Proc. Natl. Acad. Sci. USAVol. 92, pp. 7455-7459, August 1995Biochemistry

Identification of the Bacillus subtilis pur operon repressor(purine repressor/gene regulation/protein-DNA interaction/adenine phosphoribosyltransferase/phosphoribosyl pyrophosphate)

MANLI WENG, PETER L. NAGY, AND HOWARD ZALKINDepartment of Biochemistry, Purdue University, West Lafayette, IN 47907

Communicated by Charles Yanofsky, Stanford University, Stanford, CA, May 10, 1995 (received for review March 29, 1995)

ABSTRACT Transcription of the Bacillus subtilis puroperon is repressed in response to a signal of excess adenine.We have purified the repressor protein and have identified,cloned, and overexpressed the purR regulatory gene thatcontrols transcription initiation of the operon. B. subtilispurRencodes a 62-kDa homodimer that binds to the pur operoncontrol region. The PurR binding site which overlaps thepromoter encompasses "110 bp. The protein-DNA interac-tion is inhibited by 5-phosphoribosyl 1-pyrophosphate. Amutation that deletes the repressor binding site or one thatdisruptspurR abolishes binding activity in vitro and repressionof transcription in vivo in response to the excess adeninesignal. These results lead to a model in which an excess-adenine signal is transmitted to PurR via the 5-phosphoribo-syl 1-pyrophosphate pool. In addition, purR is autoregulated.There is no structural or mechanistic similarity between theB. subtilis and Escherichia coli purine repressors.

The Bacillus subtilispurEKBC(orf)QLFMNHD operon (whereorf is an open reading frame of unknown function), hereincalled thepur operon, encodes the 10 enzymes required for denovo synthesis ofIMP (1). Transcription of the 12-gene operonfrom a oA-dependent promoter is initiated 242 nt upstream ofthepurE, the first structural gene. Transcription of the operonis subject to dual regulation. Addition of adenine to cellsresults in repression of transcription initiation, whereas gua-nine addition signals premature transcription termination inthe mRNA leader region preceding the purE gene (1, 2). Aputative repressor protein that binds to the pur operon 5'flanking region has been partially purified (3). Althoughbinding of the protein was specific for pur operon 5' flankingDNA, it was independent of putative effector molecules suchas adenine, adenosine, or adenine nucleotides. The DNA siteto which the protein bound was mapped to nt -145 to -29relative to the start of transcription. A deletion ofpur operon5' flanking DNA from -193 to -64 abolished repression byadenine whereas guanine-mediated regulation was retained.This finding supported the idea that the extended proteinbinding site identified in vitro was in fact a control site forrepression of transcription. We now report a definitive iden-tification of the B. subtilispur operon repressor and a model forregulation.

MATERIALS AND METHODSBacterial Strains. The B. subtilis stains that were used

included DEl, the prototrophic wild type (1); DE64, a Purlstrain withpur operon control-site deletion from -193 to -64(relative to +1, the start of transcription) (3); DElCZ, DE1,purC-lacZ CmR (3); DE64 CZ, DE64 purC-lacZ CmR (3);MWR, DE1 purR NmR, MWRCZ, DElCZ purR CmR NmR;MWR64CZ, DE64CZpurR CmR NmR. The purC-lacZ fusionwas integrated into the pur operon, resulting in a Pur-

phenotype. Strain MWRZ1 is DEl purR-lacZ purR+ CmR. ApurR-lacZ translational fusion was integrated by homologousrecombination into purR. Strain MWRZ2 is MWRZ1purE::neo CmR NmR. Bacteria were grown in minimal (1) orLB (5) medium.

Purification of pur Operon Repressor (PurR). Repressorwas purified from strain DE1 cells that had been grown inminimal medium and stored at -20°C. All steps were carriedout at 4°C. Cells (5 g) were disrupted by a French pressure cellin 20 ml of buffer A (50 mM potassium phosphate, pH 7.0/0.1mM EDTA/2 mM dithiothreitol/1 mM phenylmethanesulfo-nyl fluoride). The cell extract was initially centrifuged at 15,000x g for 30 min and then at 180,000 x g for 5 hr. The top 18 mlof the supernatant and the pellet were discarded while thebottom 2.0 ml was retained for fractionation by (NH4)2SO4.The protein fraction that precipitated between 45% and 65%saturated (NH4)2SO4 was dissolved in 1.0 ml of buffer A,dialyzed against buffer A, and chromatographed on a column(1.1 cm x 82 cm) of Sephacyl S-300 in buffer A. DNA-bindingactivity was recovered in the exclusion volume due to associ-ation of the repressor with DNA. A 5-ml pooled fractioncontaining repressor was treated with 1% (wt/vol) streptomy-cin sulfate to dissociate the protein from DNA and thencentrifuged at 15,000 x g. The supematant was dialyzedagainst buffer B [25 mM Hepes, pH 7.6/1 mM dithiothreitol/20% (vol/vol) glycerol, 0.1% (vol/vol) Nonidet P-40] contain-ing 0.1 M KCl. The final step was purification by DNA affinitychromatography (6). The dialyzed sample was divided in halfand 1.8 ml was adsorbed to 2 ml of the affinity matrix (6) inbuffer B. The repressor was eluted between 0.5 and 0.6M KCl.After regeneration of the affinity matrix (6) the remainingsample was purified. The DNA affinity column was preparedby coupling 350 ,g of syntheticpur operon DNA fragment (nt-116 to -21 to which GATC 5' ends had been added) to 5 mlof CNBr-activated Sepharose 4B (6). Protein-DNA bindingactivity was monitored throughout the purification by gelretardation assay (3) using a -147 to -21 pur operon DNAprobe. A unit of binding activity is defined by the conversionof half of the free DNA probe (10 fmol) to the bound form.Specific binding activity is units per mg of protein. Protein wasdetermined by the method of Bradford (7) prior to the affinitypurification. The protein concentration was not determinedafter purification. Purity of fractions was determined by SDS/PAGE. Protein bands were stained with silver (8).

Protein Analyses. Repressor for sequence analysis was pu-rified from 15 g of cells by three repititions of the proceduredescribed -above. The pooled affinity-purified protein wasconcentrated and glycerol and KCI were removed from thebuffer by addition of water and several cycles of centrifugalconcentration using Centriprep-30 and Centricon-30 mem-branes (Amicon). NaHCO3 was added to 0.1 M, the samplewas concentrated to 0.1 ml, and the protein was precipitatedwith ethanol. The entire sample (protein amount not deter-mined) was electrophoresed in an SDS/12% polyacrylamidegel and electroblotted to a poly(vinylidene difluoride)-type

Abbreviation: PRPP, 5-phosphoribosyl 1-pyrophosphate

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Proc. Natl. Acad. Sci. USA 92 (1995)

membrane (9). Protein bands, visualized by staining withCoomassie blue, were cut out for microsequencing (9). Thequantity of protein was estimated by comparison of theintensity of the stained protein band with known quantities ofbovine serum albumin and cytochrome c in adjacent lanes.

Native molecular weight was estimated by gel filtration on acalibrated column (1.55 cm x 85 cm) of Sephacryl S-200.Ultracentrifuged extract prepared as described above wastreated with DNase I, applied to the calibrated column, andeluted with buffer A. Proteins were detected by absorbance at280 nm and binding activity by gel retardation assay (3).

Cloning and Overexpression. The purR gene was cloned byPCR using chromosomal DNA from strain DE1 and 30-nt 5'and 3' primers. An Nde I site overlapping the initiator ATGcodon was incorporated into the 5' primer and a Hindlll sitewas incorporated into the 3' primer 100 bp downstream of thetranslation stop codon. The resulting 960-bp fragment wasligated into plasmid pT7-7 (10) to yield pMW10. PlasmidpMW10 was transformed into Escherichia coli strainBL21(DE3) (10) forpurR overexpression. Strain BL21(DE3)/pMW10 was grown in LB medium with ampicillin (50 ,ug/ml)at 37°C to mid-logarithmic phase. T7 polymerase was inducedby addition of 2% lactose and growth was continued for 17 hrat 30°C. Frozen cells were stored at -20°C prior to preparationof the extract.Gene Disruption. purR in pMW10 was disrupted at codon

108 by insertion of a NmR gene from pBEST 501 (11) that isfunctional in both B. subtilis and E. coli. The plasmid with thepurR disruption was named pMW11. The disrupted gene wasintegrated into the B. subtilis purR locus by homologousrecombination, and the purR disruption in neomycin-resistantstrains was verified by Southern blotting. A similar procedurewas used to disrupt orf2, a gene that is contiguous with purR(see Table 2).

f3-Galactosidase Assay. pur operon expression was deter-mined by measurement of jB-galactosidase in a strain with apurC-lacZ fusion integrated into the chromosomalpur operon(12). Bacteria with purC-lacZ fusions are purine auxotrophsand were grown with a limiting (xanthine) or excess (adenine,guanine) purine supplement as described (12). The mediumcontained either chloramphenicol (strains DElCZ andDE64CZ) or neomycin (strains MWRCZ and MWR64CZ) at5 jug/ml. ,B-Galactosidase was assayed exactly as described(12), in SDS/chloroform-treated cells.

RESULTSPurification of the Repressor ofpur Operon Transcription.

The PurR was purified from B. subtilis as a route to genecloning for further analysis of the regulatory system as well asto facilitate protein overproduction. For purification, repres-sor activity was monitored by its capacity for specific bindingtopur operon DNA (3). A summary of the purification is givenin Table 1. There were two key purification steps. First, afterultracentrifugation of the cell extract a substantial fraction ofthe repressor was of high molecular weight as a result ofbinding to DNA and could be separated from most cellularproteins by gel filtration on Sephacryl S-300. Second, afterrelease from DNA by treatment with streptomycin sulfate the

Table 1. Summary of repressor purification from 5 g of cells

Total Total SpecificVolume, protein, activity, activity,

Fraction ml mg units units/mgExtract 2.0 54 8000 148(NH4)2SO4 1.0 7.9 8000 1,013Gel filtration 5.0 0.070 1690 24,140DNA affinity 4.8 ND 600 ND

ND, not determined.

repressor was further purified by DNA affinity chromatogra-phy. Data in Table 1 indicate >160-fold purification throughthe gel filtration step. After the final purification step, DNAaffinity chromatography, protein concentration was not deter-mined, due to limited availability. As a result, the specificbinding activity of the purified protein was not calculated andthe final purification factor is not known. Purification wasmonitored by SDS/PAGE. Protein fractions from gel filtrationand DNA affinity chromatography are shown in Fig. 1. Thepurified preparation contained a major protein of about 32.5kDa as well as other less abundant proteins.Amino-Terminal Amino Acid Sequence Determination. Af-

finity-purified repressor was electrophoresed in an SDS/12%polyacrylamide gel and electroblotted to a poly(vinylidenedifluoride)-type membrane. The -32-kDa protein band wasexcised for sequence determination. The amino acid sequenceobtained from 25 cycles of Edman degradation was Met-Lys-Phe-Arg-Arg-Ser-Gly-Arg-Leu-Val-Asp-Leu-Thr-Asn-Tyr-Leu-Leu-Thr-His-Pro-(Val/Ala)-Glu-Leu-Ile-(Ala/Thr).This sequence corresponds in 23 of 25 positions with an openreading frame of unknown function at coordinates 118,041-118,898 in a recently reported 180-kb region near the B. subtilisorigin of replication (GenBank accession no. D26185) (13).Assignments at cycles 21 and 25 do not match the genesequence and are assumed to be incorrect. Further character-ization reported in this paper indicates that the gene atcoordinates 118,041-118,898 encodes thepur operon repressorand is designated purR.ThepurR Region. A map and description of thepurR region

are given in Table 2. The purR gene is flanked upstream anddownstream by unidentified open reading frames. A proteindatabase search using BLAST (14) did not provide clues to thefunctions of the flanking genes, although orf2 is highly con-served in E. coli, Azotobacter vinelandii, yeast, and rat. Thetranslation termination codon for purR and the translationstart for orf2 overlap, ATGA, indicative of translational cou-pling and perhaps related function. ThepurR region is flankedby sspF and spoVG, genes that have been mapped to 6° on theB. subtilis chromosome (15). There is a highly significant matchto a consensus B. subtilis orA-dependent promoter in the 56-bpintercistronic space between orfl and purR, 33 bp upstreamof the purR translation start codon. TTGATA...(17 bp)...TATATT. Transcription from this putative promoter

A B

106 -

80 -

49.5-

32.5 - 4 PurR

27.5 -

FIG. 1. SDS/PAGE of pur operon repressor after gel filtration(lane 1 0.55 ,ug) and after DNA affinity chromatography (lane 2)(protein quantity not determined). Molecular mass markers are givenin kilodaltons. The SDS/10% polyacrylamide gel was stained withsilver. Arrow points to the repressor protein.

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Proc. Natl. Acad. Sci. USA 92 (1995) 7457

Table 2. Description of the purR region

No. of No. ofGene Coordinates* base pairs amino acids

sspF 116,783-116,968 186 61orfl 117,116-117,985 870 289purR 118,041-118,898 858 285orf2 118,895-119,272 378 125spoVG 119,466-119,759 294 97

The gene organization is sspF-(147 bp)-orfl-(56 bp)-purRorf2-(193bp)-spoVG.*Obtained from GenBank accession no. D26185. Coordinates includethe translation stop codon.

would yield a bicistronicpurRorj2 mRNA that may terminateat a factor-independent transcription termination motif '30nt following the orf2 translation stop.

Cloning ofpurR and Overproduction of the Repressor. ThepurR gene was cloned from B. subtilis chromosomal DNA byPCR. Plasmid pMW10, in which purR is transcribed from aphage T7 promoter, was used to transform E. coli BL21(DE3).Overproduction of the repressor protein in E. coli is shown inFig. 2. 0Overexpression ofpurR from plasmid pMW10 yieldeda protein band of -32 kDa that was not obtained from cellscarrying the vector control. Approximately one-third of theoverproduced repressor protein remained soluble after cen-trifugation of the extract at 15,000 x g for 30 min.

Properties ofthe PurR. SDS/PAGE of the repressor proteinpurified from B. subtilis (Fig. 1) and overproduced in E. coli(Fig. 2) indicates a protein subunit of -32 kDa, consistent witha molecular mass of 31.2 kDa calculated from the gene sequence.A molecular mass for the native protein of -65 kDa was obtainedby gel filtration, indicative of a dimer (data not shown).

Protein database searches with BLAST (14) indicated limitedsimilarity of the pur operon repressor with adenine phospho-ribosyltransferase from E. coli (16), Saccharomyces cerevisiae(17), and human (18). An alignment of the repressor with thethree phosphoribosyltransferases gave 16% identity (29 of 183invariant residues) in the four sequences over the 183-aaoverlapping region. This compares with 23% identity (43 of183 invariant residues) for the three adenine phosphoribosyl-transferases. Although the limited sequence identity is insuf-ficient to establish homology between thepur operon repressor

1 2 3 4: S . _ | -- 80.:1$;E_1 _ ~49.5

_l;j = = _ ~~~32.5PurR _"

. _ w --- ~27.5

-18.5

FIG. 2. Overproduction of B. subtilis pur operon repressor in E.coli. BL21(DE3)/pMW10 cell extract was centrifuged at 15,000 x g for30 min. The supernatant fraction (50 ,ug of protein) and a correspond-ing amount of the pellet were fractionated in an SDS/12% polyacryl-amide gel. Lane 1, pellet, vector control; lane 2, pellet, pMW10(purR+); lane 3, supernatant, vector control; lane 4, supernatant,pMW10 (purR+). Molecular mass markers are given in kilodatons.Arrow points to the overproduced repressor at 32.5 kDa.

and adenine phosphoribosyltransferase, the alignment identi-fied a putative binding site for 5-phosphoribosyl 1-pyrophos-phate (PRPP) in the pur operon repressor. Fig. 3 shows analignment of a 13-bp PRPP binding motif (22) in the puroperon repressor, three adenine phosphoribosyltransferases,and the PRPP binding sites of three phosphoribosyltrans-ferases of known structure (19-21). The PRPP binding motifis also conserved in PRPP synthetases (23).Binding ofPurR to DNA.A protein that binds specifically to

the 5' region flanking thepur operon promoter was previouslydetected in extracts of B. subtilis and was partially purified (3).We have now shown that this protein is the repressor of thepuroperon. Protein binding from B. subtilis cell extract to a puroperon DNA probe is shown in Fig. 4, lane 1. Two protein-DNA complexes were detected, as reported previously (3).Two experiments have established the correspondence of theDNA-binding activity with the product of thepurR gene. First,disruption of the purR gene eliminated binding activity in theextract (Fig. 4, lane 2). Second, overexpression of the purRgene in E. coli resulted in the appearance of DNA-bindingactivity in the E. coli cell extract. E. coli cells do not containproteins that bind to the B. subtilis pur operon control region(Fig. 4, lane 5). However, overexpression ofpurR resulted inbinding activity that yielded the same protein-DNA doublet asobtained with the B. subtilis extract (Fig. 4, lane 4).

Since pur operon transcription is repressed by addition ofadenine or adenosine to cells, we examined the effect ofpurines, nucleosides, and nucleotides on the repressor-DNAinteraction in vitro. Adenine, adenosine, AMP, ADP, ATP,IMP, guanine, GMP, GDP, GTP, and UMP at a concentrationof 100 ,uM were without effect on the binding of repressor tocontrol-region DNA (data not shown). Since the sequencealignment shown in Fig. 3 indicates that the repressor mostlikely contains a PRPP binding site, we examined the effect ofPRPP on repressor-DNA interaction. Results summarized inFig. 5 show that PRPP inhibited repressor binding to puroperon control region DNA. The PRPP concentration re-quired for half maximal inhibition of protein-DNA bindingwas 5 ,uM (Fig. 5). Ribose 5-phosphate, PPi, or Pi at aconcentration of 100 ,uM had no effect on this protein-DNAinteraction. The purine bases, nucleosides, and nucleotideslisted above did not modulate inhibition by PRPP.The in vitro repressor-DNA interaction was further probed

by DNase I footprinting and by deletion analysis. The DNaseI footprint covered about 110 bp, extending from nt -136 to-26, and was similar to that reported previously (3). Thecharacteristic feature of the footprint is a central 20-bp pro-tected core, nt -86 to -66, flanked by protected and hyper-sensitive segments having -10-bp periodicity that would cor-respond to five upstream and four downstream helical turns ofprotein-DNA interaction (data not shown). The central corewas more strongly protected from DNase I than flanking DNAat either end of the footprint. These features are reminiscentof a protein-DNA complex formed by wrapping of DNA

PurRAPRT-hsAPRT-ecAPRT-scGPATOPRTHGPRT

VLI IDDFMKAGGTVVVVDDLLATGGTVLVVDDLLATGGTVVVVDDVLATGGTVVMVDDS IVRGTTVMLVDDVITAGTAVLIVEDI IDTGKT

FIG. 3. Alignment of a PRPP binding motif in B. subtilis PurR andin adenine phosphoribosyltransferase from human (APRT-hs) (18), E.coli (APRT-ec) (16), and S. cerevisiae (APRT-sc) (17); glutaminePRPP amidotransferase (GPAT) (19); orotate phosphoribosyltrans-ferase (OPRT) (20); and hypoxanthine (guanine) phosphoribosyl-transferase (HGPRT) (21). Residues conserved in six of the sevenproteins are emphasized in bold type.

Biochemistry: Weng et al.

Proc. Natl. Acad. Sci. USA 92 (1995)

1 2 3 4 5

.. :__ _B v .* . : ' e r |F *

I ..I.

.~~~~~~~~i .:_

..I..4-~ ~~~~~~~~~~~~~~~~~~~~~~~~~~~~t _

*,I,.

90-80-70-

0 60-) 50-

z 40-a-.o 30-

20-10-

FIG. 4. Binding of PurR to control-region DNA. Gel retardationassay was carried out with approximately 25 jig of cell extract from B.subtilis or 25 ng of extract from E. coli and 10 fmol of DNA probe (nt-147 to -21). Lane 1, B. subtilis DE1 (purR+); lane 2, B. subtilis MWR(purR-); lane 3, no extract; lane 4, E. coli BL21(DE3)/pMWR(purR+); lane 5, E. coli BL21(DE3)/pT7-7 (vector control). Arrow-heads point to free (F) and bound (B) forms of the pur operon DNAprobe.

around a protein (24). Gel shift assays with various 5' flankingsequences of the pur operon indicated that the central coreregion is necessary but not sufficient for protein-DNA inter-action. Maximal binding affinity appears to require a segmentof DNA that overlaps the central core with an overall lengthof 100 bp or more. These results establish that the recombinantpur operon repressor binds to DNA as previously described forthe protein in the B. subtilis cell extract (3).We have found binding of PurR to the 5' flanking regions

of two additional genes,purA (25) andpurR (data not shown).The interaction of PurR with these genes was similar to thatdescribed for the pur operon, including inhibition of PRPP.

In Vivo Repression. The regulatory role ofpurR was exam-ined by assay of pur operon expression from a genomicpurC-lacZ fusion strain. Data in Table 3 show 15-fold regula-tion of the pur operon by adenine and 22-fold regulation byguanine in apurR+ strain. A deletion of the upstream controlregion to position -64 had little effect on expression butresulted in loss of regulation by adenine; 11-fold regulation ofguanine was retained. These results confirm the earlier reportson regulation of the pur operon by addition of adenine andguanine to cells (3, 12). Data in Table 3, line 3, show that thepurR mutation in strain MWRCZ abolished repression of thepur operon by adenine, whereas regulation by guanine wasretained. Repression by adenine was likewise abolished in thepurR/cis control-region double mutant. These data thereforeestablish that purR controls expression of the operon inresponse to the uptake of adenine by the cells. The 15-foldregulation of adenine and 22-fold regulation by guanine instrain DElCZ results from dual control of transcription ini-

**l * S e_^

I :,... ..... : _i_E...............1 ....... _

_ ,

0 5 10 15 20 25 30 35 40 45 50

PRPP, iM

FIG. 5. Inhibition by PRPP of PurR binding topur operon control-region DNA. Binding mixtures contained 4.4 fmol (0.22 nM) of puroperon DNA probe, 48 ng of E. coli BL21(DE3)/pMWR cell extract,and varied PRPP concentrations. Binding was determined by gelretardation. Inset shows the autoradiogram. Free and bound DNAwere quantitated by scintillation counting. The leftmost lane shows thecontrol with no PRPP, followed by PRPP concentrations of 0.25, 0.50,1.0, 2.5, 5.0, 10, 15, 20, 25, 35, and 50 ALM (left to right). The rightmostlane shows a control with no protein. The saturation curve was fit tothe Michaelis-Menten equation by ULTRAFIT software (Biosoft, Cam-bridge, U.K.).

tiation and transcription termination (1) and is influenced byadenine-guanine nucleotide interconversions (4). Regulationof 11- to 13-fold by guanine was seen when repression byadenine was eliminated.

Expression of thepurR gene was determined in apurR-lacZfusion strain (Table 3). By this assay expression of purR was<2% that ofpurC. Addition of adenine to cells repressedpurRby a factor of 3. The purR gene is thus autoregulated. Excessguanine in the growth medium had no effect onpurR expres-sion. In addition, disruption of orJ2 had no effect on theexpression or regulation ofpurR or the pur operon (data notshown).

DISCUSSIONWe have identified, cloned, and overexpressed the purR reg-ulatory gene, which controls transcription initiation of the B.subtilispur operon. ThepurR gene was described previously asan open reading frame of unknown function at the 60 regionof the chromosome (GenBank accession no. D26185). Basedon predictions from the nucleotide sequence, purR wouldappear to be transcribed from a orAdependent promoter toyield a bicistronic mRNA coding for thepur operon repressorand a 125-aa protein of unknown function.The purine repressor is a 62.4-kDa dimer with identical

subunits of 285 aa. Based on an alignment with three adeninephosphoribosyltransferase sequences as well as with threephosphoribosyltransferases of known structure, a 13-aa PRPP

Table 3. Regulation ofpur operon and purR expression

Fold,3-Galactosidase activityt regulation

Fusion Strain Operator* purR Xan Ade Gua Ade + Gua Ade Gua

purC-lacZ DElCZ + + 4120 ± 106 273 ± 17 185 ± 3 46 ± 1 15 22DE64CZ A + 3650 ± 53 3750 ± 62 330 ± 8 313 ± 24 0.97 11MWRCZ + - 3014 ± 141 3150 ± 31 246 ± 13 210 ± 28 0.96 12MWR64CZ A - 3340 ± 525 3310 ± 464 248 ± 53 239 ± 51 1.0 13

purR-lacZ MWRZ2 + + 60 ± 32 20 ± 0.2 65 ± 25 20 ± 0.8 3.0 0.9

*The cis control region is either wild type (+) or deleted (A).tBacteria were grown in medium supplemented with xanthine (Xan), adenine (Ade), guanine (Gua), or Ade + Gua as described (12). Activityin Miller units (5) is the average ± SD of two or three independent determinations each in duplicate.

7458 Biochemistry: Weng et aL

Biochemistry: Weng et al. ~~~~Proc. Natl. Acad. Sci. USA 92 (1995) 7459

binding motif was identified at residues 199-211 of the re-pressor. It is therefore possible that the PurR PRPP site couldbe derived from an adenine phosphoribosyltransferase-likeprotein. Although the overall identity between adenine phos-phoribosyltransferase and pur operon repressor is insufficientto establish homology, the sequence similarity suggests astructural organization for the repressor. The repressor may bea two-domain protein with a 76-aa amino-terminal DNA-binding domain joined to a 209-aa domain related to adeninephosphoribosyltransferase.How does PurR mediate repression of target genes by excess

adenine? Upon uptake, adenine is converted into the adenine5' nucleotides (4), consuming PRPP in the process. ADP is theprimary allosteric inhibitor of PRPP synthetase, and inhibitionof ADP is augmented at elevated ATP concentrations (26).Inhibition of PRPP synthetase coupled with the consumptionof PRPP for salvage of adenine could explain the capacity ofadenine to lower the intracellular PRPP concentration (4).Thus, a signal of adenine excess is communicated to PurR viathe decreased PRPP pool. The decreased PRPP concentrationpermits binding of repressor to the 5' control site of the puroperon, as well as purA and purR PRPP is thus an importantsignal molecule for regulating adenine and guanine nucleotidesynthesis. In addition to regulating gene expression, PRPPcompetes with AMP for binding to the glutamine PRPPamidotransferase catalytic site (19). This competition is in-strumental for the end-product control of glutamine PRPPamidotransferase, the key regulatory enzyme for de novopurine nucleotide synthesis.The unique feature of thepur repressor-DNA interaction is

the -1410-bp control site that is bound. A DNase I footprintobtained with cell extract containing 1000-fold enriched DNA-binding activity relative to that in aB. subtilis extract confirmedthe earlier work (3) and supports the proposal that binding isto a central 20-bp DNA core region flanked by five helicalturns of contiguous upstream DNA and four helical turns ofcontiguous downstream DNA. We do not know what accountsfor protein-DNA binding specificity.

Finally, it is important to emphasize that the B. subtilis puroperon repressor has no structural or mechanistic similarity tothe E. coli pur regulon repressor. E. coli PurR is a dimericprotein having a 60-aa DNA-binding domain joined to a 280-aadomain with functions for corepressor binding and dimeriza-tion (27, 28). The corepressor-dimnerization domain is struc-turally related to E. coi periplasmic binding proteins and theintact PurR to the LadI family of repressors (28, 29). E. coliPurR binds to a 16-bp palindromic recognition sequenceutilizing major- and minor-groove interactions between helicesof the DNA-binding domain and the 16-bp operator.

We thank Sihong Chen for determining the DNase I footprint of thePurR-pur operon control region. This work was supported by GrantGM24658 from the U.S. Public Health Service. Synthesis of oligonu-cleotides and protein microsequencing were performed by the PurdueLaboratory for Macromolecular Structure, which is supported by Dia-

betes Research and Training Grant DK20524. This is Journal Paper 14653from the Purdue University Agricultural Research Station.

1. Ebbole, D. J. & Zalkin, H. (1987)1J. Biol. Chem. 262,8274-8287.2. Ebbole, D. J. & Zalkin, H. (1988) 1. Biol. Chem. 263, 10894-

10902.3. Ebbole, D. J. & Zalkin, H. (1989)1J. Biol. Chem. 264, 3553-3561.4. Nygaard, P. (1993) in Bacillus subtilis and Other Gram-Positive

Bacteria: Biochemistry, Physiology, and Molecular Genetics, eds.Sonenstein, A. L., Hoch, J. A. & Losick, R. (Am. Soc. Microbiol.,Washington, DC), pp. 359-378.

5. Miller, J. H. (1972) Experiments in Molecular Genetics (ColdSpring Harbor Lab. Press, Plainview, NY).

6. Kadonaga, J. T. (1991) Methods EnzymoL. 208, 10-23.7. Bradford, M. M. (1976) Anal. Biochem. 72, 248-254.8. Sasse, J. & Gallagher, S. R. (1994) in Current Protocols in

MolecularBiology, eds. Susubel, F. M., Brent, R., Kingston, R. E.,Moore, D. M., Seidman, J. G., Smith, J. A. & Struhl, K. (Greene& Wiley, New York), pp. 10.6.1-10.7.3.

9. Moos, M., Jr. (1994) in Current Protocols in Molecular Biology,eds. Susubel, F. M., Brent, R., Kingston, R. E., Moore, D. M.,Seidman, J. G., Smith, J. A. & Struhl, K. (Greene & Wiley, NewYork), pp. 10.19.1-10.19.12.

10. Tabor, S. & Richardson, C. C. (1985) Proc. Natl. Acad. Sci. USA82, 1074-1078.

11. Itaya, M., Kondo, K. & Tanaka, T. (1989) Nucleic Acids Res. 17,4410.

12. Ebbole, D. J. & Zalkin, H. (1989)1J. Bacteriol. 171, 2136-2141.13. Ogasawara, N., Nakai, S. & Yoshikawa, H. (1994) DNA Res. 1,

1-14.14. Altschul, S. F., Gish, W., Miller, W., Myers, E. W. & Lipman,

D. J. (1990)1J. MoL. Biol. 215, 403-410.15. Anagnostopoulos, C., Piggot, P. J. & Hoch, J. A. (1993) in

Bacillus subtilis and Other Gram-Positive Bacteria: Biochemistry,Physiology, and Molecular Genetics, eds. Sonenshein, A. L., Hoch,J. A. & Losick, R. (Am. Soc. Microbiol., Washington, DC), pp.425-461.

16. Hershey, H. V., Gutstein, R. & Taylor, M. W. (1982) Gene 19,89-92.

17. Yuryev, A. & Corden, J. L. (1994) Yeast 10, 659-662.18. Hikada, Y., Tarle, A., O'Toole, T. E., Kelley, W. N. & Pallella,

T. D. (1987) Nuckeic Acids Res. 15, 9086.19. Smith, J. L., Zaluzec, E. J., Wery, J.-P., Niu, L., Switzer, R. L.,

Zalkin, H. & Satow, Y. (1994) Science 264, 1427-1433.20. Scapin, G., Grubmeyer, C. & Sacchettini, J. C. (1994) Biochem-

istry 33, 1287-1294.21. Eads, J. C., Scapin, G., Xu, Y., Grubmeyer, C. & Sacchettini,

J. C. (1994) Cell 78, 325-334.22. Hove-Jensen, B., Harlow, C. J., King, C. J. & Switzer, R. L.

(1986)1J. Biol. Chem. 261, 6765-6771.23. Nilsson, D., Hove-Jensen, B. & Arnvig, K. (1989) MoL. Gen.

Genet. 218, 565-571.24. Wahle, E. & Kormberg, A. (1988) EMBO J. 7, 1889-1895.25. Maints&lai, P. & Zalkin, H. (1992)1J. BacterioL. 174, 1883-1890.26. Arnvig, K., Hove-Jensen, B. & Switzer, R. L. (1990) Eur. J.

Biochem. 192, 195-200.27. Choi, K. Y. & Zalkin, H. (1992)1J. Bacteriol. 174, 6207-6214.28. Rolfes, R. J. & Zalkin, H. (1988)1J. Biol. Chem. 263,19653-19661.29. Schumacher, M. A., Choi, K. Y., Zalkin, H. & Brennan, R. G.

(1994) Science 266, 763-770.

Biochemistry: Weng et aL