that the two promoter regions differ in their
susceptibility to nuclease cleavage, indicating that the DNA
conformations are different.
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Fig. 1.
a, regulatory region of
bacteriophage Mu mom gene. The 10 and
35 elements of
momP1 are overlined (top strand). The
10
hexamer and the proposed UP element for momP2 are
underlined (bottom strand). The transcription start sites
for both momP1 and momP2 are indicated with
arrows; the T6 run (top strand) is enclosed in
an open rectangle. Regions protected by RNAP in
momP1 and momP2 are indicated. b,
sequence of the momP1 promoter. Substitution mutations in
the T6 run of the spacer region of the momP1
promoter are indicated. The T residues at positions
17 through
12
are designated T1 through T6, respectively.
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Fig. 2.
Nuclease sensitivity pattern of WT and mutant
momP1 promoters. The (OP)2Cu cleavage
reactions of the WT (pLW4, lanes 1) and tin7
mutant (ptin7, lanes 2) promoters and the
sensitivity pattern of the top (a) and the bottom
(b) strands are shown. c, DNase I cleavage
reactions of WT (lane 1), tin7 (lane
2), T2G (lane 3), T3G (lane 4), and T2G T3G
(lane 5) promoters in the top strand is shown. d,
DNase I cleavage reactions in the top strand of WT (lane 1),
tin7 (lane 2), and T4C (lane 3) mutant
promoters. Hyper-reactive residues are indicated with
arrowheads and asterisks. G,
A, T, and C refer to Sanger's dideoxy
sequencing ladder of the region of pLW4 using end-labeled
mom forward and reverse primers.
14T was cleaved more
frequently in T2G, whereas
12T,
14T, and
16T were hypersensitive
in T3G, and
12T and
14T were hypersensitive in T2GT3G. The narrower
minor groove of the A/T tract is altered by the G substitutions,
leading to its widening at these positions. As a consequence, different
DNase I-hypersensitive sites are observed (marked by
asterisks in Fig. 2c) in each of the mutants;
(OP)2Cu footprinting analysis gave analogous results (not
shown). Because both DNase I and (OP)2Cu-mediated DNA
cleavages are in the minor groove (39, 40), the T6 run
mutations appear to change the DNA conformation in the minor groove.
This is further supported by an altered migration pattern of DNA
fragments in polyacrylamide gels (data not shown).
-galactosidase activity in cells harboring these plasmids. Moreover,
the C-activated level was analyzed in cells also harboring a compatible
C-producing plasmid, pVN184.
-galactosidase activity comparable with the
WT momP1 promoter in the absence and in the presence of C
protein (Table I). The variable increase in momP1 promoter
activity among these mutants could be due to differences in their
perturbations of DNA structure as shown in Fig. 2. None of the mutant
promoters showed activity as high as that of T4G (tin7),
which showed an increase that was between 46- and 80-fold depending on
the type of fusion examined (Tables I and
II). These results suggest that in
addition to DNA distortion, an alternative mechanism might be operating
in tin7, most likely having an extended
10 promoter
because of the specific base substitution at
14 position (discussed
further below).
Production of -galactosidase activity in E. coli DH10B cells
containing a pLW4-momP1 promoter-lacZ fusion derivative ± compatible plasmid pVN184
Production of -galactosidase activity in E. coli LL306 cells
containing a pLC1-momP1 promoter mutant plasmid
-galactosidase activity. As seen in Table II, the substitutions
generated variable increases in enzyme level, in good agreement with
the results observed with the pLW4 plasmid system. Most interesting are
the three T4X substitutions. First, the T4G mutant had the highest
level of C-independent expression, 80-fold above the WT. In contrast,
T4A had a 6-fold increase, whereas T4C showed no increase. Thus, the
three different T4X substitutions produced three different phenotypes.
We suggest that the high level of constitutive expression by T4G
(tin7) is due to its having an extended
10 promoter, in
addition to the alteration in DNA conformation. In contrast, the T4A
(as well as the T3A) substitution appears to only affect
momP1 DNA conformation, indicating that T-A to A-T base pair
alterations can also affect conformation. At first glance, it was
surprising that the T4C mutant did not show increased momP1
expression; however, as will be shown below, the T4C mutant does not
exhibit any structural difference from the WT based on in
vitro cleavage. Finally, it should be noted that the T2G and T3G
mutations create a TG at positions
17 and
16 and at positions
16
and
15, respectively. Although these mutations exhibited enzyme
levels severalfold higher than the WT, they do not appear to provide
extended
10 functional capability.
-galactosidase levels with certain
T6 mutant momP1 promoters indicate increased
transcription initiation capability. Interaction of RNAP at a promoter
can be ascertained by assessing open complex formation using an
in vivo KMnO4 footprinting technique (36).
E. coli DH10B cells harboring a T6 mutant pLW4
plasmid in the absence or the presence of the C protein-producing
plasmid, pVN184, were probed (see "Experimental Procedures"). In
accordance with its high level of constitutive promoter activity in the
absence of C protein, tin7 showed hypersensitive bands (Fig.
3, lane 4) characteristic of
open complex formation; the observed pattern was in good agreement with
the results of Balke et al. (17). Open complex formation in
the absence of C protein was also observed with mutants T2G and T2GT3G
(Figs. 3, lane 12, and
4a, lane 10). However, open complexes were not detected with T3G, T3C, and T2C (Fig.
3, lanes 8, 10, and 14, respectively),
which correlates with their relatively lower levels of
-galactosidase expression (in the absence of C protein). As could be
predicted from the promoter strength analysis (Table I), the WT
momP1 promoter and the T4C mutant were unable to produce
detectable open complexes (Fig. 3, lanes 2 and 6,
respectively). In the presence of C protein, however, all promoters
showed open complex formation (Fig. 4, a and b).
This result rules out an artifactual inability to detect open complexes
with the mutant constructs used in these experiments.
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Fig. 3.
In vivo KMnO4
footprinting analysis. The presence (+) or absence ( ) of
rifampicin (Rif) to trap RNAP in the open complex is
indicated. OC refers to the bottom strand hypersensitive
sites upon open complex formation in momP1. Sequencing lanes
are shown as G, A, T, and
C.
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Fig. 4.
Open complex formation by T6 run
mutants. The presence (+) or absence ( ) of C protein
(C) and rifampicin (Rif) are indicated.
OC indicates the hypersensitive sites produced upon open
complex formation in the bottom strand. G, A,
T, and C refer to sequencing lanes. Analysis with
tin7, T4C, T2GT3G (a) and T2G, T3G, T3C
(b) were carried out.
-galactosidase activity showed open complex
formation, we employed a more direct method of assessing promoter
strength. For this purpose, we assayed for momP1-specific
mRNA transcripts in total RNA isolated from WT, T4G
(tin7), and T6 mutant (T2G, T3G, T3C, and
T2GT3G) plasmid-containing cells (see "Experimental Procedures").
The results of such an experiment are shown in Fig. 5a. In all of the mutants
examined the transcription start site was identical to that of the wild
type momP1 promoter, indicating that the mutations did not
lead to the formation of new promoters. Those mutants (e.g.
T3G and T3C) that failed to show open complexes in the
KMnO4 probing experiments did produce increased amounts of
momP1-specific transcripts compared with the WT promoter
(Fig. 5). There was a good correlation in the fold increase in
momP1-specific transcript levels and the relative promoter
strengths of these mutants with respect to the WT levels (compare
Tables I and II and Fig. 5b).
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Fig. 5.
momP1 transcript levels produced
by T6 run mutants. Primer extension reactions were
carried out using mom reverse primer and reverse
transcriptase as described under "Experimental Procedures."
G, A, T, and C refer to
sequencing lanes. a, autoradiograph showing the
momP1 transcript and ampicillin (Amp) signals.
b, levels of momP1 specific RNA (with respect to WT levels
after normalizing with the ampicillin signal).
10 Hexamer Do Not Increase
Activity of the WT (or T6 Run Mutant) momP1
Promoter--
The results presented above support the view that
alteration in DNA conformation caused by disruption of the
T6 run results in increased basal activity of the
momP1 promoter. However, the scenario is somewhat
complicated by the fact that the mom regulatory region has
two overlapping divergent promoters, momP1 and
momP2. The T6 run substitution mutations
generated in momP1 also disrupt the A6 tract in
the complementary strand (Fig. 1), which is proposed to function as
part of an UP element directing leftward transcription from the
momP2 promoter (18). Hence, an alternate possibility for the
increased activity of momP1 in tin7 and other
T6 run mutants could be due to weakening of the UP element
of momP2. Therefore, momP2 transcript levels were
measured by isolating total RNA and extending it with end-labeled
mom forward primer using reverse transcriptase. The results
are shown in Fig. 6, where
momP2 transcripts were detected with tin7, as
well as with some other T6 (T2G, T3G, and T3C) run mutants.
Thus, the substitution mutations in the T6/A6
run did not abolish momP2 activity while having increased momP1 activity. This conclusion was supported by
results from independent experiments in which synthetic duplexes having
mutations in the T6/A6 run (corresponding to
T2G or T3G or T4G) were cloned into pLC1 in an orientation where
lacZ gene transcription was under control of
momP2 (in these constructs the momP1
10 hexamer was also altered so as to reduce its potential expression). We observed
that each of the single-base substitution mutations lowered the enzyme
level less than 2-fold (data not shown). Thus, T6 run mutations that increase momP1 transcription do not do so by
reducing momP2 transcription.
View larger version (96K):
[in a new window]
Fig. 6.
Detection of the momP2
transcript by T6 run mutants. The experiment was
carried out as described under "Experimental Procedures" using
equal amounts of total RNA (20 µg) in all of the lanes and
end-labeled mom forward primer for primer extension.
10 hexamer was
mutated in the WT, tin7, and T2GT3G mutant constructs (Fig.
7a). In these mutants, loss of
momP2 function was confirmed by measuring leftward
transcript levels produced in vivo by the parental and
disrupted momP2 promoters, using primer extension assays
with total RNA extracted from cells harboring these plasmids (Fig.
7b). The results in Fig. 7c show that there was
no increase in the WT, tin7, or T2GT3G momP1
promoter activity in the momP2
10 disrupted background.
These results indicate that the overlapping momP2 promoter
plays, at most, only a minor role in momP1 activity, unlike
other overlapping promoters. We conclude that mutations in the
T6 run that increase momP1 expression function
by alleviating DNA distortion.
View larger version (42K):
[in a new window]
Fig. 7.
Effect of momP2 10 hexamer
mutations on momP1 activity. a, the
sequence of the mom regulatory region is shown indicating
the mutation in three different promoters (WT, tin7, and
T2GT3G). b, detection of momP2-specific mRNA
in the wild type mom promoter (lanes 1 and
3) and in a disrupted momP2
10 hexamer
background (lanes 2 and 4) using end-labeled
mom forward primer. Lane 1, 15 µg of total RNA;
lane 2, 15 µg of total RNA; lane 3, 30 µg of
total RNA; lane 4, 30 µg of total RNA. c,
momP1 promoter strength (measured by
-galactosidase
activity) in a disrupted momP2
10 hexamer
background. The values plotted are the averages of four
experiments.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
10 element could produce an unfavorable
conformation for RNAP occupancy. Different T to G substitutions in this
run showed different sensitivity patterns to nucleases as compared with
the WT momP1 promoter (Fig. 2). This is attributed to a
difference in the DNA structure caused by each substitution. These
substitutions in the T6 run also produce variable increases
in the basal activity of mutant momP1 promoters (Tables
I and II). The increase in the promoter strength of some of the mutants
was correlated with the formation of detectable open complexes and the
levels of momP1-specific transcripts in the absence of
trans-activator protein C (Figs. 3-5).
13 to
16) led to the increase in
promoter activity. Moreover, substitutions elsewhere in the spacer do
not increase the activity, including the mutant T1C, whose substitution
still retains the run of T5 nucleotides. A point to be
remembered and shown here is that momP1 wild type promoter
is unable to form an open complex on its own (Ref. 17 and Fig. 3). This
is not due to the suboptimal spacer length (see the Introduction and
Fig. 1) because single-base and two-base deletions in the spacer (18 and 17 bp, respectively) do not lead to any increase in promoter
activity (data not shown). Thus, it is difficult to visualize RNAP
contacting each and every residue between
13 to
16 when it is
unable to make favorable contacts in an optimally spaced promoter. An
altered DNA structure of the various T6 run mutants leads
to the formation of an open complex at these promoters. As an
additional support for altered DNA conformation detected in nuclease
probing experiments, the wild type promoter fragment (226 bp) migrated
slower than the T2G mutant promoter fragment (226 bp) in a gel
electrophoretic mobility assay. Taken together, we conclude that the
transcription from promoters having substitutions in the T6
run is due to the removal of an unfavorable distortion.
G at position
14, produces a
15T,
14G, which is characteristic of extended
10 promoters (28, 41).
Extended
10 promoters are usually constitutive, and they do not
require a
35 element or an activator protein. In contrast to the T4G, the corresponding T4C substitution did not increase expression of
momP1 nor alter promoter DNA conformation compared with the WT. All three
15T (T3X) substitutions exhibited an
increase in momP1 basal activity compared with WT. However,
although the T3G mutation created a TG at positions
16 and
15, it
did not produce the same high level of expression exhibited by T4G
(tin7); this is to be expected because the former TG is not
positioned properly to create an extended
10 promoter. Thus, we
suggest that a combination of both DNA conformational alteration and
extended
10 promoter characteristics contribute to the T4G
(tin7) phenotype, but the increase in basal activity of the
other T6 run mutants is due to the removal of an
unfavorable distortion in momP1 promoter DNA. Once this
distortion is ameliorated, an otherwise very weak promoter can be
transcribed in the absence of activator protein C. However, these
promoters are still dependent on C for full activity, as shown by both
the C-mediated increases in
-galactosidase activity (Table I) and
the formation of open complexes (Fig. 4).
10 and
35 regions) that is important for activity of a promoter
(42, 43). It has also been demonstrated that the sequences located
either upstream or downstream of the
10 and
35 regions determine
the kinetics of association of promoter with RNAP and efficiency of
transcription initiation (44, 45). It is believed that the spacer DNA
holds the
10 and
35 regions in the proper orientation for their
recognition by the RNAP holoenzyme complex without having any specific
contacts with RNAP. However, characterization of mutants of the
PRM promoter of phage
bearing dC9·dG9 sequences in a stretch of the spacer
DNA separating the contacted
10 and
35 regions showed reduced
promoter activity both in vitro and in vivo (46,
47). These mutations were interpreted as altering the structure of the
spacer DNA and, as a consequence, leading to a change in the
orientation or local structure of the contacted
10 and
35 elements
of the promoter. A library of synthetic promoters of Lactococcus
lactis having randomized 17-bp optimal length spacer in between
the consensus
10 and
35 elements was assayed for activity both in
L. lactis and E. coli (48). In both host
backgrounds, a large variation (~400 fold) in promoter activity was
observed because of variations in the spacer sequence context. It seems
that the overall three-dimensional topological structure of the
promoter DNA that arises from a particular nucleotide sequence could be
important for the activity of a promoter.
10 hexamer did not lead to an increase in the
basal level activity of momP1 (Fig. 7c). On the
other hand, disruption of the T6 run in the spacer region
of momP1 promoter led to increased rightward transcription,
indicating the importance of its role as a cis-acting negative element. Another possible role attributed to momP2
is to act as a sink for capturing RNAP in the vicinity of
momP1, so that RNAP is ready for occupancy at
momP1 at the right time of mom expression (17).
Our results do not exclude that possibility.
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ACKNOWLEDGEMENTS |
---|
We thank Nivedita Mitra for the help in
generating some of the mutants and the -galactosidase assays, J. Jacob for the technical assistance, and other members for discussions.
![]() |
FOOTNOTES |
---|
* This work was supported by a grant from the Department of Science and Technology, Government of India (to V. N.) and by United States Public Health Service Grant GM29227 from the National Institutes of Health (to S. H.).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.
§ Recipient of a Senior Research Fellowship from the University Grants Commission, Government of India.
To whom correspondence should be addressed: Dept. of
Microbiology and Cell Biology, Indian Inst. of Science, Bangalore 560 012, India. Tel: 80-360-0668 or 80-309-2598; Fax: 80-360-2697; E-mail: vraj@mcbl.iisc.ernet.in.
Published, JBC Papers in Press, March 6, 2001, DOI 10.1074/jbc.M011790200
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ABBREVIATIONS |
---|
The abbreviations used are: RNAP, RNA polymerase; bp, base pairs; WT, wild type.
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