The development of genetic competence in Bacillus subtilis is regulated by a complex signal transduction cascade, which resultsin the synthesis of the competence transcription factor, encodedby comK . ComK is required for the transcription of the late competence genes that encode the DNA binding and uptake machineryand of genes required for homologous recombination . In vivoand in vitro experiments have shown that ComK is responsiblefor transcription activation at the comG promoter . In this study,we investigated the mechanism of this transcription activation.The intrinsic binding characteristics of RNA polymerase withand without ComK at the comG promoter were determined, demonstratingthat ComK stabilizes the binding of RNA polymerase to the comG promoter . This stabilization probably occurs through interactions with the upstream DNA, since a deletion of the upstream DNAresulted in an almost complete abolishment of stabilizationof RNA polymerase binding . Furthermore, a strong requirementfor the presence of an extra AT box in addition to the commonComK-binding site was shown . In vitro transcription with B.subtilis RNA polymerase reconstituted with wild-type -subunits and with C-terminal deletion mutants of the -subunits was performed,demonstrating that these deletions do not abolish transcriptionactivation by ComK . This indicates that ComK is not a type Iactivator . We also show that ComK is not required for open complexformation . A possible mechanism for transcription activation is proposed, implying that the major stimulatory effect of ComK is on binding of RNA polymerase.

 
Genetic or natural competence is a physiological differentiation state in which bacteria are able to take up exogenous DNA fromthe medium . This phenomenon has been best studied in the gram-positive soil bacterium Bacillus subtilis . Competence development is initiated at the onset of stationary growth as a result of acomplex regulatory cascade . Through quorum sensing, environmentalsignals such as nutrient availability and cell density are sensedand interpreted [16, 34] . Regulation by this cascade leads tothe synthesis of the competence transcription factor [CTF],encoded by comK. Via an autoregulatory loop, ComK stimulatesthe expression of its own gene . In addition to this, ComK isrequired for the transcription of the late competence genes, comC, -E, -F, and -G, which encode the DNA binding and uptakemachinery and addAB and recA, which are necessary for DNA recombinationand integration [14, 31, 32, 37, 50, 69].

Previously, we have described the mechanism of binding of ComKto the promoter regions of specific competence genes [35, 69].A transcriptional fusion of the comG promoter with lacZ showedthat in vivo expression of comG was completely abolished ina comK deletion mutant [68] . In vitro transcription studiesconfirmed that ComK alone is capable of activating transcriptionat the comG promoter [35].

Transcriptional regulation by activators has been shown to affect transcription initiation at one or more of the following steps:[i] stimulation of RNA polymerase [RNAP] binding, [ii] stimulationof the isomerization to an open promoter complex, and [iii]helping in promoter clearance [1, 2, 44, 45] . The free energyof reaction intermediates of transcription initiation can belimiting at any of these steps . Activator interactions couldfunction in lowering the energy barrier of the rate-limitingstep or steps in order to accelerate the overall transcriptioninitiation reaction [27].

The possible interactions at the promoter site that could leadto transcriptional activation are generally divided into threeseparate levels: [i] direct protein-protein contacts betweenthe activator protein and RNAP; [ii] conformational changestransmitted by the DNA upon binding of the activator; and [iii]additional contacts with other DNA segments than the core promotersequence, such as interactions between the DNA sequence upstreamof the activator binding site and the backside of RNAP [15, 17] . It has been postulated that the mechanism of activation depends on the architecture of the promoter as well as on the steps that are rate limiting in transcription initiation forthat promoter [38].

In the experiments reported here, we investigated the mechanismof ComK-dependent stimulation of transcription at the comG promoter. We demonstrate that RNAP binding to the promoter is stimulated by ComK and that stabilization of binding requires the presenceof the upstream region of the promoter DNA . Furthermore, weshow that C-terminal deletions in the -subunit of RNAP do notabolish transcription activation by ComK . Isomerization to theopen complex promoter is shown to be independent of ComK . Theimplications of these results for the transcription activationmechanism of ComK at the comG promoter are discussed.

 
Strains, plasmids, and plasmid construction. The plasmids and bacterial strains used in this study are listedin Table 1.

 
Plasmid pAN-G+6 was constructed by long-range PCR using primersG+6F and G+6R with pAN-G as the DNA template . Ligation of thesubsequent PCR product yielded pAN-G with a 6-bp insertion inbetween the ComK-binding sites and the promoter -35 sequence.Primers were designed to create a unique HindIII restrictionsite at the place of insertion . Both the wild-type and mutantcomG promoter fragments were cloned into the pBTK2 amy-locus integration vector [46] as a BamHI-EcoRI restriction fragment.The resulting plasmids, pBTK-G and pBTK-G+6, were transformedto B . subtilis 8G5, and ß-galactosidase assays wereperformed as described previously [67].

The pAN-G-AT-GC plasmids were constructed by PCR with pAN-Gas the DNA template and with primers G2trn-XhoI and G1trn-XhoI [for AT2] or G3trn-XhoI [for AT3] . The PCR products were digested with XhoI and ligated, resulting in plasmids in which the upstream DNA of comG is deleted and replaced by high-GC [55%] DNA from the pUC origin . The pAN-G-AT-sipS plasmids were constructed by PCR with primers G2trn-XhoI and AT-AT2 [for AT2] or AT-AT3 [for AT3] . The PCR products were digested with XhoI and BpiI. In this fragment, a PCR product was ligated, made with primers SipS-XhoI and SipS-BpiI on chromosomal DNA of B . subtilis 8G5as a template, and digested with XhoI and BpiI . In the resultingplasmids, the comG-specific upstream DNA is replaced by upstreamDNA of sips [61% AT] . The plasmid pAN-G-AT-codY was constructedby PCR on pAN-G as a DNA template with primers G2trn-XhoI andG1trn-XhoI or G3trn-XhoI for AT2 and AT3 respectively . Intothis fragment, an internal gene fragment of Lactococcus lactiscodY was ligated, made on chromosomal DNA with primers cod20and cod21 . In the resulting plasmids, the upstream DNA of comGis replaced by high-AT [60%] DNA of codY origin . Use of primerG3trn or AT-AT3 leads to the deletion of one possible AT boxin the upstream region of comG.

DNA manipulations and materials. Standard molecular biology methods [3] were used unless otherwisespecified . Enzymes were purchased from Boehringer Mannheim,New England Biolabs, Promega, or Pharmacia . DNA oligonucleotideswere synthesized by Gibco BRL or Invitrogen . Radiolabeled nucleotideswere obtained from Amersham . The media for growth of B . subtilisand Escherichia coli have been described previously [3, 70].B . subtilis chromosomal DNA was isolated and purified as describedpreviously [70] . ComK was purified in this laboratory by themethod of Hamoen et al . [35].

PCR amplifications. PCRs were carried out as described by Innes and Gelfand [40]by use of Pwo DNA polymerase [Boehringer Mannheim GmbH] andB . subtilis 8G5 chromosomal DNA, L . lactis chromosomal DNA,or plasmid pAN-G as a template . The primers used in PCRs arelisted in Table 2 . Probes for use in electrophoretic mobilityshift assays were made by PCR . A combination of the primersG1 and G2 was used to create a comG promoter fragment . A combinationof the primers G2 and G1trn and primers G2 and AT-AT3 resultsin a truncated comG promoter fragment with two or three AT boxes, respectively . Probes with longer upstream DNA sequences weremade with primers G7 and G2 for the wild type, retG-1 and G2for high-GC DNA, and SipS-XhoI or cod20 with G2 for high-ATDNA of sipS or codY origin.

 
Purification of ^A-specific RNA polymerase and of ^A factor. To purify RNAP, an overnight culture [5 µg of neomycin per µl] of B . subtilis NIG2001 [25] was diluted 100 timesand grown at 37°C in 2x tryptone-yeast medium and harvestedat time 0 . All subsequent procedures were performed at 4°C.Cells were collected by centrifugation for 10 min at 6,000 x g and washed with ice-cold buffer A [20 mM Tris-HCl [pH 8.0], 0.2 M NaCl, 1 mM 2-mercaptoethanol, 10% glycerol, 10 mM MgCl₂]. Cells were broken with a French press in buffer A containing 0.5 mM phenylmethylsulfonyl fluoride [PMSF] . Cell extracts were obtained by centrifugation for 20 min at 20,000 rpm in a SW50rotor, after adding an additional 0.5 mM PMSF . The supernatantwas diluted up to 10 times in buffer A and applied to a Talonresin column [Clontech] or Ni-nitrilotriacetic acid [Qiagen].The loaded column was washed with buffer A and buffer B [bufferA containing 5 mM imidazol] to remove nonspecifically boundproteins . Bound proteins were eluted by increasing the concentrationof imidazol up to 50 mM in buffer A . Protein-containing fractionswere diluted in low-salt buffer [20 mM Tris-HCl [pH 8.0], 10mM MgCl₂, 20% glycerol, 1 mM 2-mercaptoethanol] and appliedto a prepacked disposable 5-ml heparin-agarose column [Pharmacia].After extensive washing with low-salt buffer, RNAP was elutedby increasing the concentration of NaCl in buffer A up to 1.2M . Finally the sample was dialyzed against cold dialysis buffer[10 mM Tris-HCl [pH 8.0], 7.5% glycerol, 1 mM 2-mercaptoethanol]. ^A was purified from inclusion bodies in E . coli as describedby Chang and Doi [13] . Before use in gel retardation or in vitro transcription reactions, holoenzyme was reconstituted on ice for at least 10 min by mixing RNAP and ^A in a 1:1 molar ratio.

Gel retardation analysis. Gel retardation with ComK and RNAP was carried out essentiallyas described previously [69] . The PCR-generated DNA probes wereend labeled with T4 polynucleotide kinase by use of [-³²P]ATP. The purified proteins and probes were premixed on ice in 20µl of binding buffer, consisting of 20 mM Tris-HCl [pH8.0], 5 mM MgCl₂, 100 mM KCl, 0.5 mM dithiothreitol [DTT], 0.05-mg/mlpoly[dI-dC], 0.05-mg/ml bovine serum albumin [BSA], and 8.7%glycerol . All reactions were performed in the presence of 200µM ATP and 200 µM GTP, with the exception of thoseproducing the data shown in Fig. 5, when indicated . Binary complexeswere formed by incubation for 15 min at 37°C . To distinguishopen RNAP-promoter complexes, 2 µl of a 0.3% heparin solutionwas added directly prior to electrophoresis on a nondenaturing4% polyacrylamide gel . Gels were run in TAE buffer [40 mM Tris-acetate[pH 8.2], 2 mM EDTA] at 100 V, dried, and autoradiographed.

 
In vitro transcription assays. Reaction mixtures for in vitro transcription analyses containedthe following [in 25 µl]: 25 mM Tris-HCl [pH 7.5]; 10mM MgCl₂; 100 mM KCl; 1 mM DTT; 45 mM ammonium sulfate; 200µM [each] UTP, ATP, and GTP; 80 µM [-³²P]CTP [2µCi]; 1 µg of poly[dI-dC]; 1 µg of BSA; and9 nM template DNA . The templates used were supercoiled pAN-Gand derivatives [34] and a 260-bp DNA fragment containing the phage 29 C2 promoter [47], which give rise to transcripts of 360 and 98 nucleotides, respectively . Nonreconstituted RNAPand RNAPs reconstituted with deletion mutants of the -subunit [15, 37, and 59] were used as described by Mencia et al . [47].ComK protein was added to a final concentration of 0.35 µM.Reactions were performed at 37°C and processed as describedpreviously [51, 54] . Transcripts were separated by denaturing polyacrylamide gel electrophoresis and quantified by using aFuji BAS-IIIs image analyzer.

 
ComK stimulates binding of RNAP at the comG promoter. The basal prokaryotic promoter consists of four critical elements: i.e., the -35 and -10 hexamers, the spacer length between thesetwo hexamers, and upstream auxiliary elements . DNA sequencesthat resemble the consensus of such a core region are efficientbinding sites for RNAP . Nevertheless, they may be poor promoterswithout the presence of activator proteins [7, 20, 24] . Thepresence of a -35 consensus hexamer for RNAP is important forefficient binding of ^A-RNAP to the promoter, since ^A makes specificinteractions with DNA at this region [30] . In general, the homologyscore of promoter sequences correlates closely with the in vitrobinding affinity of ^A-RNAP [9, 20, 24] . Since the comG promotershows good homology to the ^A consensus promoter [Fig . 1], thetranscription properties of ^A-RNAP at the comG promoter wereanalyzed by in vitro transcription studies [35] . It was shownthat in the absence of ComK, hardly any transcripts are formed,while in the presence of ComK, transcription is stimulated upto 50-fold, showing that ComK is sufficient and is requiredto activate transcription at the comG promoter.

 
Binding properties of ^A-RNAP at the comG promoter were analyzedwith electrophoretic mobility shift assays . RNAP was shown tobind to the comG promoter also in the absence of ComK . In thepresence of ComK, the amount of complexes formed increased two-to fivefold [Fig. 2], resulting in a supershifted complex . This result suggests that ComK stimulates binding of RNAP to thecomG promoter.

 
RNAP binding is stabilized by ComK through the upstream DNA region. When a truncated comG promoter fragment, lacking the DNA upstream of the ComK-binding sites, was used in an electrophoretic mobility shift assay under the same conditions, the stabilization ofthe RNAP-promoter complex was abolished [Fig . 3A], although binding of ComK or RNAP alone was not disturbed . Therefore,it can be concluded that for stabilization of the complex, theDNA upstream of the ComK boxes is important.

 
Stimulatory effects of upstream DNA on transcription activationare known for several promoters [17, 61, 62] . Often, a specificactivation sequence, the UP element, can be distinguished, consistingof an AT-rich region located between -40 and -60 relative tothe transcription start site [22, 23, 29] . In the case of thecomG promoter, this region is occupied by ComK binding, butthe DNA-bending capacities of ComK suggest a possible specificsequence to be located further upstream of the promoter [59]. To test whether the importance of the upstream DNA in the case of stable RNAP binding at the comG promoter is a result of the presence of a specific sequence or of the structural presenceof DNA, mutants were constructed in which the upstream DNA ofcomG was replaced by nonspecific DNA, with either a high GCcontent or a high AT content . Furthermore, two types of constructswere tested, which differed in the number of possible ComK-bindingsites upstream of the promoter . Commonly, one K box, consistingof two AT boxes is present upstream of a ComK-activated gene[35] . In the case of comG, an extra AT box is located one helicalturn upstream of the common K box . In one set of mutants, calledAT3, all three AT boxes were present, while in the other setof mutants, called AT2, only two boxes were present [Fig . 3C].

The different promoters were tested by electrophoretic mobility shift assays and in vitro transcription assays [Fig . 3B], showingthat deletion of the third AT box resulted in an almost completeloss of stabilization of RNAP binding and transcription in thepresence of ComK . This result indicates that the presence of the third AT box is of great importance for transcription activation at the comG promoter . However, the box alone is not sufficient to stabilize RNAP binding, since stabilization is still almost completely abolished when a truncated comG promoter lacking the DNA upstream of the three AT boxes is used . The replacementof the comG-specific upstream DNA with either high-GC or high-AT DNA showed only a slight reduction of stabilization of RNAPbinding, which never exceeded a 2- to 2.5-fold difference.

ComK is not a type I transcriptional activator. An important class of prokaryotic transcription factors mediatestranscription activation through direct contacts with the RNAP.A preferred activation target is the C-terminal domain of the -subunit of RNAP [6, 21] . In general, those activators bindingat or upstream from position -60 relative to the transcriptionstart site normally interact with the -subunit [41] . To investigatewhether ComK stimulates transcription through contacts withthe -subunit, in vitro transcription assays were performed usingRNAPs reconstituted with either wild-type -subunit or -subunits lacking the last 15, 37, or 59 amino acids from the carboxyl-end, respectively . Equivalent amounts of the reconstituted RNAPs were added to the transcription reaction mixtures, and the reaction products were separated by electrophoresis . The results demonstrated that the RNAPs containing deletion mutants of the -subunit werestill stimulated by ComK [Fig . 4A], suggesting that direct protein-proteincontacts between ComK and the -subunits are not required fortranscription activation . However, the maximum level of transcriptionby RNAP reconstituted with the mutant -subunits was reducedapproximately twofold compared to that of the wild-type polymerase[Fig . 4B] . This suggests that the C-terminal domain of the -subunit is important for optimal transcription activity, as will be discussed.

 
Using electrophoretic mobility shift assays, it was also shownthat ComK did not promote the binding of purified -subunits [wild type and deletion mutants] nor that of purified ^A to thepromoter [results not shown] . Thus, it can be concluded that direct protein-protein contacts between ComK and the - or -subunits of RNAP are not required for stabilizing RNAP binding to the promoter.

Open complex formation at the comG promoter is independent of ComK. The second step in transcription initiation is open complex formation . Competitor resistance is widely used as a functional assay for open complex formation [66, 71] . Heparin challengeexperiments indicated that the presence of ComK is not requiredfor formation of open complexes at the comG promoter [Fig . 5A].Open complex formation was shown to be dependent on the presenceof the initiating nucleotides . Only when ATP and GTP were addedto the reaction mixtures did RNAP-promoter complexes becomeresistant to a heparin challenge . Upon addition of both nucleotides,an additional stabilizing effect on RNAP binding and an additionalshift were observed, compared with the complexes formed in theabsence or presence of only one of the nucleotides . This couldbe caused by the fact that in the presence of both initiatingnucleotides a short abortive transcript can be formed [66].Likely, the formation of a short transcript stabilizes the bindingof RNAP and causes a slightly altered migration pattern uponelectrophoresis.

Normal open complex formation was also seen when the truncated comG promoter fragment with two AT boxes was used in the heparin challenge experiments [results not shown] . These results confirmthat when RNAP is bound to the promoter, open complex formationoccurs upon the addition of nucleotides and independent of ComK.

Transcription activation is helix face dependent. To investigate whether the orientation of the ComK-binding sitesis important for transcription activation, a mutant comG promoter fragment was created that contained a 6-bp insertion betweenthe ComK box and the -35 hexamer . In this situation, the ComK-bindingsite is located on the opposite face of the DNA helix, and asa consequence, the bound ComK dimers are on the opposite faceof the helix compared to the downstream RNAP . It has been foundthat ComK induces a bend in the DNA upon binding [35] . In thecomG+6 construct, this ComK-induced bend is present in the opposite direction compared to the wild-type situation.

The comG+6 promoter fragment was used in in vitro transcription assays, which showed abolishment of activation of transcription by ComK . In vivo, this promoter was placed in front of the lacZ gene in the amy locus of the B . subtilis chromosome . ß-Galactosidaseexpression was abolished to the same level as in a comK deletionmutant [68; results not shown].

Electrophoretic mobility shift assays with comG+6 showed that stabilization of RNAP binding was disturbed, a situation comparable to that with the truncated comG promoters . Still, the initial level of RNAP binding in the absence of ComK was the same as that in the wild-type comG promoter, as was open complex formation [Fig . 5B] . Binding of RNAP seemed to be lost when ComK boundto the other side of the helix . These results indicate a strict helix face dependency for transcription activation by ComK.It has been proposed that intrinsic or protein-induced DNA bends immediately upstream of a promoter site can activate transcriptionby looping the upstream DNA sequences around to interact withthe backside of RNAP [56, 58, 59] . This would explain both therequirement for the upstream region of the comG promoter andthe helix face dependency.

 
Transcription initiation frequently requires the interactionof several DNA binding proteins that ultimately modulate theactivity of RNAP . In competence development in B . subtilis,comK encodes the central regulator, also known as the CTF . ComKactivates and binds specifically to the promoters of the latecompetence genes and the genes required for recombination . Invitro studies have shown that purified ComK alone is capableof activating transcription at the comG promoter . In this report,we describe the mechanism of this transcriptional activation.

In order to see in which step of transcription initiation ComKis involved, several approaches were taken . Using electrophoretic mobility shift assays, it was shown that RNAP can bind to thecomG promoter also in the absence of ComK, but that the amountof closed complexes is stimulated up to fivefold when ComK ispresent [Fig. 2].

Stabilization of the RNAP-promoter complex in the presence ofComK was shown to be dependent on the DNA upstream of the ComK-binding sites . When this upstream DNA was deleted, binding of ComK andRNAP alone to the fragment was not disturbed [Fig . 3A], but the supershifted RNAP-promoter complex was no longer stabilized, suggesting that stabilization of RNAP binding is a result of bending of the upstream DNA by ComK, thereby enabling interactions between the DNA and RNAP.

Replacement of the comG-specific upstream DNA by either high-GC or high-AT DNA resulted in only a slight reduction in stabilization of RNAP binding and transcription [Fig . 3C] . Several sequencecomparisons were made between the upstream DNA of comG and thoseof other ComK-activated genes . No clear conserved sequences could be indicated, but a major difference between comG and other ComK-activated genes is the presence of an extra AT box upstream of comG . Binding assays and in vitro transcription studies comparing promoter fragments with either two or threeAT boxes upstream of the comG gene showed the requirement ofthe third box for stabilization of RNAP binding and transcriptionin vitro [Fig . 3C] . Previous footprinting studies by Hamoen et al . demonstrated that all three AT boxes are protected by ComK [35] . The presence of this extra AT box might be the determinantthat results in the large transcription at the comG promoter.Array studies indicated that comG transcription is the highestof all ComK-activated genes, and in vitro transcription studieswith ComK-activated genes have thus far only been successful for comG [4, 33, 55] . Studies with a truncated comG promoter that still contained all three AT boxes but lacked the upstreamDNA no longer showed stabilization of RNAP binding, indicatingthat, in addition to the third AT box, the presence of moreupstream DNA is required.

The requirement for upstream DNA correlates with the resultsshown in in vitro transcription assays with RNAPs reconstitutedwith the wild type or C-terminal deletion mutants of the -subunit. The results indicated that a direct interaction between the -C-terminal domain [CTD] and ComK is not required for RNAP activation[Fig. 4], since ComK could still stimulate transcription by mutant RNAPs . Electrophoretic mobility shift assays showed that ComK is not able to recruit purified -subunit or ^A to the comGpromoter, another indication that no significant contacts betweenComK and RNAP are involved in activation.

Although transcription activation was not abolished in the reconstituted mutant RNAPs, a twofold reduction in maximal transcription was observed . Rowe-Magnus et al . [63] reported a similar observationfor the transcription of the spoIIG promoter by Spo0AP . Theysuggested an effect on the interaction of RNAP with promoter DNA by the -subunit mutation . The CTD of the -subunit is knownto interact with additional promoter sequences [UP elements] to stabilize polymerase-DNA interactions at some promoters [17, 22, 23, 29, 61, 62] . Although in the upstream region of comG,a clear UP element could not be demonstrated, it still is possiblethat specific AT-rich stretches in the upstream DNA interactwith RNAP . In E . coli, the same residues of the -CTD were foundto be involved in interaction with activators such as CRP andpromoter UP elements [52] . If the -subunit CTD would indeedhelp to stabilize the binding of RNAP to the promoter, it wouldexplain why deletions in this domain disturb optimal transcriptionactivity and why the presence of upstream DNA is important foroptimal RNAP binding.

Although we cannot totally rule out the possibility that ComK interacts with RNAP through some other region of the enzymethan the -subunits or ^A, like the ß- or ß'-subunit,we currently favor the notion that ComK activation of transcriptionfrom the comG promoter is mediated via stabilization of RNAPbinding through the upstream region of the promoter DNA.

Each step in the transcription initiation process is in principle a target for regulation by transcriptional activators [27]. Activation can involve multiple interactions between a single activator molecule and the transcription machinery, each interaction being responsible for a specific mechanistic consequence . Infact, such multiple interactions have become a commonly observedfeature in transcription activation [11, 38, 53] . To advanceour understanding of the effect of ComK on the transcriptioninitiation process at the comG promoter, several experimentswere performed to investigate in which step of transcriptioninitiation ComK is involved.

When we inverted the orientation of the ComK- and RNAP-binding sites in the comG+6 promoter construct, stabilization of RNAP binding to the promoter was abolished . In the presence of ComK,no basal level of RNAP binding was observed, suggesting thatbinding of ComK to the opposite face of the helix hinders RNAPbinding to the promoter . In addition, in vitro and in vivo transcriptionfrom this promoter was lost . Helix face dependency has beentaken as evidence for cross-talk between RNAP and the activatorprotein [28] . Since protein-protein interactions with the - or -subunits of RNAP are not involved in transcription activationby ComK, it is likely that the orientation of the DNA bend causedby ComK binding is responsible for the helix face dependency.It has been proposed that activator-induced bending of the DNAupstream of the promoter facilitates caging of RNAP to optimizethe promoter [2, 10, 59] . We conclude that the mechanism of activation relies on contacts between the DNA upstream of the ComK-binding sites and the backside of RNAP . Similar findingshave been reported for the gal and lac promoters [15] and forthe CRP-dependent malT promoter [18].

For the in vitro transcription assays, supercoiled templateswere used, because we found them to be approximately 20-foldmore efficient in transcription than runoff transcription assaysusing the linear template [results not shown] . The supercoiledstate of the chromosome is known to affect the activity of manypromoters [57] . It is a fairly common phenomenon among prokaryoticpromoters to be stimulated by DNA superhelicity [5, 60] . Thestimulatory effect of superhelicity of the template on transcriptionefficiency is also in agreement with our model . The influenceof DNA bending on regulatory processes may be modulated by DNAsuperhelicity [26] . Specifically, supercoiling and bending maysynergistically enhance polymerase contacts by creating a definedDNA topology at the promoter site, a view also put forward byZinkel and Crothers [72] . Alternatively, DNA supercoiling mayoptimize the three-dimensional geometry of the DNA for correctalignment of the proteins and/or DNA sites, thus lowering energybarriers in transcription initiation [43].

The comG promoter has a strong resemblance to the B . subtilis consensus promoter sequence for ^A-dependent promoters [Fig.1] . In general, such consensus-like promoters stably bind RNAPand require alterations to accelerate the late steps of thetranscription initiation pathway [19, 39] . Therefore, ComK mightalso influence transcription initiation in one of the laterstages after closed complex formation . The stabilization ofthe closed complex by ComK will, of course, contribute to acceleratethe overall transcription process.

The second step in transcription initiation, open complex formation, was found to be independent of ComK . Addition of initiating nucleotides was sufficient to induce a heparin-resistant promoter complex . The formation of an open promoter complex is not disturbed when half a helical turn is inserted in between the promoterand the ComK boxes . All of this clearly indicates that opencomplex formation is not a rate-limiting step for transcriptioninitiation at the comG promoter.

It has been suggested that consensus ^A promoters that efficientlybind RNAP and that exhibit strong open complex formation maybe limited in the subsequent movement of the polymerase to theelongating complex [12, 39] . RNAP binding at these promotersgenerates a nucleoprotein complex that is too stable to allowpromoter clearance [39, 51] . Melting of the DNA strands in thepromoter region in the presence of NTPs leads to an initiating complex that is trapped in short abortive transcript synthesis [48] . The escape from this complex into an elongating transcriptionmachinery involves major conformational changes, including lossof the promoter-specific contacts and the release of the factor[42, 49, 65] . Escape from abortive initiation has been foundto be rate limiting at several other prokaryotic promoters [48, 64] . In the case of the comG promoter, initial experiments wereperformed to elucidate the role of ComK in promoter escape ofRNAP . To distinguish between an effect of ComK on RNAP bindingor promoter escape, ComK had to be added after the binding step.In this case, involvement of ComK in promoter clearance could not be demonstrated, since transcription levels were severely decreased when ComK was added in a later stage of the initiation process than in the binding step . Further research will be required to investigate whether bending of the upstream DNA around ComK results not only in stabilization of RNAP binding, but alsoin creating optimal conditions for later steps in the transcription initiation process, like promoter escape.

The proposed model for the role in transcription activationby ComK is summarized in Fig . 6 . Although RNAP is capable of binding to the comG promoter in the absence of ComK, binding is stimulated when ComK is present [step 1] . In the case ofthe comG promoter, ComK can bind to three AT boxes, resultingin bending of the upstream DNA around ComK . This DNA probablyinteracts with the RNAP, thus stabilizing the RNAP-promotercomplex [step 2] . Further studies should be performed to seewhether interactions between the upstream DNA and the backsideof RNAP also help to induce conformational changes in the promoterDNA and/or RNAP that are required for promoter clearance.

 
In this study, we have investigated the mechanism of transcription activation at the comG promoter, which differs from most other ComK-activated genes by containing a third AT box . However,we suggest that the transcription activation mechanism at promoters containing only two AT boxes is comparable to the model presentedin this study . It is likely that the major effect of ComK is stabilization of RNAP binding via the upstream DNA region . Wesuggest that the function of the third AT box is mainly enhancing transcription levels at the comG promoter . This view is supported by the fact that the level of expression of the comG operon is the highest of all ComK-activated genes.

 
We thank M . Fujita and Y . Sadaie for their kind gift of B . subtilis NIG2001, used for purification of RNA polymerase; and B . Chang and R . Doi for their kind gift of plasmid pCD2, used for overexpression and purification of ^A . We also thank Sierd Bron, Caroline Eschevins,and Wiep Klaas Smits for helpful discussions.

 
* Corresponding author . Mailing address for L . W . Hamoen: Sir William Dunn School of Pathology, University of Oxford, South Parks Rd., Oxford OX1 3RE, United Kingdom . E-mail: L.W.Hamoen@biol.rug.nl . Mailing address for O . P . Kuipers: Department of Genetics, University of Groningen, NL-9751 NN Haren, The Netherlands . Phone: 31[0]50.363.2093 . Fax: 31[0]50.363.2348 . E-mail: O.P.Kuipers@biol.rug.nl.

Present address: Unilever Bestfoods Nederland B.V., NL-3071JL Rotterdam, The Netherlands.

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