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INTRODUCTION |
CooA is a CO-sensing transcription activator from
Rhodospirillum rubrum, and it activates the expression of
the cooFSCTJ and cooMKLXUH operons in response to
CO (1-3). These two adjacent operons encode proteins that oxidize CO
to CO2 with concomitant reduction of H+ to
H2 and allow growth of this organism on CO as a sole energy source (4-6). cooA lies immediately 3' of the
cooFSCTJ operon, and its expression does not depend on CO.
cooA insertion and deletion mutants fail to accumulate
mRNA from either the cooFSCTJ or cooMKLXUH
operons in response to CO (1, 2). Sequence analysis suggested that CooA
is a homolog of cAMP receptor protein
(CRP),1 a well characterized
transcription factor in Escherichia coli (1). The proposed
helix-turn-helix DNA binding motif of CooA is highly similar to that of
CRP, whereas the effector-binding domain displays lower similarity with
CRP. CooA is not abundant in R. rubrum, but overexpression
and purification of CooA were achieved in both R. rubrum and
E. coli. CooA purified anaerobically from R. rubrum was indistinguishable spectroscopically or in terms of
activity from CooA purified aerobically from either R. rubrum or E. coli (7, 8). CooA is homodimeric under all
conditions and contains approximately 2 mol of protoheme/dimer. CO
directly binds to the heme of the reduced form of CooA, but not to the oxidized form, and this CO binding induces a conformational change in
CooA that allows it to bind DNA in a sequence-specific manner (8).
Oxidized CooA can be readily reduced, acquiring the ability to bind CO,
and the mechanism for this reversible redox reaction involves an
unusual switch of protein ligands on one side of the heme (8, 9).
Because the heme of CooA is 6-coordinate when reduced and when bound by
CO, CO binding apparently displaces one of the axial ligands to the
heme. It is our working hypothesis that this protein ligand
displacement serves as the trigger for the conformational change in the
CooA structure upon CO binding (8, 9).
E. coli RNAP is composed of
2
'
subunits; the role of
is to bind
10 and
35 promoter elements.
At promoters with A + T-rich sequences upstream of the
35 hexamer,
termed "UP elements," the C-terminal domain of the
subunit
(
-CTD) makes specific DNA contacts that enhance transcription
initiation (13). Two regions of the
-CTD (including residues
Leu262, Arg265, Asn268,
Cys269, Gly296, Lys298, and
Ser299) were found to be important for UP
element-dependent transcription and DNA binding (14, 15).
The
-CTD is an independently folding domain of RNAP that is joined
to the N-terminal domain of the
subunit by a flexible linker that
allows the
-CTD to occupy different positions at different promoters
(10-12). The
-CTD is also known to make specific contacts with a
range of activator proteins (16, 17).
E. coli CRP controls the transcription of numerous genes
involved in carbon source utilization. Upon the binding of its
allosteric effector, cAMP, CRP undergoes a conformational change that
allows a dimer of CRP to bind to a specific 22-base pair sequence at target promoters (consensus sequence is
5'-AAATGTGATCTAGATCACATTT-3', in which the most
important bases for CRP recognition are in bold) and to activate
transcription at those promoters (18). CRP-dependent promoters can be grouped into two classes based on the position of the
CRP-binding site relative to the start of transcription as well as on
the mechanism for transcription activation (19). At class I promoters,
the DNA-binding site for CRP is upstream of that for RNAP and is
centered at position
61.5,
71.5,
82.5, or
92.5. At class II
CRP-dependent promoters, to which the
CooA-dependent promoters are analogous, the binding site
for CRP is centered at
41.5, overlapping the
35 region, and the
-CTD binds to DNA upstream of the CRP dimer.
Direct interaction between CRP and RNAP plays a pivotal role in
transcription activation at both promoter classes (20, 21). In
particular, transcription activation at class II promoters requires two
distinct contacts between CRP and the
subunit and a third contact
between CRP and
70. One interaction is between
activating region 1 (AR1) of the upstream subunit of the CRP dimer and
the
-CTD. This interaction increases initial binding of RNAP to the
promoter (22). Recently, residues 285-288 and 317 of
-CTD have been
shown to comprise the surface that interacts with AR1 of CRP at class
II promoters (23). The second contact, between activating region 2 (AR2) of the downstream subunit of CRP and the N-terminal domain of the
subunit, facilitates isomerization of the closed complex to the
open complex (24). The residues in AR1 and AR2 of CRP are not conserved
in CooA, which suggests that there might be certain differences in the
interactions of CooA and CRP with
. The third activator region (AR3)
in CRP, formed by residues 52-58, interacts with
70 of
RNAP (20). Because the AR3 region in CRP is highly similar to an
analogous region in CooA, this region in CooA might serve an AR3-like function.
The two CooA-regulated R. rubrum promoters, PcooF
and PcooM, contain 2-fold symmetric DNA sequences that serve as
CooA-binding sites and are similar to the CRP consensus sequence. This
is consistent with the similarity between CooA and CRP in their
helix-turn-helix motifs (2, 3). The CooA-binding sites lie at the
43.5 and
38.5 positions relative to the transcription start sites
in PcooF and PcooM, respectively, overlapping with the
35 region (2, 3). This overlap suggests that both CooA-regulated
promoters are analogous to class II CRP-dependent promoters. We chose PcooF for this study because it is the
stronger promoter based on the amount of primer extension product and
level of coo-encoded proteins synthesized in vivo (1, 2).
Although CooA shares some common features with CRP, such as DNA binding
properties and effector-induced activation, it displays striking
differences from CRP in the effector-binding domain and in regions AR1
and AR2. We were interested to know whether CooA was necessary and
sufficient for CO-dependent activation of transcription and
whether the mechanism of activation by CooA was similar to that of CRP.
In this work, we used in vivo CooA reporter systems and
in vitro transcription assays to examine the properties of CooA in transcription activation. Because of the particular questions we wished to address, we performed the bulk of the work with RNAP from
E. coli, so that any differences between CooA and the CRP detected would reflect properties of CooA. The nature of transcription activation by CooA was investigated through the study of the
interaction between CooA and RNAP and, in particular, the interaction
between CooA and the
-CTD.
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EXPERIMENTAL PROCEDURES |
Construction of a System for CooA Expression and a Reporter of
CooA Activity in E. coli--
CooA was overexpressed from a vector,
pYHA1, created as follows. A 1.9-kilobase
PvuI-BamHI fragment containing
Ptac-cooA-rrnBT1T2, was isolated from pKK223-3 (8), digested with PvuI, mung
bean nuclease, and BamHI, and cloned into the
EcoRV and BamHI sites of pACYC184. A plasmid
containing a PcooF-lacZ fusion, pYHF4, was
constructed by inserting a polymerase chain reaction-amplified EcoRI-HindIII fragment, extending from positions
250 to +70 of PcooF, into plasmid pMSB1 (25), which created
pMSBPcooF. The reporter region was recombined from plasmid
pMSBPcooF into
phage RS468 (26) in strain DH5
containing
pYHA1. Lysogens were screened by the blue color of plaques on Luria
broth (LB) + X-gal plates incubated anaerobically in the presence of
CO. The promoter region of integrated PcooF-lacZ
fusion in the chromosome was confirmed by DNA sequencing.
In Vitro Transcription Assays--
Polymerase chain
reaction-amplified EcoRI-HindIII fragments from
positions
250 to +70,
90 to +70, and
60 to +70 of PcooF were cloned into pRLG770, which contains transcription terminator rrnBT1T2 downstream of the
multicloning site (27), to yield plasmids pYHF1, pYHF2, and pYHF3,
respectively. The supercoiled plasmids used as DNA templates for these
assays were purified with the Midi Kit from Qiagen. The RNAPs used in
these experiments were E
70 purified from E. coli or a Rhodobacter sphaeroides RNAP preparation enriched for the E
70 homolog (28). Standard
multiple-round transcription assays (13) were modified as described
below to accommodate the requirement of CooA for an anoxic environment
to bind CO (7). The sealed tubes containing 25-µl reactions (0.2 nM supercoiled plasmid, 3.5 nM RNAP, 40 nM CooA dimer, and a buffer (30 mM KCl, 40 mM Tris acetate, pH 7.9, 10 mM
MgCl2, 1 mM dithiothreitol, 100 mg/ml bovine
serum albumin, 200 mM ATP, 200 mM CTP, 200 mM GTP)) were degassed and filled with argon in the head
space. After the addition of dithionite to 1.7 mM to
scavenge any free oxygen, CO was added, and the reactions were
incubated at room temperature for 15 min. This step served to activate
CooA and allow the activated CooA to interact with the promoter and
RNAP to form a predicted 21-nucleotide transcript by incorporating ATP,
GTP, and CTP. The reactions were then exposed to air, and 10 mM UTP and 5 µCi of [32P]UTP (DuPont) were
added to extend the mRNA at room temperature for 20 min. Reactions
were terminated and electrophoresed as described (29). The signal
intensities of transcripts were quantified using a PhosphorImager
(Molecular Dynamics) and ImageQuant software.
DNase I Footprinting Assays--
A DNA fragment containing the
PcooF sequence from position
90 to +70 was polymerase chain
reaction-amplified with an unlabeled bottom strand primer and a top
strand primer labeled with [
-32P]ATP and
polynucleotide kinase. The amplified fragment was purified by
polyacrylamide gel electrophoresis, followed by an Elutip Minicolumn (Schleicher & Schuell). The labeled fragment was incubated with CooA,
or RNAP, or both in the presence of CO and under the stringent anoxic
conditions described previously (2), except that 40 nM pure
CooA and 3.5 nM RNAP were used in a 20-µl binding
reaction. The reactions were treated with 2 units/ml RQ RNase-free
DNase I (Promega) for 30 s. DNase I cleavage products were
separated on a 6% (w/v) polyacrylamide-urea gel. Neither heparin nor
any nucleotides were added into the reactions.
CO Induction of PcooF Expression in E. coli and
Measurement of
-Galactosidase Activity in Vivo--
Strains with a
PcooF-lacZ reporter were grown aerobically in LB
medium containing 100 mg/ml ampicillin and 35 mg/ml chloramphenicol for
12 h to reach stationary phase. In stoppered test tubes, 20-µl
cultures were diluted into 2 ml of LB medium supplemented with 20 mM glucose and the same antibiotics. The air in the head
space was replaced by argon and 2% CO, and the cultures were grown
anaerobically to an A600 of approximately 0.45.
-Galactosidase activity was determined according to Miller (30).
Protein Purification and Reconstitution of RNAP--
N-terminal
His-tagged wild-type and mutant
subunits were overexpressed from
plasmid pHTT7f1-NH
(31) or derivatives constructed by gene
replacement of the EcoRI-BamHI fragment with
fragments encoding the desired alanine substitutions (14). Purification of
subunits by Ni2+ affinity chromatography was
performed as described in Tang et al. (31). Preparation of
inclusion bodies of
,
', and
70 from strains
XL1-Blue (pMKSe2), BL21 DE3 (pT7
'), and BL21 DE3 (pLHN12
),
respectively, and reconstitution of RNAP were carried out as described
(31). CooA was purified from an overproducing strain of R. rubrum (UQ459) by the method of Shelver et al. (7).
 |
RESULTS |
CooA Is Necessary for CO-dependent Expression in
Different Organisms--
CooA has been shown to be necessary for the
CO-dependent expression of the two coo operons
of R. rubrum (1, 2), and purified CooA binds DNA in a
CO-dependent manner (7). These data are consistent with the
hypothesis that CooA is both necessary and sufficient for sensing and
activating transcription in response to CO, but they are not
conclusive. We therefore examined the requirement for CooA in two
heterologous systems. We chose R. sphaeroides because it is
related to R. rubrum, yet does not appear to have the
coo system, as judged by the absence of CO dehydrogenase activity and a failure to hybridize to probes of the coo
genes (2). In this organism, a pRK404-based plasmid carrying
cooFSCTJ with its normal promoter failed to produce
detectable CO dehydrogenase activity (the product of cooS)
in response to CO. However, when cooA was added to the
plasmid in its normal position at the 3' end of the cooFSCTJ
operon, exposure of the R. sphaeroides strain carrying this
plasmid to CO produced easily detectable CO dehydrogenase activity
(2).
We then examined CO- and CooA-dependent transcription in
E. coli, an organism that is less related to R. rubrum and also lacks any evidence of a coo system. For
this test, a reporter system was constructed that contained a
PcooF-lacZ fusion in the chromosome and a plasmid
overexpressing CooA. In this system, we detected a substantial increase
of
-galactosidase activity upon CO induction (200 Miller units in
the presence of CO and 1.6 units in its absence), suggesting that CooA
is sufficient for activating the transcription of PcooF in
E. coli. Similar results with CooA reporters in E. coli have recently been reported by others (9). These results
establish that CooA is necessary for the transcriptional response to CO
and that it is able to associate productively in vivo with
RNAPs from both E. coli and R. sphaeroides.
CooA Is Sufficient to Activate the Transcription of
PcooF in Vitro--
The above results indicate that CooA
is necessary, but only in vitro analysis can establish
whether it is sufficient for CO-dependent transcriptional
activation or whether additional factors are required for this
activation. The ability of CooA to activate PcooF was studied
by monitoring RNA synthesis in a purified system containing only DNA,
RNAP, CooA, nucleotide triphosphates, and the proper buffer. To
investigate the nature of CooA-mediated activation at the PcooF
promoter, we modified the standard in vitro transcription
assay, because CooA is only able to bind CO when reduced. The modified
assay, detailed under "Experimental Procedures," was kept anoxic in
the presence of CO to support activation by CooA until the formation of
a 21-nucleotide transcript from PcooF. At this point, the
reaction was exposed to air and extended aerobically for technical
convenience. When the reactions were maintained strictly anoxically
throughout the entire experiment, a quantitatively similar result was
obtained, indicating that the in vitro transcription assay
conditions we employed were sufficient for maximal CooA activity (data
not shown).
The in vitro transcription assays were performed with a
supercoiled DNA template (pYHF1) containing the PcooF and extending
250 base pairs upstream and +70 base pairs downstream of
transcription start site. The reactions were carried out in the
presence or absence of CO. As shown in lanes 2 and
4 of Fig. 1,
CO-dependent transcripts were detected using RNAP from both R. sphaeroides and E. coli. The observed size of
the transcript from PcooF correlated well with the predicted
size of 240 nucleotides. In the absence of CO, no transcripts from
PcooF were seen, whereas transcription of the control (RNA-1)
was not affected by CO (Fig. 1, lanes 1 and
3).

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Fig. 1.
In vitro transcription of CooA
with wild-type or -CTD-truncated RNAPs. A
supercoiled DNA template containing PcooF sequences from 250
to +70 of the transcription start site was used in this assay. The
RNAPs used in each pair of reactions are wild-type (WT) RNAP
from E. coli and R. sphaeroides (R. s)
and the mutant E. coli RNAP with a truncation of the C
terminus of starting at residue 235 ( -235). The + and on
the line labeled "CO" refer to the presence or the absence of CO in
the headspace of the reaction tubes. The positions of transcripts
initiated at the PcooF and the RNA-1 promoter are indicated by
arrows.
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These results demonstrate that CooA is sufficient for
CO-dependent transcriptional activation and that no other
factors are required. The results with the E
70 from
E. coli indicate that PcooF can be recognized by
70 when activated by CooA.
CooA-activated Transcription Requires the
-CTD of
RNAP--
Because the
-CTD makes specific DNA contacts at some
promoters and specific protein contacts with a number of transcription activators including the CooA homolog CRP (13, 20), we wished to test
whether there were similar contacts between
-CTD and either CooA or
PcooF. We first examined whether the C-terminal domain of the
RNAP
subunit is required for transcription activation of
PcooF by CooA. To address this question, we assayed the ability
of a reconstituted E
70 containing a truncation of the
C-terminal domain of
to direct transcription from
CooA-dependent promoter PcooF in vitro. Use of E. coli RNAP with the
-CTD truncation resulted in
a dramatic reduction of transcription from PcooF (Fig. 1,
lanes 5 and 6). The enzyme activities of
wild-type and mutant RNAPs were similar as demonstrated by
transcription from the RNA-1 promoter. With a longer exposure of the
x-ray film, we were able to detect a low level of a
CO-dependent transcript from PcooF using RNAP with
-235 (data not shown). Because the
-CTD is known to make contacts
with activators and UP elements, the ineffectiveness of the
-CTD-truncated RNAP for PcooF transcription suggests that
the
truncation disrupts the binding of
to CooA, to a UP
element, or to both.
CooA Utilizes Protein-Protein Contacts with
-CTD to Facilitate
RNAP Binding to PcooF--
To analyze potential
protein-protein interaction between CooA and RNAP and to specifically
address interactions between
-CTD and CooA, we performed DNase I
footprinting experiments using wild-type RNAP or the
-CTD-truncated
RNAP in the presence or absence of CooA. As shown in Fig.
2, lanes 2-4, CooA alone
protected PcooF on the top strand from positions
55 to
31
relative to the start site of transcription, in agreement with our
previous observations (2, 7). In contrast, neither the wild-type nor
the
-CTD-truncated RNAPs alone protected PcooF from DNase I
(lanes 5 and 7). This result indicates that RNAP
does not form a stable complex at PcooF in the absence of CooA.
When both CooA and wild-type RNAP were incubated together with the DNA
fragment, the protected region extended from the CooA-binding site in
both directions, upstream to
68 and downstream to at least
1
(lanes 8 and 9). A similar result was obtained when R. sphaeroides RNAP was used, indicating that the RNAPs
from these two different organisms function similarly in interacting with CooA at PcooF (lanes 10-12). In contrast to
the results obtained with the wild-type RNAP, the
-CTD-truncated E. coli RNAP showed no evidence of forming a stable complex
even in the presence of CooA (lane 6). This suggests that
CooA activates transcription by enhancing the initial and stable
binding of RNAP to PcooF through direct protein-protein contact
with the
-CTD.

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Fig. 2.
DNase I footprinting analysis of CooA
interacting with RNAP. A 3'-32P-end-labeled polymerase
chain reaction fragment containing PcooF from position 90 to
+70 was incubated with various concentrations of CooA and 3.5 nM RNAP in the presence of CO and under stringent anoxic
conditions. The filled bar on the right indicates
the region protected by CooA, whereas the open plus the
filled bar represents the region protected by wild-type
(WT) RNAP plus CooA. Lane 1, no protein;
lanes 2-4, CooA protein; lane 5, the RNAP
variant with the -CTD truncation ( -235); lane 6, CooA
plus the RNAP variant with the -CTD truncation; lane 7,
wild-type RNAP from E. coli; lanes 8 and
9, CooA plus wild-type RNAP from E. coli;
lane 10, wild-type RNAP from R. sphaeroides
(R. s); lanes 11 and 12, CooA plus
wild-type RNAP from R. sphaeroides.
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Analysis of CooA-regulated Promoter PcooF--
As
noted above, the presence of CooA and wild-type RNAP, but not the
-CTD-truncated variants, caused DNase I protection to extend
upstream and downstream of that region protected by CooA alone. The
region upstream of the CooA-binding site is A + T-rich (Fig.
3) relative to most of the R. rubrum genome, reminiscent of the A + T-richness of UP elements in
E. coli promoters that contact the
-CTD to increase
transcription (13, 38). To test whether
interacts with this region
in a sequence-specific manner, we created PcooF constructs
differing only in the extent of that region. PcooF sequences
from positions
90 to +70 and
60 to +70 were cloned into the
transcription assay vector pRLG770, resulting in the constructs pYHF2
and pYHF3, respectively. These plasmids were tested for
CooA-dependent in vitro transcription activity
with wild-type RNAP. The level of transcripts from those constructs was
quantitatively compared with that of the construct pYHF1 containing the
PcooF sequences from
250 to +70. As shown in Fig.
4, these three templates yielded similar
amounts of CooA-dependent transcripts, indicating that the
specific sequences upstream of position
60 in PcooF do not
contribute significantly to promoter activity. This also suggests that
the upstream A + T-rich sequence of PcooF does not act as a UP
element. Although we cannot exclude the possibility that some portion
of the protection upstream of the CooA-binding site is due to a
conformational change in CooA induced by the presence of RNAP, the
interaction of
-CTD with DNA upstream of CooA is consistent with the
sequence-nonspecific interactions between DNA and
-CTD
observed at class II CRP-dependent promoters
(23).

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Fig. 3.
Sequence of PcooF region. The
figure shows the upper strand sequence of PcooF. The
transcription start is at +1. The underlined sequences
represent the putative 10 region, the 2-fold symmetrical CooA-binding
site, and the A + T-rich sequence upstream of CooA-binding site as
marked, respectively. The region protected by CooA in DNase I
footprinting experiments is indicated by the filled bar. The
open bar represents the region protected by wild-type RNAP
plus CooA.
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Fig. 4.
In vitro transcription analysis of
PcooF deletions. The transcription reactions were
performed in the same manner as described in Fig. 1. All of the
reactions contained wild-type RNAP from E. coli, CooA, and
CO. The supercoiled DNA templates used in the assay differ only in the
extent of the upstream sequence of PcooF. Lane 1,
pYHF3 containing PcooF sequences from 60 to +70; lane
2, pYHF2 containing PcooF sequences from 90 to +70;
lane 3, pYHF1 containing PcooF sequences from 250
to +70. The positions of transcripts initiated at the PcooF and
the RNA-1 promoter are indicated by arrows.
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Identification of Residues in
-CTD That are Critical for
CooA-dependent Activation at PcooF--
We
wished to determine whether CooA made similar contacts with the
-CTD
as seen with its homolog, CRP, and also to test whether the extended
DNase I protection upstream of CooA reflected the direct contact of
-CTD with DNA. To address these questions, we measured
CooA-activated transcription using a CooA reporter system containing a
PcooF-lacZ fusion integrated into the E. coli chromosome, a plasmid expressing CooA, and a library of
plasmids encoding single alanine substitutions throughout the entire
-CTD (23). The effects of the
-CTD mutants in this screen were
small and somewhat variable (data not shown), but they did identify a
few potential mutants that were then assayed for their effects on
CooA-dependent transcription in vitro.
We tested reconstituted RNAPs in vitro containing single
alanine substitutions for Thr285, Val287,
Glu288, and Arg317 (the patch on
that interacts with CRP at class II CRP-dependent promoters (23)), Arg265 (the residue most important for
interaction of
with DNA (14)), and a few additional mutants
suggested by the in vivo screen, including
Val306, Leu307, and Ser313.
The results of the in vitro transcription analysis with a
subset of the variant RNAPs are shown in Fig.
5. The activity of each RNAP preparation
was normalized to the transcription from the RNA-1 promoter. The R265A,
L307A, and V287A RNAPs were defective in CooA-dependent
transcription of PcooF, providing 13, 34, and 37% of wild-type
RNAP activity, respectively (Fig. 5, lanes 2, 3,
and 7). Because R265A (14) and
L307A2 affected UP
element-dependent transcription and RNAP extended the DNase
I protection upstream of the CooA-binding site, this strongly suggested
that
-CTD makes contacts with DNA upstream of CooA and that these
contacts are important for CooA-meditated transcription activation.
V287A was also defective in CooA-dependent transcription,
although the other
-CTD variants important for class II
CRP-dependent transcription (T285A, E288A, and R317A) had
little or no effect. These results suggest that the CooA contact site
in the
-CTD of RNAP shares some determinants of, but is not
identical to, the site for CRP contact at class II-type promoters. RNAPs containing
mutants V306A and S313A, which were also suggested by the in vivo screen, were not defective in
CooA-dependent transcription in vitro (data
not shown).

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Fig. 5.
In vitro transcription analysis
of -CTD variants. The E. coli
RNAPs were reconstituted with either wild-type (WT) subunit or the subunits containing alanine substitutions at the
positions indicated in the figure. All of the reactions were carried
out in the presence of CooA, CO, and the supercoiled DNA template with
PcooF sequences from 250 to +70. The positions of transcripts
initiated at PcooF and the RNA-1 promoter are indicated by
arrows. Based on the quantitation of transcript
accumulations, R265A, L307A, and V287A show 13, 34, and 37% of
wild-type RNAP activity for PcooF, respectively. These
percentages represent the averages of three independent experiments in
which all values were within 18% of the average.
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|
 |
DISCUSSION |
Many transcription activators directly contact the
subunit of
RNAP (16, 17). In this study, we determined that CooA is sufficient for
directing RNAP to initiate transcription of PcooF and that
CooA-activated transcription of PcooF requires the C-terminal
domain of the RNAP
subunit. Consistent with these observations,
CooA facilitates the binding of wild-type RNAP to PcooF but not
that of
-CTD-truncated RNAP. These observations suggest that direct
protein-protein contact between CooA and the
-CTD of RNAP plays an
essential role in transcription activation of PcooF.
We suspect that the
-CTD/CooA interaction plays a role similar to
that between AR1 of CRP and
-CTD at class II
CRP-dependent promoters. In class II
CRP-dependent promoters,
-CTD makes nonspecific contacts
with the DNA segment immediately upstream of the CRP site (16, 23). In
this study, we found that wild-type RNAP, but not RNAP with the
-CTD
truncation, extends DNase I protection upstream of that protected by
CooA alone. Furthermore, we determined that
R265A and L307A, which
decrease UP element-dependent transcription, also affect
PcooF activity. Because the specific DNA sequence upstream of
the CooA-binding site is not critical for PcooF activity, we
propose that
-CTD makes nonspecific contacts with the DNA
immediately upstream of the CooA site and that this protein-DNA
interaction is important for CooA-activated transcription of
PcooF.
Val287 is also important for CooA-activated transcription,
although the other tested
residues that contact CRP at class II promoters are not critical for CooA-dependent transcription
of PcooF. Therefore, a different set of side chains, but probably the same region of
-CTD, might be important for activation by CRP and CooA. This hypothesis is consistent with the low
similarity between CRP and CooA in activating region 1 (22).
In the DNase I footprinting analysis, RNAP does not completely protect
the DNA downstream of PcooF +1, even in the presence of CooA
and in the absence of heparin (Fig. 2). In addition, the majority of
the CooA-RNAP-PcooF complexes detected in gel-shift assays are
heparin-sensitive (data not shown). This partial downstream protection
and the heparin sensitivity suggest that the detected ternary complex
(CooA-RNAP-PcooF) may be a mixed population of closed and open
complexes. Although most CRP-dependent promoters, such as
lacP1, melT, galP1, form stable open
complexes with RNAP in the presence of CRP (32-35), other promoters,
such as rrnB P1 even in the presence of its activator protein Fis (36), form an unstable open complex with RNAP (37).
Because basal level transcription of PcooF in the absence of
active CooA is not detectable, it is formally possible that the
deletion of
-CTD affects basal level transcription of PcooF
and not its activation by CooA. However, the requirement for at least
one residue in
-CTD (Val287) that has no effect on DNA
binding (23) suggests that the
-CTD requirement involves, at least
in part, a protein-protein interaction between CooA and
-CTD.