From the Department of Pharmacology, School of Medicine, Wayne State University, Detroit, Michigan 48201
Received for publication, November 20, 2000, and in revised form, December 15, 2000
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We have previously demonstrated that sequential
activation of the bacterial ilvIH-leuO-leuABCD gene
cluster involves a promoter-relay mechanism. In the current study, we
show that the final activation of the leuABCD operon is
through a transcriptional derepression mechanism. The
leuABCD operon is transcriptionally repressed by the
presence of a 318-base pair AT-rich upstream element. LeuO is required
for derepressing the repressed leuABCD operon. Deletion analysis of the repressive effect of the 318-bp element has led to the
identification of a 72-bp AT-rich (78% A+T) DNA sequence element, AT4,
which is capable of silencing a number of unrelated promoters in
addition to the leuABCD promoter. AT4-mediated gene silencing is orientation-independent and occurs within a distance of
300 base pairs. Furthermore, an increased gene-silencing effect was
observed with a tandemly repeated AT4 dimer. The possible mechanism of
AT4-mediated gene silencing in bacteria is discussed.
The leu-500 mutation is an A to G transition in the
The intervening promoter that relays the distant interaction between
pilvIH and pleu-500 is the leuO
promoter (pleuO). In addition to transcriptional activity
from pleuO, the leuO gene product, LeuO, is also
required to provide a trans-acting function for activation of
pleu-500 (6). It appears that the functional pleuO (or other replaced promoter) and LeuO are coupled in
activating pleu-500. The molecular basis for
pleu-500 activation by the combined action of
pleuO and LeuO is still a mystery.
There is a stretch of 434 base pairs
(bp)1 that is AT-rich DNA
flanked by the divergently arrayed leuO and
leuABCD (14). Besides the promoter sequences of the
flanking genes, the function of the remaining 318-bp AT-rich (69% A+T)
DNA is unknown (illustrated in Fig. 1). By monitoring
pleu-500 activation, we found that the 318-bp AT-rich
intervening DNA appears to repress the short-range interaction (11)
between the two flanking promoters. Interestingly, LeuO relieves the
repression. The repressive effect of the AT-rich intervening DNA on the
short-range promoter-promoter interaction (pleuO and
pleu-500) could potentially be due to anchoring of the
AT-rich DNA to a large mass, which restricts DNA rotation and thereby
abolishes short-range promoter-promoter interaction via DNA
supercoiling. However, detailed analysis to search for DNA rotation
blockage has ruled out this anchorage possibility.
The repressive activity of the 318-bp AT-rich intervening DNA has been
narrowed down to a 72-bp AT-rich (78% A+T) DNA, referred to as AT4 in
this work. AT4 is located at the pleuO end of the 318-bp
AT-rich DNA. AT4 can repress promoter activity within a 300-bp
distance. This repression is independent of the orientation of AT4.
AT4-mediated repression of the promoter activity appears to be promoter
nonspecific, because all promoters tested are repressed by AT4. These
results support a role for AT4 as a gene silencer in bacteria.
Plasmids and Bacterial Strains--
pWU802T, pEV101, pSO1000,
pAO, pJW270, and pBR322 have been previously described (6, 15-18).
pWU812T was derived from pWU802T. To construct pWU812T, a 320-bp
promoterless non-AT-rich (47% A+T) DNA was generated by polymerase chain reaction (PCR) from the coding region of human cathepsin B gene
(19), with the primers introducing HincII and
BstXI sites at the ends. The digested
HincII-BstXI fragment was then used to replace
the 318-bp HincII-BstXI segment containing the
native AT-rich sequence (69% A+T) between the divergently transcribing ptac and pleu-500 in pWU802T. The native 318-bp
AT-rich intervening DNA was also PCR-amplified with primers containing
AatII restriction sites. The AatII-digested AT
DNA fragment was inserted into the unique AatII site on pAO
to yield pAO-AT and pAO-ATR. The plasmid carrying an AT DNA insert,
with the leuO end of the DNA orientated proximal to
pbla, was designated pAO-AT. The plasmid carrying the
DNA insert in the opposite orientation was designated as pAO-ATR. Similarly, the 72-bp DNA located near the pleuO end of the
318-bp AT-rich DNA was PCR-amplified with primers so that the 72-bp AT4 DNA was flanked by AatII restriction sites on both ends. The
AatII-digested AT4 DNA was inserted at the unique
AatII site on pAO to yield pAO-AT4 (the leuO end
of the DNA insert was proximal to pbla) and pAO-AT4R
(the opposite orientation). The AT4 DNA sequence was
5'-CACAATCATACACCAAGTGAATGATCATTTAAGTTTCAATTAAATGTTTATATTATTAATAGCTAAAAAGTT-3'. The nucleotide sequence of the rest of the intervening DNA between the
divergently arrayed leuO and leuABCD genes can be
obtained from the GenBankTM data base (accession number AF106956).
Other testing plasmids were all derived from the above-described
plasmids and were individually described in the experiments. The
following 72-bp DNA sequence from the lacZ coding region was
used to replace the AT4 DNA on pWU802T as described in Fig. 6.
5'-AACCATCGAAGTGACCAGCGAATACCTGTTCCGTCATAGCGATAACGAGCTCCTGCACTGGATGGTGGTACC-3'.
The following four synthetic DNA oligomers, consisting of nucleotide
sequences of the lacZ coding region, were used to
sequentially extend the distance between the AT4 insert and
pbla in pAO-AT4 as described in Fig. 8:
5'-GCGATCTTCCTGAGGCCGATACTGTCGTCGTCCCCTCAAACTGGCGGATCCAA-3'; 5'-GATCCACGGTTACGATGCGCCCATCTACACCAACGTAACCTATCCCATGAATTC-3'; 5'-AATTCAATCCGCCGTTTGTTCCCACGGAGAATCCGACGGGTTGTTACTGGTACC-3'; and
5'-CATTTAATGTTGATGAAAGCTGGCTACAGGAAGGCCAGACGCGAAAGCTTGTAC-3'.
CH601(topA+) and
CH582(topA Primer Extension--
Primer extension was carried out as
previously described (11). Several DNA oligomers were used as primers
in the study. 5'-TCTGGGTGAGACAAAACAGGAAGGC-3' was used for detecting
pbla-mediated transcripts;
5'-AGAATTCTCATGTTTGACAGCTTATCATCG-3' was used for detecting
pleu-500-mediated transcripts;
5'-CCTATAAAAATAGGCGTATCACGAGGCCCT-3' was used for detecting
ptac-mediated transcript; 5'-CTACAGCATCCAGGGTGACGGTGCC-3' was used for detecting ptetA-mediated transcripts. All
primers hybridize only with plasmid DNA sequences so that no
transcripts from the chromosomal genes would interfere with the primer
extension results. With the exception of Figs. 1 and 6 (see below), an
individual primer was mixed with 100 µg of total RNA in the primer
extension reactions. Two primers were mixed in the primer extension
reactions for simultaneous detection of the bla and
leu-500 transcripts in Fig. 1 and the leu-500 and
tac transcripts in Fig. 6. The initiation sites of RNA
transcripts were verified based on the specific sizes of the primer
extension DNA products that run off the 5'-end of RNA transcripts. A
DNA sequence ladder was prepared using each individual primer for
verifying the initiation site at a DNA sequence level. The
radioactivity of primer extension DNA product was visualized and
quantified using a Storm imaging system (model 840, Molecular Dynamics). The reported quantification is the average of at least two
experiments. The signals were normalized based on the total plasmid DNA
content in the harvested cells. 1.5-ml aliquots of bacterial culture
were saved at the harvest and used to prepare total plasmid DNA. The
total plasmid DNA in the 1.5-ml sample was analyzed by agarose gel
electrophoresis followed by Southern blotting using a
32p-labeled probe that specifically hybridizes with plasmid DNA.
Analysis of DNA Topoisomers by Two-dimensional Gel
Electrophoresis--
DNA samples were separated by 1% agarose gel
electrophoresis in the first dimension in 1× Tris-phosphate-EDTA (TPE)
buffer (containing 80 mM Tris phosphate and 8 mM EDTA, pH 8.0), the 20- × 20-cm gel was then soaked in 4 µM chloroquine (Sigma) for 2 h. The soaked gel was
turned 90° and electrophoresed in the second dimension in 1× TPE
buffer containing 4 µM chloroquine. The two-dimensional gels were subjected to in situ Southern hybridization
(described in Ref. 21) using pAO-specific 32p-labeled probe
so that the coexisting pEV101 was not visualized.
Immunoblotting Detection of Overexpressed LeuO in a
leuO The 318-bp AT-rich Intervening DNA Represses Short-range
Promoter-promoter Interaction--
The 318-bp AT-rich DNA is located
between the two divergently arrayed promoters, pleuO and
pleuABCD. Previous studies have demonstrated that this
region is involved in the promoter relay mechanism for
pleu-500 activation (5, 6). To test the effect of the 318-bp
AT-rich intervening DNA on the interaction between pleuO and
pleuABCD, we have constructed two plasmids, pWU802T and
pWU812T (Fig. 1). pWU802T was constructed
from a plasmid that contains the entire region of pleuO,
318-bp AT-rich DNA, and pleuABCD in their corresponding
chromosomal context, by replacing pleuO with an
IPTG-inducible tac promoter, ptac (25). The
leu-500 mutant was included so that a nearly on-off
pleu-500 activity change (6) could be used as indication of
the short-range ptac-pleu-500 interaction (11).
pWU812T was constructed from pWU802T by replacing the 318-bp AT-rich
intervening DNA with a 320-bp promoterless and non-AT-rich DNA sequence
from human cathepsin B cDNA (19). The leuO coding region
in both plasmids was truncated so that no functional LeuO was generated
in cis. Upon IPTG induction, LeuO was produced in
trans from a coexisting expression vector, pEV101 (6). The
absence of the 318-bp AT-rich DNA in pWU812T resulted in an
IPTG-inducible pleu-500 activation in S. typhimurium CH582 (topA The 318-bp AT-rich Intervening DNA-mediated Repression Is Not Due
to Restriction of the Rotational Motion of the DNA Helix--
The
short-range interaction between the two divergently arrayed promoters,
ptac and pleu-500, is almost certainly due to
transcription-driven DNA supercoiling (11, 13). The repressive effect
of the 318-bp AT-rich intervening DNA on the short-range
promoter-promoter interaction is intriguing. One possible explanation
is that the AT-rich intervening DNA may be organized into a large
structure (e.g. anchored to a large mass) so that DNA
rotation along its helical axis is restricted (schematically
illustrated in Fig. 2). To test this
possibility, a DNA supercoiling assay based on previously established
methodology (15) was used. For these experiments, the 318-bp AT-rich
DNA was inserted at the unique AatII site in pAO so that the
major transcription unit,
This assay was designed based on the theory that if an anchor was
formed at the inserted AT-rich DNA, the accumulation of transcription-driven supercoiling would be intensified due to the lack
of a diffusional pathway to dissipate DNA supercoils (15). Under such a
condition, inhibition of DNA gyrase would result in a relatively more
rapid increase in the DNA linking number (positive DNA supercoiling) in
both monomeric and dimeric plasmid DNAs as previously demonstrated
using lac operator as a model system (15). When such an
experiment was performed in cells harboring pAO-AT, which contained the
318-bp AT-rich DNA instead of the lac operator, no
accumulation of the positively supercoiled DNA topoisomers was observed
(Fig. 2C). In fact, the topoisomer distribution of pAO was
almost identical to that of pAO-AT (Fig. 2, compare A and
C), suggesting that the 318-bp AT-rich DNA insert did not
significantly restrict DNA helix rotation and therefore could not block
DNA supercoils generated by transcription of the bla gene.
In addition, IPTG induction (the production of LeuO protein from
pEV101) did not affect the topoisomer distribution of either pAO or
PAO-AT (Fig. 2, B and D). These results suggest that the 318-bp AT-rich DNA-mediated repression is unlikely to be due
to restriction of DNA helix rotation.
The 318-bp AT-rich DNA Represses Transcription of Adjacent
Promoters--
The 318-bp AT-rich DNA could repress transcription from
one of the flanking promoters (either ptac or
pleu-500) and thereby abolishes short-range
promoter-promoter interaction. To test this possibility, we examined
the transcription activity of ptac in pWU802T and pWU812T.
Primer extension results indicated that the ptac activity
(Fig. 3) strikingly correlated with the
pleu-500 activity (Fig. 1). The ptac functioned
normally if the 318-bp AT-rich DNA was replaced with a neutral DNA
sequence of similar size as in pWU812T (lanes 6 and
8 in Fig. 3). In the presence of the native 318-bp AT-rich
DNA, the ptac activity on pWU802T was severely impaired
(Fig. 3, lane 4). On the same plasmid, the ptac
activity was partially restored if LeuO was provided in
trans (Fig. 3, lane 2). This result strongly supports
the notion that the 318-bp AT-rich intervening DNA is a negative
regulatory element for transcription.
A 72-bp DNA Element Within the 318-bp AT-rich DNA Silences Adjacent
Genes--
To test whether or not the repressive effect of the 318-bp
AT-rich DNA element on transcription can be observed with other promoters, the 318-bp DNA was inserted at the unique AatII
site located 99 bp upstream of the bla promoter
(pbla) in pAO. In either orientation (pAO-AT and pAO-ATR),
the 318-bp AT-rich DNA insert caused an ~45% reduction of the
pbla activity (Fig.
4A, compare lanes 2 and 3 with lane 1). Deletion analysis using
pAO-AT had located a predominant gene-silencing effect (more than 80%
reduction on pbla activity) in AT4, a 72-bp AT-rich DNA
located near the pleuO end of the 318-bp DNA (lane
6 in Fig. 4A). Those DNA inserts containing the 72-bp
AT4 plus all or part of the rest of 318-bp AT-rich DNA (AT, ATR, and
AT2) exerted lesser gene-silencing effects (lanes 2,
3, and 4 in Fig. 4A). Furthermore, the
AT1 DNA segment, which represents the 146-bp pleu-500 end of
the 318-bp AT-rich DNA, enhanced the pbla activity
(lane 5 in Fig. 4A). These results indicate that,
although the 72-bp AT4 exhibits a clear gene-silencing effect, a
complex transcriptional effect is present in the rest of the 318-bp
AT-rich DNA. The stronger silencing effect that is associated with AT4
may be due to elimination of other complex and opposing effects within
the 318-bp AT-rich intervening DNA.
AT4-mediated Gene Silencing Is Additive, Orientation-independent,
and topA Genetic Background-independent--
Inversion of the AT4 DNA
insert did not significantly affect gene silencing (compare lanes
8 and 10 in Fig. 4A). The reduction of
pbla activity was ~80% with either orientation.
Furthermore, the gene-silencing effect was additive. In either
orientation, an ~95% reduction was achieved when the AT4 DNA insert
was tandemly repeated (lanes 9 and 11 in Fig.
4A). Thus far, characterization of the 72-bp gene silencer
had been carried out in the S. typhimurium topA LeuO Protein Negates AT4-mediated Gene Silencing--
The
trans-acting LeuO protein was shown to relieve 318-bp AT-rich
DNA-mediated repression of the short-range pleuO
(ptac)-pleu-500 interaction in pWU802T (Fig. 1).
To test whether or not LeuO can also suppress AT4-mediated gene
silencing, the effect of LeuO on AT4-mediated silencing of
pbla was examined in an E. coli
leuO
However, the effect of LeuO on transcription could be nonspecific. To
test whether or not the effect of LeuO is specific for AT4, a 72-bp DNA
consisting of a DNA sequence from the lacZ coding region was
synthesized and used to replace the 72-bp AT4 DNA in pWU802T
(illustrated in Fig. 6). In the absence of LeuO (i.e. the
absence of pEV101), this replacement resulted in a LeuO-independent pleu-500 activation on the mutant plasmid (Fig.
6B, lane 2). A significant
ptac-mediated transcription activity was also detectable in
the mutant plasmid (Fig. 6A, lane 2). With the
native 72-bp AT4 in place, a significantly reduced ptac
activity was detected in pWU802T (Fig. 6A, lane
1). Apparently, the reduced ptac activity was too weak
to activate pleu-500 at this distance (Fig. 6B,
lane 1). Our study, thus far, has clearly indicated that
LeuO relieves 318-bp intervening DNA-mediated repression (Fig. 1) by
specifically negating AT4-mediated gene silencing.
The Effect of Adjacent Transcriptional Activity on AT4-mediated
Gene Silencing--
The 72-bp gene silencer, AT4, is located at the
5'-ends of the divergently arrayed leuO and
leuABCD genes. Such a chromosomal organization may not be a
coincidence, because transcription-generated negative DNA supercoiling
is known to accumulate in such a topological domain (13, 21). To test
whether or not AT4 functions most effectively when placed between
divergently arrayed promoters, we inserted AT4 at the unique
AatII site in pBR322 DNA so that AT4 was flanked by the
divergently arrayed bla and tetA genes (illustrated in Fig. 7). As expected, AT4
caused reductions in both pbla and ptetA
activities (Fig. 7, A and B). However, the gene-silencing effect of AT4 in pBR322 (Fig. 7A, lanes
2) was lower than that in pAO-AT4 (Fig. 4). The anti-tet
transcription activity (16) that read through the AT4 insert in pBR322
could be the reason for this reduction. Despite the reduced effect, AT4-mediated gene silencing on pBR322 was still additive in the presence of a tandemly repeated AT4 dimer (Fig. 7, A and
B, lanes 3). Due to the simultaneous
gene-silencing effects on both flanking promoters, it remained unclear
whether or not the bacterial gene silencer was affected by an adjacent
transcription activity.
To test the effect of adjacent transcription on AT4-mediated gene
silencing, pJW270-based plasmid constructs were used. pJW270-based plasmids are essentially the same as pBR322, except that the
tetA promoter is replaced by an IPTG-inducible
lacUV5 promoter and that an iq
promoter-controlled laci gene was inserted at the 5'-end of
the lacUV5 promoter (Refs. 17 and 21, and plasmid
maps in Fig. 7). The opposite orientation of the laci
gene in pJW270 and pJW270II was designed to test the effect of an
adjacent transcription activity on AT4-mediated gene silencing. As
expected, AT4-mediated gene silencing on pbla was observed
in pJW270II-AT4 and pJW270II-AT4R when laci was transcribing
away from the AT4 DNA inserts (Fig. 7C, lanes
4-6). Strikingly, the gene-silencing effect was abolished when
laci was inverted in pJW270-AT4 and pJW270-AT4R (Fig.
7C, lanes 1-3). With a location at the 3'-end of
an adjacent transcription unit, AT4 not only exerted no gene-silencing
effect on the pbla, but the transcription activity of
pbla was actually increased (Fig. 7C, lanes
2 and 3). This result demonstrates that a parallel (convergently) arrayed adjacent transcription unit abolished
AT4-mediated gene silencing. Transcription-driven positive DNA
supercoiling at the 3'-end of a transcription unit could be the reason
why AT4 function was abolished at such a location. Another
interpretation is that AT4 is functional only when it is located at the
5'-end of a transcription unit. For maximum gene-silencing activity, adjacent transcription activities should be transcribing away from the
gene silencer. Read-through transcription activity and a 3'-end
location of a transcription unit will either weaken or impair
AT4-mediated gene silencing.
AT4-mediated Gene Silencing Is Effective Up to a 300-bp
Distance--
The chromosomal location of AT4 was centered at the pleu-500 activation has been suggested to be regulated
in a complex manner involving sequential promoter activation in the ilvIH-leuO-leuABCD gene cluster. Lilley and Higgins (26)
have suggested that the transcriptional activity of leuO may
be responsible for pleu-500 activation. The present study
has experimentally demonstrated the importance of the leuO
gene in pleu-500 activation. We have shown that both
pleuO and the leuO gene product are required for
pleu-500 activation. The function of LeuO is to reverse
silencing mediated by the AT4 DNA sequence element, and the function of transcription from pleuO is to provide short-range
promoter-promoter interaction for activation of pleu-500
(11).
The AT-rich DNA that is flanked by the divergently arrayed
leuO and leuABCD is found in both S. typhimurium (GenBankTM accession number AF106956) and E. coli (GenBankTM accession number AF 106955). These noncoding,
AT-rich DNAs appear to be the regulatory regions for sequential gene
activation of the ilvIH-leuO-leuABCD gene cluster (24).
However, these AT-rich intervening DNAs show little DNA sequence
homology (14). AT richness appears to be the only similarity between
these DNAs. Previous studies have demonstrated that both the AT-rich
DNA as well as the leuO gene are important for
pleu-500 activation (6). However, the precise role(s) of the
AT-rich DNA had been unclear. The present study has identified a 72-bp
AT4 bacterial gene silencer in the AT-rich DNA.
How does a 72-bp A/T-rich DNA sequence element cause the
transcriptional repression? AT4 is unlikely affecting transcription activity via a currently known regulatory mechanism, because bacterial transcription-negative regulation is usually functional at a short distance (e.g. within 100 bp). In fact, the binding site of
the bacterial regulator often maps directly in the promoter region. For
example, the lac repressor binding site (lac
operator) overlaps with the What is the mechanism whereby AT4 silences an adjacent transcription
activity at a distance of 300 bp? LeuO-mediated reversal effect (Fig.
5) has provided a possible clue. Overexpression or underexpression of
LeuO has been linked to a number of
hns H-NS has been known to bind preferentially to curved DNA (36, 37). Once
H-NS is recruited to the local site (the AT-rich DNA sequence element,
AT4), the binding cooperativity of H-NS may cause a cis-spreading
(oligomerization) of H-NS to the promoter region. The H-NS oligomer may
physically block RNA polymerase complex from accessing the promoter
( A similar direct transcriptional repressor role of H-NS was proposed by
Ueguchi and Mizuno (40). They have shown in vitro that H-NS
inhibits proV (proU)-mediated transcription by
directly binding to the promoter region. The repressive H-NS complex is strikingly local and highly specific to the DNA sequence in the proV promoter, because H-NS does not affect transcription
from ptac on the same DNA molecule (40). One or more
cis-elements in the proV promoter must be responsible for
H-NS recruitment to the local site. The 72-bp AT4 DNA may contain one
or more similar elements that trigger H-NS localization in the
proV promoter. Because DNA structure rather than the
specific DNA sequence is known to be important for H-NS localization
(36, 37 and reviewed in Ref. 41), no DNA sequence homology is expected
between the promoters that utilize such a mechanism in their
transcriptional silencing.
The idea that DNA structural elements could serve as signals for the
formation of a transcriptional repressive nucleoprotein structure may
also be applicable to explain the well known eukaryotic heterochromatic
gene-silencing mechanism, because high A+T composition and
repetitiveness are the two common features for DNA structural elements
involved in heterochromatin formation. The LINE-1 element in X
chromosome inactivation (42 and reviewed in Ref. 43), the satellite
DNAs in the centromeric- or telomeric-heterochromatin (reviewed in Ref.
44), and the silencers I and E in the yeast MAT loci (reviewed in Ref.
45) are all AT-rich. Combinations of any pair of the yeast silencers,
HMR-E, HML-R, and HML-I, can result in inactivation of any gene
activity flanked by the AT-rich DNA elements (46). However, the
mechanistic link between DNA structural elements in the eukaryotic
silencers and the formation of heterochromatin has been unclear.
Detailed studies of the 72-bp AT-rich DNA-mediated gene silencing in
bacteria could shed light on the mechanism of gene silencing in both
prokaryotes and eukaryotes.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
10 region of the promoter of the Salmonella typhimurium
leuABCD operon (1). The transcriptional activity of the mutant
promoter is DNA supercoiling-dependent (2). The mechanism
whereby the leu-500 promoter (pleu-500) is
activated in the topA mutants is intriguing (3-7). Previous
studies using a plasmid system have demonstrated that activation of
plasmid-borne pleu-500 in topA mutants requires an upstream transcriptional activity transcribing away from
pleu-500 (8-11). This notion has been confirmed in a recent
study using the chromosomal setting (12). Transcriptional activation of the ilvIH promoter (pilvIH) located 1.9 kilobases
upstream of pleu-500 was shown to be responsible for
pleu-500 activation (5). Transcription-driven DNA
supercoiling (13) has been suggested to play a role in this long-range
promoter-promoter interaction.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
), an isogenic pair of S. typhimurium strains, were described previously (3) and provided by Dr. David Lilley. AS19, an Escherichia coli B strain that is
permeable to drugs such as novobiocin (20) was previously used in
studies involving gyrase inhibition (15, 21). The
leuO
strain, MF1, was derived from MC4100, an
E. coli K-12 strain (22). TO2 is a
leuO strain due to the replacement of the
BstXI-ApaI fragment of the leuO coding
region with a DNA fragment carrying the cam
(cmr) gene (23). MF1 was prepared by introducing the
leuO::cam mutation into MC4100 (recipient strain)
by P1 transduction using TO2 as a donor strain. Disruption of the
leuO in the chromosome in MF1 was confirmed by the
restriction enzyme cleavage pattern and the size of the DNA product of
polymerase chain reaction (PCR). Bacteria were grown in Luria-Bertani
(LB) medium at 37 °C with aeration. 50 µg/ml ampicillin or 12.5 µg/ml tetracycline was added as needed.
Strain--
A LeuO-specific antiserum was raised by
injecting the purified overexpressed S. typhimurium
His-tagged LeuO into a rabbit. The affinity-purified IgG (1.4 mg/ml)
from the antiserum was used at a dilution factor of 1:5000 as the
primary antibody to detect the cellular LeuO protein. The secondary
antibody was anti-rabbit IgG conjugated to alkaline phosphatase. The
blot was developed by ECL in a modification of a previous procedure
(24) using an ECF Western blotting kit (Amersham Pharmacia Biotech).
The chemifluorescence signal was detected and quantified by the Storm 840 imaging system (Molecular Dynamics).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
), regardless of
the presence or absence of LeuO (Fig. 1, lanes 6 and
8). In contrast, with the 318-bp native AT-rich intervening DNA in place, pleu-500 failed to be activated in pWU802T in
the absence of LeuO (Fig. 1, compare lanes 3 and
4). Consistent with the previous results (6), IPTG induction
resulted in activation of pleu-500 on pWU802T when LeuO was
provided in trans (Fig. 1, compare lanes 1 and
2). These results demonstrate a repressive effect of the
318-bp AT-rich intervening DNA on the short-range pleuO(ptac)-pleu-500 interaction. The
trans-acting LeuO relieves the repression.
View larger version (46K):
[in a new window]
Fig. 1.
The 318-bp AT-rich intervening DNA represses
the interaction of the flanking promoters. pWU802T containing the
native 318-bp AT-rich intervening DNA and pWU812T containing a 320-bp
non-AT-rich DNA replacement were assayed for pleu-500
activation in CH582 using primer extension. Upon IPTG induction, the
coexisting pACYC-based plasmid, pEV101, provided both LeuO and
lac repressor (LacI), whereas pSO1000 provided only LacI in
trans. LacI is required for controlling expression from the
IPTG-inducible ptac in pWU802T, pWU812T, and pEV101 in the
lac repressor-free CH582. The presence of either one of the
coexisting plasmids and the IPTG treatment is indicated with "+"
sign. The " " sign or blank indicates the absence of
the treatment or coexisting plasmid in the experiment. The DNA sequence
ladders are included to locate the transcription initiation sites of
leu-500 and bla transcripts. The bla
transcript serves as an internal control.
-lactamase gene (bla), is
transcribing away from the AT-rich DNA insert (illustrated in Fig. 2).
The resultant plasmid, pAO-AT, and the parental plasmid, pAO, were used
in the assay.
View larger version (48K):
[in a new window]
Fig. 2.
The 318-bp AT-rich DNA does not restrict the
rotational motion of DNA helix. pAO-AT is identical to pAO except
that it carries the 318-bp AT-rich DNA insert. AS19 harboring either
pAO (A and B) or pAO-AT4 (C and
D) with (B and D) or without
(A and C) the coexisting pEV101 was treated with
100 µg/ml novobiocin 30 min prior to cell harvest. To provide LeuO in
trans, cells harboring pEV101 were induced with 1 mM IPTG
(1 h) prior to the novobiocin treatment (30 min) (B and
D). Plasmids were isolated and analyzed by two-dimensional
gel electrophoresis. The locations of the negatively supercoiled DNA
topoisomers (a or a') and the positively
supercoiled DNA topoisomers (b or b') were marked
in the arc shape patterns of monomeric or dimeric DNAs, respectively.
No preferential positive supercoiling accumulation in pAO-AT (compare
the b/a ratio for monomeric DNAs or
b'/a' ratio for dimeric DNAs) was observed.
View larger version (18K):
[in a new window]
Fig. 3.
The 318-bp AT-rich DNA possesses
gene-silencing activity. Primer extension was employed for
detecting RNA transcripts initiated from ptac on pWU802T or
pWU812T in a similar set of experiments as described in Fig. 1. The
primer extension results were quantified and shown in arbitrary
units.
View larger version (54K):
[in a new window]
Fig. 4.
AT4 DNA sequence element is responsible for
gene silencing. As illustrated, the whole or the various parts of
the 318 AT-rich DNA was inserted in either orientation at the unique
AatII site located at the 99 position of bla in
pAO. Plasmids were assayed in CH582. Primer extension results were
quantified and shown. All quantified results were compared with the
pbla activity on pAO (A, lanes 1 or
7), which was arbitrarily set as 1. The identified
AT4-mediated gene-silencing effect was tested in CH601(B).
Again, pbla activity on pAO in either CH601 (lane
1) or CH582 (lane 3) was quantified and served as the
standard for comparison with the pbla activity in pAO-AT4 in
either CH601 (lane 2) or CH582 (lane 4),
respectively.
strain, CH582, where pleu-500
activation was originally studied. The topA
genetic background has been shown to enhance short-range
promoter-promoter interaction such as activation of a plasmid-borne
pleu-500 (11). To examine whether AT4-mediated
gene-silencing effect was dependent on the
topA
genetic background, pAO-AT4 was tested in
an S. typhimurium topA+
strain, CH601, which is the parental strain of CH582. The same degree of gene silencing (~80% reduction of the pbla
activity) was observed in both the topA+ and the
topA
strains (Fig. 4B).
strain, MF1 (Fig.
5). A slightly stronger gene-silencing
effect (~88% reduction of the pbla activity) was found in
the LeuO-free strain (Fig. 5A, compare lanes 1 and 2). When LeuO was provided in trans in MF1
from a coexisting expression vector, pEV101, AT4-mediated gene
silencing was nearly abolished even without IPTG induction (Fig.
5A, lane 3). This was probably due to the leakage
of LeuO from the expression vector, pEV101. Such a leakage was
evidenced from immunoblotting analysis (Fig. 5C, lane
2; 8.6 ng of LeuO was detected in the 100 µg of total protein
loaded). Upon further increase of cellular LeuO due to IPTG induction
(Fig. 5C, lanes 3-6), AT4-mediated gene
silencing was completely eliminated (Fig. 5A, lanes
4-7). The pbla activity was fully restored with 50 µM IPTG treatment (Fig.
5A, compare lanes 5 and 1). In a control experiment using the parental plasmid
pAO, the pbla activity was unaffected by IPTG treatment
(data not shown). These results indicate that LeuO negates AT4-mediated
gene silencing.
View larger version (54K):
[in a new window]
Fig. 5.
LeuO protein negates AT4-mediated gene
silencing. pAO or pAO-AT4 was assay for AT4-mediated gene
silencing in E. coli leuO strain, MF1. The
presence of the coexisting pEV101 and the IPTG treatment is indicated
with "+" sign. The "
" sign indicates the absence of the
treatment or coexisting plasmid in the experiment. 1.5-ml aliquots of
bacterial culture from the IPTG titration experiment were saved at
harvest and prepared for the total protein lysates. 100 µg of total
protein from each lysate was loaded along with the LeuO standard, 10 ng
of purified LeuO (lane C) and the size markers (lane
M) on SDS-polyacrylamide gel electrophoresis. The LeuO standard
was not visible in the Coomassie Blue-stained gel shown in
B. The overexpressed LeuO bands were quantified and compared
with the LeuO standard in the immunoblotting result of the gel
(C). The detected LeuO amount (nanograms) in 100 µg of
total protein at each IPTG titration point was shown below
each lane. In the absence of pEV101, there was no immunologically
detectable LeuO in the leuO
strain, MF1 (lane
1).
View larger version (25K):
[in a new window]
Fig. 6.
AT4 represses the short-range interaction
between ptac and pleu-500.
pWU802T(AT4)R was derived from pWU802T by replacing the 72-bp AT4 DNA
element with a promoterless neutral DNA of same size derived from the
lacZ coding region. pWU802M(AT4)R was derived from
pWU802T(AT4)R by deleting the 66-bp ptac. Plasmids were
assayed in CH582, in which ptac activity is not required
IPTG induction due to the lack of lac repressor in S. typhimurium. The transcription activities of both ptac
and pleu-500 were simultaneously detected using primer
extension (A and B).
View larger version (37K):
[in a new window]
Fig. 7.
An adjacent transcription activity affects
AT4-mediated gene silencing. AT4-mediated gene silencing was
analyzed by placing the AT4 DNA insert at the unique AatII
site on pBR322, pJW270, and pJW270II, respectively. Both flanking
pbla and ptetA activities on various pBR322
vectors in CH582 were detected using primer extension and shown in
A and B, respectively. The constructs were:
lane 1, pBR322 vector with no insert; lane 2,
pBR322 with AT4 insert; lane 3, pBR322 with a tandemly
repeated AT4 dimer. The pbla activity of pJW270 or pJW270II
vector in CH582 with or without the AT4 insert in either orientation
was detected by primer extension and shown in C.
83
position of leuO and at the
351 position of the
divergently arrayed leuABCD. In pBR322-AT4, AT4 was located
at the
135 position of bla and the
159 position of
tetA. Thus it was unclear whether or not AT4 could directly
silence pleuABCD at its more distal location (351 bp). If
AT4-mediated gene silencing reaches a distance of 351 bp, LeuO may
cause pleu-500 activation directly rather than indirectly
via a subsequent short-range
pleuO(ptac)-pleu-500 interaction. To
address this issue, four promoterless DNAs consisting of DNA sequences
from the coding region of lacZ were used to sequentially extend the distance between AT4 DNA insert and pbla in
pAO-AT4 (illustrated in Fig. 8). The
location of AT4 in pAO-AT4 was at the
135 position of bla.
The four insertions resulted in plasmids with AT4 located at positions
188,
242,
296, and
350 of bla, respectively. Primer
extension results indicated that AT4-mediated gene silencing was
slightly reduced but remained effective up to a distance of 296 bp
(Fig. 8, lanes 3-5). The gene-silencing effect was
completely abolished at the distance of 350 bp (Fig. 8, lane
6).
View larger version (40K):
[in a new window]
Fig. 8.
AT4-mediated gene silencing can reach a
distance of 300 bp. A series of promoterless DNAs consisting of
DNA sequences from the lacZ coding region were inserted at
the DraIII site between the AT4 DNA insert and
bla in pAO-AT4 as illustrated. The insertions resulted in a
series of plasmids where AT4 DNA is located at the positions centered
at 188,
242,
296, and
350 (counted from the +1 position) of
bla, respectively. Using primer extension, the
pbla activity was measured from pAO, pAO-AT4, and the
plasmid series in CH582. The primer extension results were quantified.
The pbla activity in pAO was arbitrarily set as 1.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
10 sequence of lac promoter
(27). Hence, the bacterial repressor is usually gene-specific. Acting
at a distance (e.g. 1000 bp) is possible via an intervening
DNA looping mechanism. Two examples of this include DNA
looping-mediated transcriptional activation in nitrogen
(ntr) regulation (28) and DNA looping-mediated transcriptional repression of the araBAD promoter (29).
However, if DNA looping due to protein-protein interaction is important for AT4-mediated gene silencing, the optimum distance for gene silencing should be about 500 bp (30). Using the lac
operator as a model system, it has been experimentally demonstrated
that, starting from a distance of ~150 bp, DNA looping mediated by
repressor binding to the operator increased dramatically when the size
of the intervening DNA increased. This effect peaked at a distance of
500 bp (30). In contrast, AT4 retained its transcriptional repressive
effect up to a distance of 300 bp. The repressive effect was abolished
at a distance longer than 350 bp (Fig. 8). Starting from a distance of
188 bp up to the 300-bp limit, the repressive effect was slightly
reduced rather than dramatically enhanced as one might expect for the
above-discussed protein-protein interaction at a distance scenario.
This clear difference argues against the possibility that AT4 functions
as a binding site for a repressor "acting at a distance," which
represses transcription via a direct contact with the RNA
polymerase complex at a distance.
-associated phenotypes (23, 31, 32).
Mizuno's group has shown that LeuO relieves bgl silencing
in E. coli (23). Both H-NS and AT-rich DNA flanking the
bgl promoter have been shown to be responsible for
bgl silencing (33-35). In addition, using the
pleu-500 activity as a reporter, we have shown genetically that H-NS plays a repressive role in the transcriptional
regulation.2 Together, these
results suggest a possible involvement of H-NS in AT4-mediated gene silencing.
35 and
10 sequences). The binding cooperativity of H-NS may
determine the size of the H-NS oligomer and hence the 300-bp distance
limit of the AT4-mediated gene-silencing effect. The proposed mechanism
is similar but distinct from the nucleoprotein filament model for the
bacterial centromere site-mediated transcriptional silencing, which
affects genes within several kilobases (38). Our model is also
different from the DNA sequestration-mediated gene silencing model
(39).
![]() |
ACKNOWLEDGEMENT |
---|
We are in debt to Dr. Ray Mattingly for his critical reading of the manuscript.
![]() |
FOOTNOTES |
---|
* This work was supported by a National Institutes of Health Grant GM-53617.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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF106956 and AF106955.
To whom correspondence should be addressed: Dept. of Pharmacology,
Wayne State University, School of Medicine, 540 E. Canfield Ave.,
Detroit, MI 48201. Tel.: 313-577-1584; Fax: 313-577-6739; E-mail:
haiwu@med.wayne.edu.
Published, JBC Papers in Press, December 19, 2000, DOI 10.1074/jbc.M010501200
2 M. Fang and H.Y. Wu, unpublished data.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
bp, base pair(s);
PCR, polymerase chain reaction;
IPTG, isopropyl-1-thio--D-galactopyranoside;
H-NS, histone-like nucleoid structuring protein.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Mukai, F. H., and Margolin, P. (1963) Proc. Natl. Acad. Sci. U. S. A. 50, 140-148 |
2. | Trucksis, M., Golub, E. I., Zabel, D. J., and Depew, R. E. (1981) J. Bacteriol. 147, 679-681[Medline] [Order article via Infotrieve] |
3. | Richardson, S. M. H., Higgins, C. F., and Lilley, D. M. J. (1984) EMBO J. 3, 1745-1752[Abstract] |
4. | Richardson, S. M. H., Higgins, C. F., and Lilley, D. M. J. (1988) EMBO J. 7, 1863-1869[Abstract] |
5. | Wu, H.-Y., Tan, J., and Fang, M. (1995) Cell 82, 445-451[Medline] [Order article via Infotrieve] |
6. |
Fang, M.,
and Wu, H.-Y.
(1998)
J. Bacteriol.
180,
626-633 |
7. |
Fang, M.,
and Wu, H.-Y.
(1998)
J. Biol. Chem.
273,
29929-29934 |
8. | Chen, D., Bowater, R. P., Dorman, C. J., and Lilley, D. M. J. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 8784-8788[Abstract] |
9. | Chen, D., Bowater, R. P., and Lilley, D. M. J. (1993) Biochemistry 32, 13162-13170[Medline] [Order article via Infotrieve] |
10. | Chen, D., Bowater, R. P., and Lilley, D. M. J. (1994) J. Bacteriol. 176, 3757-3764[Abstract] |
11. | Tan, J., Shu, L., and Wu, H.-Y. (1994) J. Bacteriol. 176, 1077-1086[Abstract] |
12. | El Hanafl, D., and Bossi, L. (2000) Mol. Microbiol. 37, 583-594[CrossRef][Medline] [Order article via Infotrieve] |
13. | Liu, L. F., and Wang, J. C. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 7024-7027[Abstract] |
14. | Haughn, G. W., Wessler, S. R., Gemmill, R. M., and Calvo, J. M. (1986) J. Bacteriol. 166, 1113-1117[Medline] [Order article via Infotrieve] |
15. | Wu, H.-Y., and Liu, L. F. (1991) J. Mol. Biol. 219, 615-622[CrossRef][Medline] [Order article via Infotrieve] |
16. | Stuber, D., and Bujard, H. (1981) Proc. Natl. Acad. Sci. U. S. A. 78, 167-171[Abstract] |
17. | Giaever, G. N., Schneider, L., and Wang, J. C. (1988) Biophys. Chem. 29, 7-15[CrossRef][Medline] [Order article via Infotrieve] |
18. | Oehler, S., Eismann, E. R., Krämer, H., and Müller-Hill, B. (1990) EMBO J. 9, 973-979[Abstract] |
19. | Cao, L., Taggart, T., Berquin, I. M., Fong, D., and Sloane, B. F. (1994) Gene 139, 163-169[Medline] [Order article via Infotrieve] |
20. | Sekiguchi, M., and Iida, S. (1967) Proc. Natl. Acad. Sci. U. S. A. 58, 2315-2320[Medline] [Order article via Infotrieve] |
21. | Wu, H.-Y., Shy, S.-H., Wang, J. C., and Liu, L. F. (1988) Cell 53, 433-440[Medline] [Order article via Infotrieve] |
22. | Casadaban, M. J. (1976) J. Mol. Biol. 104, 541-555[Medline] [Order article via Infotrieve] |
23. |
Ueguchi, C.,
Ohta, T.,
Seto, C.,
Suzuki, T.,
and Mizuno, T.
(1998)
J. Bacteriol.
180,
190-193 |
24. | Fang, M., Majumder, A., Tsai, K.-J., and Wu, H.-Y. (2000) Biochem. Biophys. Res. Commun. 276, 64-70[CrossRef][Medline] [Order article via Infotrieve] |
25. | De Boer, H. A., Comstock, L. J., and Vasser, M. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 21-25[Abstract] |
26. | Lilley, D. M. J., and Higgins, C. F. (1991) Mol. Microbiol. 5, 779-783[Medline] [Order article via Infotrieve] |
27. | Miller, J. H. (1980) in The Operon (Miller, J. H. , and Reznikoff, W. S., eds) , pp. 31-88, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY |
28. | Merrick, M. J., and Edwards, R. A. (1995) Microbiol. Rev. 59, 604-622[Abstract] |
29. | Lobell, R. B., and Schleif, R. F. (1990) Science 250, 528-532[Medline] [Order article via Infotrieve] |
30. | Mossing, M. C., and Record, M. T., Jr. (1986) Science 233, 889-892[Medline] [Order article via Infotrieve] |
31. | Shi, X., and Bennett, G. N. (1995) J. Bacteriol. 177, 810-814[Abstract] |
32. | Klauck, E., Böhringer, J., and Hengge-Aronis, R. (1997) Mol. Microbiol. 25, 559-569[Medline] [Order article via Infotrieve] |
33. | Schnetz, K. (1995) EMBO J. 14, 2545-2550[Abstract] |
34. |
Schnetz, K.,
and Wang, J. C.
(1996)
Nucleic Acids Res.
24,
2422-2428 |
35. | Mukerji, M., and Mahadevan, S. (1997) Mol. Microbiol. 24, 617-627[CrossRef][Medline] [Order article via Infotrieve] |
36. | Yamada, H., Yoshida, T., Tanaka, K., Sasakawa, C., and Mizuno, T. (1991) Mol. Gen. Genet. 230, 332-336[Medline] [Order article via Infotrieve] |
37. | Owen-Hughes, T., Pavitt, G. D., Santos, D. S., Sidebotham, J. M., Hulton, C. S., Hinton, J. C., and Higgins, C. F. (1992) Cell 71, 255-265[Medline] [Order article via Infotrieve] |
38. |
Rodionov, O.,
Lobocka, M.,
and Yarmolinsky, M.
(1999)
Science
283,
546-549 |
39. |
Kim, S.-K.,
and Wang, J. C.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
8557-8561 |
40. | Ueguchi, C., and Mizuno, T. (1993) EMBO J. 12, 1039-1046[Abstract] |
41. | Pettijohn, D. (1996) in Escherichia coli and Salmonella Cellular and Molecular Biology, volume 1 (Neidhardt, F. C. , Curtiss, R., III , Ingraham, J. L. , Lin, E. C. C. , Low, K. B. , Magasanik, B. , Reznikoff, W. S. , Riley, M. , Schaechter, M. , and Umbarger, H. E., eds) , pp. 158-166, American Society for Microbiology, Washington, DC. |
42. |
Bailey, J. A.,
Carrel, L.,
Chakravarti, A.,
and Eichler, E. E.
(2000)
Proc. Natl. Acad. Sci. U. S. A.
97,
6634-6639 |
43. |
Lyon, M. F.
(2000)
Proc. Natl. Acad. Sci. U. S. A.
97,
6248 |
44. | Murphy, T. D., and Karpen, G. H. (1998) Cell 93, 317-320[Medline] [Order article via Infotrieve] |
45. | Haber, J. E. (1998) Annu. Rev. Genet. 32, 561-599[CrossRef][Medline] [Order article via Infotrieve] |
46. | Shei, G. J., and Broach, J. R. (1995) Mol. Cell. Biol. 15, 3496-3506[Abstract] |