Biochimie (1992) 74, 627-634 © Soci6t6 franqaise de biochimie et biologie mol6culaire / Elsevier, Paris

627

The effect of supercoiling on the in vitro transcription of the spollA operon from Bacillus subtilis T Bird 2, D Burbulys I, JJ

Wu

I,

M A Strauch l, JA H o c h 1, GB Spiegelman 2.

1Department of Molecular and Experimental Medicine, Research Institute of Scripps Clinic, 10666 North Torrey Pines Road, La Jolla, CA 92037, USA; 2Department of Microbiology, University of British Columbia, 6174 University Boulevard, Vancouver, BC V6T ! W5, Canada (Received l I December 1991; accepted 20 February 1992)

Summary - - The spollA operon codes for an alternative sigma factor which appears in the early stages of a sigma factor expression cascade during sporulation in Bacillus subtilis. We have used a single round in vitro transcription assay to probe requirements for transcription initiation at the spollA promoter. Core RNA polymerase or holoenzyme containing 6 A was reconstituted with a n protein and used to transcribe the spollA promoter. Formation of heparin resistant transcription initiation complexes required that the spollA template be supercoiled. Topoisomers of the spollA template were created and transcribed at various temperatures. Changes in the superhelicity of template DNA had a significant influence on the amount of transcription complexes formed at the spollA promoter. Bacillus subtilis I sporulation I in vitro transcription / sigma factors / supercoiling

Introduction The genetic program that directs endospore development in Bacillus subtilis integrates a variety of regulatory mechanisms which operate at all levels of gene expression [1]. The temporal control of sporulation genes is now known to be achieved largely through the sequential expression of alternative sigma factors each of which directs RNA polymerase to transcribe sets of sporulation genes as they are required [2-4]. The spollA operon is of integral importance to the sigma factor cascade since it encodes o F, one of the first sporulation specific sigma factors to appear during spore development. The operon also codes for two products that act synergistically to regulate o F activity [5]. Expression of the spollA operon is regulated at the level of transcription with m R N A synthesis evident 60 rain following the initiation of sporulation [6]. The induction of spollA transcription is dependent on the stage zero sporulation genes including the proposed response regulator, Spo0A [6]. Transcription of the spollA operon has been studied by Wu et al [7] who defined the in vivo transcription *Correspondence and reprints

start site and showed that in vivo transcription of the operon was dependent on spoOH. The spoOH gene codes for oH, an alternative sigma factor necessary for the expression of a number of genes associated with stationary phase [4, 8]. We have used an in vitro transcription system to investigate the properties of transcription initiation at the spollA promoter by purified RNA laolymerase containing the a n sigma factor. These experiments confirmed that the spoliA promoter is transcribed by o n holoenzyme. In addition, the formation of transcription complexes at the spolIA promoter was found to require a supercoiled template with the level of complexes formed very sensitive to changes in superhelical density.

Materials and methods Plasmid constructs Plasmid p2AB3 was constructed by insertion of the 900 bp AvaI-BamHI fragment (spollA sequences from +200 to -700; [ 15]) from pPPI 15 (Wu and Piggott, unpublished observations) into AvaI-BamHI digested pUCI8. The plasmid pPS-28 is a pUCI8 derivative that carries a l138-bp PvuII fragment encoding the rho independent tandem terminators from the rrnB rRNA operon of E coli (obtained from pKK232-8 [9]) cloned into the pUCI8 HincII site. pIIA-28 is a derivative of

628 pPS-28 carrying a 950-bp Hindlll-Aval fragment encoding the spollA promoter 161 cloned upstream of the terminators. In vitro transcription from pllA-28 perfomled on a circular template would yield a transcript 350 nucleotides in length if RNA elongation ended at the first terminator downstream of the promoter. The Hindlll-Aval fragment was isolated from p2AB3 by cleaving with Aval, filling in the extended ends with the Klenow enzyme and releasing the promoter containing fragment with Hindlll. The fragment was isolated by electrophoresis and ligated to pPS-28 which had been cut with Pstl, treated with Klenow enzyme to fill in the Pstl end and then cut with Hindlll. Plasmid structure was confirmed by sequencing. For transcription studies, plasmid DNA was isolated by performing cleared lysate and CsCI gradient purification, essentially as described by Sambrook et al [ 101. In vitro transcription assay

Transcription reactions were carried out in 40 mM Hepes (pH 8.0), 10 mM magnesium acetate, 0.1 mM dithiothreitoi, 0.1 mM EDTA and 100 lag/mi bovine serum albumin in a final reaction volume of 20 lal. Assays were composed by premixing buffer and DNA in 0.65 ml Eppendorf tubes on ice. These tubes were warmed for 2 min at 37°C prior to the addition of 2 lal of RNA polymerase (combinations of core polymerase or t~A holoenzyme and t~rt protein as described in figure legends). Reactions were further incubated at 37°C for 5 min after which complexes were challenged by addition of 2 }.tl of 100 lal/ml heparin and incubation for 5 min. Elongation from surviving open complexes was facilitated by addition of 2 lal of a nucleotide mix that added GTP, ATP and CTP to a final concentration of 500 taM and UTP to 10 taM (and 0.015 mCi/mmol; [ct32P]UTP: AmershamL Transcript elongation was carried out for 5 min after which reactions were stopped with 5 lal of loading buffer (8.0 M urea, 180 mM Trizma base, 180 mM boric acid, 0.004 mM EDTA, 0.02% bromophenyl blue and 0.02% xylene cyanole). Samples were run through a 5e~ denaturing polyacr.ylamide gel. Promoter activity was determined by measuring the Cerenkov radiation in gel slices containing transcript III, which had been localized by autoradiography. To determine promoter activity we assumed that transcripts I! and !!i both terminated at the site on the TI terminator indicated tbr E coli termination !111. This assumption will slightly overestimate the level of transcription. The number of transcripts produced in a spoilA transcription reaction was determined by converting counts pet" minute (cpm) to counts per transcript by calculating the cpm per UTP in the reaction and multiplying by 100, the number of UTP residues in transcript 11. Isolation of core RNA polymerase

Vegetative RNA polymerase holoenzyme (o A) was isolated from B subtilis strain 168S essentially as reported iL, [i2] with the following differences. After the glycerol gradient step, peak enzyme activity factions were pooled, concentrated and loaded onto a heparin-Sepharose column (5 cc) that had been equilibrated with 50 mM NaCI, l0 mM Tris-Cl, l0 mM EDTA, l0 mM MgCl2, 10% glycerol, l0 mM ~-2-mercaptoethanol and 60 taM phenylmethyl sulfonyl fluoride. The column was washed with l0 ml of the same buffer except that 0.1 M potassium glutamate was substituted for NaCl. The polymerase was eluted with !.2 M potassium glutamate. To isolate core polymerase, active fractions from the heparin-Sepharose column were pooled, concentrated and diluted I:10 with P cell buffer (0.05 M Hepes (pH 7.9), 10 mM EDTA, 10 mM ~-2-

mercaptoethanol, 60 taM phenylmethyl suifonyi fluoride, 20% glycerol) before loading onto a phospho-cellulose column (10 cc) that had been equilibrated with P cell buffer containing 0.1 M potassiu.~rg!utamate. The column was washed with five volumes of buffer containing 0.1 M potassium glutamate and then core polymerase was eluted with a 100-ml linear gradient of 0.1 to 1.5 M potassium glutamate in P cell buffer. Core polymerase was found to elute between 0.30-0.45 M potassium glutamate. Fractions that contained core enzyme, as determined by sodium dodecylsulfate-polyacrylamide gel electrophoresis, were combined and concentrated. The glycerol content of the buffer was made up to 50% and the core was stored at 70°C. Purification methods for o H have been described [271. Primer extension assays

RNA from in vitro reactions as described above was recovered from polyacrylamide gel slices used to separate the various transcripts, as seen in figure 1, by electroelution. The RNA was purified by adding 10 lag of yeast RNA and extracting with phenol/chloroform. The RNA was precipitated with ethanol twice and redissoived in 10 lal of sterile distilled water. Aliquots of the RNA were used as templates for avian myeloblastosis virus reverse transcriptase (BRL) using reaction

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,ia, O Fig 1. Transcription products from the spollA promoter. The products from in vitro transcription reactions were separated on a 5% polyacrylamide, 7 M urea gel and the gel was then exposed to Kodak X-OR-MAT film overnight. Transcription products are marked with I, II and l i t designations. Supercoiled plIA-28 plasmid DNA was used as template for the transcription reactions shown in lanes 1, 2, 4 and 5 while pIIA-28 which had been linearized with HindIII was used as template for reactions in lanes 3 and 6. Reactions in lanes I and 3 contained core RNA polymerase and purified o H protein while the reaction in lane 2 contained only core polymerase. Reactions in lanes 4 and 6 were performed with RNA polymerase holoenzyme containing o A (Eo A) and purified o H while the reaction in lane 5 contained only E o A holoenzyme.

629 conditions recommended by the supplier for cDNA synthesis, and a primer (5'-GGCGAATATCATCCTTCTCC-3') which had been labelled with polynucleotide kinase (BRL) and ['t32p]ATP (ICN). The DNA sequencing ladder used the same primer, double stranded pIIA-28 and the Sequenase sequencing kit (USB). The products of the extension reaction and the DNA sequence were electrophoresed through a 6% polyacrylamide, 7 M urea gel which was then dried and exposed to Kodak X-OR-MAT film for 18 h.

Generation of topoisomers Relaxation reactions were carried out in 100-~! volumes that contained 40 mM KCI, 10 mM Tris-HCl, pH 7.4, l mM EDTA and 4 mM MgCl2. Purified pIIA-28 plasmid DNA (25 l.tg) was mixed with various amounts of ethidium bromide (final concentrations ranged from 0 to 5.5 ~tM). The relaxation was initiated by adding 6 l~i of a 100S extract made from Drosophila Kc cells [13] that was found to exhibit topoisomerase activity (unpublished results). Following 2 h incubation at 37°C, the reactions were phenol-extracted three times to remove protein and ethidium bromide, and the DNA was precipitated with ethanol. The topoisomers were resuspended in l0 mM Tris-Cl, pH 7.2, ! mM EDTA, and the DNA was quantitated by spectrophotometry (A26o) and the concentrations given by this method were checked by electrophoresis through agarose to compare reiative amounts of DNA. The degree of supercoiling present in each topoisomer preparation was ascertained by electrophoresing DNA samples though 1.4% agarose gels in 40 mM Tris-Cl (pH 7.9), 5 mM sodium acetate and 1 mM EDTA at 4 V/cm for 18 h. The gels were photographed after ethidium bromide staining and the average degree of supercoiling in each lane was determined by densitometric scanning performed on the photographic negatives using the procedure described by Keller [14]. To achieve resolution of the supercoils in topoisomers with high levels of superhelical density chloroquin was added to the gel and running buffer at a concentration of 0.016 I~g/ml.

e n z y m e digest, however, little or no transcription was detected in these assays. In contrast, when supercoiled template was assayed, three transcript bands were observed following electrophoresis through polyacrylamide gels (fig l). Two of the transcripts (transcripts II and III) were uniquely dependent on the presence o f o H in the transcription reaction. Primer extension m a p p i n g (fig 2) showed that transcripts I! and III originated from the same site identified in vivo as the

G A T C

II III

eA

Results

in vin'o transcription fi'om the spoliA promoter A single round in viu'o transcription assay was used to investigate transcription initiation a t the spollA promoter. The assay was begun by m i x i n g purified core R N A polymerase or h o l o e n z y m e containing o A with o a protein and spollA template DNA. Initiation c o m p l e x e s were allowed to form for 5 m i n at the reaction temperature. Heparin was then added to the reactions to sequester free R N A p o l y m e r a s e and destroy non-specific p r o t e i n / D N A contacts. After a 5 - m i n heparin c h a l l e n g e nucleoside triphosphates were added to permit heparin resistant initiation c o m p l e x e s to elongate R N A transcripts. If heparin was added to the reactions prior to D N A , no transcription was observed, indicating that the heparin was able to bind all free p o l y m e r a s e in the reaction and effectively inhibit transcription initiation. Initial experiments were performed using spollA template which had been linearized with a restriction

Fig 2. Primer extension mapping of the transcripts from the

spollA promoter. Transcripts II and III were isolated from a polyacrylamide gel by electroelution after being localized by autoradiography. A primer extension assay was performed on the purified RNA as described in Materials and methods and the same labelled 20-mer primer was used to generate the DNA sequence of the spollA promoter region shown. The DNA sequence around the start site determined using in vivo RNA (0) is indicated and the arrow on the right shows the position of the primer extension products. The DNA sequence shown is the complement of the DNA sequence actually determined in the sequencing reaction.

630 start site of transcription for the spollA promoter [7]. These data confirmed the previous report that the spoIIA promoter is recognized by polymerase containing o H [7, 15]. Since transcripts II and III share the same initiation site, they must differ at the site of termination and probably reflect heterogeneity in termination at the T1 site on vector pPS-28. The transcripts present in the transcript I band were isolated and subjected to primer extension analysis using the same primer used to map the ends of transcripts II and III; however, no extension product was detected. Transcript I was later found to include at least two high molecular weight transcripts that could be resolved when electrophoresed through low percent polyacrylamide gels. Transcription of the vector used to construct pIIA-28 (pPS-28) with o H plus o A holoenzyme and oA holoenzyme only, yielded high molecular weight transcripts (data not shown). Thus these transcripts may originate from promoter sequences within the vector portion of pIIA-28 and are likely recognized by o A holoenzyme although they have not been analyzed further.

Transcrtption of spollA topoisomers Because transcription initiation at the spolIA promoter appeared to be very sensitive to template supercoiling we examined the influence of superhelical density on spollA promoter activity. A series of topoisomers of pIIA-28 was generated (described in Materials and methods) using an extract made from Drosophila Kc cells which had been found to exhibit topoisomerase activity (Duncan and Spiegelman, unpublished results). The extract was used to relax fully supercoiled pllA-28 plasmid in the presence of various concentrations of ethidium bromide. Topoisomers with average superhelical densities ranging from 0.0 to -0.074 were obtained (fig 3). The Drosophila cell extract was found to contain some DNA nicking activity so that a small amount of plasmid was linearized during the relaxation reaction. Because our results showed linear spollA template was a very poor substrate for transcription initiation; we assume its presence had little effect on in vitro transcriptioo of the topoisomers. The spollA topoisomers were used as templates in vitro at four temperatures (fig 4). At 37°C the level of formation of transcription initiation complexes at the spollA promoter rose with increased supercoiling of the template up to a superhelical density of -0.050. The degree of stimulation was approximately seven-fold and was most dramatic at superhelical densities above -0.024. The effect of temperature on transcription complex formation appeared to be independent of the extent of template supercoiling, so that at 12°C even highly supercoiled template could not support transcription. Experiments

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9 10 11 12 13 t~

SC Fig 3. Agarose gels showing plIA-28 topoisomers, pIIA-28 topoisomers with increasing average superhelical densities were generated as described in Materials and methods. Samples of each of the topoisomer preparations were electrophoresed through a 1.4% agarose gel and then stained with ethidium bromide to visualize the DNA. The position of open circle (OC), linearized (L), or fully supercoiled (SC) plasmid DNA is indicated. Average superhelical densities calculated for each preparation are as follows: lane l, +0.04; lane 2, 0.0; lane 3, -0.004; lane 4, --0.008; lane 5, -0.012; lane 6, -0.016; lane 7, -0.024; lane 8, -0.033; lane 9, -0.041; lane 10, -0.049; lane I l, -0.057; lane 12, -0.066; lane 13, -0.074.

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~3

0.00

0.02

0.04

O.OS

008

superhelicai density (~)

Fig 4. The effect of superhelicai density and temperature on

spoilA transcription initiation. Topoi~omers shown in figure 3 (lanes 2-13) were assayed at 12°C (open squares), 20°C (closed squares), 28°C (open circles) and 37°C (closed circles) as described in Materials and methods.

631 that involved forming transcription complexes at 37°C before shifting to 16°C during the 5-min heparin challenge resulted in very low levels of transcription from fully supercoiled template (data not shown). This temperature shift experiment suggested that low temperatures destabilize preformed heparin resistant complexes. Discussion

We have demonstrated that the in ¢itro transcription of the spollA promoter requires the presence of the o n sigma factor. These data confirm previously in vitro and in vivo experiments for the function of the spoOH gene product in the initiation of the sigma cascade which dictates the pattern of gene expression during sporulation [3, 7, 15]. We found that we could add purified o H protein to isolated o A holoenzyme preparations as well as to core preparations and produce transcripts from the spollA promoter. We did not observe significant differences in the pattern of expression using t~A holoenzyme as opposed to core polymerase indicating that the presence of the ~ subunit which is present in the holoenzyme preparations [ 16] but absent in the core preparations did not affect the transcription properties of the all/core complexes. We presume that the transcription from spoIIA directed by o n added to o A holoenzyme is due to the presence of core in the a A holoenzyme preparation since it is only 50% saturated with a A, as opposed to the displacement of a A from holoenzyme in that preparation. We found that a H holoenzyme could readily form heparin resistant complexes at the spoIIA promoter in the absence of initiating nucleotides, as long as the template was supercoiled. The precise definition of a heparin resistant complex has not been made for any Bacillus polymerase-promoter complex, however circumstantial evidence suggests that the complex represents the equivalent of the open complex stage in the initiation reaction carried out by E coli RNA polymerase [17]. Transcription initiation by ~ coli RNA polymerase has four kinetically significant stages: 1) binding at the promoter to form an initial complex (usually termed as closed complex); 2) isomerization of the closed complex to a minimum of one intermediate form; 3) conversion of the intermediate form to an open complex in which the DNA has been denatured and the polymerase is able to rapidly initiate transcription; and 4) commitment to elongation [18, 19]. Our data suggest that o H holoenzyme can progress through the stages to open complexes without the presence of initiating nucleotides. This characteristic is somewhat unusual for Bacillus polymerase-promoter complexes, since in many examples

open complexes are not detectable unless at least the first two nucleotides of the nascent transcript are included in the incubation with the polymerase and the promoter [ 16: 20]. Furthermore, the ability to form open complexes in the absence of nucleotides is not a general feature for a H containing holoenzyme since we found that open complex formation at spoVG, another promoter transcribed by o n holoenzyme does not form open complexes without the presence of initiating nucleotides. In examining the stimulation of transcription by superhelical density, we found that the dependence showed a non-linear response. Changes in superhelical density between -0.024 and -0.050 significantly stimulated the formation of open complexes, whereas changes outside that range had less effect. The requirement for a superhelical template is not a general feature of a H dependent promoters since spoVG can be transcribed from linear templates [21 ]. Thus the requirement for supercoiling must represent some unusual feature of the spolIA promoter polymerase complexes. Supercoiling has been observed to influence transcription initiation from E coli promoters [22, 23]. All of the stages of the initiation reaction have been shown to be sensitive to superhelical density so that no single step is associated with superhelical density dependence. In general, however, the most likely stages to be affected by changes in superhelical density of the template are: l) the transition between open and closed complexes in which the free energy associated with superhelicity contributes to DNA melting at the initiation site [24]; and 2) the binding of the polymerase to form the closed complex where changes in superhelical density can alter the relative positions of the recognition sequences at the promoter due to deformations of the DNA helix [17]. Since the assays we have reported are equilibrium studies, they do not separate the various stages of the transcription reaction. Initial experiments on the rate of transcription initiation at spolIA have been carded out, and these suggest that the formation of heparin resistant complexes is extremely fast. These experiments also suggest that the rate of transcription complex formation is increased by supercoiling of the template but we do not know what reaction step is affected. Temperature dependence of transcription initiation is thought to primarily influence either an isomerization of the polymerase during the transition through the intermediate stage(s) of the reaction, or the DNA denaturation step during which the strands separate at the initiation site [24]. The temperature dependence of heparin resistant complex formation at the spolIA promoter showed a typical non-linear response suggesting that the temperature dependence of the a H holoenzyme complex is similar to those of B subtilis

632 a A holoenzyme and of E coli RNA polymerase complexes [24]. If the stimulation of transcription due to supercoiling of the template affected the same step of the initiation reaction as the temperature it would be expected that the temperature dependence would shift with different levels of supercoiling. Alterations in temperature profiles of this type have been seen with other promoters in which highly supercoiled templates transcribe at near maximal rates even at 15°C [25]. In the case of the spollA promoter, the temperature profile for transcription was effectively the same for all topoisomers suggesting that supercoiling could not substitute for temperature in the reaction and thus that the two reaction conditions affected different steps in the overall process. If the effect of temperature on spolIA transcription is similar to its effect at other promoters [24, 25], then temperature primarily affects the balance between closed and open complexes probably by altering the reaction rate constants for the steps involved. The data described above from a temperature shift experiment support this notion, indicating that lowering the reaction temperature after open complex formation resulted in converting open, heparin resistant complexes to closed, heparin sensitive complexes. Thus the formation of open complexes at the spoIIA promoter does not seem to be an irreversible step as is found at a number of promoters [ 18, 19]. Furthermore, if superhelical density affects a step that is separate from the step which is affected by temperature, it is likely that the supercoiling dependence of the reaction is due to an effect on polymerase binding to form a closed complex rather than on isomerization of intermediate complexes which is thought to involve protein conformational change within the polymerase itself [24]. Activation of the spollA operon has been shown to be dependent on many of the stage zero sporulation genes [7]. The dependence on spoOH is clearly due the requirement for the o 8 sigma factor. Since SpoOF, Spo0B and Spo0A participate in the phosphorelay which results in the phosphorylation of Spo0A [26] it seems likely that spolIA is controlled by Spo0A phosphorylation in vivo. We have reported in vitro evidence to support this notion elsewhere [27]. Spo0A is a member of the response regulator class of proteins [28] and as with many of the operons regulated by this class of proteins, the spoIIA operon is transcribed by RNA polymerase associated with an alternative sigma factor [29, 30]. Thus spollA is a member of a class of regulons affected by both transcription regulators and alternative sigmas. The best characterized of this class of genes are the nitrogen regulon members in the enteric bacteria [31 ]. These genes are transcribed by polymerase containing 054 and require the phosphorylated form of the response regulator NtrC. Holoenzyme containing (~54 is capable of binding to

promoters but is not capable of forming open complexes without a specific ATPase associated activity of the NtrC protein [32, 33]. Efficient formation of open complexes depends on a supercoiled template, however, the role of the supercoiling is unclear [32]. The data we have presented support the idea that Spo0A stimulation of spoIIA transcription involves a mechanism different than the stimulation of nitrogen regulon promoters by NtrC. The key difference is that o H holoenzyme is able to form open complexes at the spoIIA promoter in the absence of the response regulator and thus an ATP dependent step carded out by the response regulator is not required. This difference is not unexpected since oH holoenzyme initiates transcription at non-Spo0A dependent promoters [21, 34]. Transcription levels we observed with the spoIIA promoters were low, with less than 0.1 transcripts per template even at maximum inputs of polymerase. We have found that this level could be dramatically stimulated by adding Spo0A to the transcription reaction so that we believe that it represents a significant characteristic of the polymerase-promoter interaction rather than low specific activity of either the core or the o a preparation we used. Our current model of the interaction of the polymerase with the spollA promoter is that the formation of the closed complexes, or a very early intermediate stage in the reaction is rate limiting and that this stage is the most likely point for control by transcription regulators such as the phosphorylated form of Spo0A. Finally, we note that it has been reported that levels of DNA supercoiling change during certain steps in sporulation [35]. While our results could suggest supercoil density could be used to modulate spollA expression, they do not compel us to do so. It is interesting that the superhelical density range where transcription was most sensitive to change was close to the levels reported for DNA in vivo (-~ - 0.030) [36]. The facts that: I) o H holoenzyme can transcribe spolIA in vitro; and 2) the in vivo level of spoIIA transcription is very low at a time when o H is present [7, 37, 38] may indicate that negative as well as positive controls operate on spolIA transcription initiation. Conclusions The following conclusions were made: 1) formation of heparin resistant transcription complexes at the developmentally regulated promoter of the spollA operon m vttro requires the presence of a H, supercoiling of the DNA template but not the presence of initiating nucleotides. 2) Transcription of templates carrying the spoilA promoter which differed in

633 superhelical density s h o w e d that heparin resistant c o m p l e x formation varied non-linearly with superhelical density. 3) Transcription o f the t o p o i s o m e r s at different temperatures indicated that temperature and template superhelical density affected transcription initiation by different m e c h a n i s m s . 4) C o m p a r i s o n o f the initiation properties o f spollA and nitrogen regulon genes in enteric bacteria suggests that S p o 0 A regulates s p o l l A in a m a n n e r quite distinct f r o m NtrC activation o f the nitrogen regulon promoters.

Acknowledgments We thank L Duncan for preparation of core and o A holoenzyme. This work was supported by a grant from the Natural Sciences and Engineering Research Council to GBS, and by Grant GM 19416 from the National Institute of General Medical Sciences to JAH.

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The effect of supercoiling on the in vitro transcription of the spoIIA operon from Bacillus subtilis.

The spoIIA operon codes for an alternative sigma factor which appears in the early stages of a sigma factor expression cascade during sporulation in B...
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