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Journal of Bacteriology, January 2000, p. 513-517, Vol. 182, No. 2
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
The Amino Terminus of Salmonella enterica Serovar
Typhimurium 
54 Is Required for Interactions with an
Enhancer-Binding Protein and Binding to Fork Junction DNA
Mary T.
Kelly and
Timothy R.
Hoover*
Department of Microbiology, University of
Georgia, Athens, Georgia 30602
Received 24 June 1999/Accepted 22 October 1999
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ABSTRACT |
Transcription initiation by the 
54-RNA polymerase
holoenzyme requires an enhancer-binding protein that is thought to
contact 54 to activate transcription. To identify
potential enhancer-binding protein contact sites in 54,
we compared the abilities of wild-type and truncated forms of Salmonella enterica serovar Typhimurium 54
to interact with the enhancer-binding protein DctD in a chemical cross-linking assay. Removal of two regions in the amino-terminal portion of 54, residues 57 to 105 and residues 144 to
179, prevented cross-linking, but removal of either region alone did
not. In addition, deletion of 56 amino-terminal residues of
54 (region I) reduced the affinity of the protein for a
fork junction DNA probe.
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TEXT |
Transcription initiation by
54-RNA polymerase holoenzyme
(54-holoenzyme) requires an enhancer-binding protein
(19, 20, 22). 54-Holoenzyme binds to
promoters with consensus sequences in the 
12 and 24 regions to form
a closed complex. Enhancer-binding proteins generally bind to sites
upstream of the promoter and contact 54-holoenzyme
through DNA looping (21, 23). Productive intermediates between enhancer-binding proteins and 54-holoenzyme lead
to isomerization of the closed complex to an open complex that can
initiate transcription. Enhancer-binding proteins must hydrolyze ATP or
GTP to catalyze open-complex formation (20, 28).
Interactions between 54-holoenzyme and enhancer-binding
proteins are transient, and little is known about the nature of these interactions. The C4-dicarboxylic acid transport protein D
(DctD) is an enhancer-binding protein from Sinorhizobium
meliloti that can be cross-linked to 54 and the 
subunit of RNA polymerase (16). Some mutant forms of DctD
that fail to activate transcription also fail to cross-link to these
subunits (27), suggesting that DctD contacts these subunits
of 54-holoenzyme to catalyze open-complex formation. To
identify the region of 54 that interacts with DctD, we
generated a series of truncated 54 proteins and assessed
their abilities to cross-link with DctD.
Deletion of 179 amino acid residues from the amino terminus of
54 disrupts cross-linking to DctD.
The
cross-linking reagent succinimidyl
4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC)
can cross-link Sinorhizobium meliloti DctD and
Salmonella enterica serovar Typhimurium 54
(16). Sulfo-SMCC is a heterobifunctional cross-linking
reagent that has a maleimide group and an
N-hydroxysuccinimide ester group, which react preferentially
with sulfhydryl groups and primary amines, respectively, and are linked
by a relatively long and flexible spacer arm. Cys-307 of S. enterica serovar Typhimurium 54 is critical for
cross-linking to DctD (16), presumably because it is surface
exposed and reacts readily with the maleimide group of sulfo-SMCC. For
the cross-linking experiments in this study, we used
DctD(
1-142), which is a truncated, constitutively active form of DctD (17). As in previous studies, these
cross-linking assays were done with purified 54 proteins
and DctD(1-142) in the absence of core RNA polymerase
and DNA (16, 27).
We initially generated three amino-terminally truncated
54 proteins that had deletions of residues 2 to 56 (
2-56), 2 to 105 (2-105), and 2 to 179 (2-179) (Fig.
1). All deletions were generated by using
PCR to introduce a unique NdeI site at the desired position of rpoN, the gene encoding 54. The
NdeI site was used to clone the truncated rpoN
alleles into the expression vector pCyt3 (provided by Elliot Altman,
University of Georgia), which placed the rpoN alleles under
the control of the Escherichia coli lac promoter/operator.
The rpoN alleles were also cloned into the expression vector
pET-28a(+) (Novagen), which resulted in attachment of a hexahistidine
tag at the amino terminus of each truncated 54 protein.
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FIG. 1.
Deletion mutants of S. enterica serovar
Typhimurium 54. The three functional regions of
54 are indicated as I (stippled boxes), II (hatched
boxes), and III (open boxes). The core-binding, modulation, and
DNA-binding domains within region III are noted. Residues corresponding
to the amino-terminal or internal deletions are also indicated. The
ability of each protein to cross-link to DctD(1-142) is
indicated as a positive (+) or negative () result.
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Hexahistidine-tagged
54 proteins were overexpressed in
E. coli BL21 (DE3) [F
ompT
(
lon)
hsdSB gal
DE3::
lacI lacUV5-gene
1 (T7
polymerase)]
by growing the cells in Luria-Bertani broth supplemented
with
1 mM isopropyl-
-
D-thiogalactopyranoside (IPTG).
Cells were harvested,
resuspended in 50 mM Tris-acetate (pH 8.2)-200
mM KCl-1 mM EDTA-1
mM dithiothreitol, and lysed in a French pressure
cell at 12,000
lb/in
2. Like the full-length
hexahistidine-tagged
54 protein (
15), the
truncated hexahistidine-tagged
54 proteins were in the
insoluble fraction following centrifugation
of the cell lysates. The
insoluble fractions containing the hexahistidine-tagged
54 proteins were washed with a solution containing 1 M
NaCl and
1% Triton X-100, after which the hexahistidine-tagged
proteins
were solubilized in 50 mM Tris-HCl (pH 8.0)-50 mM NaCl-0.1
mM
EDTA-1 mM dithiothreitol-5% glycerol-1% sarcosyl as described
previously (
6). The hexahistidine-tagged
54
proteins were then purified by affinity chromatography with
nickel-nitrilotriacetic
acid resin as described previously
(
15). Preparations of the
hexahistidine-tagged
54 proteins were >90% homogeneous as judged from
Coomassie blue-stained
sodium dodecyl sulfate-polyacrylamide gels
containing samples
of the preparations (data not
shown).
The
2-56 mutant lacked region I of
54. Region I,
which consists of approximately amino acid residues 1 to 56, has
important
roles in transcriptional activation (
4,
7,
13,
14,
26,
30). A
54 protein lacking region I still binds
core RNA polymerase and
directs polymerase to promoter DNA, but the
resulting holoenzyme
cannot respond to enhancer-binding protein to form
an open complex
that is capable of transcription initiation (
4,
26). Region
I-deleted holoenzyme, however, can initiate
transcription in the
absence of an enhancer-binding protein under
solution conditions
that permit transient DNA melting or from a
premelted heteroduplex
template (
4,
26). Region I appears to
prevent polymerase
from undergoing isomerization to form an open
complex in the absence
of an enhancer-binding protein (
4).
Region I has a weak core-binding
activity, suggesting that it exerts
influence on core by direct
protein-protein interactions
(
9).
The
2-56 mutant cross-linked to DctD
(1-142) (Fig.
2, lane 6), indicating that region I is
not required for contact
with this enhancer-binding protein. These data
suggest that despite
its requirement for responsiveness to
enhancer-binding protein,
region I does not interact directly with that
protein. Consistent
with this suggestion, Buck and colleagues showed
that a polypeptide
containing region I inhibited transcriptional
activation by
54-holoenzyme in a manner that was
noncompetitive with respect to
the enhancer-binding protein
(
10).
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FIG. 2.
DctD(1-142) cross-linking assays.
Cross-linking reactions were carried out and visualized by
immunoblotting with antiserum directed against S. enterica
serovar Typhimurium 54 as described previously
(16). The black dots to the right of the gels indicate the
positions of 54 proteins, and the asterisks indicate the
positions of major cross-linked products. In addition to cross-reacting
with antiserum directed against 54, these cross-linked
products also cross-reacted with antiserum directed against DctD
(reference 16 and data not shown). The presence (+)
or absence () of sulfo-SMCC and DctD(1-142) is
indicated above each lane. The hexahistidine-tagged 54
proteins used were the wild type (lanes 1 to 3), 2-56 (lanes 4 to
6), 2-105 (lanes 7 to 9), 2-179 (lanes 10 to 12), 101-179
(lanes 13 to 15), (2-56, 144-179) (lanes 16 to 18), and
(2-105, 144-179) (lanes 19 to 21).
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The
2-105 mutant lacked region I and most of region II. Region II
of
S. enterica serovar Typhimurium
54 spans
residues 50 to 120 and is very acidic. Deletions within
region II
appear to reduce the rate of open-complex formation
(
29).
Like the region I-deleted holoenzyme, holoenzyme containing
54 deleted for regions I and II binds promoter DNA and
initiates
transcription from a premelted heteroduplex template in
the absence
of enhancer-binding protein (
2). The
2-105 mutant cross-linked
to DctD
(1-142) (Fig.
2, lane 9), indicating that regions
I and II are dispensible for
contact with DctD
(1-142).
The
2-179 mutant lacked regions I and II and part of region III.
Region III is responsible for core binding and DNA binding
and also
enhancer responsiveness (
5,
8,
11,
24,
25,
30). Core binding
by
54 appears to involve more than one region of the
protein. The minimal
core-binding domain, which spans residues 120 to
215, has the
highest affinity for core RNA polymerase and likely
directs formation
of the holoenzyme (
9). The DNA-binding
determinants are located
between residues 329 and 477, while a
modulation domain that stimulates
the DNA-binding activity of the
DNA-binding domain lies within
residues 180 to 306 (
3,
6,
18,
24).
The
2-179 mutant failed to cross-link to
DctD
(1-142) (Fig.
2, lane 12), suggesting that a
sequence between residues
106 and 179 is required for interactions with
DctD
(1-142).
To define this sequence more precisely, we
generated internal
deletions within
54. We initially
deleted residues 101 to 146 and residues 144 to
179 by using unique
restriction sites within
rpoN. Both of these
mutant
proteins,
101-146 and
144-179, cross-linked to
DctD
(1-142) (data not shown). When we deleted residues
101 to 179, the resulting
protein could also be cross-linked to
DctD
(1-142) (Fig.
2, lane
15).
Regions II and III appear to be involved in interactions with
DctD(1-142).
Because 101-179 cross-linked to
DctD(1-142) but 2-179 did not, we reasoned that a
region between residues 2 and 100 compensated for the loss of residues
101 to 179. To test this hypothesis, we generated amino-terminal
deletions in the mutant proteins 101-146 and 144-179. The
double-deletion mutants (2-56, 101-146) (data not shown) and
(2-56, 144-179) (Fig. 2, lane 18) cross-linked to
DctD(1-142). However, when residues 2 to 105 were
deleted in addition to residues 144 to 179, the resulting protein,
(2-105, 144-179), failed to cross-link to DctD(1-142) (Fig. 2, lane 21). These data suggest that a sequence at around residues 57 to 105 and a second sequence at around
residues 144 to 179 are involved in interactions with DctD(1-142), but only one of these sequences is needed for cross-linking.
In vivo DNA-binding activities of 54 deletion
mutants.
All of the mutant proteins that we generated contained
intact DNA-binding and modulation domains. We wanted to verify that the
two deletion mutants that failed to cross-link to
DctD(1-142) retained their DNA-binding activities, as
this would imply that the DNA-binding and modulation domains of these
mutant proteins were folded correctly. The modulation domain contains
Cys-307, which is critical for cross-linking of 54 to
DctD(1-142) by sulfo-SMCC (16).
We examined the abilities of the deletion mutants to repress
transcription from a phage P22
ant'-'lacZ reporter gene in
which
the
54-dependent
Sinorhizobium meliloti
nifH promoter overlapped the
ant promoter
(
1). When
54 was overexpressed from a plasmid
in an
S. enterica serovar Typhimurium
strain carrying a
chromosomal copy of the
ant'-'lacZ fusion, expression
from
this reporter gene was reduced ~25-fold (Fig.
3). All of
the deletion mutants that we
generated repressed transcription
from the
ant'-'lacZ
reporter gene when overexpressed in the same
strain, indicating that
they retained their DNA-binding activities.
The degree to which these
mutant forms of
54 repressed transcription was not as
great as that of wild-type
54, as the mutant proteins
reduced expression from the
ant'-'lacZ reporter gene by 5- to 15-fold (Fig.
3). Several of the mutant
54 proteins
were expressed at lower levels than wild-type
54,
however, and so these repression assays are not completely indicative
of the abilities of these mutant
54 proteins to repress
transcription from the
ant'-'lacZ reporter
gene (data not
shown). Some of the deletions extended into the
core-binding domain,
and these mutant proteins may have repressed
transcription by binding
directly to the
nifH promoter rather
than by directing
polymerase to this promoter. Although the mutant
54
proteins retained their DNA-binding activities, none of them
complemented the
rpoN mutation, as indicated by their
failure
to confer glutamine prototrophy.
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FIG. 3.
Repression of the ant'-'lacZ reporter gene by
the 54 deletion mutants. S. enterica serovar
Typhimurium strain TRH107 (15) was transformed with
derivatives of pCyt3 bearing the various rpoN alleles.
Following the overexpression of the 54 proteins in this
strain by the addition of 0.1 mM IPTG, -galactosidase activities
were determined as described previously (15). Values shown
are averages of data from three assays, and for each sample 1 standard
deviation is indicated by an error bar.
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In vitro DNA- and core-binding activities of 54
deletion mutants.
We examined the abilities of the deletion
proteins to bind to promoter DNA in vitro by using a 21-bp
double-stranded probe that spanned the 29 to 9 region of the
Sinorhizobium meliloti nifH promoter and a fork junction
probe that spanned the same region but had a 5' overhang on the
template strand corresponding to residues 11 to 9. Guo and Gralla
(12) showed that E. coli 54 formed
a heparin-resistant complex with this fork junction DNA probe.
Holoenzyme formed with wild-type
54 or full-length
hexahistidine-tagged
54 had a much higher affinity for
the fork junction probe than for
the double-stranded probe (Fig.
4, lanes 3 to 6). In contrast,
mutant
54 proteins with amino-terminal deletions had higher
affinities
for the double-stranded probe, both as free
54 and when associated with RNA polymerase (Fig.
4,
lanes 7 to 12
and 19 to 24). The three original amino-terminal deletion
mutants,
2-56,
2-105, and
2-179, appeared to bind the
double-stranded
probe better than wild-type
54 did, both
as free
54 and the holoenzyme forms. These data indicate
that the
54 mutant proteins retained their DNA-binding
activities, consistent
with the repression observed at the
ant'-'lacZ reporter gene.
These data also demonstrate that
region I is required for efficient
binding to the fork junction probe
but not the double-stranded
probe. This is consistent with our previous
observation that certain
amino acid substitutions within region I
reduce the affinity of
54 for the fork junction probe
(
15). Casaz and Buck (
7) reported
that deletion
of region I of
54 affects the conformation of the
carboxy-terminal DNA-binding
domain, which may account for the reduced
affinities of the region
I deletion mutants for the fork junction
probe.
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FIG. 4.
Gel mobility shift assays with 54
deletion mutants. The double-stranded probe was used in the
odd-numbered lanes, and the fork junction probe was used in the
even-numbered lanes. All reaction mixtures contained 5 nM DNA and 300 nM E. coli core RNA polymerase, and 54
proteins were present at 600 nM in lanes 3 to 18 and at 1 µM in lanes
19 to 24. Reactions were carried out as described previously
(15), and products were visualized by exposing the 5%
native polyacrylamide gels to X-ray film for 24 h. Lanes 1 and 2 contained core RNA polymerase only. The 54 proteins used
were as follows: wild-type 54 (lanes 3 and 4),
hexahistidine-tagged wild-type 54 (lanes 5 and 6),
2-56 (lanes 7 and 8), 2-105 (lanes 9 and 10), 2-179 (lanes
11 and 12), 101-146 (lanes 13 and 14), 144-179 (lanes 15 and
16), 101-179 (lanes 17 and 18), (2-56, 101-146) (lanes 19 and
20), (2-56, 144-179) (lanes 21 and 22), and (2-105, 144-179)
(lanes 23 and 24). Wild-type 54-holoenzyme shifted
>50% of the fork junction probe. Free probe is not shown. H, the
shifted species with holoenzyme bound to the DNA probe; S, the shifted
species with 54 bound to the DNA probe.
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The mutant
54 proteins retarded the mobility of the DNA
probes to different degrees, which may reflect altered mobilities of
the various mutant proteins in the native gel. Complexes of the
mutant
forms of
54-holoenzyme and the DNA probes, however, had
the same mobility.
For each mutant protein, we confirmed that the
faster-migrating
species was due to free
54 by omitting
core from the gel mobility shift assay (data not
shown). Interestingly,
the three mutant proteins with internal
deletions,
101-146,
144-179, and
101-179, retarded the mobility
of the
double-stranded probe to a greater extent than they retarded
the fork
junction probe. A possible explanation for this observation
is that
these mutant forms of
54 bend the double-stranded probe
more than they bend the fork junction
probe.
The gel mobility shift assays also allowed us to assess the abilities
of the mutant
54 proteins to bind to core RNA
polymerase. In the gel shift assays
with the deletion mutants
2-56,
2-105, and
2-179, the amount
of double-stranded probe shifted
by holoenzyme decreased with
increases in the extent of the deletion.
It was surprising that
2-179 yielded a holoenzyme-shifted species
at all given that
the deletion extended into the minimal core-binding
domain. Core
protects sequences within the DNA-binding and modulation
domains
from hydroxyl radical cleavage (
7), suggesting
possible interactions
of core with these regions of
54.
It is possible that the double-stranded DNA probe stabilized
interactions between core and these regions in the
2-179 mutant
protein. The three mutant proteins with internal deletions,
101-146,
144-179, and
101-179, also retained some
core-binding activity,
although their affinities for core appeared to
be greatly reduced
compared to that of wild-type
54.
The three double-deletion mutants bound poorly to both the fork
junction probe and core. All of these mutant proteins shifted
the
double-stranded probe as free
54, although the shifted
species with
(2-105, 144-173) resulted
in a fairly diffuse band.
It is unclear why the double-deletion
mutants appeared to have lower
affinities for core than did the
2-179 mutant. It is possible that
the sequences that remained
at the amino termini of these deletion
mutants interfered with
core
binding.
Taken together, the in vivo and in vitro DNA-binding assays indicate
that the
54 deletion mutants retained their DNA-binding
activities and, in
some cases, their core-binding activities. We infer
from these
results that the DNA-binding and modulation domains of the
54 deletion mutants were folded correctly. Therefore, it
seems likely
that the failure of
2-179 and
(2-105, 144-179) to
cross-link
with DctD
(1-142) was due to the removal of
protein
sequences important for specific interactions with this
enhancer-binding
protein.
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ACKNOWLEDGMENTS |
We thank Ellen Neidle for comments on the manuscript.
This work was supported by award MCB-963054 to T.R.H. from the National
Science Foundation.
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FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Microbiology, 527 Biological Sciences Building, University of Georgia, Athens, GA 30602. Phone: (706) 542-2675. Fax: (706) 542-2674. E-mail:
trhoover{at}arches.uga.edu.
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Journal of Bacteriology, January 2000, p. 513-517, Vol. 182, No. 2
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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