From the Laboratory of Molecular Biology, NCI, National Institutes
of Health, Bethesda, MD 20892
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INTRODUCTION |
Escherichia coli RNA polymerase
(RNAP)1 is the sole enzyme
responsible for all transcription activity in the cell. The core RNAP,
consisting of
2
' subunits, is the basic machinery
capable of transcription elongation and termination at simple
terminators. There are many RNAP-associated proteins that modulate the
activity of RNAP during different stages of transcription (1). Examples of such RNAP-associated proteins are sigma factor (1-3), NusA (4, 5),
GreA, and GreB (6-10). The two largest subunits of RNAP,
and
',
are the homologs of eukaryotic RNAP (for review, see Ref. 11 and
references therein). In addition, GreA and GreB proteins are the
functional homologs of SII, an eukaryotic RNA Pol II-associated
protein. These homologies indicate that the basic structure and
function of RNAP and RNAP-associated proteins are conserved throughout
evolution.
Here we describe the identification of a novel E. coli
RNAP-associated protein named RapA. RapA is found to be a homolog of the SWI/SNF family of eukaryotic proteins (12, 13). Yeast swi and snf genes were genetically identified to
be important for transcription activation (14-17). Homologs of the
SWI/SNF family have been reported in Drosophila (18-21) and
humans (22-24). Purified eukaryotic SWI/SNF proteins and their
homologs are able to remodel the chromatin/nucleosome structure (21,
25, 26), which could explain their role in influencing transcription
activity at some genes. The human homologs of the SWI/SNF family
interact with the transcription machinery, the HIV-I integrase, and the
Epstein-Barr virus EBNA2 (23, 27-29). In addition, some members of the
SWI/SNF family are involved in DNA repair (30-34), and mutations in
these genes are implicated in human disease (27, 34). Bacterial ORFs
have been reported to be the homologs of the SWI/SNF family (35, 36),
however, none of the prokaryotic homolog products had been purified.
One of the unsolved questions regarding the SWI/SNF family of proteins
is whether they are associated with RNAP (13). Our results have shown
that RapA is an RNAP-associated protein, suggesting that association
with RNAP is a conserved feature of this class of proteins.
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EXPERIMENTAL PROCEDURES |
Purification of RNAP--
E. coli K12 cells (MG1655)
were grown on 4× LB in a 10-liter fermenter. Cells were harvested at
A600
8 in late log growth phase (yield
~200 g of wet cell paste). About 100 g of wet cell paste was
used per purification. RNAP was purified according to Hager et
al. (37), but with some modifications. A single-stranded DNA-agarose (Pharmacia Biotech Inc.) column was used instead of DNA-cellulose, and the step of gel filtration on Sephacryl S-300 was
omitted. The single-stranded DNA-agarose column (bed volume ~25 ml)
was pre-equilibrated with TGED buffer (0.01 M Tris-HCl, pH
7.9, 5% glycerol, 0.1 mM EDTA, 0.1 mM
dithiothreitol) containing 0.2 M NaCl, and sample was
loaded on the column at 1.5-2 ml/min. After loading, the column was
washed with 200 ml of TGED containing 0.2 M NaCl, and the
RNAP fraction was eluted with TGED containing 1.0 M NaCl at
1 ml/min. A shallow gradient (from 0.3 to 0.5 M NaCl in 400 ml of TGED buffer) was used to elute RNAP from the 10/10 preparative
Mono-Q column (Pharmacia) at 0.5 ml/min. Typically, the yield for core
RNAP was 16-20 mg, for holoenzyme was 10-16 mg, and for
holoenzyme-RapA was 2-4 mg. FPLC System (Pharmacia) was used for the
protein purification, and the purification procedures were carried out
at 4 °C.
Purification of RapA--
The combined RNAP-RapA fractions
(peak 3 from the Mono-Q 10/10 column) were buffer exchanged with Buffer
A (50 mM Hepes, 0.2 mM EDTA, 0.1 mM
dithiothreithol, pH 7.5) containing 1 M
(NH4)2SO4 using Centriprep 100 concentrators (Amicon). They were loaded on the phenyl Superose HR 5/5
column (Pharmacia), which was pre-equilibrated with the above buffer at
a flow rate of 0.5 ml/min. The column was washed with 10 ml of Buffer A
containing 1 M
(NH4)2SO4 and then subjected to
reverse linear gradient of
(NH4)2SO4 (1-0 M in 80 ml of Buffer A). Fractions of 4 ml were collected and analyzed on an
8% SDS-polyacrylamide gel stained with Coomassie Brilliant Blue R-250.
The fractions that contained the 110-kDa RapA protein were diluted with
2 volumes of TGED buffer and loaded directly on the Mono-Q 5/5 column
which was pre-equilibrated with TGED buffer containing 0.2 M NaCl. After pre-elution wash with 10 ml of TGED
containing 0.2 M NaCl, RapA was eluted with a linear
gradient of NaCl (0.3-0.5 M in 80 ml of TGED buffer).
Fractions of 4 ml were collected at a flow rate of 0.5 ml/min and
analyzed on an 8% SDS-polyacrylamide gel stained with Coomassie
Brilliant Blue R-250. The combined fractions containing pure RapA were
concentrated to 0.3-0.4 mg/ml and stored in TGED buffer containing
50% glycerol and 100 mM NaCl at
20 °C. Under these
conditions no loss of ATPase activity was detected for at least 1 year.
Reconstitution of RNAP·RapA Complex in Vitro--
Formation of
the RNAP·RapA complex was studied by gel-filtration of the protein
mixtures on a Superose 6 HR 10/30 column (Pharmacia). The purified
proteins were either premixed in 150 µl of TGED containing 0.1 M NaCl, or run separately. Following 15 min of incubation on ice, the sample was loaded on a Superose 6 HR 10/30 column pre-equilibrated with the above buffer. The column was run with the
same buffer at a flow rate of 0.5 ml/min, and fractions of 1.0 ml were
collected. Each fraction was concentrated using Microcon 10 concentrators (Amicon) at 4 °C, and about half of the sample in each
fraction was analyzed.
ATPase Assays--
ATPase assay is based on measuring the amount
of [
-32P]ADP released from [
-32P]ATP.
The reaction mixtures (10 µl) contained 40 mM Tris-HCl (pH 7.4), 40 mM NaCl, 4 mM MgCl2, 1 mM dithiothreitol, 50 µg/ml bovine serum albumin, 0.2 mM ATP and 1 µCi of [
-32P]ATP. When
indicated, poly(rA) (0.25 µg), poly(dA) (0.26 µg), or
poly(dA)·poly(dT) (0.26 µg) was included in the reaction mixture. The reactions were started by the addition of the purified RapA (0.2 µM) or RNAP holoenzyme (0.1 µM) or 2:1
(mol/mol) mixture of the purified RapA (0.2 µM) and RNAP
holoenzyme (0.1 µM). After 30 min at 37 °C, the
reactions were terminated by the addition of 1 µl of 10% SDS,
lyophilized, dissolved in 5 µl of methanol and spotted onto a
poly(ethylenimine)-cellulose plate (J. T. Baker), and
chromatography was carried out in 1 M LiCl, 1 M
formic acid. Plates were autoradiographed and were also scanned on a
PhosphorImager (Molecular Dynamics) to quantitate the amount of
[
-32P]ATP hydrolyzed. For determination of other
(d)NTPase2 activity, ATP was replaced with a
corresponding (d)NTP and [
-32P](d)NTP.
Binding of Polynucleotides to Purified RapA--
The binding of
polynucleotides to RapA was studied by fluorescent titrations using a
Luminescence Spectrometer LS 50B (Perkin-Elmer). Protein (tryptophan)
fluorescence of RapA (0.01 mg/ml in 120 µl) was measured in TGED
buffer containing 0.1 M NaCl and 10 mM
MgCl2. The reductions in the fluorescence intensity of the
sample (
ex = 295 nm) in the maximum of the emission
spectrum were monitored after the additions of increasing amounts of a
polynucleotide solution (for each addition, the sample was allowed to
equilibrate for 10 min, and all additions were made in a total
volume < 8 µl).
F/
Fmax
for each polynucleotide concentration was calculated, and the
dissociation constants were determined from the Scatchard plots.
Other Biochemical Techniques and Reagents--
RNAP
concentrations were determined by UV absorbance using the extinction
coefficient data of Lowe et al. (40). RNAP and RapA
concentrations were also determined using the Bradford method (48) with
bovine serum albumin as a standard. The purity of the RNAP and RapA
preparations was estimated from overloaded SDS-polyacrylamide gels
after staining with Coomassie Brilliant Blue R-250 or silver. 8%
SDS-polyacrylamide stacking gels were run using a Mini-PROTEAN II
Electrophoresis System (Bio-Rad). Amino acid sequencing of the
N-terminal region of RapA was done by automated Edman degradation and
phenylthiohydantoin-derivative analysis using Applied Biosystems model
476A protein sequencing system. For Western blot, electroelution was
performed using Fastblot semi-dry electroblotter (Biometra) and
Immobilon-P membranes (Millipore). The polyclonal antibodies to highly
pure core RNAP,
-70 subunit of RNAP, or the 110-kDa RapA protein
(>98% pure) were raised in rabbits by Berkeley Antibody Co., CA. The
protein bands were visualized using goat anti-rabbit peroxidase-conjugated secondary antibodies (Calbiochem) and ECL Western
blotting detection reagents (Amersham Life Science, Inc.). In
vitro transcription assays were essentially performed as described (49) except that 0.2 mM ATP, GTP, and CTP and 0.02 mM UTP, including ~5 µCi of [32-P]UTP,
were used in multiple round transcription assays. The data were
quantified with a PhosphorImager (Molecular Dynamics).
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RESULTS AND DISCUSSION |
A 110-kDa Protein Copurifies with RNAP--
Using a modified
procedure for the purification of the E. coli
DNA-dependent RNA polymerase (RNAP), we identified a
110-kDa protein that consistently copurified with RNAP (Fig.
1). In the final step of Mono-Q
chromatography, from which highly pure and active RNAP was obtained
(37), the fraction that contained the RNAP holoenzyme and the 110-kDa
protein eluted as peak 3 immediately following the core RNAP
(peak 1) and holoenzyme (peak 2) in a shallow
NaCl gradient (Fig. 1, B and
C).4 A protein of
about 110-kDa had been previously observed to copurify with RNAP
(38-40); however, it was believed to be a proteolytic fragment of the
' (40) or
subunit of RNAP (41). The 110-kDa protein in our
preparation does not react with RNAP-specific polyclonal antibodies
(Fig. 2), indicating that it is not
related to the known subunits of RNAP.

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Fig. 1.
A 110-kDa protein copurifies with E. coli RNAP. A, schematic for the modified procedure for
purification of E. coli RNAP. For detail see Experimental
Procedures. B, elution profile from the Mono-Q column,
demonstrating separation of the core RNAP (Peak 1), RNAP
holoenzyme (Peak 2), and a 1:1 complex of RNAP holoenzyme
with the 110-kDa protein, RapA (Peak 3), by a shallow NaCl
gradient. C, fractions from different steps of the
purification procedure were analyzed on an 8% SDS-polyacrylamide gel
and stained with Coomassie Brilliant Blue R-250. The positions for RNAP and RapA are
indicated. Lane 1, 6 µl of the protein standards;
lane 2, 5 µl of the E. coli crude extract;
lane 3, 10 µl of the 1 M NaCl eluate from
Polymin P precipitate; lane 4, 10 µl of a single-stranded DNA agarose 1 M NaCl eluate; lane 5, 15 µl of
the Mono-Q-purified core RNAP (Peak 1); lane 6,
15 µl of the Mono-Q-purified RNAP holoenzyme (Peak 2);
lane 7, 30 µl of the Mono-Q-purified fraction containing
the RNAP holoenzyme and the 110-kDa protein, RapA (Peak 3).
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Fig. 2.
The 110-kDa protein is not related to the
known subunits of RNAP. , / ', -70 subunits of RNAP and
the 110-kDa protein were isolated separately from the Coomassie-stained
SDS-polyacrylamide gel (by passive elution with 1% SDS, 1% Triton
X-100, 6 M urea, 50 mM Tris-HCl, pH 7.4, for
2 h at 50 °C from homogenized gel slices) and loaded repeatedly
on separate tracks of two identical 8% SDS-polyacrylamide gels,
subjected to either silver staining (top) or immunoblotting
with antibodies against both core RNAP and -70 (bottom).
The 110-kDa position is indicated by an arrow. Lane
1, 2 µl of the protein standards; lane 2, 1 µg of
the Mono-Q-purified RNAP holoenzyme (Peak 2); lane
3, 1 µg of the Mono-Q-purified fraction containing the RNAP
holoenzyme and the 110-kDa RapA protein (Peak 3); lane
4, 0.2 µg of the purified ; lane 5, 0.2 µg of the purified , '; lane 6, 0.1 µg of the purified
-70 subunit of RNAP; lane 7, 0.1 µg of the purified
110-kDa RapA protein.
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The 110-kDa protein appears to be an integral component of RNAP because
(i) it is associated with RNAP in nearly equimolar amounts, as
estimated by quantitation of the Coomassie-stained protein bands on 8%
SDS-polyacrylamide gels; (ii) there is a significant amount of the RNAP
holoenzyme (20-30% in six independent protein purification
procedures) associated with the 110-kDa protein; and (iii) almost all
of the 110-kDa protein (>90%) from the cell is associated with RNAP,
judging from the amount of this protein present during different stages
of RNAP preparation, determined by immunoblotting using the 110-kDa
protein-specific polyclonal antibodies (Fig.
3). Our results indicate that this
110-kDa protein is a novel RNAP-associated
protein, and we name it RapA. RNAP containing the RapA
protein is called RNAP·RapA hereafter.

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Fig. 3.
The majority of the 110-kDa RapA protein in
the cell co-purifies with RNAP. The fractions corresponding to
different steps of the RNAP purification procedure (see Fig. 1) were
immunostained with the 110-kDa RapA protein-specific polyclonal
antibodies. Lane 1, 50 ng of the purified 110-kDa RapA
protein; lane 2, 10 µl of the E. coli crude
extract; lane 3, 10 µl of the supernatant after Polymin P
precipitation; lane 4, 10 µl of the 0.5 M NaCl wash of the Polymin P precipitate; lane 5, 10 µl of the
1.0 M NaCl wash of the Polymin P precipitate; lane
6, Polymin P pellet after 1.0 M NaCl wash; lane
7, 10 µl of the single-stranded DNA agarose flow-thru;
lane 8, 10 µl of the single-stranded DNA agarose 1 M NaCl eluate; lane 9, 100 ng of the purified
110-kDa RapA protein. RNAP-enriched fractions were in lanes 5 and 8 (see Fig. 1C). The band which migrated
below the RapA protein (lanes 2 and 3) is of
uncharacterized origin.
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Purification of the 110-kDa RapA Protein--
To study the
biochemical properties of this new RapA protein, we separated the
110-kDa RapA protein from RNAP and purified it to homogeneity in two
steps (Figs. 1A and 4). The
fractions containing RNAP holoenzyme and the 110-kDa RapA protein (Fig. 1, Mono-Q-1, Peak 3) were first subjected to hydrophobic
interaction chromatography on phenyl Superose HR column (Fig. 4,
A and B). The second peak eluted from the column
was the 110-kDa RapA protein. Another chromatography on a Mono-Q column
followed (Fig. 4, C and D), and the highly pure
RapA protein was eluted as the first peak. In four repetitions of this
purification procedure, about 100-250 µg of highly pure (>98%)
RapA was obtained from 2-4 mg of the starting material. To obtain
homogeneous RapA, the remaining contaminants (mostly RNAP) were removed
by repeating the last purification step.

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Fig. 4.
Purification of RapA. The detailed
procedure was described under "Experimental Procedures."
A, elution profile from the phenyl Superose HR 5/5 column
demonstrating separation of the 110-kDa RapA protein from the RNAP
holoenzyme. B, fractions from the phenyl Superose HR 5/5
column. An aliquot corresponding to 100 µl from each fraction was
loaded per track of an 8% SDS-gel stained with Coomassie Brilliant
Blue R-250. Lane 1, 10 µl of the protein standards
(Bio-Rad, broad range); lanes 2 and 3, combined RNAP·RapA fractions (Peak 3 from the Mono-Q 10/10 column in Fig. 1);
lanes 4-10, fractions 7, 9, 11, 13, 15, 17, and 19 from the phenyl Superose HR 5/5 column. C, elution profile from the
Mono-Q 5/5 column (Mono-Q-2), the final step of the RapA purification procedure. D, fractions from the Mono-Q 5/5 column
(Mono-Q-2) analyzed on an 8% SDS-gel stained with Coomassie Brilliant
Blue R-250. An aliquot corresponding to 80 µl from each fraction was loaded per track of an 8% SDSgel. Lane 1, 10 µl of
the protein standards (Bio-Rad, broad range); lanes 2-8,
fractions 5, 7, 9, 11, 13, 15, and 17 from the Mono-Q 5/5 column
(Mono Q-2).
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Reconstitution of the RNAP·RapA Complex--
We found that a
stable RNAP·RapA complex can be reconstituted with highly purified
110-kDa RapA protein and RNAP holoenzyme in vitro. When the
110-kDa RapA protein and the RNAP holoenzyme were mixed and passed
through a Superose 6 HR column (Pharmacia), they coeluted as a complex
(Fig. 5A). In control
experiments, when these two proteins were applied separately to the
column, RNAP and the 110-kDa RapA protein eluted at different fractions in gel filtration (Fig. 5, B and C). These
results demonstrated that the 110-kDa RapA protein forms stable complex
with RNAP, like a subunit of RNAP.

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Fig. 5.
Reconstitution of a stable complex of
purified RNAP and RapA. The stability of the RNAP·RapA complex
was studied by gel filtration of the protein mixtures on a Superose 6 HR 10/30 column as described under "Experimental Procedures." 8%
SDS-polyacrylamide gels were stained with Coomassie Brilliant Blue
R-250. The positions for RNAP and RapA are indicated. A, 2.5 µM RNAP holoenzyme + 0.6 µM RapA.
B, 2.5 µM RNAP holoenzyme. C, 0.6 µM RapA.
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The RapA Protein Is a Homolog of the SWI/SNF Family of Eukaryotic
Proteins--
Determination of the N-terminal sequence of the 110-kDa
RapA protein showed that the first 10 N-terminal amino acid residues are: Pro-Phe-Thr-Leu-Gly-Gln-Arg-Trp-Ile-Val, a sequence whose only
match is the corresponding region of the hepA ORF, located at 1.3 min on the E. coli linkage map. The E. coli
hepA ORF (42, 43) is a putative ATP-dependent helicase
(35, 36, 42) with an expected molecular weight of 110-kDa. This
hepA ORF is also homologous to the yeast SWI2/SNF2 protein
(36). We concluded that the hepA ORF encodes the 110-kDa
RapA protein. Unlike eukaryotic SWI/SNF members, which form complexes
with multiple polypeptides, this bacterial homolog RapA protein appears
to be the only polypeptide that associate with RNAP during our
purification procedure. Interestingly, yeast SWI/SNF complex is
reported to be associated with RNA Pol II (44), although a
contradictory result is also reported (26). Our finding that the RapA
protein is an RNAP-associated protein suggests that association with
RNAP is a common feature of this class of proteins.
RapA Is an ATPase and Its ATPase Activity Is Stimulated by
RNAP--
We found that RapA was capable of ATP hydrolysis (Fig.
6). The ATPase activity of RapA was not
affected by the presence of single-stranded DNA (lane 3),
RNA (lane 4), or double-stranded DNA (lane
5).

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Fig. 6.
RapA is an ATPase and its ATPase is
stimulated by RNAP. ATPase activity was determined as described
under "Experimental Procedures," and an autoradiograph of a
representative ATPase assay is shown here. The positions for substrate
ATP, the product ADP and RNA transcripts (poly(rA)) are indicated.
Lane 1, no enzyme; lanes 2-5, RapA;
lanes 6-9, RNAP; and lanes 10-13, RNAP·RapA. Lanes 2, 6, and 10, enzymes only; lanes 3, 7, and 11, poly(dA) were included; lanes 4, 8, and 12, poly(rA) were included; and lanes 5, 9, and 13, poly(dA)·poly(dT) were included.
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The ATPase activity of RapA was stimulated by the presence of RNAP
(Fig. 6, lanes 10-13), which by itself did not hydrolyze ATP or did so very poorly (lanes 6-9). Thus, RNAP·RapA
functions as an RNAP ATPase, a novel activity associated with RNAP. In
the presence of poly(dA)·poly(dT), only RNAP·RapA exhibited RNA
synthetic and ATP hydrolytic activities simultaneously (compare
lane 13 to lanes 5 and 9), with the
former being the predominant activity. The apparent
kcat of ATP hydrolysis by RNAP·RapA increased
more than 4-fold, while the Km for ATP changed very
little (Table I). The stimulation of
ATPase of other SWI/SNF members by RNAP has not been reported. The
specific activities of the ATPases of RapA and RNAP·RapA were
approximately 30 and 120 pmol of ADP released/min/microgram of RapA,
respectively (Table II), values close to
the yeast SWI/SNF ATPase (45).
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Table I
The stimulation of (d)ATPase activity of RapA by RNAP
The stimulation of (d)ATPase activity of RapA by RNAP is due to an
increase in kcat. For determining
Km and kcat of (d)ATPase, initial
velocities were measured at varying (d)ATP concentrations. The
reciprocal of the initial velocities was plotted as a function of the
reciprocal of (d)ATP concentrations to obtain the kinetic parameters.
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We believe that the RapA protein is responsible for the ATPase activity
for the following two reasons. 1) A homogeneous RapA protein that
contained no detectable impurities by Coomassie Blue R-250 or silver
stainings exhibited ATPase activity; and 2) The ATPase activity of RapA
was totally eliminated when the RapA protein was depleted in the
reactions by anti-RapA antibodies complexed with the protein A-agarose
(but not by protein A-agarose alone or by control antibodies, such as
anti-
32 antibodies complexed with the protein A-agarose;
data not shown).
RapA or RNAP·RapA was also able to hydrolyze dATP (Table II). The
kcat for dATP hydrolysis was 30-fold higher than
that of ATP (Table I). However, the Km for dATP was
more than 8-fold higher than the Km for ATP,
indicating that the affinity of RapA for dATP is much lower than that
for ATP. The hydrolysis of dATP was enhanced by RNAP due to an increase
in kcat (Table I). Because the concentration of
ATP in the cell is about 10-fold higher than that of dATP (46), it is
likely that RNAP·RapA will use both ATP and dATP with similar
efficiency. RapA or RNAP·RapA hydrolyzed other NTPs and dNTPs very
poorly (Table II), a property similar to yeast RSC, another member of the SWI/SNF family (26).
RapA Is a Polynucleotide-binding Protein--
RapA binds to
single-stranded DNA, RNA, partial DNA-DNA, and DNA-RNA duplexes (Table
III). The Kd values of
the complexes of RapA with different nucleic acids were similar,
ranging from 15 to 30 µM with a relatively better binding
to partial DNA-DNA and DNA-RNA duplexes. At present, we do not know
whether the polynucleotide-binding activity of RapA is altered when it
forms a complex with RNAP. Nucleic acid-binding activity has been
reported for other members of the SWI/SNF family (26, 47).
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Table III
Dissociation constants of the RapA-polynucleotide complexes
The polynucleotide binding was studied by quenching of the protein
(tryptophan) fluorescence as described under "Experimental Procedures."
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RapA Is Not a Helicase--
Although RapA has putative helicase
motifs, highly pure RapA showed no detectable helicase activity by
extensive helicase assays using several different substrates. However,
when RapA preparations that contained even trace amounts of RNAP
(<1%) were used, an apparent helicase activity was observed. RNAP
alone was capable of unwinding model partial duplex DNA substrates used in helicase assays, and this activity was not enhanced by RapA (data
not shown). So far, none of the members of the SWI/SNF2 family have
shown helicase activity.
In summary, we have identified a new RNAP-associated protein, RapA, in
E. coli. This bacterial homolog of the SWI/SNF family interacts with RNAP both physically (binds to RNAP) and functionally (the ATPase of RapA is stimulated by RNAP). However, the effect of RapA
on transcription using naked DNA templates (either linear or
supercoiled) in vitro is not dramatic. Fig.
7 illustrates a representative of such
experiments showing at best moderate activation in overall
transcription activity by RNAP·RapA when compared with RNAP itself.
This moderate activation of transcription (~150 to 200%) by RapA is
independent of several promoters used, and the RapA protein has no
effect on the rate of elongation and on the efficiency of termination
at both
-independent and -dependent terminators (data
not shown). Possibly, some factors are missing in our in
vitro transcription system. At present, we do not know the role of
RapA or RNAP·RapA in the cell. Apparently, the E. coli
cell that lacks a functional rapA gene is viable and able to
utilize different sugars as carbon sources.3
Currently, we are studying the expression of rapA and the
role of rapA under different conditions in the cell. Further
study of this RapA protein and its gene is necessary to understand its function in E. coli.

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Fig. 7.
RapA moderately activates transcription at
the pyrBI promoter. In vitro transcription
assays were performed using naked DNA templates containing the
pyrBI promoter as described under "Experimental
Procedures." Multiple experiments (>4) were repeated, and the data
were analyzed and presented. The transcription activities of
RNAP·RapA were normalized to that of RNAP only.
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We thank Sankar Adhya, Richard Burgess,
Susan Garges, Susan Gottesman, Howard Nash, Nancy Nossal,
Toshio Tsukiyama, Sue Wickner, Carl Wu, and Yan Ning Zhou for comments
on the manuscript. We thank Huarong Ying and Michael Maurizi for
performing amino acid sequencing of the RapA protein.