Purification, Characterization, and Reconstitution of DNA-dependent RNA Polymerases from Caulobacter crescentus*

(Received for publication, March 17, 1997, and in revised form, June 4, 1997)

Jianguo Wu , Noriko Ohta , Andrew K. Benson Dagger , Alexander J. Ninfa § and Austin Newton

From the Department of Molecular Biology, Princeton University, Princeton, New Jersey 08544

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

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 Esigma 54, Esigma 32, and Esigma 73 were reconstituted exclusively from purified C. crescentus core and sigma factors. Reconstituted Esigma 54 initiated transcription from the sigma 54-dependent fljK promoter of C. crescentus in the presence of the transcription activator FlbD, and active Esigma 32 specifically initiated transcription from the sigma 32-dependent promoter of the C. crescentus heat-shock gene dnaK. For reconstitution of the Esigma 73 holoenzyme, we overexpressed the C. crescentus rpoD gene in Escherichia coli and purified the full-length sigma 73 protein. The reconstituted Esigma 73 recognized the sigma 70-dependent promoters of the E. coli lacUV5 and neo genes, as well as the sigma 73-dependent housekeeping promoters of the C. crescentus pleC and rsaA genes. The ability of the C. crescentus Esigma 73 RNA polymerase to recognize E. coli sigma 70-dependent promoters is consistent with relaxed promoter specificity of this holoenzyme previously observed in vivo.


INTRODUCTION

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 (Esigma ; reviewed in Refs. 4 and 5). The core RNAP, composed of the alpha 2, beta , and beta ' subunits, carries out RNA chain elongation, whereas the holoenzyme, which also contains the sigma subunit (sigma ), 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 sigma 54-dependent promoters late in the cell cycle. The sigma 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 Esigma 54 holoenzyme reconstituted from purified Escherichia coli components (12, 13), the heterologous Esigma 54 holoenzyme reconstituted from the C. crescentus sigma 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, sigma 32 (rpoH; Refs. 16 and 17), sigma 54 (rpoN; Refs. 14 and 18), and sigma 73 (rpoD; Ref. 19) have been cloned and sequenced. Although sigma factors sigma 54 (14) and sigma 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 (sigma 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 Esigma 73 and Esigma 32. We also describe the purification of the C. crescentus principal sigma factor, sigma 73, after overexpression of rpoD in E. coli. The Esigma 73, Esigma 54, and Esigma 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.


MATERIALS AND METHODS

Bacterial Strains, Media, and Materials

E. coli strain DH5alpha 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. [alpha -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.

RNA Polymerase Purification

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 beta -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 (beta beta ' and alpha  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.


Fig. 1. Purification of C. crescentus RNA polymerases. The DNA-dependent RNAPs were purified from cells of C. crescentus wild-type strain CB15 as described under "Materials and Methods." SDS-PAGE gels are shown of fractions eluted from a heparin-agarose column (A), a DEAE-cellulose column (B), and a single-stranded DNA-cellulose column (C). Gels were stained with Coomassie Blue. L, protein preparation loaded onto the column; W, column wash. The positions of core RNAP subunits are indicated as beta , beta ', and alpha .
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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.

Isolation and Purification of Sigma Factors from RNA Polymerase Preparation

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 Transcription

Plasmid 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 sigma 54 activity (13). Plasmid pJW012 containing the dnaKP1 sigma 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 sigma 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 sigma 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 sigma 73 activity.


Fig. 2. Templates used for in vitro transcription assays. Construction of the six DNA templates used in the transcription assays is described under "Materials and Methods." The purified DNA fragments indicated were used in the run-off transcription assays. The supercoiled plasmid containing the fljK promoter was used in one-cycle transcription assays. The predicted sizes of individual transcripts from the templates are indicated in nucleotides (nt).
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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 M urea-PAGE with end-labeled Sau3A fragments of pUC18 as DNA size markers and visualized by autoradiography.

Assay for Core RNAP

The 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 beta -mercaptoethanol), 100 µM ATP, 10 µM UTP, and 5 µCi of [alpha -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.

Overexpression and Purification of C. crescentus sigma 73 Protein from E. coli

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 sigma 73 protein in the presence of isopropyl-beta -D-thiogalactopyranoside.

The overexpressed sigma 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 sigma 73 protein that was greater than 95% pure, as judged by Coomassie Blue staining of SDS-PAGE gels.


RESULTS

Purification of RNA Polymerases

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 beta , beta ', and alpha  subunits were pooled and applied to a single-stranded DNA-cellulose column (Fig. 1C). As shown in Fig. 1C, the majority of the RNAP beta , beta ', and alpha  subunits eluted in two distinct peaks along with several minor proteins. The fractions in peaks 1 and 2 were pooled separately ("Materials and Methods").

Identification of Esigma 73 and Esigma 32 RNAP Holoenzymes

We assayed RNAP activity in peak 1 and peak 2 using E. coli DNA templates containing either the sigma 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 sigma 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 sigma 32 holoenzyme (Esigma 32) and sigma 73 holoenzyme (Esigma 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.


Fig. 3. Identification of C. crescentus RNA polymerase holoenzymes Esigma 73 and Esigma 32. The activities of holoenzymes in peak 1 RNAP (lanes 1, 3, and 5) and peak 2 RNAP (lanes 2, 4, and 6) were determined in run-off transcription assays with DNA templates containing the sigma 70-dependent neo (lanes 1 and 2) or lacUV5 (lanes 3 and 4) promoters from E. coli and the sigma 32-dependent promoter, dnaK P1 (lanes 5 and 6) from C. crescentus, as described previously (16).
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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.

Table I. Core RNA polymerase activities assayed on poly [d(A-T)] templates


RNAPa Specific activity Activity relative to E. coli core

unitb/mg
Peak 1 RNAP 1.1  × 104 0.5
Peak 2 RNAP 3.8  × 104 1.7
E. coli core 2.2  × 104 1.0

a C. crescentus peak 1 and peak 2 RNAPs are pools of fractions shown in Fig. 1C, as described under "Results."
b Unit definition: as described under Epicentre Technologies, one unit catalyzed the incorporation of 1 nmol of ribonucleotide triphosphate into RNA in 10 min at 37 °C.

We next examined if the peak 1 RNAP core preparation could be reconstituted to give active holoenzyme. C. crescentus sigma 54 protein was used in the initial reconstitution experiments because earlier work had demonstrated that Esigma 54 RNAP holoenzyme reconstituted from E. coli RNAP core and purified C. crescentus sigma 54 specifically recognizes sigma 54-dependent promoters from both E. coli and C. crescentus (14). The reconstituted Esigma 54 holoenzyme also required the activator protein FlbD for initiation of transcription from C. crescentus sigma 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 sigma 54 and FlbD, the purified RNAP preparation from peak 1 specifically recognized the sigma 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 sigma 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 Esigma 54 holoenzyme. Consequently, we refer to the peak 1 pool as core RNAP. The peak 2 pool, which appears to contain the Esigma 73 and Esigma 32 holoenzymes (Fig. 3), as well as excess core RNAP (see below), we refer to as peak 2 RNAP.


Fig. 4. Identification of core RNA polymerase. The C. crescentus core RNAP activity was determined in in vitro transcription assays by reconstituting Esigma 54 activity from the purified C. crescentus sigma factor sigma 54 and its activator FlbD. The RNAP preparations from pooled peak 1 (lanes 1 and 2) and peak 2 (lanes 3 and 4) were assayed on supercoiled templates containing the sigma 54-dependent promoter of flagellin gene fljK, as described (13). Assays were carried out with (lanes 2 and 4) or without (lanes 1 and 3) the purified sigma 54.
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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 beta  and beta ' isolated from slice 1 and alpha  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. 


Fig. 5. Isolation of RNA polymerase subunits. Coomassie Blue-stained SDS-PAGE gels of purified C. crescentus RNAP holoenzyme (peak 2 RNAP) and isolated proteins from slices of a preparative gel of this RNAP preparation (slices 1-8; see text). Proteins were eluted from the preparative SDS-PAGE gels as described by Hager and Burgess (Ref. 23; "Materials and Methods"). The subunits of core polymerase beta  (slice 1), beta ' (slice 1), and alpha  (slice 4) as well as the sigma subunits sigma 73 (slice 2) and sigma 32 (slice 5) are indicated by arrows. Three unidentified proteins are indicated by letters A (slice 3), B (slice 3), and C (slice 6), respectively. The bovine serum albumin protein present in all slices was used as carrier during the purification.
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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 sigma 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 sigma 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 sigma 32- or sigma 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, sigma 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).


Fig. 6. Determination of the C. crescentus sigma 73 activity. Individual proteins from the peak 2 RNAP preparation were isolated by SDS-PAGE (Fig. 5; slices 1-8) and assayed after renaturation for stimulation of transcription in the presence of core RNAP (peak 1 RNAP). The activity of reconstituted Esigma 73 holoenzyme was determined by its ability to recognize and transcribe from the sigma 70-dependent E. coli neo promote.
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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 sigma 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 sigma 73 (20).


Fig. 7. Identification of E. coli sigma 70 homologue from C. crescentus. A, Coomassie Blue-stained SDS-PAGE gel of purified C. crescentus peak 1 core RNAP (lane 1), peak 2 RNAP (lane 2), and isolated C. crescentus RpoD gene product, sigma 73, overproduced and purified from E. coli (lane 3). B, Western blot analysis of C. crescentus peak 1 core RNAP (lane 1), peak 2 RNAP preparation (lane 2), and purified C. crescentus sigma 73 (lane 3) with an anti-E. coli sigma 70 antibody.
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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 sigma 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-sigma 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 sigma 73. The sigma 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 sigma 73 modification in one of the bacteria.

Functional Analysis of C. crescentus sigma 73 in Vitro

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 sigma 73 protein directed transcription from the sigma 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 sigma 70-dependent promoters, suggesting that the C. crescentus sigma 73 is a functional homologue of the principal E. coli sigma factor sigma 70.


Fig. 8. Reconstitution of C. crescentus Esigma 73 holoenzyme in vitro. The Esigma 73 was reconstituted by mixing the purified C. crescentus peak 1 core RNAP and purified C. crescentus sigma 73 protein in vitro. The Esigma 73 activity was determined by run-off transcription assays on DNA templates containing the sigma 70-dependent neo promoter from E. coli (lanes 1 and 2) or the pleC (lanes 3 and 4) and rsaA (lanes 5 and 6) housekeeping gene promoters from C. crescentus.
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We next examined the ability of purified sigma 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 sigma 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 Esigma 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. beta -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, sigma 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 sigma 70-dependent promoters.


DISCUSSION

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 Esigma 32 and Esigma 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 sigma 32 and sigma 73 from these RNAP fractions of peak 2 (Fig. 6; 16). In the presence of purified C. crescentus sigma 54 and its activator protein FlbD, core RNAP from peak 1 recognized the sigma 54-dependent promoter of the C. crescentus flagellin gene fljK (Fig. 4), as observed previously for a heterologous holoenzyme containing either C. crescentus sigma 54 or E. coli sigma 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 sigma 32- and sigma 73-dependent promoters. Possible explanations for the failure to recover active Esigma 54 include (i) an unstable sigma 54 protein that is inactivated during purification and (ii) low affinity of sigma 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 sigma 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 sigma 73, other RNAP subunits, such as delta or omega, or proteins that fortuitously fractionate with RNAP. Protein C (Fig. 5), like sigma 32 (16) and sigma 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 sigma 73 and sigma 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 sigma  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 sigma 54-dependent fljK promoter by the addition of purified sigma 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 sigma  factors, including sigma 54 (14), sigma 32 (16), and sigma 73 (Fig. 7) for the reconstitution of specific RNAP holoenzymes reported in this study.

C. crescentus recognizes E. coli sigma 70-dependent promoters in vivo (20), and our results demonstrate that the purified (Figs. 3 and 6) and reconstituted (Fig. 8) C. crescentus Esigma 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 sigma 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 sigma 73 has less promoter specificity than its E. coli counterpart sigma 70.

The availability of core RNAP from C. crescentus and the capability of reconstituting active holoenzymes using purified sigma 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.


FOOTNOTES

*   This work was supported in part by Public Health Service Grant GM22299 from the National Institutes of Health (to A.N.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Dagger    Present address: Dept. of Food Science and Technology, University of Nebraska, Lincoln, NE 68583-0919.
§   Present address: Dept. of Biological Chemistry, University of Michigan, Ann Arbor, MI 48109-0606.
   To whom correspondence should be addressed. Tel.: 609-258-3854; Fax: 609-258-6175; E-mail: anewton{at}molecular.princeton.edu.
1   The abbreviations used are: RNAP, RNA polymerase; E, core polymerase; Esigma , holoenzyme; nt, nucleotide; bp, base pair; PAGE, polyacrylamide gel electrophoresis.
2   G. Ramakrishnan and A. Newton, unpublished observations.
3   N. Ohta, J. Wu, and A. Newton, unpublished observations.
4   J. Wu and A. Newton, unpublished observations.
5   N. Ohta, A. Ninfa, and A. Newton, unpublished observations.

ACKNOWLEDGEMENTS

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 sigma 70 antibody, S. Inouye for the M. xanthus rpoD clone, and J. Smit for plasmid pSSA41 containing the rsaA promoter.


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