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A Cis-spreading Nucleoprotein Filament Is Responsible for the Gene
Silencing Activity Found in the Promoter Relay Mechanism*
Received for publication, October 18, 2004, and in revised form, December 3, 2004
Published, JBC Papers in Press, December 6, 2004, DOI 10.1074/jbc.M411840200
Chien-Chung Chen, Meng-Yun Chou, Chun-Hao Huang, Arundhati Majumder,
and Hai-Young Wu‡
From the Department of Pharmacology, School of Medicine, Wayne State University, Detroit, Michigan 48201
Transcription-generated DNA supercoiling plays a de-
cisive role in a promoter relay mechanism for the coor-
dinated expression of genes in the Salmonella typhi-
murium ilvIH-leuO-leuABCD gene cluster. A similar
mechanism also operates to control expression of the
genes in the Escherichia coli ilvIH-leuO-leuABCD gene
cluster. However, the mechanism underlying the DNA
supercoiling effect remained elusive. A bacterial gene
silencer AT8 was found to be important for the repres-
sion state of the leuO gene as part of the promoter relay
mechanism. In this communication, we demonstrated
that the gene silencer AT8 is a nucleation site for re-
cruiting histone-like nucleoid structuring protein to
form a cis-spreading nucleoprotein filament that is re-
sponsible for silencing of the leuO gene. With a DNA
geometric similarity rather than a DNA sequence spec-
ificity, the E. coli gene silencer EAT6 was capable of
replacing the histone-like nucleoid structuring protein
nucleation function of the S. typhimurium gene silencer
AT8 for the leuO gene silencing. The interchangeability
between DNA geometrical elements for supporting the
silencing activity in the region is consistent with a pre-
vious finding that a neighboring transcription activity
determines the outcome of the gene silencing activity.
The geometric requirement, which was revealed for this
silencing activity, explains the decisive role of tran-
scription-generated DNA supercoiling found in the pro-
moter relay mechanism.
DNA supercoiling has been known to play important roles in
transcriptional regulation (1–7). By using a bacterial transcrip-
tion regulation model system, we have demonstrated that tran-
scription-generated DNA supercoiling is a crucial driven force
that triggers the sequential activation of genes in the Salmo-
nella typhimurium ilvIH-leuO-leuABCD gene cluster (8 –12).
This rather complex sequential gene activation process was
named the promoter relay mechanism (11, 12). The exact mo-
lecular detail that underlies the effect of transcription-gener-
ated DNA supercoiling on the sequential activation of genes at
this locus remains unclear. The direct DNA supercoiling effect
on activating promoters of genes in this region has been ruled
out. Instead, the effect appears to mediate through cis-ele-
ments within the locus control regions (LCRs
1
illustrated in
Fig. 1) located between genes in the ilvIH-leuO-leuABCD gene
cluster (8).
Although not ruling out the possible involvement of other
cis-acting elements in the transcription regulation, we have
identified two cis-elements in the LCR-I that are important for
the promoter relay mechanism as follows: a bacterial gene
silencer, termed AT8; and a LeuO protein-binding site, termed
AT7 (13, 14). The bacterial gene silencer AT8-mediated tran-
scriptional silencing is integral to the gene expression regula-
tion and is responsible for the repressed state of the leuO gene.
LeuO protein-mediated derepression, which relieves the re-
pression of leuO gene, is also a crucial part of the promoter
relay mechanism. Transcription-generated DNA supercoiling
is likely to play its roles in the promoter relay mechanism via
modulating the processes of the repression-derepression con-
trol (10). To better understand the DNA supercoiling effect, we
investigated the basic molecular criteria of the repression ele-
ment (gene silencer) and derepression element (LeuO-binding
site). We revealed that it is the geometric features of the DNA
rather than the specific sequences of the transcription ele-
ments that are important for their transcription regulatory
functions. The striking DNA geometric requirement is consist-
ent with the involvement of transcription-generated DNA su-
percoiling in the transcription regulatory process.
The revealed DNA geometric features of the gene silencer
also provided clues for the possible involvement in the tran-
scription regulation of chromosome architectural proteins (e.g.
HU, H-NS, Lap, and IFS, etc.) that usually recognize DNA
structure rather than specific DNA sequence for their bindings
(15–17). Indeed, a genetic screening has led to the identifica-
tion of a histone-like nucleoid structuring protein (H-NS) for its
role in the gene silencing mechanism. Functionally, we demon-
strated that the gene silencer AT8 is indeed an H-NS nucle-
ation site that triggers the formation of a nucleoprotein fila-
ment structure in the region. With the assistance of the
neighboring AT-rich DNA in LCR-I, the transcriptional repres-
sive nucleoprotein structure reaches (cis-spreads) to the pro-
moter region of the leuO gene and results in the repression of
the gene. Despite the low DNA sequence homology, the Esch-
erichia coli gene silencer EAT6, but not a same size neutral
DNA sequence, was capable of replacing the S. typhimurium
gene silencer AT8 for its H-NS nucleation function in S. typhi-
murium LCR-I. Therefore, it is clear that regardless of the low
DNA sequence specificity, either the S. typhimurium gene si-
lencer AT8 or the E. coli gene silencer must provide the crucial
DNA geometry for triggering the H-NS nucleation. The re-
cruited H-NS, along with other nucleoproteins, appear to form
* This work was supported by National Institutes of Health Grant
GM-53617. The costs of publication of this article were defrayed in part
by the payment of page charges. This 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: Dept. of Pharmacol-
ogy, 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.
1
The abbreviations used are: LCRs, locus control regions; H-NS,
histone-like nucleoid structuring protein; b-caa, branched-chain amino
acids; EMSA, electrophoresis mobility shift assay.
THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 280, No. 6, Issue of February 11, pp. 5101–5112, 2005
© 2005 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.
This paper is available on line at http://www.jbc.org 5101
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a cis-spreading nucleoprotein filament that represses the ac-
tivities of promoters located within the proximity.
EXPERIMENTAL PROCEDURES
Plasmids and Bacterial Strains—Plasmid constructs: pAO, pEV101,
and pWU204 have been described previously (12, 18). A 75-bp DNA
(positions ⫹48 to ⫺27 of ilvIH), including the ⫺10 sequence of the
promoter of ilvIH, was deleted from pWU204 and resulted in pWU205;
otherwise pWU205 is identical to pWU204.
The 393-bp E. coli LCR-I was generated using PCR. Primers 5⬘-
GTCAACCCTGACGTCATAAAAACGTCC-3⬘and 5⬘-GAATGAGT-
CATTTACGACGTCATAATAATCCATAATG-3⬘were used in the PCR.
The primers contain mismatches (the underlined sequences) for pro-
ducing AatII restriction sites on both ends of the PCR product. The
AatII-digested E. coli LCR-I was inserted into the unique AatII site on
pAO. Other pAO-based testing plasmids were also derived by using
similar strategies, and those DNA inserts involved are individually
described in the experiments.
The plasmid, pWU802, was derived from pWU804 (8) by deleting a
1.4-kb BamHI-NsiI fragment that includes the coding region of leuO
gene and the downstream pilvIH. To replace AT8 DNA in S. typhi-
murium LCR-I on pWU802 with EAT6 DNA, the following two DNA
oligomers were chemically synthesized: 5⬘-taaatatataaattaattattaaata-
agcacatttaatcATCATTCACTTG-3⬘and 5⬘-GTGAATGATgattaaatgtgctt-
atttaataattaatttatatattta-3⬘; the lowercase DNA sequence is EAT6
DNA. The annealed DNA was used to replace the 58-bp HpaI-DraIII
fragment containing AT8 in S. typhimurium LCR-I on pWU802. With a
similar approach, the following two synthetic DNA oligomers, consist-
ing of a neutral DNA sequence (part of the nucleotide sequences of the
coding region of lacZ), were used to replace AT8 on pWU802: 5⬘-aacc-
atcgaagtgaccagcgaatacctgttccgtcatagcgataacgATCATTCACTTG-3⬘and
5⬘-GTGAATGATcgttatcgctatgacggaacaggtattcgctggtcacttcgatggtt-3⬘;
the lowercase is the neutral DNA sequence used. The procedures res-
ulted in the precise replacement of the AT8 DNA sequence with either
the E. coli repression element EAT6 or the neutral DNA sequence to
maintain the flanking DNA sequences in LCR-I as intact. For plasmids,
pWU802-LA and pWU802-LH were derived from pWU802. DNA olig-
omers containing the 27-bp lac repressor binding sequence (lac opera-
tor) 5⬘-CGGAATTGTGAGCGGATAACAATTTCG-3⬘and the flanking
restriction sites were synthesized, annealed, and ligated at the AatII
site or the HindIII site on pWU802 to generate pWU802-LA or pWU-
802-LH, respectively.
Plasmid pWU902OZ was derived from pWU802. The leu-500 muta-
tion (A to G transition) at the ⫺10 region of the promoter of the leu-
ABCD operon was converted back to the wild type ⫺10 DNA sequence,
otherwise the LCR-I DNA, which controls the expression of the downs-
tream leuO promoter, stayed the same as that in pWU802. The entire
coding sequence of lacZ gene, along with its upstream ShineDelgarno
sequence, was positioned downstream of the transcription-initiation
site of the leuO gene so that the leuO promoter can control the expres-
sion of lacZ gene product as a reporter for leuO promoter activity.
Plasmid pCH501S-(AT8)
2
was derived from pCH301-(AT8)
2
, which
has been described in a previous study (14). The coding region of the
sacB gene was used to replace the coding region of gfpUV of pCH301-
(AT8)
2
. The coding region of the sacB gene was amplified using PCR in
the presence of DNA template pEX100T (19), which was kindly pro-
vided by Dr. Herbert Schweizer (Colorado State University, Fort
Collins, Colorado), and two primers 5⬘-AACGTAGATCTAGAAAAAGG-
AGACATGAACG-3⬘and 5⬘-TCGACACTAGTCTCTTTTGCGTTTTTAT-
TTG-3⬘. Subsequently, the synthetic promoter, psyn(5/6;5/6), of
pCH301-(AT8)
2
was replaced with the annealed DNA product of two
oligomers, 5⬘-CACTTGGTGTATGATTGTGTATTCGCCATAGTTATG-
GTTATATTGCTTGCA-3⬘and 5⬘-GATCTGCAAGCAATATAACCATAA-
CTATGGCGAATACACAATCATACACCAAGTGCATG-3⬘, that consists
of the minimum promoter sequence of the S. typhimurium leuO gene.
The resultant plasmid, pCH501S-(AT8)
2
, contains the sacB gene con-
trolled by the expression of the leuO promoter, which is repressed by
(AT8)
2
, the tandem repeat sequence of the gene silencer AT8. PSO1000
is a pACYC-based (low copy number) plasmid carrying the laci gene for
expressing the lac repressor (20).
E. coli MC1060, a lac repressor deletion strain (21), has been de-
scribed previously (22). The leuO
⫺
strain, MF1, and its parental strain,
MC4100, have also been described previously (18). Dr. Chiharu Ueguchi
(Nagoya University, Nagoya, Japan) kindly provided us with CU284
(23), which is a hns::neo strain derived from MC4100. SC1(leuO
⫺
;hns
⫺
)
was constructed by introducing the hns::neo mutation into MF1 (recip-
ient strain) by P1 transduction using CU284 as a donor strain. Hence,
the MF1 and SC1 pair was leuO::cam hns
⫹
and leuO::cam hns
⫺
iso-
genic. Bacteria were grown in Luria-Bertani (LB) medium at 37 °C with
aeration or in a synthetic (chemically defined) medium base SSA sup-
plemented with all amino acids except for the three branched-chain
amino acids isoleucine, leucine, and valine (18). Growth in the synthetic
medium was at 32 °C with aeration. Ampicillin (50
g/ml) and tetracy-
cline (6.25
g/ml) were added, as needed.
Primer Extension—Primer extension was carried out as described
previously (13) with the following modifications. The following primers
were used: (i) 5⬘-TCTGGGTGAGACAAAACAGGAAGGC-3⬘for detect-
ing pbla-mediated transcripts; (ii) 5⬘-GAAACCATTATTATCATGACA-
TTAACC-3⬘for detecting E. coli pilvIH-mediated transcripts; (iii) 5⬘-G-
CATATAAAATAAGAAAAAGCAAAATGAGTAAAATTCG-3⬘for detect-
ing E. coli pleuO-mediated transcript; and (iv) 5⬘-CGGAAAACATAAA-
GACGCTGACAGAGAC-3⬘for detecting S. typhimurium pleuO-medi-
ated transcripts. Each primer extension reaction consisted of 100
gof
total RNA and DNA primer(s). Two primers were mixed in the primer
extension reactions for simultaneous detection of the bla and ilvIH
transcripts in the results as shown in Fig. 1Dand the ilvIH and leuO
transcripts in the results as shown in Fig. 1E. In other primer extension
reactions, only one primer was used for detecting an individual RNA
transcript. The primer extension results were visualized and quantified
by using a STORM imaging system 840 (Amersham Biosciences). The
quantification was normalized against the content of plasmid DNA in
each experiment.
Northern Hybridization—The total RNA preparations and the
procedures for Northern hybridization were conducted as described
previously (11).
Electrophoresis Mobility Shift Assay (EMSA)—As described previ-
ously (14), all testing DNA segments were annealed pairs of synthetic
DNA oligomers consisting of DNA sequences complementary to each
other as illustrated in the figures. All synthetic DNA oligomers were
5⬘-end-labeled with [
␥
-
32
P]ATP using T4 polynucleotide kinase prior to
the annealing. The radioactively labeled duplex DNAs (15 pg per reac-
tion; ⬃10,000 cpm/reaction) were then mixed with the purified histi-
dine-tagged S. typhimurium LeuO protein in a binding buffer (40 mM
Tris-Cl, pH 8.0; 4 mMMgCl
2
;70mMKCl; 0.1 mMEDTA; and 0.1 mM
dithiothreitol). The binding reaction mixtures were incubated at 37 °C
for 30 min. The mixtures were then analyzed on 5% native polyacryl-
amide gels in TBE buffer (89 mMTris borate, pH 8.3; 1 mMEDTA).
Results were visualized using a STORM imaging system 840 (Amer-
sham Biosciences).
DNase I Footprinting—PCR was used to generate DNA templates
employed for DNase I footprinting studies. Each involved DNA tem-
plate is described in the legend of each figure. Reaction mixtures (50
l/reaction) containing radioactively labeled DNA (1 ng/reaction equiv-
alent to ⬃20,000 cpm/reaction) plus increasing amounts of either puri-
fied (His)
10
-SLeuO or purified H-NS (kindly provided by Dr. Sylvie
Rimsky, IGR, Villejuif Cedex, France) were incubated at room temper-
ature for 30 min to a protein-DNA binding equilibrium. After the
incubation, 5
lof1mMMgCl
2
and 0.5 mMCaCl
2
was added into each
reaction for continued incubation at room temperature for 1 min. DNase
I (units used per reaction is indicated in each figure legend) was then
added to the reactions for a 90-s incubation at room temperature. The
DNase I footprinting reactions were stopped by adding 140
l of stop
solution consisting of 192 mMsodium acetate, 32 mMEDTA, 0.14% SDS,
and 64
g/ml yeast RNA. The samples were then phenol-extracted,
ethanol-precipitated, and resuspended in gel loading buffer. To mark
precisely the positions of the protein-mediated DNase I protection sites,
primers used for PCR were individually used in the DNA sequencing
reactions (Sequenase sequencing kit, U. S. Biochemical Corp.) for pre-
paring DNA sequence ladders. By using a chemical cleavage reaction
(24), a G ⫹A marker was also prepared from the radioactively end-
labeled DNA. Along with one of the position markers (the DNA se-
quence ladders or the G ⫹A marker), the DNA products prepared from
the footprinting reactions were analyzed on 7% acrylamide, 7 Murea
denaturing PAGE.
Tn5 Transposon Insertion-mediated Random Mutagenesis and the
Reverse Screening Procedure—1
l of EZ::TN
TM
具KAN-2典] Tnp Trans-
posome
TM
(Epicenter), which contains the Tn5transposon and the
kanamycin-resistant gene (kan
r
), was mixed in a 0.2-cm electroporation
cuvette with 40
l of electrocompetent E. coli DH5
␣
cells harboring
pCH501S-(AT8)
2
that were prepared by serial washes with cold 10%
sterile glycerol during the mid-exponential growth phase (A
600
⫽0.6).
Electroporation was performed in a Bio-Rad Gene Pulser apparatus (set
up at 25 microfarads, 200 ohms, and 2.50 kV). After adding 1 ml of SOC
medium, the components are 20 g of bactotryptone,5gofyeast extract,
and 0.5 g of NaCl, pH 7.5, in 1 liter of H
2
O (final concentrations of 20
H-NS-mediated leuO Gene Silencing5102
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mMMgSO
4
and 20 mMglucose were added into the medium after
autoclaving), the electroporated cells were transferred to a culture tube
and incubated at 37 °C for 1 h. After incubation, cells were plated on LB
agar plates containing 50
g/ml ampicillin and 25
g/ml kanamycin.
Once colonies formed, they were replicated onto plates with or without
5% sucrose supplement.

-Galactosidase Assay—The

-galactosidase assay was measured by
hydrolysis of o-nitrophenyl

D-galactosidase to produce o-nitrophenol in
permeabilized bacterial cells as described previously (25).
Direct Genomic DNA Sequencing—Chromosome DNAs were isolated
from the selected Tn5 knock-out strain and used for direct genomic
DNA sequencing. Primers that hybridize to the ends of the inserted
transposon, Tn5, were used for a bi-directional outward sequencing that
reads into the genomic DNA, flanking the Tn5 insert. The obtained
DNA sequences were used to pin-point the insertion site on the bacte-
rial genome using the Blast search (NCBI Data Bank). The direct
genomic DNA sequencing was performed at the facility of Fidelity
Systems Inc. (Gaithersburg, MD).
RESULTS
Genes in the E. coli ilvIH-leuO-leuABCD Gene Cluster Are
Regulated via the Promoter Relay Mechanism—The promoter
relay mechanism was found based on the coordinated expres-
sion of genes in the S. typhimurium ilvIH-leuO-leuABCD gene
cluster (12). By using a plasmid-borne S. typhimurium DNA
context in E. coli hosts, we initially demonstrated that trans-
acting protein factors important for the promoter relay mech-
anism are functionally available in E. coli strains. This is based
on the observed activation of a plasmid-borne leu-500 pro-
moter, the hallmark of the promoter relay mechanism, in the
heterogeneous (E. coli protein factors acting on S. typhimurium
DNA context) assay system (data not shown). The result of this
initial test prompted us to directly monitor the mRNAs of ilvIH
and leuO in MC4100, an E. coli relA1 strain under an experi-
mental condition that triggers a severe starvation for
branched-chain amino acids (b-caa), isoleucine, leucine, and
valine, during the log phase of bacterial growth. The leuO gene
in E. coli relA1 strain is normally silent and activated in re-
sponse to the starvation for b-caa during exponential growth in
17-amino acid SSA, a synthetic medium supplemented with all
amino acids except the three b-caa (18). The b-caa starvation
causes a 2-h growth arrest (the slow-down period between
points 2 and 3 shown in the growth curve in Fig. 1) prior to the
growth resumption (the growth rate increase after point 3 in
the growth curve shown in Fig. 1). We have demonstrated that
the LeuO protein is required during the growth stress for cells
to resume their growth after the 2-h growth arrest (growth
stress) because the leuO knock-out strain, MF1, failed to re-
sume its growth after the arrest (18).
According to the promoter relay mechanism, the production
of LeuO in MC4100 cells during growth stress is presumably
because of the activation of the leuO gene triggered by the
transcription activity of ilvIH promoter. The ilvIH transcrip-
tion activity-dependent leuO gene activation has been demon-
strated previously in S. typhimurium cells that are entering
the stationary phase (25); however, this has not been directly
demonstrated in E. coli strains. Hence, we monitored the
mRNAs of ilvIH and leuO during the growth of MC4100 in
17-amino acid SSA medium. Indeed, both ilvIH and leuO
mRNAs were only detectable at the end of the 2-h growth
arrest prior to the cell growth resumption (Fig. 1, Band C,
lanes 3). Both mRNAs were not detected at the time points
during the exponential growth (Fig. 1, Band C,lanes 1), and at
the beginning of the 2-h growth arrest (Fig. 1, Band C,lanes 2).
This Northern result indicated that the ilvIH operon is indeed
activated during growth stress (2-h growth arrest). The tran-
scriptional activity of the ilvIH operon is expected to
subsequently activate the leuO gene via the promoter relay
mechanism (11, 12).
The co-detection of ilvIH and leuO mRNAs during the 2-h
growth arrest can best be explained by the promoter relay
mechanism. Therefore, we expected that the deletion of ilvIH
promoter activity would also abolish the activity of the leuO
FIG.1. Genes in the E. coli ilvIH-leuO-leuABCD gene cluster
are regulated via the promoter relay mechanism. The arrange-
ments of genes in the ilvIH-leuO-leuABCD gene cluster of E. coli and S.
typhimurium are illustrated. The AT-rich DNA sequences flanking the
leuO gene are important for the expression regulation of genes in the
gene cluster. The AT-rich DNAs, upstream and downstream of leuO, are
named locus control regions, LCR-I and LCR-II, respectively. Illus-
trated in the 318-bp S. typhimurium LCR-I are the two identified
cis-elements, the repression element AT8, and the derepression ele-
ment AT7. E. coli relA1 strain, MC4100, was grown in 17-amino acid
SSA medium. The absorbance reading at 600 nm was measured and
plotted against post-inoculation time in the growth curve. Three time
points when samples were collected for Northern analysis are marked
on the growth curve. Ais the ethidium bromide-stained gel for confirm-
ing the equal amount of RNA loading of the subsequent Northern
hybridization. Band Care the Northern blots for detecting transcripts
expressed from either the ilvIH operon or the leuO gene, respectively.
The lane numbers in A–C correspond with three time points marked in
the growth curve. Dand Eare the primer extension studies that detect
the activities of pilvIH,pleuO, and pbla on the plasmids, as indicated.
H-NS-mediated leuO Gene Silencing 5103
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gene under the same experimental condition (MC4100 cells
under the starvation for b-caa). Hence, the ilvIH transcription
activity-dependent activation of leuO was tested using a pair of
multicopy plasmids, pWU204 and pWU205, that both carry the
E. coli promoter relay DNA sequence (NCBI GenBank/EBI
data bank accession number AF106955). These two plasmids
are identical except that the ilvIH promoter (–10 sequences of
ilvIH operon) has been deleted from the E. coli promoter relay
DNA sequence on pWU205, whereas the promoter of the ilvIH
operon on pWU204 remains intact (Fig. 1). The plasmids were
tested in MC4100 cells grown in 17-amino acid SSA for the
activation of the ilvIH promoter in response to the starvation
for b-caa (Fig. 1, Dand E). By using a primer extension study,
ilvIH promoter activity was detectable on pWU204 (Fig. 1D,
lane 1). Under the same testing condition, ilvIH promoter ac-
tivity was not detectable on pWU205, the ilvIH promoter-less
plasmid (Fig. 1D,lane 2). The activity of the promoter of the

-lactamase gene (bla) was simultaneously monitored as an
internal control in the primer extension studies (Fig. 1D) and
found to be identical. This internal control clearly shows that
the different ilvIH promoter activities on pWU204 and
pWU205 are not due to copy number differences between the
two testing plasmids. Further primer extension studies (Fig.
1E) demonstrated that the ilvIH promoter activity is required
for activation of the leuO gene. In the presence of the intact
ilvIH promoter, the leuO promoter activity was detectable on
pWU204 (Fig. 1E,lane 3). In contrast, because of the lack of
ilvIH promoter activity on pWU205, the identical leuO gene on
that plasmid failed to be activated (Fig. 1E,lane 4). These data
clearly support a promoter relay mechanism for the activation
of the leuO gene at the ilvIH-leuO-leuABCD gene locus in
E. coli.
The Promoter Relay Mechanism Is Regulated by Cis-elements
with Little DNA Sequence Specificity—Two cis-elements that
are important for the promoter relay mechanism, the 47-bp
repression element AT8 and the 25-bp derepression element
AT7, were identified in the S. typhimurium ilvIH-leuO-
leuABCD gene cluster (14). The two elements are located in the
locus control region I (LCR-I) upstream of the divergently ar-
rayed leuO and leuABCD (illustrated in Fig. 1). These two
elements are responsible for the repression and the derepres-
sion states of the leuO gene as an integral part of the promoter
relay mechanism. Because the promoter relay mechanism is
also responsible for the transcriptional regulation of the genes
in the E. coli ilvIH-leuO-leuABCD gene cluster, we expected to
identify similar elements in the LCR-I of the E. coli ilvIH-leuO-
leuABCD gene cluster. Therefore, it was puzzling that homol-
ogous DNA sequences to the identified S. typhimurium ele-
ments AT7 and AT8 could not be found in the E. coli LCR-I.
This was, however, consistent with a previous observation that
the DNA sequence in the region (LCR-I) is AT-rich but other-
wise shares little DNA sequence homology between the two
closely related enteric bacteria (26). Because it was impossible
to identify the cognate transcription elements in E. coli LCR-I,
based on the known DNA sequences of S. typhimurium tran-
scription elements, we searched for transcription elements in
E. coli LCR-I based on the known properties of the transcrip-
tion elements.
E. coli Repression Element—Based on previously established
criteria (13, 14), we assayed for gene silencing activity (repres-
sion element) in the 393-bp E. coli LCR-I (the AT-rich DNA
located between the ⫺20 position and the ⫺412 position of the
E. coli leuO gene) flanked by leuO and leuABCD (illustrated in
Fig. 2). The results showed that gene silencing activity is lo-
cated near the leuO promoter end of the regulatory region,
because both EAT1 and EAT3 did not show any significant
gene silencing activity in the assay (Fig. 2, lanes 3 and 5). The
gene silencing activity was finally narrowed down to EAT6, a
39-bp AT-rich DNA sequence located between ⫺84 and ⫺122
positions of the leuO gene. This location is consistent with the
FIG.2. The identification of a repression element in E. coli LCR-I. The E. coli LCR-I (a 393-bp DNA segment that consists of DNA
sequences between the ⫺412 and ⫺20 position of the E. coli leuO gene) was divided into several segments as illustrated. Each DNA segment was
individually inserted at the AatII site, which is located 95 bp upstream of the transcription initiation site (⫹1) of the

-lactamase gene (bla) on pAO.
Primer extension was used to monitor the activity of pbla for searching the transcriptional repression activity in the E. coli LCR-I. The quantified
pbla activity shown at the bottom of each lane for three repeated experiments is expressed as the mean within the range of ⫾0.004 (S.D.).
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possibility that the E. coli repression element EAT6 is also
directly responsible for the repression state of the E. coli leuO
gene in a manner analogous to that of the S. typhimurium
repression element AT8, in control of the S. typhimurium il-
vIH-leuO-leuABCD gene cluster (14).
E. coli Derepression Element—Again, by using our previ-
ously established criteria (14), we searched for the derepres-
sion element (LeuO-binding site) in the region between the
transcription start site (⫹1 position) of the E. coli leuO gene
and the repression element EAT6 (illustrated in Fig. 3).
Based on the results of EMSA, by tracing down the binding
region, a LeuO-binding site, 29-bp EAT16, was identified at a
position downstream of the E. coli repression element EAT6
and upstream of the transcription initiation site of the E. coli
leuO gene (Fig. 3). A DNase I footprinting experiment (Fig. 4)
revealed that the LeuO protein-dependent DNase I protec-
tion was indeed initiated at the LeuO-binding site EAT16
(⫺38 to ⫺66 position of E. coli leuO gene) in LCR-I (Fig. 4A,
lane 2). Upon the increase of LeuO protein concentration, the
LeuO-mediated DNase I protection was extended toward the
regions (zones 1 and 2) flanking EAT16 (Fig. 4A,lanes 3 and
4). Although the LeuO-mediated DNase I protection in the
center region of EAT16 is less clear on the complementary
strand of the LCR-I DNA (Fig. 4B), a similar DNase I pro-
tection in zones 1 and 2 was observed on the complementary
strand in the presence of a high concentration of LeuO pro-
tein (Fig. 4A, lane 1). Whether the extension of LeuO protein-
mediated DNase I protection beyond the LeuO-binding site
EAT16 at a very high protein/DNA ratio has any biological
significance in the transcription regulation, remains to be
tested in the future. Nonetheless, the DNase I footprinting
result (Fig. 4) and the EMSA data (Fig. 3) together supported
that LeuO binding in E. coli LCR-I was initiated at the
LeuO-binding site EAT16. This is consistent with the relative
positions of the repression element, derepression element,
and the targeting leuO promoter found in the S. typhimurium
ilvIH-leuO-leuABCD gene cluster (14). Hence, a similar re-
pression-derepression process may indeed be responsible for
the repression and the transient activation (derepression) of
the E. coli leuO gene as an indispensable part of the promoter
relay mechanism in the E. coli ilvIH-leuO-leuABCD gene
cluster (10).
Functional Replacement of the DNA Sequence Heterogeneous
E. coli and S. typhimurium Elements in the Repression-Dere-
FIG.3.Identification of a LeuO-binding site located between E. coli repression element and the promoter of the E. coli leuO.DNA
oligomers consisting of the DNA sequences of the various DNA segments in the region (as illustrated) were chemically synthesized and annealed
for interacting with an increasing amount of purified histidine-tagged LeuO protein in EMSA. The results of EMSA are summarized along with
the list of the name of each DNA segment. The ⫹sign indicates a positive result of the LeuO binding assay for the DNA segment. The ⫺sign
indicates a negative result of the LeuO binding assay for the DNA segment. The EMSA results of several key DNA segments are shown.
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pression Process—The identified E. coli repression-derepres-
sion elements shared no obvious DNA sequence homology with
the cognate S. typhimurium elements. The DNA sequence ho-
mology between the derepression elements is 28%, and the
homology between the two repression elements is 31% (Fig.
5A). The similarity observed was that both E. coli and
S. typhimurium repression elements are extremely AT-rich
(85% A ⫹T for AT8 and 90% A ⫹T for EAT6). The derepression
elements, S. typhimurium AT7 and E. coli EAT16, are rela-
tively less AT-rich (about 60% AT). The best possible DNA
sequence alignment, allowing gaps for the alignment between
sequences (Fig. 5A), revealed some homologous motifs between
the cognate S. typhimurium and E. coli elements. However, the
limited homologous regions do not explain the transcription
activities exerted by the elements because all the identified
elements are the minimal DNA sequences that are absolutely
required for retaining the transcriptional regulatory functions.
Although the DNA sequence homology is limited, we found
DNA geometrical similarity between the elements. In both
gene silencers, intrinsic DNA curvatures were predicted using
a SDAB computer program that predicts local bending by elas-
tic models that incorporate sequence-dependent anisotropic
bendability (27, 28). The bend centers of the predicted DNA
curvatures of S. typhimurium AT8 and E. coli EAT6 are indi-
cated in Fig. 5A. We also found a similar size DNA palindrome
in both gene silencers (Fig. 5A,underlined sequences). We
tested whether these transcription elements, with the de-
scribed DNA geometrical similarity, could functionally replace
each other’s activity in the repression-derepression process.
The repression-derepression regulatory activity was assayed
by its effects on pbla, the promoter of the

-lactamase gene
(bla) on pAO when the LeuO protein was provided in trans (Fig.
5B). Consistent with the previous findings (14), the S. typhi-
murium repression element AT8 represses the activity of pbla
as evidenced in a primer extension assay (Fig. 5B, compare
lanes 1 and 2). The placement of the S. typhimurium derepres-
sion element AT7 within the proximity of the S. typhimurium
repression element AT8 resulted in the expected transcrip-
tional derepression, thus restoring the activity of pbla (Fig. 5B,
lane 3). Most strikingly, the E. coli derepression element
EAT16 was able to replace the function of the S. typhimurium
derepression element AT7 to provide almost the same degree of
transcriptional derepression (Fig. 5B,lane 4).
The compatibility of the heterogeneous elements for repression
was then tested in the following experiments using the E. coli
repression element as the primary element (Fig. 5B,lanes 5–7).
The E. coli repression element EAT6 represses the activity of
pbla (Fig. 5B, compare lanes 1 and 5). The placement of the
E. coli derepression element EAT16 within the proximity of the
E. coli repression element EAT6 resulted in the expected tran-
scriptional derepression, which restores the activity of pbla (Fig.
5B,lane 6). The S. typhimurium derepression element AT7 was
capable of replacing the function of the E. coli derepression ele-
ment EAT16 to support LeuO protein-mediated transcription
derepression (Fig. 5B,lane 7). The nearly perfect repression-
derepression functions of the reconstituted heterogeneous groups
demonstrated that the heterogeneous transcription elements
performed their cognate functions in the repression-derepression
process despite the DNA sequence heterogeneity.
The E. coli Repression Element Is Functional for Regulation
of leuO Expression in the Context of S. typhimurium LCR-I—In
prior experiments, the activities of the repression-derepression
elements were assayed with a reconstituted plasmid DNA con-
text. To confirm the functional compatibility of the transcrip-
tion elements in a native environment (the DNA context of S.
typhimurium LCR-I with the flanking leuO and leuABCD pro-
moters), we replaced the S. typhimurium repression element
AT8 on pWU802, either with a same-size neutral DNA se-
quence (in the case of pWU802NS) or with the E. coli repres-
sion element EAT6 (in the case of pWU802ES), and we assayed
for the effect of these replacements on the activity of the pro-
moter of the leuO gene (pleuO) by using primer extension (Fig.
6A). As expected, the S. typhimurium repression element AT8
is indeed responsible for the repression of the leuO gene in its
native context (S. typhimurium LCR-I), because the replace-
ment of the S. typhimurium repression element AT8 with a
same-size neutral DNA abolished the repression of pleuO ac-
tivity (Fig. 6A,lane 3). Most strikingly, the transcriptional
repression was restored when the E. coli repression element
EAT6 was used to replace the S. typhimurium repression ele-
ment AT8 (Fig. 6A,lane 2). This is a strong piece of evidence
that the E. coli repression element exerts a reasonable tran-
scriptional repression activity in context of the S. typhimurium
ilvIH-leuO-leuABCD gene cluster, despite the lack of apparent
DNA sequence homology between the E. coli and S. typhi-
murium LCR-I. The gene silencing effect appears to extend
some distance because the promoter of the

-lactamase gene
(pbla), which is located 392 bp downstream, was also affected
when either the S. typhimurium repression element AT8 or the
E. coli repression element EAT6 was present (Fig. 6B).
The results therefore showed that both repression and dere-
pression elements were functionally interchangeable for the
control of the expression of genes in the ilvIH-leuO-leuABCD
gene cluster. Instead of DNA sequence homology, DNA geomet-
rical similarities may be important for the transcription regu-
latory functions of the repression and derepression elements.
FIG.4.LeuO protein-dependent DNase I protection initiates at
the LeuO-binding site EAT16. A DNA segment consisting of the
upstream region (⫹31 to ⫺150 position) of the E. coli leuO gene was
synthesized by PCR and radioactively labeled either at the ⫹31 position
(A)oratthe⫺150 position (B). The end-labeled DNA was incubated
with LeuO at a concentration indicated above each lane and exposed to
DNase I (0.5 unit per reaction) for the footprinting assay. The DNA
sequence ladders were included for marking the positions of LeuO-
mediated DNase I protection regions.
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This complex mechanism involving DNA geometrical changes
stabilized by transcription-generated DNA supercoiling is
likely to be responsible for the repression-derepression process.
If so, this is a novel transcription regulatory mechanism that
deserves further investigation to define the underlying molec-
ular details. As the first step, we focused on the repression
mechanism. Although the cis-acting repression element (gene
silencer) has been well characterized thus far, the trans-acting
protein factors responsible for gene silencing remain unknown.
A Genetic Approach for the Identification of Genes Required
for Bacterial Gene Silencing—A two-step screening procedure
involving an initial reverse selection (as described under “Ex-
perimental Procedures”) followed by a positive screen was used
to identify gene(s) important for the bacterial gene silencing
mediated by the gene silencer AT8. Bacillus subtilis sacB gene
encodes levansucrase, which catalyzes the hydrolysis of sucrose
resulting in the synthesis of levans (29). In the presence of 5%
sucrose, expression of sacB in Gram-negative bacteria such as
E. coli is lethal (30). The E. coli harboring “suicide” plasmid,
pCH501S-(AT8)
2
, carrying the coding region of the sacB gene
under the control of the leuO promoter, which is repressed by
the direct repeat of the bacterial gene silencer AT8, was used in
the first step screening for genes that are important for the
gene silencing. The AT8 dimer (AT8)
2
provided a very tight
gene silencing effect as demonstrated in our previous study
(14). Because the promoter of the leuO gene is one of the
natural promoters regulated by the gene silencer, expression of
the lethal gene sacB on the suicide plasmid is strongly re-
pressed in the presence of (AT8)
2
. To screen for genes required
for silencing, Tn5 transposon insertion mutagenesis (as de-
scribed under “Experimental Procedures”) was used to ran-
domly knock-out genes throughout the bacterial genome. If a
Tn5 insertion knocked out a gene important for the gene si-
lencing mechanism, E. coli harboring the suicide plasmid
would no longer survive on LB plates containing 5% sucrose
because of the relief of gene silencing. However, that mutant
FIG.5. E. coli elements are func-
tionally interchangeable with the S.
typhimurium elements for their cog-
nate activities in transcription re-
pression-derepression. Aillustrated
the DNA sequence alignments of E. coli
and S. typhimurium elements. Asterisks
mark the positions with identical DNA
sequences. Palindromes are marked with
a pair of underlined arrows pointed to-
ward each other. The bend centers of the
computer program-predicted intrinsic
DNA curvatures are marked. For testing,
various combinations of E. coli and S. ty-
phimurium elements were placed either
individually or as a group at the AatII site
on pAO. The testing plasmids were as-
sayed in MF1 harboring pEV101, which
provided LeuO protein upon 50
Misopro-
pyl 1-thio-

-D-galactopyranoside induc-
tion. The activity of pbla on the testing
plasmid was monitored using primer ex-
tension and shown in B. The quantified
pbla activity shown at the bottom of each
lane for two repeated experiments is ex-
pressed as the mean within the range of
⫾0.01 (S.D.).
FIG.6. Gene silencing activity of
the E. coli repression element in the
context of S. typhimurium LCR-I. The
linear map illustrates the LCR-I context
on pWU802 plasmid and the modifica-
tions (the 47-bp S. typhimurium repres-
sion element AT8 is replaced by either a
same size neutral DNA sequence or the
39-bp E. coli repression element) in the
LCR-I that result in pWU802NS or
pWU802ES, respectively. The relative po-
sitions and orientations of the key ele-
ments on the plasmid are illustrated.
E. coli leuO
⫺
strain MF1 harboring one of
the pWU802 plasmid series was used for
testing the effect of either the S. typhi-
murium element AT8 or the E. coli re-
pression element EAT6 on the activity of
the plasmid-borne leuO promoter, pleuO
(A)orbla promoter, pbla (B). The quanti-
fied data shown at the bottom of each lane
for three repeated experiments are ex-
pressed as the mean within the range
of ⫾0.02 (S.D.).
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can still form a colony on LB without sucrose. By using this
reverse selection procedure, we obtained 398 clones out of a
total 2,296 colonies from this negative screening. Because Tn5
transposon may also target plasmid DNAs, we excluded the
clones containing plasmids that carry the Tn5 inserts from the
398 clones. This exclusion resulted in 300 potential positive
clones selected from the first-step reverse selection procedure.
Because the reporter gene sacB is very toxic for Gram-neg-
ative bacteria used in the reverse selection, we expected many
of these 300 selected clones might contain knock-out genes
important for sucrose metabolism or sugar transport, rather
than genes directly relevant to AT8-mediated gene silencing. A
preliminary titration experiment to determine the appropriate
concentration of sucrose (titration range, 10 to 0.5% sucrose in
LB) to be used for the initial reverse selection procedure had
indicated that the sucrose toxicity is very stringent. Whereas
5% sucrose was chosen as a toxicity threshold in the initial
reverse selection, we noticed that even a small leakage of the
activity of the promoter that controls the expression of sacB
gene could result in toxicity in the presence of as little as 0.5%
sucrose. To be sure that we did not exclude possible candidate
genes, which may have minor effects on the bacterial gene
silencing in the first step selection, a relatively high toxicity
(5% sucrose) was used for the initial screening that resulted in
the 300 potential clones. We then lowered the toxicity to 0.5%
sucrose for the second-step screening. According to preliminary
testing, we expected that bacterial strains harboring pCH501S-
(AT8)
2
should not experience any toxicity at all in LB contain-
ing 0.5% sucrose if the Tn5 insertion had affected genes that
are irrelevant to the gene silencing mechanism. This is based
on the fact that the silencer repeat, (AT8)
2
, is capable of pro-
viding sufficient repression (a minimum leakage) on the sacB
gene of pCH501S-(AT8)
2
. However, any major relief of the
bacterial gene silencing was expected to result in severe toxic-
ity in LB even supplemented with only 0.5% sucrose. Hence,
the 300 potential positive clones were grown in LB supple-
mented with 0.5% sucrose as a second-step screening proce-
dure. Most strikingly, we found that with the exception of one
clone, the rest of the 299 clones survived in the culture of LB
supplemented with 0.5% sucrose. Although it is possible that
some of the 299 excluded clones may be genes that play minor
roles in the silencing mechanism, the single positive clone
isolated in the second-step screening procedure must contain a
knock-out gene crucial for the bacterial gene silencing. This
was confirmed using pWU902OZ to report the gene silencing
activity in the isolated Tn5 knock-out clone. Plasmid
pWU902OZ carries the entire regulatory region (LCR-I) of the
S. typhimurium leuO gene and the leuO promoter that controls
the expression of the downstream coding sequence of lacZ
reporter gene. Because of the effect of the gene silencer located
in the LCR-I region, the expression of the reporter lacZ gene is
repressed. If the Tn5 insertion had knocked out a gene that was
truly important for the silencing activity in the LCR-I, then we
expected an increase of the expression of the reporter lacZ
gene. Indeed, compared with pWU902OZ harboring DH5
␣
(the
parental strain of the Tn5 knock-out strain), a 16-fold increase
of

-galactosidase activity was found in the isolated Tn5 knock-
out strain harboring pWU902OZ (data not shown).
H-NS Is the Trans-acting Factor Responsible for the Tran-
scriptional Repression Mediated by the Gene Silencers—Direct
genomic DNA sequencing was used to identify the site of Tn5
insertion on the chromosome of the positive clone isolated. The
DNA sequencing result indicated that the Tn5 insertion is
located at the ⫹59 position of the coding region of the hns gene.
The insertion is likely to cause either early termination or
truncation of the translation product of the gene because it is in
FIG.7. Gene silencer-mediated
transcriptional repression is H-NS-
dependent. The pAO testing plasmid se-
ries with or without the insertion of AT4
DNA at the AatII site was used for testing
AT4-mediated transcriptional repression
in MF1 and SC1, an isogenic hns
⫹
/hns
⫺
pair (A). The pWU802 plasmid series used
in Fig. 6 was used for testing the S. typhi-
murium AT8 silencer-mediated or E. coli
silencer EAT6-mediated transcriptional
repression in the same isogenic hns
⫹
/
hns
⫺
pair (B). Primer extension was used
for detecting the activity of pbla or pleuO
in the studies. The quantified data shown
at the bottom of each lane for three re-
peated experiments is expressed as the
mean within the range of ⫾0.03 (S.D.).
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the N terminus of the coding region. Hence, H-NS, the gene
product of hns, is most likely to be the protein factor important
for the gene silencing mechanism.
The possibility was further confirmed in experiments using a
pair of isogenic hns
⫹
/hns
⫺
strains (Fig. 7). Two testing plas-
mids were used in the experiments. First, two testing plasmids,
pAO with or without the gene silencer sequence (AT4), which
had been used to characterize the bacterial gene silencer in our
previous studies (13, 14), were used to confirm the involvement
of H-NS in the bacterial gene silencing (Fig. 7A). The results
showed that a reduced gene silencing activity was observed
when pAO-AT4 was tested in the hns
⫺
strain (Fig. 7A,lane 4)
compared with the silencing activity detected in the hns
⫹
strain (Fig. 7A,lane 2). The 4-fold reduction on the gene si-
lencer AT4-mediated gene silencing must be due to the absence
of H-NS in the hns
⫺
strain because the genetic background of
the two bacterial strains is identical except for the hns gene. In
a second experimental set, we directly monitored the effects of
the gene silencer on the promoter of the leuO gene on the
testing plasmid pWU802 series (Fig. 6). The presence of either
the S. typhimurium silencer AT8 or the E. coli silencer EAT6 in
the S. typhimurium LCR-I was able to repress the promoter
activity of the leuO gene on the pWU802 plasmid series (Fig.
7B,lanes 1 and 2). The gene silencing effect was clearly H-NS-
dependent because the repression of leuO promoter activity
was significantly reduced in the hns
⫺
strain (Fig. 7B,lanes 4
and 5). Moreover, the repressive effect of H-NS on the promoter
activity of leuO gene must be mediating through either the
S. typhimurium silencer AT8 or the E. coli silencer EAT6,
because the replacement of the gene silencer with a neutral
sequence failed to affect the promoter activity of leuO gene in
either testing condition (Fig. 7B,lanes 3 and 6). The gene
silencing effect on the relatively distal bla promoter was also
H-NS-dependent because a 2-fold reduction on the gene silenc-
ing effect was found in the hns
⫺
strain (Fig. 7B, compare lanes
7and 8with lanes 10 and 11). The repression activity assay
was carried out in the absence of the interference of LeuO
protein because the leuO gene was knocked out in the isogenic
pair of hns
⫹
/hns
⫺
strains used in the experiment.
The Gene Silencer Important for the Promoter Relay Mecha-
nism Is an H-NS Nucleation Site—Because the H-NS-depend-
ent gene silencing activity is absolutely dependent on the pres-
ence of either the S. typhimurium gene silencer AT8 or the
E. coli gene silencer EAT6 in LCR-I, H-NS must affect the
promoter activity with a biological event specifically initiated
at the gene silencer. The DNA sequence required for triggering
the initiation process of H-NS-mediated gene silencing has
been termed the H-NS nucleation site (31, 32). Based on this
definition, an H-NS nucleation site is distinct from other ordi-
nary H-NS-preferred binding DNA sequences. H-NS nucleation
is absolutely required for the formation of a well organized
nucleoprotein structure, which is shown to be transcription-
repressive. Other ordinary H-NS-binding DNA sequences may
lead to the binding of H-NS, but in the absence of an H-NS
nucleation site, the bound H-NS is not necessarily well orga-
nized and hence is not transcriptionally repressive (31).
DNase I footprinting assays were used to investigate
whether the identified gene silencer (AT8 or EAT6) is in fact an
H-NS nucleation site. The results showed that the S. typhi-
murium gene silencer AT8 was the first region to be protected
by H-NS in the DNase I footprinting experiment (Fig. 8, left
panel,lane 3). With increasing H-NS concentration in the
DNase I footprinting experiment (Fig. 8, left panel,lanes 2 and
1), the protected region gradually extended toward the pro-
moter of the leuO gene. A detailed titration of H-NS concentra-
tion was carried out to better demonstrate the gradual exten-
sion of the H-NS-dependent protection in the DNase I
footprinting experiment (Fig. 8, right panel). Clearly, the
S. typhimurium gene silencer AT8 is the site occupied by H-NS
as the first step. Although the DNase I protection was also
slightly extended toward the 5⬘direction (Fig. 8, right panel,
lanes 1– 4), the 3⬘end extension was much more significant
(Fig. 8, right panel,lanes 1– 4), and the DNase I protection
eventually reached the promoter of the leuO gene (Fig. 8, left
panel,lanes 1–3). Hence, this DNase I footprinting result
demonstrated that the S. typhimurium gene silencer AT8 is
indeed an H-NS nucleation site, which initiates the formation
of a transcriptionally repressive nucleoprotein structure. This
structure has a potential to extend (cis-spread) toward either
direction; however, the remaining AT-rich DNA sequence in
LCR-I may confer directionality to the cis-spreading nucleo-
protein structure. In this case, the transcriptionally repressive
nucleoprotein structure preferentially extended toward the
promoter of the leuO gene.
Similar H-NS nucleation and unidirectional cis-spreading
were also observed when E. coli LCR-I was tested in a similar
DNase I footprinting experiment (Fig. 9). H-NS-dependent
DNase I protection was also initiated near the E. coli gene
silencer EAT6 (Fig. 9, lane 3), was spread in both directions,
and eventually extended preferentially toward the promoter of
the E. coli leuO gene (Fig. 9, lanes 1–3). Hence, both the
S. typhimurium gene silencer AT8 and the E. coli gene silencer
EAT6 are important for the promoter relay mechanism and are
most likely the H-NS nucleation sites. This possibility was
further tested using the replacement plasmid constructs where
the S. typhimurium gene silencer AT8 was replaced with either
FIG.8.H-NS-dependent DNase I protection in S. typhimurium
LCR-I. A unique end radioactively labeled DNA segment consisting of
the promoter and the upstream region (⫹40 position to ⫺269 position)
of the S. typhimurium leuO gene was generated by PCR. The end-
labeled DNA was incubated with H-NS at a concentration indicated
above each lane and exposed to DNase I (0.3 unit per reaction). The
positions of relevant elements and restriction sites in the region are
marked. A similar DNase I footprinting using a more detailed titration
range (200 – 800 nM) of H-NS is shown on the right.
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the E. coli gene silencer EAT6 (the case in pWU802ES) or a
same size neutral DNA sequence (the case in pWU802NS). The
previous functional studies (Figs. 6 and 7B) clearly indicated
that EAT6 was able to replace the gene silencing activity of
AT8 in the S. typhimurium LCR-I for the repression of the
promoter of the downstream leuO gene and even the more
distal bla gene. Based on this observed functional replacement,
we also anticipated observing similar H-NS nucleation on the
replacement plasmid construct, pWU802ES. Indeed, the 39-bp
EAT6 DNA sequence was the first region occupied by H-NS as
evidenced in the DNase I footprinting experiment (Fig. 10, left
panel). Although the DNase I protection was not as efficient as
had been observed in the experiment using the original
S. typhimurium LCR-I DNA sequence (Fig. 8), the H-NS-de-
pendent DNase I protection also eventually extended toward
the promoter of the leuO gene (Fig. 10, left panel,lane 1). The
lower efficiency is most likely due to imperfect compatibility of
the E. coli gene silencer EAT6 in the context of S. typhimurium
LCR-I DNA. The less-than-perfect compatibility is evidenced
on the DNase I-mediated cleavage pattern of the DNA of the
replacement plasmid construct pWU802ES. There is a stretch
of intensive DNase I-mediated cleavages located near the junc-
tion of the 5⬘end EAT6 DNA sequence and the context of the
S. typhimurium LCR-I (the dense cleavage pattern shown in
Fig. 10, left panel). Nevertheless, the DNase I footprinting
result demonstrated that the E. coli gene silencer EAT6 is able
to replace the H-NS nucleation function of the S. typhimurium
gene silencer AT8 in S. typhimurium LCR-I. The function that
is replaced must be H-NS nucleation because the H-NS nucle-
ation was not observed upon the replacement with a same size
neutral DNA sequence in the context of S. typhimurium LCR-I
in plasmid pWU802NS. Because the gene silencing activity
observed in the functional assay (Fig. 6) correlates highly with
the H-NS nucleation observed in the DNase I footprinting
experiments (Figs. 8 –10), we concluded that the gene silencer
AT8 or EAT6, important for the promoter relay mechanism,
must be an H-NS nucleation site rather than an ordinary
H-NS-preferred binding DNA sequence.
A Cis-spreading Nucleoprotein Filament Is Responsible for
the Bacterial Gene Silencing—In addition to the nucleation
site, the rest of the AT-rich DNA sequence in S. typhimurium
or E. coli LCR-I appears to also possess a certain binding
preference for H-NS because, with increasing H-NS concentra-
tions, some of this AT-rich DNA was occupied by H-NS as well
(Figs. 8 –10). This H-NS binding preference may be required for
the propagation of H-NS polymerization (as the step II sug-
gested in Ref. 33). The AT-rich DNA may also determine the
directionality of the transcriptionally repressive nucleoprotein
structure. In both cases, the H-NS binding preference led to a
cis-spreading preferentially toward the target promoter, the
FIG.9.H-NS-dependent DNase I protection in E. coli LCR-I. A
unique end radioactively labeled DNA segment consisting of the up-
stream region (⫺8 position to ⫺170 position) of E. coli leuO gene was
generated by PCR. The end-labeled DNA was incubated with H-NS at
a concentration indicated above each lane and exposed to DNase I (0.4
unit per reaction). The positions of relevant elements and restriction
sites in the region are marked.
FIG. 10. H-NS-dependent DNase I protection required the
presence of the DNA sequence of a bacterial gene silencer in
S. typhimurium LCR-I. A unique end radioactively labeled DNA
segment consisting of the promoter and the upstream region (⫹40
position to ⫺269 position) of the S. typhimurium leuO gene was
generated by PCR using either pWU802ES or pWU802NS. The silencer
AT8 in S. typhimurium LCR-I was replaced with the E. coli silencer in
the PCR product when pWU802ES was used as the DNA template (left
panel). The silencer AT8 in S. typhimurium LCR-I was replaced with a
same size (47 bp) neutral DNA sequence in the PCR product when
pWU802NS was used as the DNA template (right panel). The end-
labeled DNA was incubated with H-NS at a concentration indicated
above each lane and exposed to DNase I (0.5 unit per reaction). The
positions of relevant elements and restriction sites in the region are
marked.
H-NS-mediated leuO Gene Silencing5110
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promoter of leuO gene. Together, these data supported a nu-
cleoprotein filament model for the H-NS effect.
The binding of a foreign protein, such as the lac repressor or
repressor within the cis-spreading pathway of the transcrip-
tionally repressive nucleoprotein filament, was capable of
blocking the gene silencing effect found in the AT-rich DNA
sequences flanking the promoter of the bgl operon (34). To
confirm that a continuous cis-spreading nucleoprotein filament
is responsible for the silencing activity found in our model
system, a lac operator was positioned at the AatII site of the
testing plasmid pWU802. Under this testing condition, we ex-
pected that the lac repressor should block the gene silencing
activity from reaching the downstream bla promoter, whereas
the leuO promoter remained as repressed (the model illus-
trated in Fig. 11) if a continuous cis-spreading nucleoprotein
filament indeed simultaneously repressed the activities of both
the leuO promoter and the bla promoter on the testing plasmid
(as observed in Figs. 6 and 7). Indeed, we found that the
presence of a lac operator at the AatII site derepresses the
activity of the bla promoter but not the leuO promoter upon
providing the trans-acting lac repressor (Fig. 11, lanes 2 and 8).
If the inserted lac operator was not positioned between the
gene silencer and any one of the two target promoters (lac
operator was positioned at the HindIII site of the testing plas-
mid as shown in Fig. 11, lanes 3 and 9), the lac repressor did
not affect the gene silencing effect on either promoter. The
binding of the lac repressor at the operator site rather than the
inserted operator DNA must be responsible for the derepres-
sion of the bla promoter activity. This is concluded because no
effect was observed if the lac repressor is not provided (Fig. 11,
lanes 4– 6). Therefore, it is clear that a cis-spreading nucleo-
protein filament initiated from the gene silencer AT8 is respon-
sible for the repression of leuO promoter activity. The tran-
scriptionally repressive nucleoprotein filament further
extended to the downstream region as a continuous nucleopro-
tein filament structure (model illustrated in Fig. 11) and re-
sulted in the repression of the downstream

-lactamase gene
on the testing plasmid.
DISCUSSION
Clearly, the AT-rich LCRs in the ilvIH-leuO-leuABCD gene
cluster are prone to form DNA secondary structures that are
functionally important for transcription regulation (the pro-
moter relay mechanism) in the region. Consequently, the DNA
geometry rather than the specific DNA sequence of the LCRs
was conserved between the two closely related enteric bacteria,
E. coli and S. typhimurium. In the present study, the function
of one of these conserved DNA geometrical elements was char-
acterized. The characterization led to the finding that an H-NS-
mediated cis-spreading nucleoprotein filament is responsible
for the leuO gene silencing. H-NS is an abundant nucleoid
protein important for the architecture of bacterial chromo-
somes (32, 35, 36). The protein is known to affect the expression
of many genes including the hns gene itself (33, 37–39). Among
them, the silencing mechanisms of proU and bgl genes have
been relatively well characterized (34, 40 – 44).
It has been known for some time that H-NS binds to DNA in
a sequence-nonspecific manner, but it preferentially binds with
curved AT-rich DNA (45– 47). Our finding is similar to the gene
silencing activity mediated by the AT-rich DNAs found within
the proximity of the proU and bgl promoters (34, 48). In these
AT-rich DNAs, H-NS nucleation sites and flanking AT-rich
DNAs are also important for their gene silencing activities (31,
40). In our model system, the flanking AT-rich DNA deter-
mines the directionality of the cis-spreading transcriptionally
repressive H-NS filament structure toward the promoter of the
target gene leuO. This is a perfect example that the exact
function of a DNA element (the gene silencer in this case) shall
be evaluated in its natural DNA environment (the neighboring
AT-rich DNA in LCR-I in this case), as the gene silencer exerts
a clear bi-directional transcriptional repressive activity in the
testing plasmid pAO series where the gene silencer is sur-
rounded by plasmid DNA context (13, 14). The previously dem-
onstrated distance limit (300 bp) for the AT8-mediated gene
silencing effect that was determined using pAO plasmids (13)
may also be due to the flanking foreign DNA context. In the
FIG. 11. A cis-spreading nucleoprotein filament is responsible for gene silencer AT8-mediated transcriptional repression. The
plasmid, pWU802, was tested in MC1060, a lac
⫺
strain. The lac repressor was expressed from the pACYC-based pSO1000 in MC1060 when
necessary. The activities of the bla and leuO promoters on pWU802 were simultaneously monitored. The quantified promoter activity shown at the
bottom of each lane for three repeated experiments is expressed as the mean within the range of ⫾0.02 (S.D.). Illustrated in the model is the effect
of a tetrameric lac repressor located at either the AatII site or the HindIII site for blocking the cis-spreading nucleoprotein filament initiated from
gene silencer AT8.
H-NS-mediated leuO Gene Silencing 5111
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present study with the assays performed in the presence of the
surrounding LCR-I DNA sequence, AT8-mediated gene silenc-
ing was found to be able to extend to such a distance as to affect
not only the target leuO promoter but also the promoter of the

-lactamase gene, bla, which is located downstream on the
testing plasmid of the pWU802 series (Figs. 6, 7, and 11). This
could be related to a previous finding that the DNA sequence
upstream of the

-lactamase gene also shows H-NS binding
preference (48). This DNA sequence helps to extend the gene
silencing effect to a further distance. Hence, the gene silencer
AT8 (an H-NS nucleation site) serves as a crucial driving force
for triggering the transcription regulatory effect, but the rest of
the DNA sequence in the region determines the distance and
the directionality of the effect. This conclusion is basically
similar with the finding in a previous study by using the
repression of proU or bgl as a model system (31). It seems that
nature has evolved the optimal transcription controls in the
ilvIH-leuO-leuABCD gene clusters. The optimal transcription
controls do not require DNA sequence specificity in the control
region for either the E. coli LCR-I or the S. typhimurium
LCR-I. The DNA sequence-nonspecific, but functionally inter-
changeable, DNA elements may similarly be responsible for the
transcriptional regulation exerted by the long stretch of AT-
rich DNA within the proximity of the promoter of proU or bgl.
Studies thus far have clearly demonstrated that DNA geo-
metric similarity rather than the sequence specificity of the
gene silencer is responsible for its H-NS nucleation function in
this model system. Certain DNA geometric requirements are
probably also true for the flanking AT-rich DNA, which deter-
mines the cis-spreading (propagation) of the transcriptionally
repressive nucleoprotein structure. Transcription-generated
DNA supercoiling is very likely to play crucial roles in the
sequential activation of genes in the ilvIH-leuO-leuABCD gene
cluster via modulating the DNA geometrical changes of cis-
elements, including the gene silencer in the region. Although
the molecular details underlying the DNA supercoiling effect
remain to be further explored, the apparent DNA geometric
requirement for the H-NS nucleation has provided an explana-
tion for the decisive role of transcription-generated DNA su-
percoiling found in the promoter relay mechanism (9, 10).
Acknowledgments—We are indebted to Drs. Ray Mattingly and
Victoria Kimler and Eric Hales for their critical reading of the manu-
script. We also thank Drs. Chiharu Ueguchi, Herbert Schweizer, and
Sylvie Rimsky for providing crucial materials for the studies.
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H-NS-mediated leuO Gene Silencing5112
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Hai-Young Wu
Chun-Hao Huang, Arundhati Majumder and
Chien-Chung Chen, Meng-Yun Chou,
Found in the Promoter Relay Mechanism
Responsible for the Gene Silencing Activity
A Cis-spreading Nucleoprotein Filament Is
Genes: Structure and Regulation:
doi: 10.1074/jbc.M411840200 originally published online December 6, 2004
2005, 280:5101-5112.J. Biol. Chem.
10.1074/jbc.M411840200Access the most updated version of this article at doi:
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