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Journal of Bacteriology, December 1999, p. 7266-7273, Vol. 181, No. 23
Department of Microbiology, School of
Medicine, University of Virginia, Charlottesville, Virginia 22908
Received 6 July 1999/Accepted 17 September 1999
Fundamental questions in bacterial gene regulation concern how
multiple regulatory proteins interact with the transcription apparatus
at a single promoter and what are the roles of protein contacts with
RNA polymerase and changes in DNA conformation. Transcription of the
Escherichia coli uhpT gene, encoding the inducible sugar
phosphate transporter, is dependent on the response regulator UhpA and
is stimulated by the cyclic AMP receptor protein (CAP). UhpA binds to
multiple sites in the uhpT promoter between positions Transcription activation, a common
form of gene regulation in bacteria, can occur through formation of
specific contacts between a DNA-bound activator protein and RNA
polymerase (RNAP) holoenzyme. These protein contacts can recruit the
binding of RNAP to weak promoter sequences, stimulate its isomerization
from a closed complex to an open complex, or affect the rate of
promoter clearance by the transcription elongation complex (reviewed in
reference 32). Activator proteins can induce changes
in DNA conformation to increase promoter effectiveness. Several
portions of the RNAP subunits have been identified as having roles in
promoter activation. The C-terminal domain of CAP provides a prime example of activation through multiple RNAP
contacts. When complexed with cAMP, CAP binds to a specific DNA target
sequence in promoters of more than 100 genes in Escherichia coli (reviewed in references 6 and
22). CAP can directly activate transcription when it
binds to sites that are centered between bp We address here the involvement of RNAP subunits in transcription
activation at the uhpT promoter. Expression of the UhpT sugar phosphate transporter is induced by external glucose 6-phosphate (Glu6P) through the action of an atypical two-component system (20). The uhpT promoter lacks a To investigate the action of UhpA and CAP, we developed an in vitro
transcription system that reproduces the dependence on UhpA and the
stimulation by CAP that is seen in vivo (30). Here, this in
vitro expression system was used in combination with in vivo expression
systems to evaluate the effect on uhpT transcription of
alanine substitutions throughout Bacterial strains and plasmids.
E. coli K-12 strains
and plasmids used in this work are listed in Table
1. Plasmid-bearing cells were grown in
the presence of ampicillin (200 µg/ml). Plasmids encoding
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
RNA Polymerase
and 
70 Subunits
Participate in Transcription of the Escherichia coli
uhpT Promoter
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
80
and 32 upstream of the transcription start site, and CAP binds to a
single site centered at position 103.5. The role in uhpT
transcription of portions of RNA polymerase E
70
holoenzyme which affect regulation at other promoters was examined by
using series of alanine substitutions throughout the C-terminal domains
of RpoA (residues 255 to 329) and of RpoD (residues 570 to 613).
Alanine substitutions that affected in vivo expression of a
uhpT-lacZ transcriptional fusion were tested for their
effect on in vitro transcription activity by using reconstituted
holoenzymes. Consistent with the binding of UhpA near the 35 region,
residues K593 and K599 in the C-terminal region of RpoD were necessary for efficient uhpT expression in response to UhpA alone.
Their requirement was overcome when CAP was also present. In addition, residues R265, G296, and S299 in the DNA-binding surface of the C-terminal domain of RpoA (
CTD) were important for uhpT
transcription even in the presence of CAP. Substitutions at several
other positions had effects in cells but not during in vitro
transcription with saturating levels of the transcription factors. Two
DNase-hypersensitive sites near the upstream end of the UhpA-binding
region were seen in the presence of all three transcription factors.
Their appearance required functional CTD but not the presence of
upstream DNA. These results suggest that both transcription activators
depend on or interact with different subunits of RNA polymerase,
although their role in formation of proper DNA geometry may also be crucial.
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
70
(CTD) contains a putative helix-turn-helix motif (region 4.2, residues 570 to 613) which recognizes the 35 promoter element (15) and some of the transcription activators that bind near the 35 region, including 
cI, PhoB, Ada, cyclic AMP (cAMP)
receptor protein (CAP)-activating region-3 (AR3), FNR, and phage Mu
Mor proteins (1, 21, 23-25). The C-terminal domain of the
subunit (CTD) interacts with A+T-rich UP (upstream activating
region) elements in the 60 to 40 region (31) and with
many transcription regulators that bind upstream of the RNAP-binding
site, including CAP, OmpR, OxyR, Ada, and BvgA (4, 5, 10, 23,
38).
41.5 and 92.5 upstream
of the transcription start site and have proper helical phasing
relative to the binding site for RNAP (14, 42). Different
surfaces of CAP contact different parts of RNAP, depending on the
location of the CAP-binding site in the promoter. Contact between a
surface loop around residue 157 in CAP, called activating region-1 or
AR1, and CTD is important for CAP action at any effective site. A
second surface of CAP, AR2, contacts the amino-terminal domain of RpoA
when CAP is bound at position 41.5, adjacent to the site of the RNAP
(29). A third surface element of CAP, AR3, is normally
silent but can allow activation in place of AR1 or AR2 when altered by
mutation. The AR3 region contacts CTD (5).
35 element and
depends absolutely on the response regulator UhpA (11, 44).
UhpA is activated by its phosphorylation on aspartate-54 by the
membrane-bound UhpBC sensor kinase complex in response to extracellular
Glu6P. UhpA binds to a dyad element centered at position 64, and
phosphorylated UhpA can then bind to low-affinity sites that extend to
position 32 (7). Catabolite repression of uhpT
transcription results from the 10- to 15-fold stimulation caused by the
binding of CAP-cAMP to a DNA site centered at position 103.5
(27). Since CAP cannot independently activate transcription
from so far upstream (2, 42), it was of interest to test
whether UhpA or CAP action was related to specific contacts with RNAP.
CTD and CTD. Key residues in an
activator response segment of CTD and in the DNA-binding surface of
CTD were found to be important for uhpT transcription. CTD contributes to the action of UhpA alone, but not to that of UhpA
plus CAP, whereas CTD is important for CAP action. In addition,
CTD is important for protein-dependent changes in promoter DNA
conformation that may combine with protein-protein contacts to enhance
promoter folding around RNAP (30).
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
70 alanine substitution mutants with mutations at
residues 574 and 590 through 613 were described by Lonetto et al.
(25) and are variants of pGEX2 in which the coding region of
glutathione S-transferase (GST) is fused to RpoD residues 8 to 613 and expressed from the tac promoter. Plasmids
encoding 70 alanine substitution mutants with mutations
at residues 570 and 575 were obtained from P. Landini, University of
Birmingham (23). For overexpression and purification of
these RpoD variants, the rpoD coding sequence was cloned
into the vector pGEM-His6 so that the coding region for a
His6 tag is fused to RpoD residues and expressed from the
tac promoter. Plasmids encoding CTD alanine substitution
mutants with mutations between residues 255 and 329 were described by
Savery et al. (34) and encode derivatives of RpoA without or
with a His6-coding region inserted between codons 1 and 2.
TABLE 1.
Bacterial strains and plasmids used in this work
Screening of rpoD mutants in vivo.
Two host
strains were used to screen for rpoD mutant alleles
affecting UhpA-dependent transcription of uhpT. A plasmid
library carrying mutations resulting in single alanine substitutions at 19 positions between residues 570 and 613 of the C-terminal region of
70 (obtained from C. A. Gross, University of
California, San Francisco [25], or from P. Landini
[23]) was introduced by transformation into strains
RK1309 and IO697. Both strains are single lysogens of RZ5
uhpT-lacZ. Transformants of RK1309 were grown in minimal medium A supplemented with 1% glycerol, 0.5% Casamino Acids, and 200 µg of ampicillin per ml. Transformants of IO697 were grown in minimal
medium A supplemented with 1% glycerol, 20 µg of
L-tryptophan per ml, 0.5% Casamino Acids, and 500 µg of
ampicillin per ml. 
-Galactosidase levels were measured after
induction with 0.25 mM Glu6P for 40 min for RK1309 or 2 h for IO697.
-D-thiogalactopyranoside)-inducible promoter.
However, addition of IPTG, even to cells expressing wild-type RpoD,
resulted in marked inhibition of uhpT induction and of cell
growth, perhaps owing to nonspecific inhibition of RNAP assembly or
function (25). Hence, only basal-level expression of
rpoD was used, which results in levels of protein comparable
to those with expression from the chromosomal
rpoD+ gene. The effect of the RpoD substitutions
on transcription in RK1309 was expected to be less prominent than that
in IO697 or in the studies described by Lonetto et al. (25),
because the latter strains have reduced competition from the
chromosome-encoded wild-type RpoD protein, which is expressed from a
tryptophan-repressible trp promoter.
Screening of rpoD and rpoA mutants in
vivo.
To screen for rpoA mutants affecting
uhpT transcription, a plasmid library of rpoA
mutants expressing alanine substitutions at 69 positions between
residues 255 and 329 of CTD (obtained from T. Gaal and R. Gourse,
University of Wisconsin) (12) under lac control
was screened in strain RK1309 or TM37 in a similar manner as described
above, except that 1 mM IPTG was present throughout the growth of the
culture for induction of rpoA gene expression.
-Galactosidase assays.
As described previously
(28), cells were grown in minimal medium A supplemented with
1% glycerol and 0.5% Casamino Acids, without or with 0.25 mM Glu6P
for the indicated times. Cells were permeabilized with
CHCl3-sodium dodecyl sulfate. The time course of hydrolysis
of o-nitrophenyl--D-galactopyranoside was
measured at 415 nm in a 96-well microplate reader (Molecular Devices)
at 37°C and was normalized for culture density (optical density at 660 nm). Each assay was performed in triplicate.
Protein purification and reconstitution of RNAP with mutant
subunits.
For reconstitution of RNAP with wild-type
70 subunit or the alanine substitution mutants,
His6-tagged proteins were purified using
Ni-nitrilotriacetic acid columns (Qiagen, Valencia, Calif.), and
GST-tagged proteins were purified on glutathione agarose columns (8). Purified factors were added at a 4:1 ratio to core
RNAP (Epicentre Technologies, Madison, Wis.) (23) for the
His-tagged subunits and at 10:1 for the GST fusion proteins prior to in
vitro transcription assays.
subunits were expressed
from plasmid pHTT7f1-NH (40) or derivatives constructed by gene replacement of the EcoRI-BamHI fragments
encoding the desired alanine substitutions (12).
Purification of subunits by Ni2+ affinity
chromatography; preparation, solubilization, and renaturation of
inclusion bodies of , ', and 70 subunits; and
reconstitution of RNAP holoenzyme were carried out essentially as
described previously (12, 39).
Cells carrying plasmids pMKSe2, pT7', and pLHN12 were induced for
expression of RpoB, RpoC, and RpoD, respectively. Wild-type E. coli UhpA protein was purified as described previously
(7). CAP was purified by cAMP affinity chromatography
(46). Both proteins were >95% homogeneous.
DNase I protection.
DNase I footprinting reactions were
performed by the method of Galas and Schmitz (13) as
described previously (30). The 5'-32P-end-labeled linear DNA fragments containing the
uhpT promoter region were obtained by PCR with pRJK10 DNA as
a template (43). The primers used were IOPT +50 (positions
+50 to +26), IOPT-250 (250 to 226) (30), IOPT-179, and
IOPT-121; the sequences of these primers are available upon request.
Primer IOPT+50 was 5' end labeled by using T4 polynucleotide kinase and
[
-32P]ATP (3,000 Ci/mmol). PCR products were purified
by using the QIAquick PCR purification kit (Qiagen).
Promoter-containing DNA fragment (50,000 cpm) at a final concentration
of 1 nM was incubated with the specified amounts of purified UhpA, CAP,
and RNAP in 20 µl of TXN buffer, which comprises 40 mM Tris-HCl (pH
8.0), 50 mM KCl, 10 mM MgCl2, 10 mM dithiothreitol, and 200 µM cAMP. After 30 min at 37°C, DNase I digestion was begun by
addition of 2 µl of TXN buffer containing 25 mM CaCl2, 25 mM MgCl2, and 0.5 µg of DNase I per ml. After 30 s
at room temperature, 4 µl of stop solution {0.18 M EDTA, 0.34 µg
of poly[d(I:C)] per ml, 30% glycerol} was added. DNA was
precipitated and washed with 70% ethanol, dried under vacuum,
dissolved in loading buffer, and resolved by electrophoresis on
sequencing gels (5% polyacrylamide-7 M urea gels in 1×
Tris-borate-EDTA buffer) (33). Radioactive DNA fragments
were detected with a PhosphorImager (Molecular Dynamics, Sunnyvale,
Calif.).
In vitro transcription.
Transcription assays were performed
as described by Olekhnovich et al. (30) at 25°C in TXN
buffer (see above). The indicated amounts of the DNA template fragment,
UhpA, and CAP were incubated for 10 min in 10 µl of TXN buffer prior
to the addition of RNAP (5 µl). After 3 min at 25°C, 5 µl of TXN
buffer containing nucleoside triphosphate (NTP) substrates and heparin
was added to yield the following final concentrations: 1 nM DNA
template, 220 nM UhpA; 20 nM CAP; 5 nM RNAP; 50 µg of heparin per ml,
200 µM (each) NTPs (ATP, CTP, and GTP), and 40 µM
[-32P]UTP (2.5 Ci/nmole). After 10 min, the reaction
was terminated by addition of 5 µl of transcription stop solution (7 M urea, 0.1 M EDTA, 0.4% sodium dodecyl sulfate, 40 mM Tris-HCl [pH
8.0], 0.5% bromphenol blue, and 0.5% xylene cyanol). After
electrophoresis in sequencing gels, the products were analyzed by
autoradiography or PhosphorImager analysis with the ImageQuant program
for quantitative comparisons.
380 and +233, generated by
PCR on pRK10 as template, or carried the lacUV5 promoter
(34).
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Effect of changes in CTD on uhpT-lacZ
expression.
Binding of UhpA to the 32 region of the
uhpT promoter appears to be important for its action. It was
thus of interest to examine the role in uhpT transcription
of residues 570 to 613 in the C-terminal end of 70,
which are involved both in the recognition of the 35 element and in
the action of some activators that bind near position 40. Plasmids
expressing RpoD or GST-RpoD proteins with the wild type or 19 mutants
with alanine substitutions at residues 570, 574, and 575 and from
residues 590 to 613 (kindly provided by C. A. Gross and P. Landini) (23, 25) were introduced into strain RK1309,
carrying a single-copy uhpT-lacZ transcriptional fusion. The
plasmid-coded RpoD proteins compete with the chromosome-encoded wild-type RpoD for assembly into RNAP holoenzyme. Decreased
Glu6P-induced uhpT-lacZ expression should occur in cells
expressing an RpoD variant that is defective in uhpT
transcription. In this strain, none of the alanine substitutions
affected expression, except for substitutions at residues 570 and 575, which affect action of the PhoB and Ada proteins and caused a 40%
reduction in uhpT-lacZ expression (Fig.
1A).
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CTD only in
the absence of their respective coactivators, CAP or NarL (25), we tested the effect of some alanine substitutions in CTD on uhpT-lacZ expression in the 
crp
strain TM37. Whereas expression of the RpoD-D570A or -E575A variant in
the crp+ strain decreased uhpT-lacZ
expression, these variants had little effect on the activity remaining
in the crp strain (Fig. 2A and B). Similar results were obtained with
the R596A, K597A, H600A, S602A, and R603A variants (data not shown).
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crp
uhpT-lacZ strain, the chromosomal rpoD+
allele is replaced with 
(Cm)trpP-rpoD, which allows the
effect of the RpoD variants to be tested with reduced competition from the wild-type protein and in the absence of the activator CAP. Expression of rpoD is repressed by addition of tryptophan,
and cell growth is dependent on the tryptophan competitor
indole-3-acrylic acid, unless a complementing plasmid-borne
rpoD variant is present. In IO697, plasmids expressing the
E591A, L595A, and L598A variants did not allow indole-3-acrylic
acid-independent growth, indicating the inability of these variants to
substitute for the wild-type RpoD. The Glu6P-induced -galactosidase
levels in the remaining strains were very low, at most about 3% of
that in strain RK1309, owing to the absence of CAP function and the
poor growth of the cells. The K593A and R599A variants of RpoD
conferred a two- to threefold reduction in expression, whereas the
D570A and E575A variants conferred expression comparable to that for
the wild type and all other alanine substitution mutants. Thus, several residues in CTD appear to be important for UhpA activation.
Effect of changes in CTD on in vitro uhpT
expression.
To test the effect of the alanine substitutions in
RpoD which affected cellular expression, the His6-tagged
D570A and E575A RpoD proteins and the GST-tagged E591A, K593A, L598A,
and R599A proteins were purified. The purified proteins were
reconstituted with core RNAP to form E70 holoenzymes.
The reconstituted RNAPs were used for single-round in vitro
transcription reactions with a linear uhpT promoter DNA template (positions 380 to +233) (1 nM) and the purified UhpA (220 nM) and CAP (20 nM) proteins (Fig. 3).
Transcription from the CAP-independent lacUV5 promoter was
used to measure the RNAP activities of the reconstituted enzymes. DNA
templates were preincubated at 25°C with UhpA in the absence and
presence of CAP at concentrations previously found to be maximally
stimulatory for uhpT transcription (30). The
preincubated templates were then incubated with the RNAP holoenzymes
for 3 min prior to addition of the NTP substrates and heparin (50 µg/ml) to block further transcription initiation. Both RpoD and RNAP
core enzyme were required for transcription from either promoter (data
not shown). Expression from the lacUV5 promoter was
decreased by the E591A and L598A substitutions, suggesting a general
defect. In the former case, this decrease was associated with reduced
amounts of the RpoD protein, perhaps as a result of protein
instability.
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-galactosidase expression was an indirect effect. The
E591A variant showed uhpT transcription that was reduced to
almost the same degree as was lac transcription (Fig. 3A,
lanes 4 and 5), consistent with a general defect. In contrast, the
K593A, L598A, and R599A variants were strongly impaired for
uhpT transcription relative to lac (Fig. 3A,
lanes 6 to 8).
The effect of CAP on uhpT transcription was tested (Fig.
3B). Under these assay conditions, CAP stimulated in vitro expression by 3.2-fold, in comparison to the 10- to 15-fold stimulation of uhpT-lacZ expression by CAP. Although UhpA-dependent
transcription by the reconstituted holoenzymes containing RpoD-K593A,
-L598A, and -R599A was strongly reduced by 70 to 90%, it was strongly stimulated by CAP. In comparison to the 3.5-fold stimulation with wild-type RpoD, CAP stimulated uhpT transcription by these
variant holoenzymes by 7- to 18-fold, up to levels approaching that of the wild-type. These results show that the K593A and R599A
substitutions interfere with UhpA-dependent transcription activation
but are only moderately impaired for transcription stimulated by UhpA plus CAP. Note that CAP alone does not allow any uhpT
transcription in the absence of UhpA.
Effect of changes in CTD on uhpT-lacZ
expression.
The involvement of CTD in uhpT
expression was examined. First, plasmids expressing RpoA+
or RpoA-256, in which CTD is deleted from residue 256, were introduced into crp+ and crp
cells. Glu6P-induced expression of the uhpT-lacZ fusion was
measured after induction of the variant subunits with IPTG (Fig. 2C and
D). Expression of RpoA-256 resulted in a fivefold reduction of
uhpT expression in the crp+ strain,
indicating a key role of CTD in uhpT transcription. Expression of either RpoA+ or RpoA-256 caused a similar
20-fold decrease in uhpT expression in the absence of CAP.
This result suggested that overexpressed RpoA interferes with
uhpT expression and that CTD participates in
transcription activation by CAP but perhaps not by that of UhpA alone.
CTD important for uhpT
transcription, we examined the effect of expression of RpoA variants
carrying alanine substitutions throughout CTD from residues 255 to
329 (provided by T. Gaal and R. Gourse). Alanine substitutions at numerous positions in CTD affected uhpT expression (Fig.
4). Three RpoA mutants, R265A, G296A, and
S299A, had the strongest effects and reduced expression by 2.5- to
5-fold. Less pronounced reductions resulted from other substitutions,
including those at residues 262, 264, 278, 281, 284, 289, and 291 and
at many sites distal to residue 307. Some substitutions resulted in
elevated expression.
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Effect of changes in CTD on expression in vitro.
We
examined the in vitro transcription activity and the degree of CAP
stimulation by using 12 reconstituted RNAP holoenzymes. Wild-type RpoA
and variants with alanine substitutions at 11 positions which affected
expression in vivo were purified and mixed with the other wild-type
components to form RNAP E70 holoenzymes. Each
reconstituted RNAP holoenzyme was used in single-round runoff
transcription reactions of the uhpT and lacUV5
promoter fragments, as described above.
CTD are required for effective uhpT transcription but that most are not. It is also clear
that some changes in CTD were more detrimental to transcription than was the absence of CAP, indicating that the effect of CTD extends beyond its effect on CAP action alone.
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Changed DNA geometry depends on CTD but not upstream DNA.
To look for the sites of RNAP binding at the uhpT promoter
and for possible changes in DNA geometry caused by the binding of CAP
and CTD, we examined the DNase I digestion pattern of the
uhpT promoter fragment (positions 250 to +50) in the
presence of the three transcription factors (Fig.
6A). As seen previously (7,
30), UhpA protected sites in the 80 to 32 region, and CAP
protected or enhanced cleavage between positions 114 to 93. Surprisingly, no footprint could be assigned to RNAP, whether alone or
with UhpA and CAP. The lack of strong binding by RNAP is consistent
with the lability of the open complex to dissociation by polyanions
(30).
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80 and 74 only in
the presence of all three factors, i.e., RNAP, UhpA, and CAP (Fig. 6A,
lane 6). These sites are near the most-upstream UhpA-binding region,
close to the CAP-binding site. Footprinting was carried out with the
reconstituted RNAP carrying the wild-type or the R265A or S299A form of
RpoA. The formation of the hypersensitive sites in the ternary complex
did not occur with the R265A variant and was markedly reduced with the
S299A variant (compare lanes 6 to 8 in Fig. 6A). Finally, we tested
whether the appearance of the DNase-hypersensitive sites required the
presence of upstream DNA. Footprinting was carried out with three DNA
fragments whose upstream ends were at positions 121, 179, and 250
(Fig. 6B). DNase digestion was carried out with free DNA (lanes 1) or
in the presence of UhpA plus CAP (lanes 2) or UhpA plus CAP plus RNAP
(lanes 3). All three DNA fragments displayed comparable amounts of both
hypersensitive sites in the presence of all three protein components.
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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UhpA is absolutely required for uhpT transcription in vivo and in vitro (30, 36). CAP is required for efficient transcription, but maximal in vitro transcription can occur in the absence of CAP with longer incubation of the template with UhpA and RNAP. CAP has multiple effects on the formation of the open complex; namely, it enhances the binding of UhpA and RNAP to the promoter, accelerates open complex formation, and slows open complex dissociation (30). The instability of the open complex at the uhpT promoter is indicated both by the susceptibility of the transcription complex to polyanions (30) and by the absence of a DNase protection footprint.
The present study identified protein contact sites important for
transcription at the uhpT promoter. Since UhpA binds near the 35 region, we expected that UhpA might contact the CTD of RpoD,
as found for some transcription activators that bind in a similar
position. The structure of the CTD may resemble that of the
C-terminal helix-turn-helix DNA-binding domain of NarL and UhpA
(25). Portions of one helix recognize the 35 element and
are preceded and followed by residues that affect activation by
proteins that bind near 35, such as activation of PRM
by cI protein (24), of the ada and
aidB promoters by Ada (23), of the pho
regulon promoters by PhoB (21), and of the phage Mu middle
promoter by Mor protein (1). Alanine substitutions for K593,
K597, R599, and H600 reduced activation by CAP in vivo and in vitro
(19, 25) but only when CAP action depended on AR3. Alanine
substitutions for E591, K593, and R596 had moderate effects on
AraC-dependent transcription but only in the absence of CAP
(25). Residues 593, 596, 597, and 600 affected transcription stimulation by Fnr at the narG promoter but only in the
absence of the activator P-NarL.
Owing to its importance for transcription, in vivo assays of the effect
of alanine substitutions in CTD are difficult. Overexpression of
RpoD is toxic, but the mutant forms insufficient amounts to compete
with the wild type for incorporation into the RNAP holoenzyme. Indeed,
we found no effect of the alanine substitutions in CTD on
uhpT-lacZ expression in a wild-type strain, except for the indirect action of the 570A and 575A variants not seen in vitro. Although uninduced expression of the plasmid-coded GST-RpoD proteins is
equivalent to the expression of the chromosomal allele, these proteins
are probably unable to compete effectively for incorporation into the
holoenzyme. The requirement for CTD residues 593 and 599 was clearly
seen only in cells lacking crp and with repression of the
chromosomal rpoD allele by excess tryptophan. The role of
these residues was verified by in vitro transcription assays, showing
that residues 593, 598, and 599 were required for UhpA activation as
well as other transcription activators. As seen at other promoters,
these CTD residues are needed for the action of the transcription
factor bound near position 35, in this case UhpA. This promoter was
strongly activated by UhpA plus CAP even though UhpA-dependent
transcription was greatly impaired. Because CAP cannot activate
uhpT transcription by itself, UhpA must activate transcription in two ways, one involving interaction with CTD and
the other involving interaction with CAP.
CTD participates in the function of many transcriptional activators,
including CAP (34), OmpR (38), SoxS
(17), Ada (23), and BvgA of Bordetella
pertussis (4), as well as in antitermination by N
protein (35). Some residues in this domain, such as R265 and
S299, are needed for the action of many of these transcription
activators and for stimulation by the UP element. Other positions are
needed only for function of specific activators. It is now apparent
that CTD interacts with both DNA and the activator proteins, that
mutations affecting either function can interfere with transcription
activation, and that CTD can have positive and negative effects at
certain promoters. The structure of CTD (18) revealed
that the residues necessary for the action of the UP element and many
transcription factors comprise its DNA-binding face. The residues that
are needed only for certain transcription factors lie on protein
contact surfaces. Mutations that interfere with the DNA-binding
activity of CTD are usually more deleterious than those that affect
the activator interaction surfaces.
Alanine substitutions at numerous sites between residues 255 and 329 affected Glu6P-induced uhpT-lacZ expression. The R265A change in the DNA-binding surface was the most severely affected both
in vivo and in vitro, as seen on most CTD-dependent activators. The
degree of impairment of the G296A and S299A substitution mutants was
substantially reduced by prolonged incubation with RNAP, a condition
which also reduces the degree of stimulation by CAP. This result
suggests that CAP participates with CTD in the recruitment of RNAP
to the promoter, rather than for open complex formation or promoter
clearance. Although CAP can interact with CTD at other promoters, it
binds too far upstream to contact RNAP at the uhpT promoter
(14, 42). In studies of an artificial promoter containing
two CAP-binding sites, CAP bound at position 102.5 could stimulate
transcription when another molecule of CAP was bound at position 41.5
(2). In this situation, both CAP dimers contacted CTDs
and promoted their binding to adjacent DNA. This result suggests that
CAP sites that are too far upstream for effective contact with RNAP can
be brought into position by the DNA-bending activity of the downstream
CAP dimer. Perhaps UhpA plays a similar role at the uhpT
promoter to alter DNA geometry and allow contact of RNAP and CAP. Our
results show that both CTD and CTD are important for optimal
transcription at the uhpT promoter. Similar dependence on
both RNAP subunits has been found for the action of Ada protein and the
Mor protein of phage Mu (1, 23). Although it seems most
likely that CTD contacts UhpA, the CTD contacts are not yet known.
Because the effect of the removal of CTD or the R265A substitution
mutant was more deleterious than was the absence of CAP, it is likely
that CTD is needed for more than just allowing CAP to act. Two
possibilities are that CTD interacts directly with both CAP and UhpA
or that CTD binds to nonspecific DNA upstream of the CAP site which
is brought into reach of RNAP by the DNA-bending activity of CAP. None
of the changes in the ARs of CAP that affect transcription at other
CAP-dependent promoters had any significant effect on uhpT
transcription (26, 27), suggesting that CAP action may be
through changes in DNA geometry rather than protein contacts.
Although RNAP did not produce a DNase footprint at the uhpT
promoter, the requirement for all three proteins, i.e., RNAP, UhpA, and
CAP, for maximal formation of the DNase-hypersensitive sites at
positions 74 and 80 indicates that RNAP binds to the promoter along
with UhpA and CAP. The DNA distortion leading to DNase hypersensitivity
could result from the binding of CTD to this region or from the
production there of a sharp bend resulting from DNA looping that
requires all three proteins. Because formation of the hypersensitive
sites required CTD but not upstream DNA, we favor the view that
CTD binds between the CAP and UhpA sites. Future experiments using
affinity-directed DNA cleavage might provide evidence for this hypothesis.
The weak binding of RNAP may reflect the weak binding of UhpA to its target sites. It is likely that the weak yet specific binding of UhpA to the DNA is a general adaptation designed to allow genome-bound transcription factors to maintain communication with their cognate membrane-bound sensor kinase. Frequent dissociation of UhpA from the DNA can allow adjustment of its level of phosphorylation to reflect the current state of occupancy of the receptor for Glu6P and hence to allow efficient regulation.
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ACKNOWLEDGMENTS |
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We are indebted to Carol Gross, Paolo Landini, Tamas Gaal, and Rick Gourse for providing the strains and advice that made this work possible. John Dahl contributed to initial stages of this work. We are especially indebted to the anonymous reviewers of the manuscript who insisted on more extensive analysis of the involvement of RpoD.
This work was supported by research grant GM38681 from the National Institute of General Medical Sciences.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Microbiology HSC#441, University of Virginia, Charlottesville, VA 22908. Phone: (804) 924 2532. Fax: (804) 982 1071. E-mail: rjk{at}virginia.edu.
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Journal of Bacteriology, December 1999, p. 7266-7273, Vol. 181, No. 23
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