(Received for publication, March 17, 1997, and in revised form, June 4, 1997)
From the Department of Molecular Biology, Princeton University, Princeton, New Jersey 08544
Cell differentiation in the Caulobacter
crescentus cell cycle requires differential gene expression that
is regulated primarily at the transcriptional level. Until now,
however, a defined in vitro transcription system for the
biochemical study of developmentally regulated transcription factors
had not been available in this bacterium. We report here the
purification of C. crescentus RNA polymerase holoenzymes
and resolution of the core RNA polymerase from holoenzymes by
chromatography on single-stranded DNA cellulose. The three RNA
polymerase holoenzymes E54, E
32, and
E
73 were reconstituted exclusively from purified
C. crescentus core and sigma factors. Reconstituted
E
54 initiated transcription from the
54-dependent fljK promoter of
C. crescentus in the presence of the transcription
activator FlbD, and active E
32 specifically initiated
transcription from the
32-dependent promoter
of the C. crescentus heat-shock gene dnaK. For
reconstitution of the E
73 holoenzyme, we overexpressed
the C. crescentus rpoD gene in Escherichia coli
and purified the full-length
73 protein. The
reconstituted E
73 recognized the
70-dependent promoters of the E. coli
lacUV5 and neo genes, as well as the
73-dependent housekeeping promoters of the
C. crescentus pleC and rsaA genes. The ability
of the C. crescentus E
73 RNA polymerase to
recognize E. coli
70-dependent promoters is consistent
with relaxed promoter specificity of this holoenzyme previously
observed in vivo.
Caulobacter crescentus is a dimorphic, Gram-negative bacterium with a well defined cell cycle that generates two different daughter cells, a motile swarmer cell with a single polar flagellum and a nonmotile stalked cell. Formation of the new swarmer cell and its subsequent differentiation into a stalked cell result from a series of discrete morphogenic events, including flagellar biosynthesis, flagellum rotation, loss of motility, and stalk formation at one pole during the cell cycle (reviewed in Refs. 1 and 2). Early experiments demonstrated that this sequence of developmental events depends on de novo RNA synthesis (3) and suggested that differential gene transcription, presumably involving RNA polymerase and its accessory proteins, plays a central role in regulating the developmental program.
Bacterial RNA polymerases
(RNAP)1 are multi-subunit
enzyme complexes that can be purified as the core polymerase (E) and
the holoenzyme (E; reviewed in Refs. 4 and 5). The core RNAP, composed of the
2,
, and
subunits, carries out
RNA chain elongation, whereas the holoenzyme, which also contains the
sigma subunit (
), recognizes specific promoter sequences. Multiple sigma factors with unique promoter specificities have been identified in many eubacteria, and the use of alternative sigma subunits is a
fundamental mechanism for reprogramming RNAP specificity and
controlling complex patterns of gene transcription (reviewed in Refs.
6-8).
The most extensive study of transcription regulation in C. crescentus has been carried out on the genes in the flagellar gene hierarchy (reviewed in Ref. 9). Flagellum formation requires the
temporally controlled transcription of approximately 50 genes (10) that
are organized in a regulatory hierarchy containing four classes of
genes (I to IV). The Class II genes, which are expressed early in the
cell cycle and encode basal body and switch components, contain a
unique promoter consensus. Recent results have shown that transcription
from the Class II promoters is regulated in vivo by the
response regulator CtrA (11). The class II gene products are required,
in turn, for transcription of class III and class IV genes that are
transcribed from 54-dependent promoters late
in the cell cycle. The
54 factor and transcription
activator FlbD, which are encoded by Class II genes rpoN and
flbD, are required for transcription of the Class III and IV
genes (reviewed in Ref. 9).
Biochemical studies of transcription in vitro have employed
either the E54 holoenzyme reconstituted from purified
Escherichia coli components (12, 13), the heterologous
E
54 holoenzyme reconstituted from the C. crescentus
54 and the E. coli core RNAP
(14), or a partially purified C. crescentus RNAP (15). The
genes encoding three C. crescentus sigma factor subunits,
32 (rpoH; Refs. 16 and 17),
54
(rpoN; Refs. 14 and 18), and
73
(rpoD; Ref. 19) have been cloned and sequenced. Although
sigma factors
54 (14) and
32 (16) have
now been overexpressed in E. coli and purified, the lack of
a purified core RNAP has prevented the reconstitution of a
transcription system exclusively from purified C. crescentus components. In addition, the principal C. crescentus sigma
factor, which is required for transcription from the housekeeping
promoters (19) and predicted to contain 653 amino acids with a
molecular mass of 72,623 Da (
73; 20), had not been
isolated.
We report here the first purification and characterization of the
C. crescentus core RNAP, as well as the two holoenzymes E73 and E
32. We also describe the
purification of the C. crescentus principal sigma factor,
73, after overexpression of rpoD in E. coli. The E
73, E
54, and
E
32 holoenzymes have been reconstituted exclusively from
purified C. crescentus proteins, and the transcriptional
specificity of these RNAP preparations has been examined. The
availability of a defined, reconstituted transcription system will
allow detailed analysis of the roles of RNAP and accessory factors in
the transcriptional regulation of developmental genes during cell
differentiation and division in this bacterium.
E. coli
strain DH5 was used for propagating plasmids and cultured in ML
medium supplemented with ampicillin (100 µg/ml) or tetracycline (10 µg/ml) as necessary. C. crescentus wild-type strain CB15
(ATCC19089) was used for the purification of RNAP and grown in PYE
(peptone yeast extract; Ref. 21) medium at the fermentation facility of
the Waksman Institute. Restriction enzymes were purchased from either
New England Biolabs or Boehringer Mannheim. T4 DNA ligase and T4 DNA
polymerase were obtained from Boehringer Mannheim.
[
-32P]UTP was obtained from Amersham Corp.
Oligonucleotides were synthesized by the Princeton University SynSeq
facility. Heparin-agarose, single-stranded DNA-cellulose, and
DEAE-cellulose were purchased from Bio-Rad. Poly[d(A-T)] was
purchased from Sigma. E. coli core RNAP was purchased from
Epicentre Technologies.
Most procedures of the
purification are based on the methods described by Burgess and
Jendrisak (22). All steps were carried out at 4 °C unless noted
otherwise. A block of 100 g of frozen C. crescentus
cells were broken into small pieces and placed in a 1-liter Warring
Blender with 300 ml of grinding buffer (0.05 M Tris-HCl (pH
7.9), 5% (v/v) glycerol, 2 mM EDTA, 0.1 mM
dithiothreitol, 1 mM -mercaptoethanol, 0.233 M NaCl, 23 µg/ml phenylmethylsulfonyl fluoride, and 130 µg/ml lysozyme). The cells were blended to allow lysis and shearing
of the DNA. The sample was diluted with 500 ml of TGED (0.02 M Tris-HCl (pH 7.9), 5% (v/v) glycerol, 0.1 mM EDTA, and 0.1 mM dithiothreitol) + 0.2 M NaCl,
blended, and then centrifuged for 30-40 min at 7000 rpm. The
supernatant was collected as crude extract.
Crude extract was precipitated with Polymin P at a final concentration of 0.3% and centrifuged at 7000 rpm in Sorvall to collect the pellet. Proteins were eluted from the pellet with TGED + 1 M NaCl after washing the pellet once with TGED + 0.2 M NaCl. The Polymin P extract was then precipitated with ammonium sulfate at 50% saturation. The pellet obtained after centrifugation was resuspended in TGED and dialyzed twice against TGED + 50 mM NaCl.
Dialyzed sample was subjected to column chromatography as follows. It
was first applied to a heparin-agarose column pre-equilibrated with
TGED + 50 mM NaCl and eluted with a NaCl gradient of
50-600 mM in TGED. Fractions were examined by sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and peak
fractions containing RNA polymerase ( and
subunits), as
visualized by Coomassie Blue staining (Fig. 1A), pooled and
dialyzed as above. Dialyzed peak fractions were further purified on a
DEAE-cellulose column by fractionating with a linear NaCl gradient of
50-600 mM. After dialysis, the pooled peak fractions (Fig.
1B) were finally fractionated on a single-stranded DNA
cellulose column also with a linear NaCl gradient of 50-600
mM.
Two peak fractions, peak 1 and peak 2 (Fig. 1C), were pooled
separately and dialyzed against storage buffer (TGED + 0.05 M NaCl with 50% (v/v) glycerol). The dialyzed enzyme
preparations were aliquoted and stored at 80 °C for future
use.
Samples of RNAP holoenzyme were applied to SDS-PAGE gels (10% acrylamide), and after staining with KCl the gel was sectioned into 8 slices corresponding to the positions of visible proteins. Proteins were eluted from the gel slices, and SDS was removed by acetone precipitation, dissolved in 6 M guanidine HCl, and renatured, as described by Hager and Burgess (23).
Construction of DNA Templates Used for in Vitro TranscriptionPlasmid pNEO containing the promoter of neomycin
phosphotransferase neo gene was constructed by cloning the
HindIII/BglII fragment of transposon
Tn5 (24, 25) in pUC18 restricted with HindIII and
BamHI. The linear fragment obtained by restricting with
HindIII and EcoRI was used as a template in the
in vitro transcription experiment, and the length of the
expected transcript is 84 nucleotides (Fig. 2). The fragment containing
the lacUV5 promoter-operator region of E. coli
lac gene was cloned from the expression vector pINIIA2 (26) as a
305-bp HinfI/BamHI fragment. The DNA fragment
from plasmid pINIIA2 was subcloned into pUC18 to yield plasmid
pLACUV5. The DNA template was obtained from
pLACUV5 with HindIII and BamHI that
should produce a 79-nt transcript (Fig. 2; 26). Plasmid pAKC8 was
constructed as described previously (13). The fljK
transcription start site lies 451 bp upstream of the T7 terminator site
(Fig. 2). Supercoiled plasmid DNA was used in the one-cycle
transcription assay to determine the 54 activity (13).
Plasmid pJW012 containing the dnaKP1
32-dependent promoter has been described
previously (16). The SacI/SalI DNA fragment was
used as template in the run-off transcription assay to test the
32 activity. Plasmid pSSA41 was a gift of John Smit. The
HindIII/ClaI DNA fragment containing the
rsaA gene promoter was inserted into HindIII/AccI sites of pUC19. The
HindIII/NarI fragment from this plasmid was used
as template in the run-off transcription assay to determine the
73 activity. A 600-bp
HindIII/BamHI fragment containing the C. crescentus pleC gene promoter was subcloned into
HindIII and BamHI sites of pBluescript to yield
plasmid pJW014. The 636-bp HindIII/SalI fragment
from this plasmid was used as a DNA template in the run-off transcription assay to test the
73 activity.
In Vitro Transcription Assays
The formation of open complexes at different promoters from C. crescentus and E. coli was measured in single cycle or run-off transcription assays, as described previously (13, 16). The RNA polymerase holoenzymes were reconstituted by the addition of purified C. crescentus sigma factors to the purified C. crescentus core RNA polymerase (final concentration of 1 nmol) and incubated for 10 min at 4 °C. Transcripts obtained were fractionated on 7 M urea-PAGE with end-labeled Sau3A fragments of pUC18 as DNA size markers and visualized by autoradiography.
Assay for Core RNAPThe core RNAP activity was determined
using poly[d(A-T)] as template based on the method described by Berg
et al. (27). Each reaction of a 100-µl volume contained 20 µl of assay solution (200 mM Tris-HCl (pH 8.0), 50 mM MgCl2, and 50 mM
-mercaptoethanol), 100 µM ATP, 10 µM
UTP, and 5 µCi of [
-32P]UTP. RNA synthesis was
initiated by adding RNA polymerase and terminated after 10 min at
37 °C with 3 ml of ice-cold 3.5% perchloric acid containing 0.1 M sodium pyrophosphate. The precipitates were collected on
Whatman cellulose filter paper (3MM) and washed three times with cold 1 M HCl containing 0.1 M sodium pyrophosphate and
finally with cold ethanol. The radioactivity on the dry filter was
determined by scintillation counting.
The cloned rpoD
gene was identified and
cloned2 by colony
hybridization to its Myxococcus xanthus homologue (28). DNA
sequence on analysis confirmed that the open reading frame in this
recombinant plasmid was identical to that published for the C. crescentus rpoD gene (20). A SmaI-SstI DNA
fragment from plasmid pGIR210, which contains the rpoD gene,
was subcloned into the mutagenesis vector pAlter-1 (Promega), and an
NdeI restriction site was introduced at the first codon ATG
of the rpoD gene open reading frame by site-directed
mutagenesis. The 2.7-kilobase pairs NdeI-HindIII DNA fragment was then subcloned into the NdeI and
HindIII sites of the expression vector pRSET(A) to yield
plasmid pJW41 in which the entire open reading frame of rpoD
gene is translationally fused to the first codon of the T7 gene 10. E. coli strain BL21 (DE3) carrying the plasmid pJW41 was
used to overproduce 73 protein in the presence of
isopropyl-
-D-thiogalactopyranoside.
The overexpressed 73 protein was purified by a
previously described method (29, 30). The RpoD protein was not soluble
and formed inclusion bodies that were solubilized with 6 M
guanidine HCl in TGED buffer. The solubilized protein was renatured by
dialysis against the TGED buffer and then further purified by
chromatography on a DEAE-cellulose column. This method yielded
73 protein that was greater than 95% pure, as judged by
Coomassie Blue staining of SDS-PAGE gels.
Cellular RNAP from C. crescentus was purified by fractionation of cell extracts with
Polymin P, ammonium sulfate precipitation, and chromatography on
heparin-agarose (Fig. 1A) and
DEAE-cellulose (Fig. 1B), which removed many of the
contaminating proteins. Peak fractions from the DEAE-cellulose column
containing the RNAP ,
, and
subunits were pooled and applied
to a single-stranded DNA-cellulose column (Fig. 1C). As
shown in Fig. 1C, the majority of the RNAP
,
, and
subunits eluted in two distinct peaks along with several minor
proteins. The fractions in peaks 1 and 2 were pooled separately
("Materials and Methods").
We assayed RNAP activity in peak 1 and peak 2 using
E. coli DNA templates containing either the
70-dependent neo promoter or the
lacUV5 promoter (Fig. 2;
"Materials and Methods"). Both of these promoters are recognized
in vivo by C. crescentus3 (see below).
A third DNA template (Fig. 2) contained the
32-dependent, dnaKP1 heat-shock
promoter of C. crescentus (31). The RNAP preparation from
peak 2 recognized all three promoters, and specific transcripts of the
predicted sizes were obtained from each template (Fig.
3, lanes 2, 4, and
6). Thus the peak 2 preparation contained
32
holoenzyme (E
32) and
73 holoenzyme
(E
73) activities. No detectable transcripts were
observed when the RNAP preparation from peak 1 was assayed using the
same DNA templates (Fig. 3, lanes 1, 3, and 5).
These data suggest the possibility that peak 1 contained either
inactive RNAP holoenzyme or only core RNAP.
Identification of C. crescentus Core RNAP
We assayed for RNAP core activity directly in an in vitro transcription assay using poly[d(A-T)] as template as described by Berg et al. (27). The results summarized in Table I indicate that both peak 1 and peak 2 contained active core polymerase, although the specific activity was ~3-fold higher in the peak 2 RNAP. Interestingly, peak 2 RNAP was also more active on the poly[d(A-T)] template than purified E. coli core polymerase.
|
We next examined if the peak 1 RNAP core preparation could be
reconstituted to give active holoenzyme. C. crescentus
54 protein was used in the initial reconstitution
experiments because earlier work had demonstrated that
E
54 RNAP holoenzyme reconstituted from E. coli RNAP core and purified C. crescentus
54 specifically recognizes
54-dependent promoters from both E. coli and C. crescentus (14). The reconstituted
E
54 holoenzyme also required the activator protein FlbD
for initiation of transcription from C. crescentus
54-dependent promoters, as had been observed
both in vitro and in vivo (13).
The work described here demonstrates that in the presence of C. crescentus 54 and FlbD, the purified RNAP
preparation from peak 1 specifically recognized the
54-dependent fljK promoter and
produces a transcript of the expected size from this template (Fig. 2
and Fig. 4, lanes 2 and
4). This result confirms that peak 1 fractions contain an
active RNAP core enzyme that can be used for assays of sigma factor
activity. In the absence of added
54, however, RNAP in
neither peak 1 nor peak 2 recognized the fljK promoter (Fig.
4, lanes 1 and 3), indicating that none of these fractions contained an active E
54 holoenzyme.
Consequently, we refer to the peak 1 pool as core RNAP. The peak 2 pool, which appears to contain the E
73 and
E
32 holoenzymes (Fig. 3), as well as excess core RNAP
(see below), we refer to as peak 2 RNAP.
Isolation and Identification of Sigma Factor Subunits from the RNAP Holoenzyme Preparation
Individual proteins in the peak 2 RNAP
preparation (Fig. 1C; peak 2 pool) were isolated
after electrophoresis on preparative SDS-PAGE gels as described by
Hager and Burgess (23). The gel was sectioned into 8 slices
corresponding to the positions of bands visualized with KCl, with slice
1 containing bands at the top of the gel. Proteins were eluted from the
gel slices, and a portion of each eluted protein sample was then
analyzed on a second SDS-PAGE gel (Fig.
5). The peak 2 RNAP preparation contained several proteins in addition to core subunits and
isolated from slice 1 and
isolated from slice 4. Potential sigma factors were the 75-kDa protein in slice 2 and the 34-kDa protein in slice 5. Unidentified proteins A and B of molecular masses ~55 and 50 kDa,
respectively, were found in slice 3, and a third unknown protein C of
~28 kDa was detected in slice 6.
A portion of the proteins eluted from the SDS-PAGE gel slices were
renatured (see "Materials and Methods") and combined with C. crescentus core RNAP (peak 1) to determine their ability to direct
transcription. Assay of the 34-kDa protein renatured from gel slice 5 (Fig. 5) on the dnaK P1 template produced a run-off transcript of the expected size (62 nt; Fig. 2), which is consistent with the identification of this protein as the 32 factor
(16). When renatured proteins from gel slices 1-8 were assayed
individually in the presence of the peak 1 core RNAP using the
neo template, a run-off transcript was detected only in the assay containing proteins from slice 2 (Fig.
6). This transcript was of the size
expected (84 nt; Fig. 2) from the
70-dependent neo promoter. A
transcript of the same size was produced by peak 2 RNAP but not by peak
1 core RNAP alone (Fig. 6). No transcriptional activity was detected by
proteins eluted from slice 2 when they were assayed with peak 1 core
RNAP on DNA templates with
32- or
54-dependent
promoters.4 These results
indicate that the 75-kDa protein isolated from peak 2 RNAP preparation
specifically recognized the E. coli housekeeping promoters
and is a functional homologue of the E. coli principal sigma
factor,
70. Micro-sequencing of this protein yielded the
amino-terminal sequence (M)NNSSAETE, which is identical to that of the
translated DNA sequence of the C. crescentus rpoD gene
(20).
Isolation and Purification of the C. crescentus rpoD Gene Product
To further characterize the principal C. crescentus sigma factor identified in the reconstitution
experiments (Fig. 6), we overexpressed the rpoD gene in
E. coli and purified the full-length 73
protein to near-homogeneity (see "Materials and Methods";
Fig. 7A; lane 3). The C. crescentus rpoD gene has been shown to encode a predicted
polypeptide of 653 amino acids with a molecular mass of 72,623 Da and
designed as
73 (20).
The size of the overexpressed protein (Fig. 7A, lane 3) is
similar to the very faint protein band at ~75-kDa in the peak 2 RNAP
preparation (Fig. 7A, lane 2) and close to the predicted 72,623-Da size of the rpoD gene product (20). The purified
protein was also examined by Western blot analysis. An anti-E.
coli 70 antibody cross-reacted with the major
protein band at ~75 kDa, as well as with several smaller bands that
presumably result from proteolysis of RpoD (Fig. 7B, lane
3). The anti-
70 antibody can also recognize the
protein at ~75 kDa present in the peak 2 RNAP (Fig. 7B,
lane 2) but failed to recognize any proteins in the peak 1 RNAP
preparation (Fig. 7B, lane 1). These results further support
our assignment of peak 1 as core enzyme and the 75-kDa protein present
in the peak 2 RNAP as
73. The
73 present
in the peak 2 RNAP (Fig. 7B, lane 2) displays a slightly different mobility from that overproduced from the C. crescentus rpoD gene in E. coli (Fig. 7B, lane 3),
perhaps as a result of
73 modification in one of
the bacteria.
We examined the sigma factor activity of the purified
rpoD gene product in reconstitution experiments with core
RNAP using in vitro transcription assays (Fig.
8). Purified 73 protein
directed transcription from the
70-dependent
promoter of E. coli neo gene in the presence of peak 1 core
RNAP (Fig. 8, lane 2), whereas the core enzyme alone did not
(Fig. 8, lanes 1). These results and those in Fig. 6
demonstrate that the C. crescentus RpoD protein isolated
either from the peak 2 RNAP preparation or E. coli cells
overexpressing the cloned the C. crescentus rpoD gene
recognizes E. coli
70-dependent
promoters, suggesting that the C. crescentus
73 is a functional homologue of the principal E. coli sigma factor
70.
We next examined the ability of purified 73 to confer
transcriptional specificity in the recognition of two promoters,
rsaA (32, 33) and pleC (34; Fig. 2), that have
been used to define the
73 promoter consensus for
C. crescentus (19). Both of these C. crescentus
promoters have been characterized in vivo (19) and shown to
contain a
35 consensus sequence similar to that in E. coli
and a
10 consensus divergent from that in E. coli. The
rsaA and pleC promoters were recognized in the
in vitro transcription assays by the reconstituted
E
73 holoenzyme (Fig. 8, lanes 4 and
6). The more efficient transcription from the neo
promoter in these experiments (Fig. 8, lane 2) is consistent
with measurements of promoter strength in vivo using transcription fusions.
-Galactosidase assays of C. crescentus wild-type strains carrying either the
neop-lacZ (6876 Miller units) or the rsaAp-lacZ
(1545 Miller units) fusions indicates that the neo promoter
is 4- to 5-fold stronger than the rsaA promoter under these
conditions.4
The sizes of the rsaA and pleC transcripts
observed in vitro (Fig. 8) were those expected from the
transcription start sites mapped in vivo for the two genes,
i.e. 110 and 62 nt, respectively (Fig. 2; Ref. 19).
Transcription initiation was RpoD-dependent, since the peak
1 core RNAP alone did not recognize either promoter (Fig. 8,
lanes 3 and 5). These results confirm that the
purified rpoD gene product, 73, is the
principal C. crescentus sigma factor and that it is capable of recognizing the C. crescentus housekeeping gene
promoters, as well as E. coli
70-dependent promoters.
Many developmental events in C. crescentus are dependent on differential gene expression regulated at the level of transcription. Unlike Bacillus subtilis, where an extensive cast of alternative sigma factors and regulatory proteins governing sporulation have been identified through genetic and biochemical analysis (reviewed in Ref. 8), a defined in vitro transcription system has not been available in C. crescentus. The purification of C. crescentus RNAP was described in early studies (35, 36), but the transcriptional specificity of these enzyme preparations was not characterized. More recently, a partially purified RNAP preparation was used for the study of class II flagellar gene regulation (15). However, this is the first report of the purification and resolution of the holoenzymes and core RNAP and the reconstitution of RNAP holoenzymes exclusively from C. crescentus components.
Heparin-agarose chromatography, which has been used successfully for isolation of RNA polymerase from a number of bacterial species, including E. coli, B. subtilis, B. stearothermophilus, Lactobacillus casei, L. plantarum, and Clostridium pasteurianum (reviewed in Ref. 4), provided a great enrichment of C. crescentus RNAP (Fig. 1A). Chromatography on single-stranded DNA agarose (37) or on phosphocellulose (38) has also been reported to resolve E. coli holoenzyme and core RNAP. In our hands single-stranded DNA-cellulose chromatography was crucial for resolving C. crescentus RNAP into its core and holoenzyme fractions (Fig. 3C). Phosphocellulose and Bio-Rex 70 were not effective in resolving core and holoenzyme, although these earlier attempts were hampered by the lack of a purified sigma factor to assay for core activity.5
The fact that the first peak from the DNA-cellulose column (peak 1;
Fig. 1C) contained active core RNAP and the second peak (peak 2; Fig. 1C) contained E32 and
E
73 holoenzymes, as well as core RNAP (see below), was
demonstrated by in vitro transcription assays of the pooled
fractions (Fig. 3; Table I) and the isolation of active
32 and
73 from these RNAP fractions of
peak 2 (Fig. 6; 16). In the presence of purified C. crescentus
54 and its activator protein FlbD, core
RNAP from peak 1 recognized the
54-dependent
promoter of the C. crescentus flagellin gene fljK
(Fig. 4), as observed previously for a heterologous holoenzyme
containing either C. crescentus
54 or
E. coli
54 and the E. coli core
RNAP (13-15). Therefore, C. crescentus core RNAP appears to
function interchangeably with its E. coli counterpart in
this transcription assay.
Our purified peak 2 RNAP preparation displayed activity only on
32- and
73-dependent
promoters. Possible explanations for the failure to recover active
E
54 include (i) an unstable
54 protein
that is inactivated during purification and (ii) low affinity of
54 for binding to core RNAP, which results in its
dissociation from the core and loss early in protein fractionation.
Consistent with the latter possibility are two observations. First,
some bacterial RNAP holoenzymes are quite unstable and dissociate early
during procedures suitable for isolation of other RNAP holoenzymes
(39), and second, full-length E. coli
54 does
not bind to E. coli core RNAP tightly (40).
Three proteins (Fig. 5; bands A, B, and C) of
unknown function are also associated with the RNAP holoenzyme
fractions. These proteins could represent additional sigma factors,
breakdown products of 73, other RNAP subunits, such as
delta or omega, or proteins that fortuitously fractionate with RNAP.
Protein C (Fig. 5), like
32 (16) and
73,
has been isolated and subjected to amino acid sequencing, but unlike
the latter two proteins, there is no similarity of its amino-terminal
amino acid sequence to any sequences deposited in
GeneBank.4 It will be interesting to determine whether any
of these three proteins, A, B, and C, are RNAP subunits or accessory
proteins that are involved in transcriptional regulation.
The relative amounts of 73 and
32 as
visualized by the intensity of staining in SDS-PAGE gels displayed
variability depending on the purified preparation examined, but we
estimated that pooled peak 2 RNAP contained less than 0.4 mol eq of
total
factor relative to the core subunits (see Fig. 5). This
result suggests that peak 2 RNAP also contains core enzyme. Consistent
with the presence of excess core in the peak 2 RNAP fractions was the
stimulation of transcription from the
54-dependent fljK promoter by the
addition of purified
54 and FlbD to the peak 2 pool
(Fig. 4) and the high activity of this RNAP pool when assayed on the
poly[d(A-T)] template (Table I).
The above results raise the question of why the core enzyme elutes from
the single-stranded DNA column in two peaks. One possibility is that
the more active core enzyme (Table I) binds more tightly to the column
and elutes with the holoenzymes in peak 2. Alternatively, the core
eluted in peak 2 could represent core that has bound tightly as the
holoenzyme from which sigma factors elute at lower salt concentrations
than the other polymerases in those complexes. Whatever the explanation
of this fractionation, our experiments have depended critically
on the isolation of the functional core RNAP. The inability to resolve
different RNAP holoenzymes from one another5 prompted us
initially to overexpress and purify C. crescentus factors, including
54 (14),
32 (16), and
73 (Fig. 7) for the reconstitution of specific RNAP
holoenzymes reported in this study.
C. crescentus recognizes E. coli
70-dependent promoters in vivo
(20), and our results demonstrate that the purified (Figs. 3 and 6) and
reconstituted (Fig. 8) C. crescentus E
73
efficiently recognized the lacUV5 and neo
promoters from E. coli, as well as promoters of the C. crescentus housekeeping genes rsaA and pleC
(Fig. 8). The
35 consensus sequence of the C. crescentus biosynthetic and housekeeping gene promoters (19) is similar to the
35 consensus of E. coli
70-dependent promoters, but the
10
sequences from these two bacteria align only poorly. Moreover, the
C. crescentus
10 and
35 sequences are more closely
spaced than in most E. coli promoters (19). These results
suggest that the principal C. crescentus sigma factor
73 has less promoter specificity than its E. coli counterpart
70.
The availability of core RNAP from C. crescentus and the
capability of reconstituting active holoenzymes using purified factors will permit biochemical analysis of gene regulation in vitro in great detail. These reagents will also be important in studying the role of RNAP and accessory factors in the temporal and
spatial regulation of developmental gene transcription during the cell
division and differentiation.
We thank G. Ramakrishnan for the original
isolation of the C. crescentus rpoD gene used in this study.
We also thank R. R. Burgess for providing anti-E. coli
70 antibody, S. Inouye for the M. xanthus
rpoD clone, and J. Smit for plasmid pSSA41 containing the
rsaA promoter.