JOURNAL OF BACTERIOLOGY,0021-9193/00/$04.0010

Aug. 2000, p. 4180–4187 Vol. 182, No. 15

Copyright © 2000, American Society for Microbiology. All Rights Reserved.

Coactivation of the RpoS-Dependent proP P2 Promoter byFis and Cyclic AMP Receptor ProteinSARAH M. MCLEOD,1 JIMIN XU,1† AND REID C. JOHNSON1,2*

Department of Biological Chemistry, UCLA School of Medicine, Los Angeles, California 90095-1737,1 andMolecular Biology Institute, University of California, Los Angeles, Los Angeles, California 900952

Received 27 January 2000/Accepted 26 April 2000

The Escherichia coli proP P2 promoter, which directs the expression of an integral membrane transporter ofproline, glycine betaine, and other osmoprotecting compounds, is induced upon entry into stationary phase toprotect cells from osmotic shock. Transcription from the P2 promoter is completely dependent on RpoS (s38)and Fis. Fis activates transcription by binding to a site centered at 241, which overlaps the promoter, whereit makes a specific contact with the C-terminal domain of the a subunit of RNA polymerase (a-CTD). We showhere that Fis and cyclic AMP (cAMP) receptor protein (CRP)-cAMP collaborate to activate transcriptionsynergistically in vitro. Coactivation both in vivo and in vitro is dependent on CRP binding to a site centeredat 2121.5, but CRP without Fis provides little activation. The contribution by CRP requires the correct helicalphasing of the CRP site and a functional activation region 1 on CRP. We provide evidence that coactivation isachieved by Fis and CRP independently contacting each of the two a-CTDs. Efficient transcription in vitrorequires that both activators must be preincubated with the DNA prior to addition of RNA polymerase.

During the transition from rapid vegetative growth to sta-tionary phase, a set of genes is induced which is involved inlong-term survival under environmental stress. One such genein Escherichia coli is proP, which encodes a transporter ofproline, glycine betaine, and other osmoprotecting compounds(12, 23, 32). While the upstream P1 promoter of proP is tran-siently induced as part of a specific stress response to osmoticshock, the downstream P2 promoter is expressed for a briefperiod as cells are about to enter stationary phase, presumablyas a safeguard from potential osmotic stress (39, 56).

Expression from the proP P2 promoter has an unusually highdependence both in vivo and in vitro on the Fis protein and thestationary-phase sigma factor, s38, encoded by rpoS (57). Fisbelongs to the general family of nucleoid-associated factorsand is a global regulator of transcription, acting as a repressorat some promoters and an activator at others (13, 19, 21, 24, 35,43, 48, 54, 55). Under rapid growth conditions, Fis is the mostabundant transcriptional regulator in the cell (1, 3). However,Fis levels decline dramatically in late exponential phase andbecome undetectable in stationary phase. In contrast, s38 lev-els do not begin to accumulate until late exponential phase(34). This results in a narrow window of P2 expression due todeclining Fis levels and a rising s38 population shortly beforecells enter stationary phase.

We have previously investigated transcriptional activation byFis at the proP P2 promoter. As shown in Fig. 1A, there are twospecific Fis binding sites, located at 241 (site I) and 281 (siteII) nucleotides from the start of transcription (56). Activationat P2 is mediated by Fis at site I, which overlaps the 235binding element for the sigma subunit of RNA polymerase,dictating that Fis is acting as a class II transcriptional activator(57). Fis binding to the weaker Site II does not significantlyaffect transcription (57). An essential activation patch on Fis

has been localized to a four-amino-acid region spanning theloop between helices B and C adjacent to the DNA bindingdomain of one subunit of the Fis homodimer (7, 38). Thisregion on the upstream subunit of Fis is believed to directlycontact the C-terminal domain of the a subunit of RNA poly-merase (a-CTD) (38).

In addition to Fis activation, there is an upstream cyclicAMP (cAMP) receptor protein (CRP) binding site located at2121.5 relative to the start of P2 transcription (57). The CRPprotein represses the P1 promoter under low osmotic growthconditions; however, the effect of CRP on P2 activation has notbeen reported (33, 58). In this report, we further investigatethe regulation of proP by examining the effects of CRP ontranscriptional activation of the P2 promoter. We find thatwhile CRP alone is a very weak activator of P2 transcription,CRP and Fis coactivate transcription synergistically. The prop-erties of CRP and Fis coactivation are explored.

MATERIALS AND METHODS

Plasmids. Plasmid pRJ4069 was used as a template for most of the single-round in vitro transcription assays (57). It contains a segment of the proPsequence from 1109 to 2200 with respect to the P2 transcription initiation siteupstream of the rrnB terminators (T1T2) in the vector pKK223-3 (Pharmacia).The DNA template with the mutation that prevents CRP binding (pRJ4070) wasderived from pRJ4069 (Fig. 1) (58). pRJ1677 (15 insertion in Fis site II) wascreated from pRJ4069 by a two-step PCR method using an oligonucleotideranging from 266 to 2100 nucleotides that contained a 5-bp insertion betweennucleotides 281 and 282 as shown in Fig. 1. For multiple-round transcriptionreactions, the plasmids used were pRJ4039 (wild type), pRJ4045 (mutations inFis site II), and pRJ4051 (mutations in Fis site I) (57). All of these plasmids areproP-104D1–lacZ fusion derivatives of the lacZ protein fusion vector pRS414(50). The sequence changes in these mutations are as noted in Fig. 1 and havebeen shown previously to strongly reduce or abolish Fis or CRP binding to theirrespective sites (56, 58).

The in vivo b-galactosidase assays were performed using two sets of plasmidscontaining CRP. pZYCRP was the parent of the activation region 1 (AR1)mutations, which were obtained from R. Ebright, Rutgers University. The AR2mutation (H19YK101E) was derived from pDCRP1, which was provided by S.Busby, University of Birmingham.

Proteins. Fis wild-type and mutant proteins were purified as described else-where (7, 22, 45). CRP was obtained from J. Krakow, Hunter College. The coreRNA polymerase enzyme was isolated by the protocol of Burgess and Jendrisak(9) and Lowe et al. (36) as previously described (57). The s38 protein wasoverproduced from plasmid pETF and purified as described elsewhere (51). Topurify the a-CTD, extracts containing overproduced levels of the a-CTD peptide

* Corresponding author. Mailing address: Department of BiologicalChemistry, UCLA School of Medicine, 10833 Le Conte Ave., LosAngeles, CA 90095-1737. Phone: (310) 825-7800. Fax: (310) 206-5272.E-mail: rcjohnson@mednet.ucla.edu.

† Present address: West Los Angeles VA Medical Center, Los An-geles, CA 90073.

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(amino acids 245 to 329) were prepared from cells containing pEBT7-aCTD(20). DNA was removed by precipitation with polyethyleimine in the presence of1 M NaCl, and the supernatant was subjected to a 50 to 80% ammonium sulfatefractionation. This material was loaded onto a phenyl-Sepharose (Pharmacia)column in 2.2 M ammonium sulfate–50 mM NaCl–20 mM Tris-HCl (pH 7.5)–10% glycerol–1 mM dithiothreitol–0.1 mM EDTA and washed extensively withthe same buffer. Near-homogeneous preparations of the a-CTD peptide wereobtained after elution with the same buffer minus ammonium sulfate.

In vitro transcription. Single-round in vitro transcription reactions were per-formed as described elsewhere (38); 0.1 pmol of supercoiled pRJ4069 was usedin all reactions unless otherwise noted. Reactions were typically carried out in 0.6M potassium glutamate–0.1 mM cAMP with 1.6 pmol of Fis protein, 1.1 pmol ofCRP, and 0.2 pmol of RNA polymerase (Es38) in a 50-ml reaction volume.Multiple-round transcription reactions were performed under the same condi-tions, and transcripts were visualized by primer extension assays as previouslydescribed (57). Fold activation was determined by quantification of the P2transcript on a phosphorimager, normalizing each lane to the amount of rna1transcript.

Determination of proP transcription in vivo. For b-galactosidase assays, CRPmutants were transformed into strains RJ7024 (MG1655 DlacX74 Dcrp zhe::Tn10lRJ4065 F9 lacIqs A4 proAB1 fzz::Tn10dcam) and RJ7025 (MG1655 DlacX74Dcrp zhe::Tn10 lRJ4066 F9 lacIqs A4 proAB1 fzz::Tn10dcam). Both lRJ4065 andlRJ4066 are recombinants of lRS45 with plasmids pRJ4065 and pRJ4066,respectively (50, 58). Plasmid pRJ4065 contains a proP-104D1–lacZ gene fusionwhere the P1 promoter is inactivated by a mutation at 212. Plasmid pRJ4066 isidentical to pRJ4065 except that it also carries a mutation that greatly reducesCRP binding as depicted in Fig. 1. Each strain was grown overnight, subculturedin Luria broth supplemented with ampicillin, and grown at 37°C for 4 h, where

the peak of proP P2 expression occurs for the crp1 strain and the AR1 and AR2mutants. b-Galactosidase activity was measured as described elsewhere (40).Each strain was grown in parallel from at least two separate transformants andassayed in duplicate. Primer extension assays of total mRNA isolated fromRJ4446 (crp1) and RJ4445 (Dcrp zhe::Tn10) (MG1655 DlacX74) were performedas previously described (58).

DNase I footprint assay. DNase I footprinting assays were performed aspreviously described (8). A uniquely 59-end 32P-labeled fragment from 2190 to1103 (with respect to P2 transcriptional initiation) of proP was used; 1.1 pmol ofCRP protein and 2.2 nmol of a-CTD were bound to the DNA fragment for 10min under the same conditions used in the in vitro transcription reaction in atotal volume of 20 ml. After binding, DNase I was added for 30 s at roomtemperature. The reaction was quenched, extracted with phenol-chloroform, andthen subjected to ethanol precipitation and electrophoresis on a sequencing gel.

RESULTS

Fis and CRP activate transcription synergistically in vitro atproP P2. A high-affinity CRP site is centered 2121.5 nucleo-tides from the start of transcription at the proP P2 promoter(33, 58). To test the effect of CRP on proP P2 regulation,single-round in vitro transcription reactions were performedwith CRP-cAMP and RNA polymerase complexed with s38

(Es38). As shown in Fig. 2, addition of CRP-cAMP had only asmall effect, stimulating transcription about twofold over thebasal level (no added activator). Fis typically activated tran-scription 20- to 30-fold. However, when both Fis and CRP-cAMP were added to the in vitro transcription reaction, thelevel of activated transcription ranged from 75- to 125-fold.This level of activation is greater than the multiplicative effectof each activator alone. Transcripts from each lane were nor-malized to the levels of rna1 from the vector promoter, whichwere unaffected by the addition of saturating amounts of CRP-cAMP or Fis. CRP coactivation was dependent on the additionof cAMP, as expected (data not shown). These data show thatFis and CRP activate transcription synergistically at proP P2.

The conditions that promote maximal coactivation by Fisand CRP in vitro were examined. Coactivation was stronglystimulated by a supercoiled template, as was observed with Fis

FIG. 1. The proP regulatory region. (A) Schematic of the proP regulatoryregion. The two Fis binding sites centered at 241 and 281 plus the CRP sitecentered at 2121.5 relative to the start of transcription from P2 (11) aredepicted (39, 56, 57). Also shown are the start of the coding region and promot-ers P1 and P2. Expression from the P2 promoter is entirely dependent on Es38,while the P1 promoter is transcribed most efficiently by Es70. Fis site II bindinghas a slightly inhibitory effect on transcription from the P1 promoter in vitro,while CRP binding strongly inhibits P1 transcription. Fis binding at site I plusCRP binding at 2121.5 coactivate transcription at P2. (B) Sequence of the proPregulatory region (15). The core recognition sequences for the Fis binding sitesare depicted as solid lines between the top and bottom DNA strands. The CRPbinding site is denoted by a dotted line between the top and bottom DNAstrands, with solid lines at the nucleotides most important for CRP binding. Theregion on both the top and bottom strands protected from DNase I digestion byCRP is shown with an open bar. Regions protected from DNase I digestion bythe addition of a-CTD peptide are shown with filled bars. Mutations that reduceprotein binding to the CRP site, Fis site II, and Fis site I are depicted above thetop DNA strand. The 15 bp insertion within Fis site II is also shown.

FIG. 2. Coactivation of proP P2 transcription in vitro by Fis and CRP-cAMP.The denaturing polyacrylamide gel displays the products of single-round in vitrotranscription of supercoiled pRJ4069 in the presence of Fis, CRP-cAMP, or bothactivators. Locations of transcripts generated by the proP P2 and rna1 promotersare indicated by arrows; fold activation over the basal level (no activator added)is shown at the bottom.

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alone (57) (data not shown). Figure 3 shows the effects on invitro transcription of different concentrations of potassium glu-tamate in the presence of Fis and CRP, both as solitary acti-vators and in combination. The weak activation by CRP-cAMPalone was constant at different concentrations of potassiumglutamate, while the most efficient coactivation by CRP-cAMPplus Fis occurs at 0.6 M. Activation by Fis alone was greatlystimulated by the high concentrations of potassium glutamate,as was observed previously (57). Generally, the conditionswhich best promote activated transcription by Fis alone are themost favorable for coactivation with CRP.

Coactivation requires the CRP site at 2121.5 and Fis site Ibut not Fis site II. Fis site II, located at 281, has previouslybeen shown to have little affect on Fis-dependent transcriptionat proP P2 (57). Since Fis site II is positioned between theessential Fis binding site I at 241 and the CRP binding site at2121.5, it seemed possible that it could influence coactivationwith CRP. To test this hypothesis, transcription reactions onthe wild-type proP P2 promoter and a template containing amutation that prevents Fis from binding at site II (56) werecompared (Fig. 4). The site II mutant template was able tosupport efficient synergistic activation with Fis and CRP, indi-cating that Fis binding to site II is not important for coactiva-tion. As expected, Fis binding to site I was essential for coac-

tivation of proP P2 (Fig. 4). We have shown previously that thesite I mutation virtually eliminates binding whereas the site IImutation abolishes binding but creates a weak binding sitecentered 7 bp upstream of site II (56). The site II mutant wasshown to have no effect on Fis activation (57). Likewise, whenthe CRP site was mutated to prevent binding (58), little coac-tivation was obtained in the presence of Fis (Fig. 5). Therefore,synergistic activation of proP P2 requires CRP binding at2121.5 and Fis binding at site I but not site II.

Coactivation is dependent on the phasing between CRP andFis at site I. To determine if the relative helical orientationbetween Fis and CRP was important for activation, a 5-bpinsertion was created between nucleotides 281 and 282 in theFis site II binding site which would shift the orientation of CRPby a half helical turn of DNA. As shown in Fig. 5, this changein the helical position of the CRP dimer abolished any contri-bution by CRP on proP P2 activation. As expected, Fis stillactivated transcription on this template in both the presenceand absence of CRP. Thus, coactivation by Fis and CRP isabsolutely dependent on the helical phasing between CRP andFis bound at site I within the promoter.

CRP coactivates proP P2 in vivo. Transcriptional activationby CRP in vivo was also examined. Figure 6A shows proP P2transcripts visualized by primer extension of total mRNA iso-lated from wild-type and Dcrp cells. A sharp peak of P2 tran-script at 3 h after subculture, typical of proP P2 transcription,was seen only in the wild-type cells, suggesting that CRP isimportant for optimal P2 expression. However, the growthdefect inherent to the Dcrp strain raised the possibility that theCRP effect on P2 transcription might be amplified in this assay.For example, Fis and/or RpoS levels may be altered in the Dcrprelative to the crp1 cells. Therefore, b-galactosidase assayswere also performed using two different proP-lacZ fusions car-ried on a l prophage as reporters. Both reporters contain a

FIG. 3. Effect of potassium glutamate concentration on coactivation of proPP2 in vitro. The bar graph shows fold activation over the basal level (no addedactivator) from in vitro transcription reactions performed with CRP, Fis, and Fisplus CRP in the presence of 0.2 to 0.6 M potassium glutamate (K1Glu).

FIG. 4. Coactivation of proP P2 transcription from DNA templates with Fisbinding site mutations. Gels show primer extension products generated aftermultiple-round in vitro transcription reactions performed in the presence of Fisand/or CRP-cAMP. The DNA templates were wild type (WT), and plasmids thatcontained mutations that reduce Fis binding to site II and site I, as indicated.Transcripts originating from proP P2 are indicated with an arrow.

FIG. 5. Coactivation of proP P2 transcription from mutant DNA templateswith a CRP binding site mutation and change in spacing between the CRP siteand the P2 promoter. Single-round in vitro transcription reactions were per-formed in the presence of Fis and/or CRP-cAMP. Panels, from left to right, showtranscripts obtained from a wild-type (WT) DNA template, from a template witha mutation that greatly reduces CRP binding, and from a template with a 15 bpinsertion at Fis site II between the CRP site and the P2 promoter. Locations ofthe transcripts generated from the proP P2 and rna1 promoters are indicated witharrows.

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P1(212) mutation that abolishes transcriptional initiationfrom the P1 promoter of proP (39, 56). One construct has anotherwise wild-type proP promoter region, while the othercarries a mutation that strongly reduces CRP binding to thepromoter in vitro as well as in vivo (Fig. 1) (58). With theseconstructs, the effect of CRP on proP P2 transcription can beevaluated without using cells lacking CRP. Figure 6B showsthat in the presence of wild-type CRP (pZYCRP1) and afunctional CRP binding site, transcription at proP P2 was stim-ulated 2.5-fold in vivo when the cells were grown in Luriabroth. The CRP site mutant had 126 U of b-galactosidase,while the natural promoter produced 391 U. Up to a sevenfoldincrease in b-galactosidase activity by CRP binding has beenmeasured in M9 glycerol medium, although the overall level ofexpression was considerably lower (80 U of b-galactosidase).The stimulation by CRP in vivo was completely dependent onthe presence of Fis; cells lacking Fis have ,5 U of b-galacto-sidase (data not shown).

CRP activates proP P2 through AR1. Two regions on theCRP protein, AR1 and AR2, have been found to be critical fortranscriptional activation at other promoters (10). Dependingon the position of the CRP binding site, either AR1 or bothregions are required for efficient activation. CRP mutants con-taining substitutions in both AR1 and AR2 were tested for theability to activate transcription in vivo at proP P2 by measuringb-galactosidase activity generated by the reporter constructs

described above. Wild-type and mutant crp genes were sup-plied on a plasmid in a Dcrp strain. Figure 6B shows that theCRP mutants T158A and H159L (52), which contain muta-tions in AR1, had similar levels of activity regardless of thepresence of the CRP binding site, indicating that they wereunable to stimulate P2 transcriptional activation. The tran-scriptional activation was reduced to 106 and 82 U, respec-tively, from the 391 U of b-galactosidase produced by thewild-type control. Immunoblotting of cell extracts indicatedthat the CRP AR1 mutants did not alter the temporal expres-sion of Fis (data not shown). In contrast to the AR1 mutants,CRP containing the substitutions H19Y K101E (47) located inAR2 had no detectable effect on P2 transcription (Fig. 6C).The twofold stimulation of transcription in the presence of afunctional CRP binding site was maintained in the AR2-CRPmutant, although overall transcription in both reporters waselevated. The reason for these higher levels of transcriptionwith the AR2 mutant is not known. Thus, AR1, but not AR2,is required for CRP activation of proP P2.

The a-CTD of RNA polymerase binds to the DNA adjacentto CRP. Due to the remote position of the CRP binding site atproP, CRP is most likely contacting Es38 through the a-CTDof polymerase, which is attached to the rest of polymerasethrough a flexible linker (6, 29). As shown above, AR1 of CRP,a region that has been shown to contact a-CTD at other pro-moters (10), is necessary for CRP activation of P2. To deter-

FIG. 6. Effect of CRP on proP P2 transcription in vivo. (A) Primer extension assays were performed with 10 mg of mRNA isolated from RJ4446 (crp1) and RJ4445(Dcrp) at 1-h intervals after subculture of an overnight culture into Luria broth. The sequencing ladder generated from the same primer used in the primer extensionassays is shown on the left; the proP P2 transcript is indicated with the arrow. (B) b-Galactosidase assays of strains containing crp1 and AR1 crp mutants (T158A andH159L) supplied on pZYCRP in a Dcrp background carrying one of two ProP-LacZ reporters. The dark bars depict b-galactosidase activities (Miller units) obtainedfrom a proP P2-lacZ reporter that contains the wild-type CRP binding site; the gray bars represent activities from a strain with the same reporter except for a mutantCRP binding site. The peak activities are given, which was 4 h after subculture for each strain. In all cases, standard deviations were less than 10%. proP P1 activityis abolished in these reporters because of a mutation at 212 within its promoter. (C) b-Galactosidase assays of the CRP AR2 (H19YK101E) mutant together with theparent crp1 plasmid, pDCRP, performed as for panel B.

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mine if CRP is contacting the a-CTD at proP, we mapped thebinding position on the proP promoter of a truncated a subunitcontaining only the last 85 amino acids comprising the C-terminal domain (20) by DNase I footprinting. The a-CTDpeptide was used for these experiments since Es38 binds poorlyto a linearized proP P2 template, consistent with the supercoil-ing requirement for transcription (57). The a-CTD peptidecaused a modest reduction in DNase I cleavages throughoutthe lanes. However, in the presence of CRP, specific protectionof DNase I cleavage was observed immediately adjacent to thedownstream side of CRP at concentrations of a-CTD used todetect binding at the rrnB P1 promoter containing a strong UPelement (6). This protection extends the CRP protected regionat least 9 bp on the top strand (Fig. 7) and 7 bp on the bottomstrand, as indicated in Fig. 1. The precise boundaries at theCRP and a-CTD junctions are not possible to determine be-cause of the lack of DNase I reactivity within this A/T-richsegment. Since the a-CTD preferentially binds to A/T-richregions in the minor groove (18), the binding site for thea-CTD may overlap the highly A/T-rich segment present at thedownstream end of the CRP protected region. No CRP-de-pendent protection by the a-CTD was observed upstream ofthe CRP site. This footprinting data is consistent with CRPstimulating transcription by the promoter-proximal subunit ofCRP directly contacting the a-CTD.

Coactivation by CRP and mutant Fis proteins. We havepreviously shown that a small region on one subunit of Fis, the

B-C loop, is required for activation of proP P2 by Fis alone.The important amino acids within this region include residuesGln 68, Arg 71, Gly 72, and Gln 74. Arginine 71 is believed todirectly contact RNA polymerase because this residue stronglyaffects cooperative binding with Es38, and most substitutionsat Arg 71 strongly reduce transcriptional activation (38). Todetermine the effect of this region on coactivation with CRP, invitro transcription reactions were performed with CRP to-gether with Fis mutants that by themselves are strongly defec-tive in P2 activation. A gel of in vitro transcription reactionsshowing coactivation by CRP and representative Fis mutantscontaining substitutions at residue 71 and 72 is shown in Fig. 8.These Fis mutants promoted little to no activated transcrip-tion. However, when these mutants were combined with CRP,transcript levels were considerably increased. Coactivationranged up to 30-fold, depending on the Fis mutant, eventhough these mutants only weakly potentiated P2 transcriptionon their own. The full level of activation that is seen with CRPand wild-type Fis is not achieved; however, it is evident that thetranscriptionally impaired Fis mutants are still competent forcoactivation with CRP.

The order of addition of activators is important for syner-gistic activation at proP P2. In the previous transcription re-actions, Fis and CRP were allowed to bind to the DNA priorto Es38 addition. To determine whether this order of additionwas important for efficient activation, we incubated one acti-

FIG. 7. DNase I footprint of DNA complexes with CRP and a-CTD. CRPprotein (1.1 pmol) and/or a-CTD (2.2 nmol) were bound to a 59-end 32P-labeledproP fragment (top strand) for 10 min prior to DNase I cleavage, as indicated atthe top. Lane G refers to the G chemical sequencing ladder; numbers to the leftare in relation to the proP P2 initiation site. Bars designate the regions protectedby CRP-cAMP and a-CTD in the presence of CRP-cAMP.

FIG. 8. Coactivation of proP P2 by Fis mutants and CRP. Single-round invitro transcription reactions were performed with CRP and Fis mutants contain-ing substitutions in the B-C loop region that is required for P2 activation by Fis.Basal indicates that no activators were added. Locations of the transcripts fromthe P2 and rna1 promoters are indicated with arrows; fold activation over thebasal level for each reaction is given at the bottom. WT, wild type.

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vator plus Es38 with the plasmid template prior to addition ofthe second activator. Single-round transcription was then ini-tiated by the addition of nucleoside triphosphates (NTPs) andheparin. In Fig. 9A, Fis plus Es38 were incubated with theDNA for 15 min followed by the addition of CRP-cAMP. Thisresulted in a 32-fold activation of transcription, which wassomewhat greater than the level for Fis alone but considerablyless than the 100-fold activation obtained by preincubatingboth activators with DNA prior to Es38 addition. Incubationfor a longer period (30 min) prior to elongation did not im-prove the efficiency of coactivation. In Fig. 9B, CRP-cAMPplus Es38 were incubated with the template for 15 min beforeaddition of Fis. Under these conditions, the degree of activa-tion was similar to that obtained with Fis alone (20-fold).Again, longer incubations prior to elongation did not improvethe efficiency of coactivation. We conclude that efficient coac-tivation by Fis and CRP requires that both activators be boundto the DNA prior to Es38.

DISCUSSION

Previously it has been shown that proP P2 expression iscompletely dependent on Fis and the stationary-phase sigmafactor, s38 (39, 56, 57). The 20- to 30-fold activation by Fis isabsolutely dependent on the a-CTD of RNA polymerase thatcontacts the B-C loop region of one of the subunits of the Fishomodimer (7, 38). In this report, we show that the CRPprotein acts synergistically with Fis to mediate even higherlevels of transcription. Binding of CRP at a site centered 121.5bp upstream of the P2 promoter gives little (,2-fold) activa-tion by itself. In combination with Fis binding at site I, which

overlaps the promoter, CRP mediates a 75- to 125-fold stim-ulation of transcription at proP P2.

While transcriptional synergy by multiple activators is oftenfound in eukaryotic organisms, there have been relatively fewreports of synergy in prokaryotes (26, 46). Examples of syner-gistic activation include the ansB promoters: in E. coli thecoactivation is dependent on both CRP and FNR, and inSalmonella enterica serovar Typhimurium coactivation is de-pendent on two CRP dimers (49). Synergy has been clearlydemonstrated in artificial bacterial promoters between CRPbound upstream of the promoter and either another CRP orlambda cI protein bound to a site overlapping the 235 element(11, 30, 31). Transcriptional activation at proP P2 is the firstexample of synergy involving the Fis protein, though the re-ports of Fis as a coregulator of transcription appear to beincreasing (14, 16, 25, 28, 37, 54). To our knowledge, a directrole in activation by CRP binding this far upstream of thepromoter has not been previously reported.

Coactivation by CRP at proP P2. To provide evidence thatCRP binding at 2121.5 directly contacts Es38 at proP P2, CRPmutants containing substitutions at residues that disrupt poly-merase-CRP interactions at either class I or class II promoterswere tested. The CRP AR1 mutants were found to stronglyreduce activation, while the AR2 mutants had no effect onactivation of transcription at proP P2. This is not surprisingbecause CRP positioned upstream of the promoter usuallydoes not mediate transcriptional activation through AR2.These results for CRP activation at proP through AR1 aresimilar to those found where CRP is acting as an upstreamactivator alone or in conjunction with either another moleculeof CRP, FNR, or the l cI protein bound in an analogousposition to Fis site I at proP P2 (11, 30, 31). However, 121 bpupstream of the transcriptional start site is an unusually largedistance for direct activation by CRP.

Because AR1 of CRP has been shown to stimulate transcrip-tion at other promoters by contacting the a-CTD of RNApolymerase, it is highly likely that CRP is contacting thea-CTD of Es38 at proP P2 (17, 27). In support of this, DNaseI footprint analysis with purified a-CTD revealed binding ofa-CTD to the DNA just downstream of the CRP binding site.This specific association of the a-CTD to the DNA is depen-dent on CRP binding, suggesting that the CRP protein, to-gether with the sequence of the DNA on the downstream side,is directing the positioning of the a-CTD. Murakami et al.measured the position of both a-CTDs of RNA polymerase atpromoters containing two CRP binding sites by affinity cleav-age of a-CTD-EDTA z Fe conjugants (41). They found that thea-CTD was unable to contact the DNA when the binding sitewas at 2113.5, even when another CRP dimer was bound at241.5. Similar results have also been obtained by hydroxylradical footprinting of CRP-RNA polymerase complexes (5).These findings differ from what we observe at proP P2, wherethe a-CTD contacts the CRP protein bound even further up-stream at 2121.5. While the localization of a-CTD by DNaseI footprinting performed here is with the a-CTD not tetheredto RNA polymerase, in contrast to the previously mentionedexperiments, CRP bound at 2121.5 nonetheless functions toactivate transcription of proP P2 in a manner dependent onAR1. In addition to the sequence of the intervening DNA, anobvious difference between these situations is that at proP theFis protein is bound at 241 instead of a second CRP molecule.However, it is not expected that Fis would induce a greaterdegree of DNA bending or a significantly different trajectory ofthe DNA from CRP bound at the same position (38, 45).

Coactivation by Fis at proP P2. An essential activation re-gion on Fis has previously been localized to the B-C loop

FIG. 9. Effect of the order of addition of activators on single-round proP P2transcription. (A) Basal (lane 1) indicates that the reaction was performed withno activators. Reactions in lanes 2 to 4 were performed under standard condi-tions (e.g., Fig. 2). Lanes 5 and 6 show transcripts produced when Fis and Es38

were incubated with the DNA template for 15 min at 37°C prior to addition ofCRP. After a further 10 or 30 min of incubation, transcription was initiated bythe addition of NTPs and heparin. (B) In vitro transcription reactions as in panelA except that in lanes 5 and 6 CRP and Es38 were incubated with the DNAtemplate for 15 min prior to Fis addition. After an additional 10 or 30 min,transcription was initiated with NTPs and heparin. Locations of transcripts gen-erated from the P2 and rna1 promoters are indicated by arrows; fold activationover the basal level for each reaction is given at the bottom.

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region (38). In transcription reactions performed in vitro withFis as the sole activator, B-C loop mutants stimulate little, ifany, activated transcription. However, together with CRP,these mutants are able to synergistically activate transcription.Therefore, with CRP bound at the promoter, the dependenceon the B-C loop of Fis appears to be alleviated. A competentB-C loop region is necessary to achieve the full level of tran-scription seen with CRP and wild-type Fis, implying that theB-C loop is still playing a role in coactivation. It is possible thatCRP may stabilize the polymerase sufficiently to partially over-come a weakened interaction by the Fis B-C loop mutants withthe a-CTD. In addition, it is possible, although we consider itunlikely, that DNA bending promoted by the B-C loop Fismutants is sufficient to stimulate transcription when CRP ispresent.

An alternative explanation for coactivation between CRPand the Fis B-C loop mutants is that a second determinantother than the B-C loop region on the Fis dimer mediates theresidual coactivation with CRP. Without a competent B-C loopregion, this potential second transcriptional activation region isunable to stimulate significant transcription when Fis is thesolitary activator (38). This postulated second patch has twopossible targets. One is CRP protein bound at 2121.5, but wehave not observed any cooperative binding between Fis andCRP at proP that could support such a model. Another expla-nation is that a second patch on Fis could contact polymerasein a manner different from the previously discovered contactbetween the B-C loop of Fis and a-CTD of RNA polymerase.Because Fis binding site I overlaps the 235 sigma subunitrecognition element, a Fis-s38 protein-protein contact is pos-sible. Other transcriptional activators such as CRP, FNR, andthe Mu Mor protein that bind to an analogous position havebeen shown to make multiple contacts with RNA polymerase(2, 4, 44, 53). Most of these activators contact the a-CTDthrough their upstream side and also make a contact on thedownstream side with either the sigma subunit or the N-ter-minal domain of the alpha subunit. An a-CTD-independentstimulation of rrnB transcription has been noted by Bokal et al.(7). Muskhelishvili et al. have also reported cooperative DNAbinding between Fis and s70 at the tyrT promoter (42).

A model of synergistic activation at proP P2. In our modelfor synergistic activation of proP P2, Fis minimally contacts onea-CTD domain, and the second a-CTD domain is bound to theCRP subunit oriented proximal to the promoter (Fig. 10). Thelack of coactivation by CRP on a DNA template with a 5-bp

insertion is consistent with the requirement for looping of theintervening DNA. One notable aspect of the coactivation seenat proP is that it is most efficient when both activators arebound to the DNA prior to Es38. This observation implies thatonce a complex is formed between the first activator and Es38,the second activator is no longer fully capable of stimulatingtranscription. This could be due to the formation of one of avariety of specific protein-DNA architectures which limit ac-cessibility to the second activator or, in the case of CRP alone,the targeting of Es38 to a nonproductive location. As men-tioned previously, an alternative model is that Fis and CRPbound to their respective sites must first interact. In order toachieve maximal coactivation, this interaction may be requiredto colocalize the two activators close to the promoter via DNAlooping prior to the binding of Es38.

ACKNOWLEDGMENTS

We thank J. Krakow for the gift of CRP protein, R. Ebright and S.Busby for CRP plasmids, and Leah Corselli for purification of thea-CTD. We also thank Ann Hochschild for valuable discussions andRichard Gourse for Fis plasmids and critical reading of the manu-script.

This work was supported by National Institutes of Health grantGM38509 to R.C.J.

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