J. Mol. Biol. (1996) 256, 436–448

The Bacillus subtilis Response Regulator Spo0AStimulates Transcription of the spoIIG Operonthrough Modification of RNA PolymerasePromoter Complexes

Terry H. Bird 2, Janet K. Grimsley 1, James A. Hoch 1 andGeorge B. Spiegelman 2,3*

1Department of Molecular Sporulation in Bacillus subtilis is dependent on the response regulatorSpo0A, which both represses and activates transcription in vitro. Theand Experimental Medicineactivity of Spo0A is increased by phosphorylation. We previouslyResearch Institute of Scripps

Clinic, 10666 North Torrey demonstrated that the phosphorylation increased the ability of Spo0A tostimulate in vivo transcription from the promoter for the spoIIG operon, onePines Road, La Jollaof the operons known to be regulated by Spo0A in vivo. In the workCA 92037, USAreported here we have examined the kinetics of transcription initiation at2Department of Microbiology the spoIIG operon promoter using a single round transcription assay and

and Immunology the kinetics of formation of spoIIG promoter–RNA polymerase complexes3Department of Medical using DNase I footprinting. Both the kinetic assays and the footprint assaysGenetics, University of indicated that the initial binding of the polymerase to the template was notBritish Columbia, 6174 dependent on the presence of Spo0A. The phosphorylated form of Spo0AUniversity Blvd, Vancouver stimulated the rate of initiation by affecting a step that occurred after theBritish Columbia, Canada initial interaction of the polymerase with the template. Phosphorylation ofV6T 1Z3 Spo0A may stimulate transcription by modifying preinitiation complexes

containing the polymerase and the promoter.7 1996 Academic Press Limited

Keywords: Bacillus subtilis; Spo0A; transcription activation; sporulation;response regulators*Corresponding author

Introduction

The spo0A gene product is required by stationaryphase Bacillus subtilis cells to enter the developmen-tal process of sporulation (Piggot & Coot, 1976;Errington, 1993; Hoch, 1993). Spo0A directlyregulates expression of a variety of genes, and bothactivates and represses transcription (reviewed byErrington, 1993; Hoch, 1993; Strauch & Hoch, 1993).Spo0A binds to a DNA consensus sequence calleda 0A box (TGTCGAA: Strauch et al., 1990; Balduset al., 1994). These sequences are found downstreamof the promoters repressed by Spo0A and upstreamof the promoters activated by Spo0A (Spiegelmanet al., 1995). Promoters stimulated by Spo0A includethose recognized by RNA polymerase with either of

two sigma factors: the major sigma vegetative factor,sA, or sH, a sigma factor required for expressionof genes in stationary phase and during sporu-lation (Errington, 1996; Hoch 1993; Moran, 1993;Haldenwang, 1995).

Spo0A is a member of the response regulatorclass of proteins (Ferrari et al., 1985; Kudoh et al.,1985; Trach et al., 1985). Transcription regulatormembers of this family typically have a two-domainstructure: a DNA binding domain, whose activitiesare repressed by a second domain, the repressordomain (Stock et al., 1989; Parkinson & Kofoid,1992). Phosphorylation of the repressor domainactivates the protein. In the case of Spo0A,phosphorylation takes place in the N-terminalregion (D56: Burbulys et al., 1991; Green et al., 1991).Furthermore, the C-terminal portion of Spo0Aalone is sufficient to activate transcription ofdevelopmentally regulated genes in vitro (Grimsleyet al., 1994) and sporulation in vivo (Ireton et al.,1993). Phosphorylation of Spo0A takes placethrough an elaborate mechanism, beginning with

Present address: T. H. Bird, Department of Biology,Indiana University, Bloomington IN 47405, USA.

Abbreviations used: Spo0A-P, phosphorylated Spo0A;Spo0A(-P), either phosphorylated or unphosphorylatedSpo0A.

0022–2836/96/080436–13 $12.00/0 7 1996 Academic Press Limited

Transcription Stimulation by Spo0A 437

either of two kinases, which phosphorylate theproduct of the spo0F gene. The phosphate on Spo0Fis transferred to another intermediate, Spo0B, andthen to Spo0A. The intermediates in the phospho-relay serve in part to provide targets for phospha-tases to intercept the signal for Spo0A activation(Ohlsen et al., 1993; Perego et al., 1994).

One of the key targets of Spo0A activation is thepromoter for the spoIIG operon. This operon isinduced after sporulation onset and encodes one ofthe first of the sporulation-specific sigma factors(Kenny & Moran, 1987; Kenney et al., 1988, 1989).Mutational analysis and in vitro footprinting studieshave shown that there are two sets of 0A boxesupstream of the spoIIG promoter. One set (termedsite 1) contains boxes centered at −84 and −94 andthe second set (site 2) contains boxes centered at −50and −40 (Satola et al., 1991; Baldus et al., 1994).Site 1 is not needed for normal regulation of thepromoter in vivo (Baldus et al., 1994). The spoIIGpromoter is used by RNA polymerase containingthe major vegetative sigma factor, sA. However, thepromoter sequence lacks a consensus −35 sequence17 to 18 bp upstream of the likely −10 sequence(Satola et al., 1991). There is a −35 consensussequence 22 bp upstream of the −10; however, thissequence would be out of the correct orientation forefficient use. It has been suggested that the unusualspacing might be the major barrier to unaidedtranscription (Satola et al., 1991).

Previous work has shown that phosphorylatedSpo0A (Spo0A-P) stimulates in vitro transcriptionfrom a DNA fragment containing the spoIIGpromoter very effectively. In contrast unphosphoryl-ated Spo0A stimulates transcription very poorly(Satola et al., 1992; Bird et al., 1993; Baldus et al.,1994; Bird, 1995). It has been reported thatphosphorylation of Spo0A increases it affinity for0A boxes (Trach et al., 1991) and specifically forweaker binding sites, such as those at site 2 of thespoIIG operon (Baldus et al., 1994). However, it is notknown how the binding of Spo0A to the 0A boxstimulates transcription and whether the differencebetween Spo0A and Spo0A-P can be attributedsolely to the differences in binding affinity.

In the experiments reported here, we have begunto investigate the mechanism of the phosphoryl-ation-dependent Spo0A stimulation of transcrip-tion. We have used DNase I footprinting andanalysis of the kinetics of formation of complexesthat are resistant to the polymerase inhibitorheparin to study initiation directed by Spo0A andSpo0A-P. The results of the two approaches bothindicate that RNA polymerase readily bindsunaided to the spoIIG promoter, but the resultingcomplex is inactive in transcription. Addition ofboth Spo0A and Spo0A-P results in formation ofdifferent RNA polymerase–promoter complexes,but only the complex induced by Spo0A-P caninitiate RNA synthesis rapidly. Thus the dataindicate that the effect of phosphorylation of Spo0Ais on its ability to modify RNA polymerase–pro-moter complexes.

Figure 1. Polyacrylamide gel electrophoresis of tran-scription products from the spoIIG promoter. The 600 bpDNA fragment containing the spoIIG promoter (2 nM finalconcentration) was incubated with varying concentrationsof either Spo0A (lanes 1 to 7) or Spo0A-P (lanes 8 to 14)in transcription buffer containing GTP and ATP for threeminutes at 37°C. RNA polymerase was added to a finalconcentration of 25 nM and allowed to form initiationcomplexes for five minutes. The complexes werechallenged with a mixture of heparin, CTP and UTP toallow heparin-resistant complexes to elongate RNA, butblock further initiation. After ten minutes of elongation,stop buffer was added and the samples were elec-trophoresed through an 8% polyacrylamide gel contain-ing 7 M urea. The gel was exposed to Kodak XAR film,overnight. The arrowhead marks the position of thespoIIG transcript and the origin of electrophoresis isindicated. The final concentration of Spo0A or Spo0A-Pwas: 0 nM, lanes 1, 8; 10 nM, lanes 2, 9; 20 nM, lanes 3,10; 50 nM, lanes 4, 11; 80 nM, lanes 5, 12; 160 nM, lanes6, 13; 320 nM, lanes 7, 14.

Results

Previous experiments showed that preincubationof the phosphorylated form of Spo0A (Spo0A-P)with a template containing the spoIIG promoterstimulated single-round transcription (Satola et al.,1991; Bird et al., 1993). An example of the stimul-ation is illustrated in Figure 1. Template DNA andSpo0A(-P) were incubated for three minutes at 37°Calong with initiating nucleotides [a-32P]GTP andATP. RNA polymerase was then added and, after anadditional five minutes, a mixture of UTP, CTP andthe RNA polymerase inhibitor heparin was added.After an additional ten minutes, to allow elongation,transcripts were separated by electrophoresis. Thetranscription reaction resulted in a single labelledproduct that could be easily quantified to determinethe number of promoter–polymerase complexescapable of elongating RNA under the heparinchallenge. The maximum stimulation of transcrip-tion was reached at approximately 200 nM Spo0A-Pand this level was maintained at higher concen-trations. Spo0A also stimulated transcription, butmuch less effectively. Four different preparations ofpolymerase were tested in similar experiments andno differences to the response to Spo0A-P weredetected. In addition, three different preparations ofSpo0A showed the same behavior.

The data in Figure 2 demonstrate that both ATPand GTP were necessary to form complexes that

Transcription Stimulation by Spo0A438

Figure 2. Requirement of initiating nucleotides forheparin resistance. DNA template (2 nM) was incubatedwith either Spo0A or Spo0A-P (400 nM in either case) intranscription buffer containing different sets of initiatingnucleotides for three minutes at 37°C. RNA polymerase(25 nM final concentration) was added and allowed toform complexes for three minutes. A mixture of heparinand nucleotides that would allow elongation was thenadded and, after five minutes, the reactions wereterminated with stop buffer and electrophoresed asdescribed in Materials and Methods. The gel was exposedto Kodak XAR film overnight. The nucleotides present inthe incubation prior to heparin addition were: ATP, lanes1, 2; ATP and GTP, lanes 3, 4; ATP, GTP and CTP, lanes5, 6. Reactions containing Spo0A are in odd-numberedlanes, those containing Spo0A-P are in even-numberedlanes. The arrowhead marks the position of the spoIIGtranscript and the origin of electrophoresis is indicated.

The effect of nucleotides on heparin-stable complexformation indicates that the spoIIG promoter is in aclass of Bacillus promoters in which heparinresistance is not attained until RNA synthesis hasbeen initiated (Whipple & Sonenshein, 1992).

Spo0A-P stimulation of the initiation rate

To investigate the role of Spo0A-P in transcriptionstimulation, we followed the kinetics of formation ofheparin-resistant complexes. Template DNA (2 nM),ATP, GTP and Spo0A(-P) (400 nM) were preincu-bated at 37°C for three minutes and then RNApolymerase (25 nM) was added. At times afterpolymerase addition, samples were removed andadded to heparin, CTP and UTP, and the level oftranscripts produced after five minutes elongationwas determined (Figure 4). In the presence ofSpo0A-P, there was a rapid rise in heparin-resistantcomplexes and the maximum level was stable over20 minutes. In the presence of Spo0A, both theinitial rate of complex formation and the maximumlevel of complexes formed were significantly lower.Thus Spo0A-P stimulated both the final extent ofcomplexes formed and the initial rate of heparin-resistant complex formation. Heparin-resistant com-plexes were stable for over 60 minutes, suggestingthat dissociation from the heparin resistant statewas negligible (data not shown).

To analyze the effect of Spo0A-P in more detail,we measured the rate of heparin-resistant complexformation at different concentrations of Spo0A(-P).In all cases, Spo0A(-P) was preincubated with thetemplate (2 nM) along with ATP and GTP for threeminutes, and the reaction was started by theaddition of RNA polymerase (25 nM). Rates wereanalyzed using a composite rate assay (Stefano &Gralla, 1982) to determine the value for kobs, theobserved overall forward rate constant for heparincomplex formation. The rate assay plots were linear,indicating that one step in the reaction was ratelimiting (Figure 5). The value of kobs was determinedfrom the absolute value of the rate plot slopes. Thevalues for kobs increased with increasing Spo0A-P

could elongate RNA when challenged with heparin.When RNA polymerase was first incubated withSpo0A-P or Spo0A, template DNA and ATP, andthen challenged with a mixture containing the threeremaining nucleotides and heparin, no transcriptswere produced (lanes 1 and 2). When GTP wasincluded with ATP, RNA polymerase and template,challenge with heparin plus CTP produced tran-scripts when Spo0A-P was present, but little or notranscripts when Spo0A was present (lanes 3 and 4).When the initiation reaction contained ATP, GTPand CTP (lanes 5 and 6) slightly more transcriptswere produced than when just ATP plus GTP wereadded. The nucleotide sequence of the spoIIGtranscript (Figure 3) showed that in the presence ofATP and GTP an 11 base RNA could be produced.

Figure 3. DNA sequence of thespoIIG promoter region, and pro-tected and exposed bases. The DNAsequence (Kenny et al., 1989) isshown with the numbering relativeto the +1 position, which is theunique start site of transcription(Kenney et al., 1989; Bird et al., 1993).The proposed −10 and −35 se-quences are enclosed (Kenny et al.,1989) and the 0A boxes are under-lined (Baldus et al., 1994). The

regions protected from DNase I by Spo0A and Spo0A-P are overlined. The regions protected by RNA polymerase areindicated by the open-ended bar. The exact 5' end of the protected region was not determined (represented by brokenlines). Bases that were more sensitive to DNase I in the presence of RNA polymerase alone are indicated by open circles.Bases that were more sensitive to DNase I in the presence of Spo0A and RNA polymerase are indicated by arrowheads.Prominent sites of DNase I cleavage near +1 that were more protected in the presence of RNA polymerase and Spo0A-Pare indicated by filled circles. The data for DNase I protection are given in Figures 8 and 9.

Transcription Stimulation by Spo0A 439

Figure 4. Time-course of formation of heparin-resistantcomplexes. Template DNA (2 nM) was incubated witheither 400 nM Spo0A (q) or Spo0A-P (w) in transcriptionbuffer containing GTP and ATP for three minutes at 37°C.At time zero, RNA polymerase was added (finalconcentration 25 nM) and at intervals, samples werewithdrawn and added to a mixture of heparin, CTP andUTP. After five minutes to allow for elongation, theproducts of the transcription reaction were elec-trophoresed through denaturing polyacrylamide gels andthe level of transcripts produced was determined asdescribed in Materials and Methods. Note that the scalesfor transcripts produced in the presence of Spo0A (right)and Spo0A-P (left) are not the same.

Figure 5. Rates of heparin-resistant complex formationin the presence of Spo0A and Spo0A-P. Initiationreactions containing 2 nM DNA, different concentrationsof Spo0A or Spo0A-P and ATP plus GTP were allowed toincubate for three minutes and then started by theaddition of RNA polymerase (25 nM final concentration).Samples were withdrawn and added to a mixture ofheparin, CTP and UTP. After five minutes to allowelongation, the products were electrophoresed thoroughpolyacrylamide gels and the level of transcripts producedwas determined as described in Materials and Methods.Duplicate samples were allowed to initiate for 15 minutesto determine the completion level (Cmax). The approach toCmax was calculated as described in Materials andMethods, and is plotted against the time of initiation. Theslope of the rate assays is the value of −kobs. Only aselection of assays carried out with Spo0A are shown, forclarity (top panel); the bottom panel shows data forSpo0A-P. The concentrations of Spo0A(-P) in the reactionswere: (q) no Spo0A; (Q) 50 nM; (R) 100 nM; (r)200 nM; (W) 400 nM; (w) 800 nM.

concentration (Figure 6). Spo0A also increased kobs,but to a much smaller degree.

The relationship of kobs to individual rateconstants that control the formation of heparin-resistant complexes depends on the reactionmechanism and on the rate-limiting step in thereaction (Strickland et al., 1975; Stefano & Gralla,1982; McClure, 1985). In general, RNA synthesisinitiation proceeds through a binary complex (theclosed complex) formed by the binding of RNApolymerase to the promoter. The initial complexthen undergoes rate-limiting isomerizations to acomplex in which the DNA strands are separatedand the polymerase can bind nucleotides andcommence polymerization of RNA very rapidly (theopen complex). The initial complex between thepolymerase and the DNA is normally assumed tobe heparin sensitive. In the assays reported here,heparin resistance required initiation of RNAsynthesis; therefore, these assays did not distinguishbetween uninitiated, open complexes and initiatedcomplexes. The effect of a transcription regulator onthese steps can be detected by comparing thedependence of the overall forward rate constant(kobs) on the polymerase concentration with andwithout the transcription activator (see Hawley &McClure, 1982; Malan et al., 1984; Shih & Gussin,1984; Spassky et al., 1984).

To begin such an analysis, we measured thedependence of the reaction rate on RNA polymeraseconcentration in the presence and absence of400 nM Spo0A-P. Reactions were started by addingRNA polymerase to template that had beenpreincubated either alone or with Spo0A-P. The

level of complexes formed after different times wasmonitored as described above, and the data wereused to determine kobs from initial rate plots such asthose seen in Figure 5. Values of tau (1/kobs = tau)are plotted versus the reciprocal of the RNApolymerase concentration in the reaction (Figure 7).

In the presence of Spo0A-P, the slope of the tauversus 1/RNA polymerase concentration did not

Transcription Stimulation by Spo0A440

Figure 7. Dependence of kobs on RNA polymeraseconcentration. Initiation rate assays contained 2 nM DNA,without Spo0A (q) or with 400 nM Spo0A-P (w), GTPand ATP, which were incubated at 37°C for three minutes.Reactions were initiated by addition of RNA polymeraseand, at various times, samples were added to a mixtureof heparin, CTP and UTP. After ten minutes of elongation,the transcripts produced were measured as described inMaterials and Methods. The values for kobs weredetermined from plots such as those shown in Figure 5and are plotted as the reciprocal of kobs (tau) versus thereciprocal of the polymerase concentration used in thereaction. The error bars represent standard error values.A line to fit the data by least-squares analysis washorizontal for the data obtained without Spo0A and hada slight negative slope for the data obtained with Spo0A-P(not shown).

Figure 6. Dependence of kobs on Spo0A(-P) concen-tration. kobs values, determined from Figure 5 are plottedagainst the concentration of Spo0A(-P) in the initiationreaction. (w) The reaction containing Spo0A-P; (q) thereactions containing Spo0A.

show the expected positive slope and was notsignificantly different than zero over the range ofpolymerase concentrations tested. The line derivedby least-squares analysis for the Spo0A-P case hada negative slope (not shown), which might occur ifthe polymerase demonstrated inhibition at highconcentrations. However, the slope was very smalland equal to zero within the experimental error.The level of transcription observed when no Spo0Awas added was very low, so the tau values had alarge error, and again there was no obviousdependence on the concentration of polymerase.The difference in ordinate intercepts of linesthrough the two data sets reflects stimulation oftranscription by Spo0A-P. One explanation for thelack of dependence on polymerase is that the initialbinding of the polymerase rapidly reached equi-librium, or completion, relative to other steps in thereaction and so did not affect the overall reactionrate. We assume that the reaction rate was notlimited by binding or polymerization of nucleotidesand that Spo0A-P must bind to the DNA promoterto stimulate the reaction rate.

Kinetic footprints

The kinetic data suggested that RNA polymerasemight rapidly equilibrate with the spoIIG promoterto form a complex and that Spo0A-P might alter thatcomplex to one that could initiate RNA synthesis.This suggested that we might be able to detect oneor more intermediates in the initiation reactionusing a structural assay. To search for earlyintermediate complexes we examined the time-course of appearance of RNA polymerase-depen-dent DNase I footprints at the spoIIG promoter,leaving out GTP from the reaction conditions. Thisensured that complex formation did not proceedbeyond the heparin-sensitive stage. Template DNAfragment that had been labelled at the BamHI endwas incubated without, or with, either Spo0A orSpo0A-P for three minutes at 37°C. Because

Spo0A-P preparations contained ATP from thephosphorelay reactions, ATP was added to allexperiments. At time zero, RNA polymerase wasadded, and then samples were removed and treatedwith DNase I for ten seconds. The DNA fragmentswere separated on denaturing polyacrylamide gels,and the gels were subject to autoradiography.

Figure 8 shows results from three kineticfootprint experiments; A shows the formation of thefootprint by RNA polymerase alone. Lanes 1 and 2show control digests of the labelled DNA fragment.In the first time point (lane 3) of the complexformation, which represents five seconds of incu-bation of the polymerase and DNA and a furtherten seconds of treatment with DNase I, generalprotection of a wide region of the promoter wasobserved. The 5' boundary of this region was atapproximately −160 (not seen in Figure 8) and thisdid not change under any of the conditions tested(see Discussion). The 3' boundary was at +20(Spiegelman et al., 1995; also not shown in Figure 8).The polymerase did not show extensive protectionof the promoter as is seen in the initial (closed)complexes formed by E. coli RNA polymerase andstrong promoters such as the T7A1 promoter (seeKrummel & Chamberlin, 1989, and referencestherein). There was a low level of backgroundcleavage at all positions, possibly indicatingreversible equilibrium with the unbound state.Addition of the polymerase caused the appearanceof a conspicuous hypersensitive site at −45 andminor increases in DNase I sensitivity at sites −33,

Transcription Stimulation by Spo0A 441

Figure 8. Early kinetics offormation of RNA polymerase–Spo0A(-P)–spoIIG promoter com-plexes. DNase I footprint reactionswere performed as described inMaterials and Methods using thePvuII-BamHI DNA fragment frompUCIIGtrpA, labelled at the BamHIend. For each panel, a large reactionmixture containing 2 nM DNA and400 nM ATP was prepared. Controlsamples were withdrawn andtreated with DNase I for ten seconds(lanes 1 and 2 in A; lane 1 in B; lane1 in C). For the remaining reactionsin A, RNA polymerase was thenadded (final concentration of100 nM) and at the times indicatedfor the various lanes, samples werewithdrawn and treated with DNaseI for ten seconds. For reactions in B,lanes 2 to 6, Spo0A (to 400 nM) wasadded to the DNA and allowed tobind for five minutes. A sample waswithdrawn and treated with DNaseI (lane 2). RNA polymerase was thenadded and at the times indicatedsamples were withdrawn andtreated with DNase I (lanes 3 to 6).For the reactions in C, lanes 2 to 6,Spo0A-P was added (to 400 nM) andallowed to bind for five minutes.One sample was withdrawn andtreated with DNase I (lanes 2). RNApolymerase was then added andsamples were withdrawn as in B(lanes 3 to 6). The positions of basesrelative to the start of transcription(+1) are indicated. The open bar to

the right of each panel shows the extent of protection by RNA polymerase. Filled areas in the open bar represent theregions protected by Spo0A or Spo0A-P. Various positions are indicated to the right of the panels. The crossed daggersshow significant positions that were not protected; the openhead pins show positions that were not protected by RNApolymerase alone, but whose protection was increased when Spo0A(-P) was added; the filled arrowheads in A showpositions that were exposed when RNA polymerase was added. The stippled arrowhead in B is in the same positionas the filled arrowhead in A. The striped arrowheads are at positions that became exposed over the time-course, inB and C.

−34, −21 and −10. The rapid appearance of the RNApolymerase footprint corroborated the kinetic data,which had suggested a rapid equilibration betweenbound and unbound polymerase. We termed thecomplex formed by the polymerase and thepromoter the CI complex.

The footprints formed in the presence of Spo0Aare shown in Figure 8 B. Lane l shows the DNaseI digestion pattern in the absence of protein, andlane 2 shows the protection pattern in the presenceof Spo0A alone. As reported earlier, unphosphoryl-ated Spo0A bound to a region around −84 to −100(site 1; Satola et al., 1992; Baldus et al., 1994). Bindingto the 0A boxes at positions −40 and −50 was notdetected with unphosphorylated Spo0A, againconfirming the observations by Baldus et al. (1994).Addition of RNA polymerase (lanes 3 to 6) resultedin protection of a broad area of the DNA within thefirst 15 seconds (five seconds of initiation and ten

seconds of DNase I treatment). The −45 position,which was hypersensitive in Figure 8A, was notunusually sensitive when Spo0A was present. Theregion between −24 and +1 was more protectedrelative to the pattern seen with RNA polymerasealone.

Within 30 seconds of adding the polymerase, thefootprint pattern had changed (lane 4, Figure 8B).The base at −45, which appeared to be unprotectedin lane 3, had been protected. In contrast, bases at−17, −27 and −28 were now more sensitive to DNaseI. Other slight increases in DNase I sensitivity wereseen at positions −33, −34 and −56 to −59. No furtherchanges were seen (lanes 5 and 6). The change inDNase I pattern seen over the first 30 secondssuggests that the polymerase bound in one fashionand then underwent a conformational change.Because the change was seen only in the presenceof Spo0A, we concluded that the change was

Transcription Stimulation by Spo0A442

induced by the interaction of the polymerase andSpo0A and we termed the complex containing thehypersensitive sites at −27 and −28 the CII complex.

The reduction in DNase I cleavage at the −45position might be explained by the binding ofSpo0A, since there is a 0A box that covers this site(Baldus et al., 1994; Figure 3). If Spo0A did bind tothis site, it must have been due to a cooperativeeffect with the polymerase, since it did not bindthere in the control (lane 2). The increase in DNaseI digestion at −27, −28 suggested that the binding ofthe polymerase and Spo0A resulted in distortion ofthe DNA in this region. The digestion pattern on theother strand of the DNA showed the same positionsexposed (Spiegelman et al., 1995), indicating thatthis was a double-strand distortion. The change inthe DNA structure did not significantly change thebinding of the polymerase to the region between−10 and +1, since positions −7, −4 and +1 were stilldigested. However, the background was reduced,suggesting stabilization of the bound complex.

To examine the effect of phosphorylating Spo0A,Spo0A-P was preincubated with the labelled DNA

before adding the polymerase, and a time-course offootprint formation was examined (Figure 8C).Comparison of lane 2 in Figure 8C with the control(lane 1) and with lane 2 in B demonstrated thatSpo0A-P bound more tightly to the region round−90 than did Spo0A, since the protection was morecomplete. In addition, unlike Spo0A, Spo0A-Pbound to the 0A boxes at −50 and −40. The sameeffects of Spo0A phosphorylation were reportedearlier (Baldus et al., 1994).

In the first time-point after adding thepolymerase, the footprint showed ratheruniform protection between −60 and +10 (lane 3,Figure 8C). The DNase I sensitivity of positions −27and −28 that was seen when Spo0A was added, wasnot observed with Spo0A-P. Bases at positions −10,−17, −21, −33, −34 and −56 to −59 showedslight increases in DNase I sensitivity. Thusphosphorylated Spo0A induced a change that eithereliminated the −27, −28 sensitivity or prevented itsformation. The complex represented by the patternseen in the presence of Spo0A-P was designatedCIII.

Figure 9. Effect of Spo0A andSpo0A-P concentration on the com-plexes formed at the spoIIG pro-moter. The PuvII-BamHI DNAfragment (labelled at the BamHIend) was incubated alone (lanes 1and 2 left panel; and lane 1, rightpanel), or in the presence of increas-ing concentrations of either Spo0A(left panel) or Spo0A-P (right panel)for five minutes. The concentrationsof Spo0A(-P) are indicated on theconcentration axis in the Figure.RNA polymerase was added to alllanes except lane 1, left panel; andafter five minutes of incubation, thesamples were treated with DNase Ifor ten seconds. Positions are num-bered relative to the start of tran-scription (+1). The openhead pinsindicate prominent sites of cleavagegradually protected by Spo0A-Pand RNA polymerase; the shadedarrowheads represent bands ex-posed by RNA polymerase, butprotected in the presence of Spo0A-P and RNA polymerase; and thestriped arrowheads show bandsexposed either by RNA polymeraseand Spo0A or by RNA polymeraseand only low levels of Spo0A-P.

Transcription Stimulation by Spo0A 443

Effect of Spo0A(-P) concentration onDNase I footprints

Since the initiation rates were lower with lowerlevels of Spo0A-P (Figure 6), we examined thecomplexes formed at different concentrations ofSpo0A(-P) (Figure 9). Lane 1, left panel, shows thedigestion pattern of the labelled DNA fragment.Lanes 2, left panel, and lane 1, right panel, show theDNase I digestion pattern when RNA polymerasewas added. Changes in the digestion patterninduced by the polymerase were more obvious inthis experiment than in the data shown in Figure 8,but the overall pattern was the same. In particular,the presence of the polymerase increased thesensitivity of bases at positions −54, −45, −34, −33,−17 and −10 compared with the control.

Different concentrations of Spo0A (lanes 3 to 7,left panel) were incubated with the DNA and thenRNA polymerase was added to the reaction. Afterfive minutes the reactions were subjected to a tensecond DNase I treatment. Addition of low levels ofSpo0A increased the DNase I sensitivity atpositions −27 and −28 and reduced the sensitivity ofmost other bases, especially −45. In other data, wehave found that above 600 nM Spo0A the hypersen-sitivity of the −27 and −28 positions began todiminish (Bird, 1995). While the −20 to −40 regionwas increasingly protected at high Spo0A, there wasstill only partial protection of the region around +1.

Lanes 2 to 6 (Figure 9, right panel) show theDNase I digestion pattern obtained when RNApolymerase and Spo0A-P were incubated with thetemplate before digestion with DNase I. At thelowest levels of Spo0A-P tested, the footprintpattern lacked the prominent cleavage at −45, −21and −17 characteristic of CI complexes and con-tained a low but significant level of enhancedcleavage at positions −27 and −28. By 200 nMSpo0A-P, the −27 and −28 positions were protectedand there was substantial protection of thepromoter in the region −13 to −60, in particular nearthe +1 position. The sensitivity of the bases atpositions −27 and −28 at low inputs of Spo0A-P andtheir disappearance at higher inputs suggested thatlow levels of Spo0A-P acted like Spo0A. We usedKMnO4 to search for unpaired bases in the CIII

complex, and found the DNA was resistant to thisreagent (data not shown). Furthermore, CIII for-mation was not temperature sensitive (Bird, 1995).We take this as evidence that the CIII complex doesnot contain unpaired DNA bases and thus is not anopen complex in the sense used for other promoters.

Discussion

We used kinetic analysis and DNase I footprint-ing to examine Spo0A(-P) stimulation of transcrip-tion initiation at the spoIIG promoter. There werefour salient results from these studies. First,composite rate assay plots, which were used tomeasure overall rate constants for transcriptioninitiation, were linear. The linearity indicates that

the forward reaction was dominated by the rate ofa single step. The overall observed rate constant(kobs) for the reaction-limiting step was increasedslightly by Spo0A and dramatically by Spo0A-P.Second, in the presence of Spo0A-P, the overallforward rate constant did not increase with thepolymerase concentration, suggesting that therate-limiting step in initiation was not dependent onbinding of the polymerase to the promoter. Third,DNase I footprinting showed that RNA polymerasealone rapidly formed a complex with the spoIIGpromoter. Fourth, the footprints observed in thepresence of Spo0A and Spo0A-P differed from eachother and from the footprint observed in thepresence of RNA polymerase alone, suggesting thatSpo0A(-P) altered the preinitiation complexes. Sincetranscription initiation was rapid and efficient in thepresence of Spo0A-P, we presume that the complexobserved with Spo0A-P is a direct precursor to theinitiated state.

On the basis of these observations, we suggest thefollowing model for Spo0A(-P) stimulation oftranscription initiation at spoIIG. RNA polymerasebinds in a rapid and reversible manner to thetemplate. This binding equilibrates rapidly relativeto other steps in the reaction, accounting for theminimal contribution of polymerase concentrationto the reaction rate and for the immediateappearance of the polymerase footprint observed inFigure 8. While both Spo0A and Spo0A-P can bindindependently to the template, they do notappreciably affect the equilibration of the poly-merase binding. This implies that Spo0A andSpo0A-P affect the rate of the reaction by interactingwith RNA polymerase that has already bound tothe template. There was some evidence of synergybetween binding of Spo0A and the polymerase,since Spo0A did not bind to site 2 alone, butappeared to do so when the polymerase waspresent (Figure 8B).

We propose that Spo0A(-P) accelerates a rate-limiting modification of the initial polymerase-promoter complexes, thus increasing the overallforward rate constant with Spo0A-P concentration.The modification induced by Spo0A (forming the CII

complex) did not permit rapid initiation, suggestingthat a major barrier to initiation was subsequent tothe formation of this complex. However, assumingthat CII can act as an intermediate in the initiationreaction, stimulation of its formation might explainthe low-level stimulation of initiation by Spo0A. Themodification induced by Spo0A-P (forming the CIII

complex) was accompanied by a high rate ofinitiation, suggesting that the CIII complex formssubsequently to the barrier to transcription in-itiation at the spoIIG promoter. We propose thatphosphorylating Spo0A endows it with the abilityto modify the initial RNA polymerase-promotercomplexes to form complexes that can initiate RNAsynthesis rapidly.

We have not rigorously proved that CI, CII and CIII

are successive stages in initiation. For example,although CI complexes are replaced by CII com-

Transcription Stimulation by Spo0A444

plexes in the time-course experiment shown inFigure 8B, this event could reflect dissociation fromCI complexes and reassociation as CII complexes.Similarly, the data in Figure 9 suggested stronglythat CII complexes are precursors to CIII complexes,since at low Spo0A-P inputs, where the rate ofinitiation was lower, the complexes had CII

characteristics. However, the apparent change incomplexes could reflect dissociation of CII complexesand re-formation of a more stable CIII complexesdirectly. Furthermore, we may have not detected CI

complexes in the time-course of CIII formation(Figure 8C), as had been seen in the formation of CII

complexes (Figure 8B), because the overall rate ofthe reaction driven by Spo0A-P was very fast.However, it is also possible that formation of CIII

complexes does not involve CI complexes. Ad-ditional work will be needed to establish conclus-ively the relationship of the complexes detected inthe footprint assays.

The Spo0A-P preparations used in our studieswere 50% phosphorylated, so all reactions containedboth Spo0A and Spo0A-P. Furthermore, Spo0A orSpo0A-P were added before the polymerase in bothrate assays and footprint assays. Since Spo0A didnot stimulate initiation effectively and induced CII

complex formation, then perhaps it should haveinhibited the activity of Spo0A-P. The following twoconsiderations may explain why the data weobtained did not show such an inhibition. First,neither Spo0A nor Spo0A-P forms tight complexeswith DNA. For example, gel retardation exper-iments with these proteins showed extensivesmearing of the retarded bands (Bird & Spiegelman,unpublished observations). Therefore, when Spo0Ais added before the polymerase, we do not expectit to bind irreversibly the template, creating asubstrate that would trap polymerase in anunproductive state. We have shown that at constantSpo0A concentration, low levels of phosphorylationof Spo0A stimulate transcription, suggesting thatunphosphorylated Spo0A is not inhibitory (Birdet al., 1993). Secondly, gel retardation experimentsalso showed that complexes of Spo0A(-P), RNApolymerase and the template were not stable untilboth ATP and GTP were added (Bird & Spiegelman,unpublished observations). This result agrees withthe test of heparin stability in Figure 2. We envisionthat before initiation there is an equilibrationbetween complexes containing various combi-nations of Spo0A(-P), RNA polymerase and thetemplate, and none of the complexes detected in thefootprinting assays (which are all heparin sensitive)would be stable endpoints. It is instructive to pointout that the spoIIG promoter is in the class of Bacilluspromoters at which RNA polymerase does not formstable complexes until after initiation of RNAsynthesis. This is characteristic of some but not allBacillus promoters (Dobinson & Spiegelman, 1985,1987; Whipple & Sonenshein, 1990, 1992; Rojo et al.,1993; Jiang & Helmann, 1994). At these promotersthe steps in initiation are rapidly reversible untilcommitment to elongation, and the DNase I

footprints do not show close contact with thetemplate until after synthesis of an oligomeric RNA.

One proposal to explain the lack of promoteractivity of the spoIIG promoter in the absence ofSpo0A-P is that the −35 and −10 consensussequences are separated by 22 bp rather than theoptimum 17 bp (Kenney et al., 1989; Satola et al.,1991). The effect of increasing spacer length in E. colipromoters appears to act through the change inrelative orientation of the two consensus sequences,which at the spoIIG promoter might be as much as180° out of optimal position (e.g. see Stefano &Gralla, 1980, 1982; Mulligan et al., 1984; Ayers et al.,1989; Warne & deHaseth, 1993). An alternative viewof the spoIIG promoter is that since the −35 sequenceis not in it is proper orientation, the promoter lacksa −35 sequence and the low level of transcriptionreflects this deficiency.

The complex formed by Spo0A–polymerase–pro-moter (CII) had an unusual structure as indicated byhypersensitivity to DNase I, which was not inducedby either Spo0A alone or by the polymerase alone(at positions −27 and −28). The complex containingphosphorylated Spo0A did not show the DNAdistortion. If we assume that the altered DNAstructure reflects the extended spacer region,Spo0A-P might resolve the distortion and stimulateinitiation by allowing the polymerase to releasecontacts with the −35 region while holding it ontothe DNA through protein–protein contacts. If thespoIIG promoter is viewed as lacking a −35consensus, then the role of Spo0A-P would be toposition the polymerase so that sA would contactthe −10 sequence. The latter model for Spo0A-P issimilar to the role of the PhoB protein of E. coli(Kimura et al., 1989; Makino et al., 1993). The twomodels are rather similar in the mechanism ofinteraction of RNA polymerase and Spo0A-P, butdiffer in the nature of the barrier to transcriptioninitiation that they overcome.

Figures 8 and 9 show RNA polymerase protectionof the DNA extended upstream of the site 1 0Aboxes. As the area of protection is probably toolarge to be covered by one polymerase, this mayrepresent the binding of a second polymerase.Because 0A boxes resemble −35 consensus se-quences, the site 1 0A boxes may also stimulatebinding of RNA polymerase. However, site 1 is notneeded for normal regulation of the spoIIG promoterin vivo (Baldus et al., 1994) and there was only oneinitiation site in vitro (Bird et al., 1993; and Fig-ures 1 and 3). The significance of the upstreambinding, if any, is unclear at present. This bindingcould be related to the apparent decrease ininitiation rate at high enzyme inputs (Figure 7).

Spo0A has significant amino acid identity withOmpR, the regulator for ompF and ompC genes inE. coli. Tsung et al. (1990) suggested that OmpRstimulated transcription by stimulating the for-mation of closed complexes. This activity would beequivalent to stimulating the formation of CI

complexes and so is clearly different from the effectwe observed with Spo0A. Instead Spo0A appears to

Transcription Stimulation by Spo0A 445

be more like phage l cI and cII transcriptionregulators, which stimulate isomerization of poly-merase-promoter complexes (Hawley & McClure,1982; Shih & Gussin, 1984). Formally, the nitrogenregulator NtrC has a similar function (reviewed byGeiduschek, 1993; Merrick, 1993; Morrett &Segovia, 1993), but the unusual nature of the sigmafactor sN imposes distinctive characteristics onNtrC. Since sN is defective in opening the DNAstrands, this function is performed in part by NtrCitself (Sasse-Dwight & Gralla, 1988; reviewed byMerrick, 1993). In contrast, for other responseregulators, the sigma function is intact, but thepolymerase is prevented from proceeding at someother step (reviewed by Adhya & Garges, 1990).

Another regulator, MerR, is proposed to distortthe promoter DNA to align −35 and −10 regions(Frantz & O’Halloran, 1990; Parkhill et al., 1993).MerR is a repressor and activator of the mer operonof transposon Tn501. When MerR has boundmercury, its binding to the mer promoter causesDNA distortion and leads to transcription acti-vation. MerR-dependent DNA distortion occurs inthe absence of RNA polymerase and, unlike Spo0A,MerR binds DNA with high affinity.

While the data we have presented indicate thatSpo0A-P modifies RNA polymerase-spoIIG pro-moter complexes, many details of the mechanismhave not been worked out. In particular, we areinterested in the relationship between the com-plexes revealed in the footprints. We are alsointerested in what subunit of the polymerase iscontacted by Spo0A. Spo0A has the property ofstimulating transcription by polymerases withdifferent sigma subunits. For example, Spo0A-Pstimulates transcription from the spoIIA operon,which is transcribed by polymerase containing thesH subunit (Wu et al., 1989, 1991; Trach et al., 1991).At spoIIA and other sH promoters, the 0A boxesare located further upstream than the −50 site atspoIIG and they are in the opposite orientation(Spiegelman et al., 1995). The significance of thesechanges has not been discovered.

Materials and Methods

In vitro transcription assays

RNA polymerase was isolated as described (Dobinson& Spiegelman, 1987). The final polymerase preparationwas free of DNase and RNase, and was greater than 98%pure as judged by staining sodium dodecyl sulfate/poly-acrylamide gels of polymerase samples with Coomassieblue. The concentration of polymerase was determinedusing the Lowry assay (Sandermann & Strominger, 1972),with bovine serum albumin (Fraction V, Sigma ChemicalCo.) as a standard. The polymerase preparations werefound to be 30 to 50% active when measured in DNAsaturation, single round transcription assays. Kineticexperiments report the concentration of polymeraseprotein added to the assays.

The template was a 600 bp DNA fragment generated byPvuII digestion of the plasmid pUCIIGtrpA. This plasmid(Satola et al., 1991) carries a DNA fragment containing the

spoIIG promoter and the trpA transcription terminator,which functions effectively in vitro. The fragment waspurified by electroelution from an agarose gel used toseparate it from the vector. The fragment contains at least100 bp of vector sequences on either side of the spoIIGsequences. The concentration of promoter-containingDNA fragment was determined using absorbancy at260 nm. Stock solutions of the isolated fragment werestored in 10 mM Hepes (pH 8.0), 20 mM potassiumacetate, 0.1 mM EDTA at 4°C.

Standard transcription assays contained as finalconcentrations: 2.0 nM template, 20 mM potassiumacetate, 0.4 mM ATP, 0.4 mM CTP, 0.4 mM UTP, 0.005 mM[a-32P]GTP (20 Ci/mmol) in 1 × transcription buffer(40 mM Hepes (pH 8.0), 10 mM magnesium acetate,0.1 mM EDTA, 0.1 mM dithiothreitol and 0.1 mg/mlacetylated bovine serum albumin (Sigma Chemical Co.).Unlabelled nucleoside triphosphates were FPLC grade(purchased from PL Biochemicals) and the radioactivenucleotide was purchased from New England Nuclear.

In transcription initiation rate assays the template DNAwas incubated with either Spo0A or Spo0A-P, as required,in 1 × transcription buffer, along with ATP and GTP forthree minutes at 37°C. Following this incubation, whichpermitted Spo0A(-P) binding to the DNA, an appropriatedilution of RNA polymerase (diluted with1 × transcription buffer containing 30% glycerol) wasadded to begin the reaction. At time-points afterpolymerase addition, samples were removed, added to amixture containing CTP, UTP (final concentrations0.4 mM) and heparin (final concentration 10 mg/ml), andincubated for a further ten minutes to allow for elongationof transcripts. The elongation reactions were stopped bythe addition of one-third volume of 8.0 M urea, 180 mMboric acid, 180 mM Trizma base, 4 mM EDTA, 0.02% eachbromophenol blue and xylene cyanol. The radioactivetranscripts were separated from unincorporatednucleotides by electrophoresis through a polyacrylamidegel (8% acrylamide; 0.21% bis-acrylamide) containing 7 Murea, 45 mM boric acid, 45 mM Trizma base, 0.5 mMEDTA. The location of the radioactive transcripts wasdetermined by autoradiography and the incorporationwas monitored by measuring the Cerenkov radiation inexcised gel slices containing the transcripts.

Observed rate constant (kobs) for transcription initiationreactions was calculated using methods previouslydescribed (Stefano & Gralla, 1982) and lines to fit eachdata set were calculated by least-squares analysis. Briefly,duplicate reactions were allowed to continue initiation forat least ten minutes and then challenged with heparin,UTP and CTP. The level of heparin-resistant complexes inthese samples was taken as the maximum level (Cmax). Thetranscription at each time-point in the rate assay (Ct) wasused to calculate ln[(Cmax − Ct)/Cmax]. The slope of the plotln[(Cmax − Ct)/Cmax] versus the time of initiation (t) is −kobs,the observed, overall rate constant for the reaction. Therelationship of kobs to individual rate constant follows thederivation due to Strickland et al. (1975), Stefano & Gralla(1982) and McClure (1985)

Phosphorylation of Spo0A

Spo0A and the other proteins required to phosphoryl-ate were isolated from Escherichia coli cells carryingrecombinant clones of the genes for these proteins inexpression vectors (Burbulys et al., 1991). Spo0A wasphosphorylated in a reaction that contained KinA(1.0 mM), Spo0F (2.0 mM), Spo0B (0.2 mM), ATP (1.0 mM)and Spo0A (8.0 mM) in 1 × transcription buffer. Reactions

Transcription Stimulation by Spo0A446

were incubated at room temperature for 90 minutes. Atthis ATP concentration phosphorelay reactions equi-librated within 60 minutes and resulted in greater than50% of the Spo0A protein becoming phosphorylated (Birdet al., 1993). Samples of non-phosphorylated Spo0A usedin the transcription reactions also contained 1.0 mM ATPin 1 × transcription buffer. Extensive tests demonstratedthat Spo0F, Spo0B and KinA had no effect in thestimulation of transcription by either Spo0A or Spo0A-P.The phosphate on Spo0A-P is unstable and is unlikely tosurvive purification and storage of the protein. We assumethat Spo0A that had not been treated in a phosphorelayreaction contained negligible levels of phosphorylation.

DNase I footprint reactions

Footprint reactions used a BamHI/PvuII DNA frag-ment from pUCIIGtrpA that had been labelled at theBamHI site with [g-32P]ATP (7000 Ci/mmol; ICN Bio-chemicals) and polynucleotide kinase (Bethesda ResearchLaboratories). The BamHI site is 135 bp 3' to the start oftranscription. The final DNA concentration in eachfootprint reaction was 2 nM. DNase I reactions werecarried out in 1 × transcription buffer at 37°C by addingDNase I (Sigma Chemical Co.) to a final concentration of0.67 mg/ml. After ten seconds the reactions were stoppedby addition of 75 ml (three volumes) of 0.1% sodiumdodecyl sulfate, 4.0 mM EDTA, 270 mM sodium chloride,40 mg/ml sonicated calf thymus DNA (Sigma ChemicalCo.). The samples were precipitated with ethanol, and theprecipitate was collected, dried and resuspended in 95%formamide, 45 mM boric acid, 45 mM Trizma base,0.5 mM, EDTA and 0.02% each bromophenol blue, xylenecyanol. Volumes containing equal cpm from each DNaseI digestion within an experiment (typically 105 cpm) wereelectrophoresed through a 5% polyacrylamide sequencinggel containing 7 M urea (Sambrook et al., 1989). The gelswere dried and exposed to film (Kodak Co.).

AcknowledgementsWe thank C. P. Moran for the clone containing the spoIIG

promoter and L. Duncan for preparation of RNApolymerase. We thank an anonymous referee for pointingout a number of improvements to the manuscript. Thiswork was supported by funds from the Natural Scienceand Engineering Research Council of Canada (to G.B.S.)and by grant GM19416 from the National Institutes ofGeneral Medical Sciences, National Institutes of Health,United States Public Health Service (to J.A.H.).

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Edited by M. Gottesman

(Received 28 February 1995; accepted in revised form 21 November 1995)