the c1 genes of p1 and p7

votume 17 Number 19 1989 Nucleic Acids Research

The cl genes of PI and P7

Francis A.Osborne, Sonja R.StovaJl and Barbara R.Baumstark*

Department of Biology, Georgia State University, Atlanta, GA 30303, USA

Received July I I , 1989; Revised and Accepted August 29, 1989 EMBL accession nos X16005, X16006

ABSTRACTThe cl genes of the heteroimmune phages PI and P7 were sequenced and their products werecompared. P7cl expression was correlated with the translation in vitro of a protein whose predictedmolecular weight (33,000 daltons) is indistinguishable from that of the Plcl repressor. The cl regionsfrom both PI and P7 were found to contain open reading frames capable of coding for a 283-aminoacid protein whose predicted secondary structure lacks the helix-turn-helix motif commonly associatedwith repressor proteins. Two Plcl amber mutations were localized to the 283-amino acid open readingframe. The Plcl and P7cl sequences were found to differ at only 18 positions, all but two of whichalter the third position of the affected codon and do not alter the amino acid sequence of the protein.Plasmids expressing the cl gene from either phage cause the repression of transcription from a clonedpromoter situated upstream of Plcl .

INTRODUCTIONThe cl genes of the plasmid prophages PI and P7 code for repressor proteins that arerequired for the establishment and maintenance of lysogeny (reviewed in 1). A proteincorresponding to the Plcl repressor has been isolated and shown to be a sequence-specificDNA binding protein that recognizes several widely dispersed sites on the phage DNA(2 — 7). The consensus DNA sequence recognized by the Plcl repressor(ATTTATTAGAGCA[A/T]T) contains no discernable bilateral symmetry, a feature thatis highly unusual among prokaryotic operator sites.

PI and P7 are heteroimmune; that is, each phage is able to establish a lytic infectionon a lysogen of the other phage. In this sense, their relationship is analogous to that ofphage X and 434, which differ in the DNA specificity of their cl repressor proteins (8).However, genetic studies indicate that Plcl and P7cl can be crossed into the heterologousphage without affecting the immunity specificity of the recipient (9). The basis for P1/P7heteroimmunity has been localized to a second regulatory gene, c4, that is unlinked tocl. The c4 gene products prevent the expression of antlreb, a closely linked gene thatinterferes with cl-mediated repression (10, 11). According to current models, P1/P7heteroimmunity results from the inability of the c4 repressor of one phage to prevent antlrebexpression from the heteroimmune phage genome (10, 11).

Because the cl genes of PI and P7 are genetically interchangeable, it is anticipated thatthe two gene products carry out similar or identical regulatory functions. The studiespresented in this paper were undertaken to investigate the biochemical basis for the apparentgenetic identity of the two cl genes. In this paper, we present the DNA sequence of thecl genes of PI and P7 and the predicted amino acid sequence of the cl repressor proteins.We report that Plcl and P7cl code for proteins of identical size (283 amino acids) and

© IRL Press 7671

Downloaded from https://academic.oup.com/nar/article-abstract/17/19/7671/2377117by gueston 10 February 2018

Nucleic Acids Research

nearly identical sequence. We report further that both repressors prevent the expressionof a promoter located immediately upstream of the Plcl open reading frame, an observationthat confirms their functional similarity and suggests an autoregulatory role for the twoproteins. Analysis of the secondary structure predicted by the open reading frames doesnot reveal the characteristic helix-turn-helix (12) or other motifs commonly associated withDNA binding proteins.

MATERIALS AND METHODSBacterial and phage strains.E. coli K336 is a SuO derivative of K140 (13). E. coli CB454 is a recA~, lacZ~derivative of K-12 (14). P I + is described by Scott (13). P7+ is the strain of Smith (15),as described by Scott (16). P7cl.l contains a missense mutation in the cl gene (17). TheP7 phage strains and the cl amber mutant phage strains Plcl.245Cm, Plcl.169andPlcl.55(11) were generously provided by June Scott.Enzymes and reagents.Restriction enzymes, T4 DNA ligase and polymerase, and the Klenow fragment of E.coli DNA polymerase were purchased from Boehringer Biochemicals or New EnglandBiolabs and reactions were carried out according to the manufacturers' instructions. DNAsequencing kits and in vitro transcription-translation kits were purchased from BethesdaResearch Laboratories and Amersham Corporation, respectively. Synthetic oligonucleotidesto be used as sequencing primers were prepared on an Applied Biosystems DNA synthesizer.Plasmid construction.pBRB7.2. pBRB7.2 (2) contains a 3.2 kb EcoVUPvuO. fragment from the cl region ofPI (Figure 1) inserted into the 2.3 kb EcoKUPvuU fragment of pBR322 that contains theorigin of replication and the jS-lactamase gene.pFAO2. P7 plasmid DNA was digested with PvuU, ligated to similarly digested pBR322,and transformed into E. coli K336. Ampicillin-resistant colonies were screened for clactivity by cross-streak complementation analysis against P7cl.l (18). pFAO2 containsa 3.5 kb insert of P7 DNA. The fragment was localized to the cl region of the P7 genomeby Southern hybridization against PI and P7 DNA that had been digested with BamHland BglU (data not shown).pBRB169.1 andpBRB55.1. The Plcl open reading frame was previously localized to a2.6 kb EcoRUBamHl fragment derived from Pl£coRI-7 (2). This fragment also containsthe wildtype allele for the conditional lethal mutation anA2> (19, 20). To clone the cl readingframe from the amber mutant phage Plcl . 169 and Plcl .55, we digested phage DNA with£coRI and BamHl, ligated the digestion products into similarly digested pBR322, andtransformed the ligation mixture into E. coli K336. Ampicillin-resistant, tetracycline-sensitive colonies were screened by cross-streak complementation analysis for their abilityto support the growth of Plam43. Plasmid DNA isolated from complementation-positivecells was shown by agarose gel electrophoresis to carry plasmids containing the 2.6 kbEcoRUBamHl fragment from the cl region. The cl mutant open reading frames wereplaced under the control of normal regulatory signals present in the cl region by digestingthe cl.55 and cl.l69-containing plasmids with BamHl and PvuII and inserting a 601 bpBamHUPvuU fragment containing the cl promoter region (2). The resulting plasmids,pBRB55.1 and pBRB 169.1, respectively, contain the 3.2 kb EcoSJ/PvuU fragmentanalogous to that present in pBRB7.2 (Figure 1).pBCB2.13-2.18. To identify cl-repressible promoters, we introduced selected fragments

7672



G R H _,_£.

P1

P7

3 . 2 1.5 1.0 0.5

- dpBRB7.2

pFA02

<—lacZC

<-lacZC

<-lacZC

pBCB2.13

pBCB2.16

pBCB2.18

Fig. 1. The cl regions of PI and P7. A restriction map is indicated by the solid line in the upper part of thefigure. Sites for EcoRl (E), PvuU (P), Nntl (N), BglQ (G), SamHI (B), and EcoRV (R) are shown The sequencingstrategy is indicated by the horizontal arrows. Letters and arrows above and below the map refer to sites andsequence analysis for PI and P7, respectively. The size of this region (in kilobase pairs) is indicated below themap. The DNA fragments present in selected plasmids are illustrated by boxes at the bottom of the figure. Thedashed line reveals the approximate position of the cl gene (2). The sites of the 76 mutations introduced intopFAO2 are indicated by asterisks. pFAO2.16 and pFAO2.26 contain insertions located 0.9 kb and 1.4 kb,respectively, from the PvuU site at the left side of the map. The direction of the lacL open reading frame inpBCB2.13-18 is indicated by the adjacent arrow.

from the cl region of PI into pCB192, a promoter-probe vector containing promoterlesscopies oflacZ and galK. extending in opposite directions from a multiple cloning site (21).The source of PI DNA for these constructions was pZHA3, a derivative of pBRB7.2 thatcontains a HindUl linker at the single EcoRV site located about 200 bps upstream of thecl open reading frame (Figure 1). pBCB2.13 contains a 460 bp fragment of PI DNAextending from the EcoRV site to a BglQ site within the cl open reading frame. pBCB2.16contains a 130 bp fragment extending from the EcoRV site to a BarniU site located about100 bps upstream of the cl open reading frame, while pBCB2.18 contains the regionextending from this BamtU site to the Bg[Q site within the open reading frame (Figure1). The orientation of the PI DNA fragments within these plasmids was confirmed byrestriction mapping and DNA sequencing.

To test for the regulation of promoter expression by c 1, we transformed pBCB2.13 andits derivatives into CB454(pBRB7.152) and CB454(pFAO2.152), two strains that expressP7cl and Plcl , respectively, from the pCB 192-compatible kanamycin-resistant vectorpDPT152 (22). Cells harboring both plasmids were selected by their resistance to bothampicillin and kanamycin. lacL expression was measured by the procedure of Miller (23).pBRB7.152 was generated by introducing Pl£coRI-7 into pDPT152. pBRB7.152 hassustained a spontaneous deletion within the EcoRl-1 fragment that results in the loss of2.5 kb of PI DNA from the far left side of the PI genetic map, but retains the 3.2 kbPvuWEcoRl fragment required for cl expression that is present in pBRB7.2 (Figure 1).

7673



Table 1. Complementation of Plcl.245

Plasmid

pBR322pBRB7.2pBRB55.1pBRB169.1pFAO2pFAO2.16pFAO2.26

PhenoCypeofcl gene

_Plcl +

P7cl +

P7cr^

by plasmids that contain cl genes.

EfficiencyofLysogeny

1.5X1CT6

2.5x10"'1.4X10"6

2.1xI0~6

8.7X1O"2

4.7xlO~6

3.1X10"6

RelativeEfficiency ofLysogeny

6.0X10"6

1.05.6X10"6

8.4XI0"6

0.351.9X10"3

1.2X1O"5

Complementation for lysogeny was carried out as described by Devlin et al. (26). The plasmids were carriedby E. coli K336. Cells were grown to mid-log phase at 37° in LB containing 50 fig/ml sodium ampicillin andinfected with Plcl .245 at a multiplicity of infection of 5 in the presence of 50 mM CaCI2. After 10 minutes,non-absorbed phage were removed by centrifugation and the infection was allowed to proceed for 2 hours at37°. The infected cells were plated on LB plates containing 50 /jg/ml sodium ampicillin, 50/jg/mlchlorampnenicol,and 40 mM sodium citrate. The efficiency of lysogeny is defined as the number of A p ^ m R cells at the endof infection divided by the number of ApR cells present at the start of the infection.

To construct pFAO2.152, we introduced a 2.8 kb EcoRV fragment of P7 DNA containing->-- ,i „=„„ rPiourp. 11 into the single EcoSl site of pDPT152 after it had been renderedblunt-ended by extension with T4 DINA poiymoio^. ^1 ~,^.~:::i— uy "aUc harhnrins eitherpBRB7.152 or pFAO2.152 was confirmed by measuring their ability to formchloramphenicol-resistant lysogens when infected with Plcl.245Cm.76 insertional mutagenesis.Insertional mutagenesis of Plcl was carried out using the yd transposon of F (24) asdescribed by Devlin et al. (25). E. coli W1485(pFAO2) was mated with the F~ strainMX648 and subsequently plated on ampicillin (to select for the plasmid) and streptomycinsulfate (to select for the recipient strain). Transconjugants which could support only lyticgrowth upon infection by P7cl.l (as scored by cross-streak analysis; 18) were assumedto have lost cl-complementing activity and were characterized further. The positions oftwo c\~ insertional mutations, carried by pFAO2.16 and pFAO2.26, were identified byrestriction mapping (Figure 1).DNA sequencing.DNA sequence analysis was carried out using the M13-dideoxy technique of Sanger etal. (26). Selected DNA fragments containing the cl wildtype or mutant genes wereintroduced into M13 mp8 or mp9. 18-nucleotide oligomers complementary to definedsequences within the cl gene were extended using the Klenow fragment of DNA polymerasein the presence of dideoxynucleotide triphosphates and analyzed by polyacrylamide-ureagel electrophoresis. The sequencing strategy is shown in Figure 1.

RESULTSLocalization of the P7cl gene.Initial localization of the P7cl gene was undertaken by subjecting pFAO2 to 76 mutagenesisand determining the map position of inserts which destroy the ability of the plasmids tocomplement a P7cl"~ mutation (as determined by cross-streak analysis). pFAO2 and theyd insertion mutants were tested further by comparing their ability to complement a Plclamber mutation with the complementation activity of plasmids containing cl genes isolated

7674



B C D E F G

68-

43-

29-

18-

14-

Fig. 2. /n vj/ro transcription-translation of plasmids carrying the cl region of PI and P7. Proteins encoded byselected plasmids were labeled with 33S methionine according to the procedure of DeVries and Zubay (27), usinga commercial in vitro transcription/translation kit from Amersham Corporation. The reaction mixtures were subjectedto electrophoresis on a 12.5% SDS-polyacrylamide gel and the labeled proteins were visualized by autoradiography.The migration of 14C-labeled protein molecular weight standards (Bethesda Research Laboratories) is indicatedat the left side of the figure. Plasmids present in each lane are: A. pBRB55.1; B. pBRB169.1; C. pBRB7.2;D. pFAO2; E. pFAO2.16; F. pFAO2.26; G. pBR322.

from PI wildtype and amber mutants. Lysogeny by cells infected with Plcl.245Cm wasscored as the growth of infected cells on ampicillin (to select for the resident plasmid)and chloramphenicol (to select for the phage genome). The values observed for the twoplasmids containing Plcl and P7cl (pBRB7.2 and pFAO2, respectively) are very similarand significantly higher than those obtained for pBR322 or for any of the plasmids carrying

Table 2. Assay

pCB192pBCB2.13pBCB2.16pBCB2.18

for lacZ expression

minus cl<pDPT152)

0.58154

1.14.0

from plasmids containing PI DNA

0-galactosidaseplus Plcl(pBRB7.152)

0.5515.21.23.4

activity (units)plus P7cl(pFAO2.152)

0.5413.6U3.9

fragments.

relative+PlcI

0.950.101,10.9

activity+P7cl

0.930.091.01.0

Cells containing derivatives of the Ap" promoter-probe plasmid pCB192 and the compatible KnR plasmidpDPT152 were grown in LB at 37°. When they reached mid-log phase, the cells were chilled, lysed, andassayed for 0-galactosidase activity according to the procedure of Miller (23). Plasmids derived from pCB192are indicated at the left side of the Table. Plasmids derived from pDPT152 are indicated in parentheses acrossthe top of the Table. The values reported are the average of two independent experiments. Relative activity isdefined as the |3-galactosidase activity measured in cells harboring plasmids expressing cl divided by the activitymeasured in cells carrying only pDPT152.

7675



GATATCCAATCAGGAGTACC GCATCACCCAAGACGACCTC GATGATCTCACTCACACAAT CSAATATCTCATGGCCACTA ACCAGCCAGACICACAATAAATGCA 105v

TtgAca TAIAATGCTAATAAATCTATTATTTTC GTTGGATCCTTCTATAATCC TGCCCAACAACTCCCAGTGT AATCCGCTGTGAGTTGTTGC CCATCTCAATTCTGGACCAGCATCA 210

b ' ' CCAGGtC

ATG ATA AAT TAT GTC TAC GGC GAA CAA CTG TAC CAG CAC TTC GTC AGC TTC AGC GAT CTC TTT CTA AAA AAA CCT GTT GCA CGC CCC CAA 300

NET II* Asn Tyr Val Tyr Gly Glu Gin Leu Tyr Gin Glu Phe Val Ser Phe Arg Asp Leu Phe Leu lys Lys Ala Val Ala Arg Ala Gin

tag<£i.55)

CAC GIT GAT GCC GCC AGC GAC GCT CCT CCT GIT CGC CCC GIT GTC CIT CTC CCC ITC AAA CAA ACC GAC AGC ATT CAG CCT CAA ATT CAT 390

His Val Asp Ala Ala S*r Asp Gly Arg Pro Vsl Arg Pro Val Val Val Leu Pro Phe Lys Glu Thr Asp S«r I I * Gin Ala Glu H e Asp

I A C A G

I I I IIAAA TGC ACA ITA ATG GCG CGC GAA CTC GAG CAG TAC CCA GAT CTC AAT ATC CCA AAS ACT AIT TTA I A I CCT CTA CCT AAC ATC CTT CGC 440

g

Lys Trp Thr Leu MET Ala Arg Clu Leu Glu Cln Tyr Pro Asp Leu Asn H e Pro Lys Thr l i e Leu Tyr Pro Val Pro Asn H e Leu Arg

A T c

GGT CTG CCT AAG GTI ACC ACT TAT CAG ACA GAA CCA CTG AAC AGC CIC AAT ATG ACC SCI CCC CGC ATI ATT CAT CTG ATT GAT AAG GAC 570

Gly Val Arg Lys Vat Ihr Thr Tyr Gin Thr Glu Ala Val Asn Ser Val Asn MET Thr Ala Gly Arg H e H e His Leu H e Asp Lys Asp

C

ATT CGC ATC CAA AAA AGC GCC GGG ATC AAT CAG CAC ACT GCG AAA TAC ATA GAG AAC CTG GAA GCA ACA AAA GAG CTA ATG AAG CAG IAC 660

lie Arg H e Cln Lys S«r Ala Gly II* Asn Glu His Ser Ala Lys Tyr II* clu Asn Leu Gtu Ala Thr Lys Glu Leu NET Lys Gin Tyr

T I

CCC CAC CAT CAA AAA ITC CCT ATG CCC GTA CAC GGC TTT AGC GAA ACA ATG CTG CGC GTC CAT TAC ATT ICC ACT ACC CCT AAC TAC AAT 750Pro Glu Asp Glu Lys Phe Arg MET Arg Val His Cly Phe Ser Glu Ihr MET Leu Arg val His Tyr H e Ser s*r Ser Pro Asn Tyr Asn

Ph*T C G T T

I I I I IGAT GGC AAA TCA GTT ACT TAC CAT STG CTG CTA TGT GGC GTG TTT ATC TGC GAT GAA ACT CIC CGA GAT CGA ATC ATC ATC AAC GCT CAA HO

* - -Asp Gly Lys Ser Val Ser Tyr His Val Leu Leu Cys Gly Val Ph* l i e Cys Asp Glu Thr Leu Arg Asp Gly H e H e H e Asn Gly Glu

ProC t * g ( £ i . 1 6 ° >

TTT CAC AAA GCA AAA TTT AGC CTT IAI GAC TCT ATA GAA CCC ATC ATC TCC GAC CGC TGG CCC CAC CCA AAA ATA TAT CCC CTG GCA GAT 930

Phe Clu Lys Ala Lys Ph« Ser Leu Tyr Asp Ser H e Glu Pro II* H * Cys Asp Arg Trp Pro Cln Ala Lys II* Tyr Arg Leu Ala Asp

T

ATT GAA AAT GTA AAA AAA CAA ATT CCC ATC ACT CCC GAA GAC AAA AAC GTC AAA TCA GCC CCA TCA GTT ACG CGC ACC CGC AAA ACT AAG 1020n

H i Glu Asn Val Lys Lys Gin l i e Ala H e Ihr Arg Glu Clu Lys Lys Val Lys Ser Ala Ala Ser Val Thr Arg Ser Arg Lys Thr Lys

AAG GGC CAG CCA GTA AAC CAC AAC CCC GAA ACC GCC CAA TAC

Lyi Gly Gin Pro Val Asn Asp Asn Pro Glu Ser Ala Gin ter

Fig. 3. DNA sequence of Plcl and P7cl. The DNA sequence of Plcl is indicated. Positions where the sequenceof P7cl differs from that of Plcl are indicated above the PI sequence. The amino acid sequence predicted bythe open reading frame is given below the sequence. The two amino acid substitutions present in P7cl are shownbelow the open reading frame. The locations of the amber mutant codons in cl .55 and cl. 169 are indicated bysmall letters above the sequence. Sites for selected restriction enzymes (£c»RV [v]; BamHl [b]; BglU [g]; £coR][e]; and Nru\ [n]) arc illustrated by dashed lines beneath the sequence. The cl repressor binding site is underlined.Inverted arrows beneath the sequence illustrate the inverted repeat sequence upstream of the open reading frame.Predicted promoter ribosome binding sites are indicated by the presence of the consensus sequences above andbelow the line, respectively. The DNA sequences of Plcl frombp 1 — 134 and bp 1-434 were reported previously(2,5).

mutant cl genes from either PI or P7 (Table 1). The efficiency with which a cloned P7clgene complements a Plcl mutation confirms previous genetic studies indicating that thesetwo genes are functionally interchangeable (9). The location of the y8 mutations that destroycl-complementing activity suggests that the P7cl open reading frame occupies a mapposition similar to that of the PI open reading frame (Figure 1).

7676



Proteins produced by fragments containing Plcl.As an initial step in the comparison of the PI and P7 repressors, we analyzed the geneproducts expressed from the cloned c\ regions. In an in vitro transcription-translationreaction, plasmids coding for the wildtype alleles of either Plcl or P7cl direct the productionof a protein with an estimated molecular weight of 33,000 daltons (Figure 2, Lanes Cand D), a size that agrees closely with the predicted molecular weight of the Plcl repressorreported previously (3,28). The loss of the 33,000 dalton protein in the c\~ 76-inducedP7 mutant plasmids (Figure 2, Lanes E and F) is consistent with its designation as theP7cl repressor. As expected, the 33,000 dalton protein is not observed when reactionmixtures contain DNA from Plcl amber mutants (Figure 2, Lanes A and B).DNA sequence analysis of the cl genes.To make a direct comparison between the Plcl and P7cl DNA sequences and to predictthe amino acid sequences of the repressor proteins, we carried out M13-dideoxy sequenceanalysis of cloned fragments carrying the cl genes. The sequences of about 1 kb of PIand P7 DNA were determined starting from a common EcoRV site predicted to lieapproximately 200 bps upstream of the cl genes. The PI and P7 sequences (Figure 3)both contain an ATG initiation codon preceded by a putative ribosome binding sequence(29) situated 211 bps downstream of the EcoRV site. In each case, the initiation codonis followed by an open reading reading frame extending for 283 codons. The PI and P7open reading frames code for proteins with predicted molecular weights (32,515 and 32,499daltons, respectively) that agree closely with the values of the proteins expressed fromthe cloned DNA fragments (Figure 2) and with results predicted independently for thepurified Plcl repressor (3-4). The localization of two Plcl amber mutations to the PIopen reading frame confirms its identification as the cl coding sequence, c 1.169 containsan amber mutation that would result in a protein fragment of 26,680 daltons, a value thatagrees well with the size of a protein fragment observed under the in vitrotranscription/translation reaction conditions (Figure 2, Lane B). The cl .55 amber mutationlies close to the N-terminal region of the protein, resulting in the production of a fragmentof 55 amino acids that is apparently too small to resolve under the electrophoretic conditionsused for separation of the proteins. Over 60% of the amino acid sequence predicted forthe Plcl open reading frame has been verified by amino acid sequence analysis of peptidefragments isolated from the purified repressor protein (see accompanying paper, reference3).

The DNA sequences of PI and P7 are identical for a 399-bp region that extends fromthe EcoRV site at the 5' side of the cl gene to a point 188 bps within the open readingframe. The sequences within the Plcl and the P7cl open reading frames differ at only18 positions, all but two of which occur in the wobble position of the predicted codon.From these results, we conclude that the functional identity of the PI and P7 cl genesis a consequence of their nearly identical amino acid sequence.Analysis of promoters upstream of the cl open reading frame.Expression of Plcl was shown previously to require sequences on the distal side of aBamHl site (2, 5) located about 100 bps upstream of the open reading frame (Figure 3).A binding site for the cl repressor has also been shown to exist close to this BamHl site(2, 5, 6). To determine whether this region contains a promoter that is detectable in vivoand, further, to determine whether this promoter can be regulated by cl repressor proteinsfrom either PI or P7, we introduced several DNA fragments from this region into thepromoter probe vector pCB192, screened for promoter activity (as monitored by lacLexpression) and checked for repression of this activity in the presence of a compatible

7677



plasmid expressing Plcl or P7cl. Cells harboring pBCB2.13 (a plasmid which carriesa 460 bp fragment of PI DNA that extends across the BamHi site upstream of cl intothe open reading frame) are dark blue in the presence of Xgal and produce significantlevels of/3-galactosidase (Table 2). In contrast, pBCB2.16 and pBCB2.18 (which eachcontain DNA from only one side of the BamHi site located in pBCB2.13) do not confera blue color on their host cell in the presence of X-Gal and express negligible amountsof/3-galactosidase (Table 2). These observations suggest that expression from the promoteridentified here requires sequences that span the BamHi site upstream of cl. Expressionof cl from a compatible plasmid in the presence of pBCB2.13 results in a 90% reductionin promoter strength (Table 2). This reduction is seen in the presence of either Plcl orP7cl, indicating that the two repressor proteins are both capable of repressing expressionfrom this promoter.

DISCUSSION.The DNA sequences of Plcl and P7cl differ at only 18 sites, all but two of which occurat the third position of the affected codon. This observation provides biochemicalconfirmation of the functional identity predicted on the basis of previous genetic analysis(9). A number of DNA binding proteins exhibit a common structural motif in which twohelices are separated by a glycine residue (12). This motif is not observed in the predictedsecondary structures (30) of the Plcl and P7cl amino acid sequences. A sequence withsome similarity to the XCro helix-turn-helix region was previously reported near the N-terminus of the Plcl protein (5); however, it was noted that the potential for helix formationis disrupted by the presence of several prolines within the region. The secondary structurepredicted for the Plcl and P7cl repressor proteins (30) does not reveal other structuralcharacteristics (e.g., Zn fingers (31), leucine zippers (32), or helix-loop-helix motifs (33))that have been associated with DNA binding activity in other systems. A search of theGenBank and EMBL databases does not reveal any other known regulatory proteins withsignificant amino acid similarity to the Plcl or the P7cl repressor sequences. Since thePlcl repressor differs from most other repressors in DNA binding specificity (i.e., inits recognition of an asymmetric operator sequence), it is not unexpected to find that theprotein does not exhibit common structural motifs at the amino acid level.

The cl-repressible promoter described in this report is located in a region just upstreamof the cl open reading frame and is oriented in the direction of cl. Because the promoteris present on a multicopy plasmid, it is not possible to make a direct calculation of promoterstrength; however, the values observed are about five-fold lower than the levels producedby a derivative of pCB192 that contains the plac promoter from pUC19 (34). Becausesequences on both sides of the BamHi site located upstream of cl are required for promoteractivity (Table 2), we suggest that the promoter spans this site. Less than 10 bps downstreamof this BamHi site is a heptanucleotide sequence (TATAATG) that is identical to the —10consensus sequence for RNA polymerase (35). If this sequence does indeed correspondto the - 1 0 region of the promoter, the —35 region would be predicted to lie on the otherside of the BamHi site in a region that overlaps a known cl repressor binding site (2—5).Analysis of this region does not reveal any sequences with significant similarity to the-35 consensus sequence. The best fit is the sequence TCTATT (Figure 3), which matchesonly two positions of the -35 consensus (TTGACA). The lack of a strong - 3 5 regionis often observed with genes that require an activator. Although a pentanucleotide sequencecorresponding to the conserved portion of the CRP protein consensus binding site (36)

7678



is located just upstream of the predicted —35 region (at position 91; Figure 3), a rolefor CRP-mediated activation in cl expression has not previously been described. Theorientation of the promoter and its cl-repressible character raise the possibility that clexpression is autoregulatory. If this is so, one potential activator would be the cl repressoritself. Expression cannot be absolutely dependent on cl-mediated activation, however,because the cloned promoter exhibits significant activity in the absence of the cl gene(Table 2). Under the conditions reported here, the presence of the cl gene results in adecrease rather than an increase in lacL expression; however, these observations do notrule out a potential activator role for the cl protein, since the ratios of repressor and operatorprovided by the multicopy plasmids may not be optimal for activation. Physiologically,the role of additional repressor binding sites in regulating cl expression also cannot bediscounted. Three potential operator sites have been identified several hundred bps upstreamof the cl open reading frame (2—5); one or more of these could be involved (possiblythrough a DNA looping mechanism; 37) in the activation or repression of cl expressionduring phage growth.

A cl-repressible promoter oriented in the direction of cl was previously reported (38)to be located entirely within PlBa/nHI-9, a fragment located upstream of cl which isbracketed by the BamHl site within pBCB2.13. Because sequences on both sides of thisBamHl site are required for the activity of the promoter in pBCB2.13, we suggest thatthe previously identified promoter is distinct from the one reported here. The promoterfrom BamHl-9 could correspond to a consensus promoter sequence that is situated about500 bps upstream of cl and overlaps a cl repressor binding site (2). If so, cl expressionis likely to be controlled by more than one promoter. Located between this promotersequence and the promoter encoded on pBCB2.13 is an open reading frame whose product(termed coi, or c-one mactivator) has been implicated in the establishment of lytic growth(1, 39; B.R. Baumstark, unpublished results). It has been suggested (2) that the decisionto enter lytic or lysogenic growth is influenced by the level of transcription initiated fromthe distal promoter (which would transcribe coi prior to the transcription of cl) relativeto that of the promoter located immediately upstream of the cl gene (which would transcribeonly cl).

A 32-nucleotide hyphenated inverted repeat sequence is located just upstream of the clopen reading frame (positions 146—188; Figure 3). It is not currently known whetherthis sequence has any regulatory effect on cl expression. Conceivably, the sequence couldserve as a recognition site for an as-yet-unidentified regulatory protein. Alternatively, itmay affect the secondary structure of the messenger RNA. A transcript extending froma promoter located upstream of the putative coi open reading frame would be capable offorming a stable stem-loop structure containing 16 bps with a single bp mismatch (AG= —33.6 Kcal) of this inverted repeat sequence. Such a structure could potentially serveas a recognition site for a regulatory factor or, alternatively, could mask such a site. Onthe other hand, transcription originating from the promoter spanning the BamHl site justupstream of cl would start at a site within the inverted repeat sequence, forming acomparatively less stable stem-loop structure of about 8 bps. The role of the inverted repeatregion in the regulation of cl expression is currently under investigation.

ACKNOWLEDGEMENTSWe thank Heinz Schuster for his review of the manuscript. This work was supported byNational Science Foundation grant DMB-8704146.

7679



Abbreviations: bp, basepairs; kb, kilobase pairs; X-GaJ, 5-Bromo-4-Chloro-3-indolyl-beta-D-galactopyranoside.

*To whom correspondence should be addressed

REFERENCES1. Yarmolinsky, M.B., and Stemberg, N. (1988). In Calendar, R., (ed.), The Bacteriophages, Plenum Publishing

Corp., NY, Vol. 1, pp. 291-438.2. Baumstark, B.R., Stovall, S.R., and Ashkar, S. (1987). Virology 156, 404-413.3. Dreiseikelmann, B., Velleman, M., and Schusler, H. (1988). J. Brai. Chem. 263, 1391-1397.4. Heinnch, J., Riedel, H.-D., Baumstark, B.R., Kimura, M., and Schuster, H. (1989). Nucleic Acids Res,

this volume.5. Eliason, J.L., and Stemberg, N. (1987). J. Mol. Biol. 198, 281-293.6. Velleman, M., Dreiseikelmann, B., and Schuster, H. (1987). Proc. Nail. Acad. Sci. USA 84, 5570-5574.7. Citron, M., Velleman, M., and Schuster, H. (1988). J. Biol. Chem. 264, 3611-3617.8. Chadwick, P., Pirotta, V., Steinberg, R., Hopkins, N., and Ptashne, M. (1970). Cold Spring Harbor Symp.

Quant. Biol. 35, 283-294.9. Chesney, R.H., and Scott, J.R. (1975). Virology 67, 375-384.

10. Wandersman, C , and Yarmolinsky, M. (1977). Virology 78, 267-276.11. Scott, J.R., West, B.W., and Laping, J.L. (1978). Virology 85, 587-600.12. Pabo, CO. , and Sauer, R. A. (1984). Ann. Rev. Biochem. 53, 293-321.13. Scott, J.R. (1974). Virology 62, 344-349.14 Schneider, K., and Beck, C.F. (1987). Methods in Enzymol. 153, 452-461.15. Smith, H.W. (1972). Nature New Biol. 238, 205-206.16. Scott, J.R. (1975). Virology 65, 173-178.17. Scott, J.R., Kropf, M.M., and Mendelson, L. (1977). Virology 76, 39-46.18. Scott, J.R. (1968). Virology 36, 564-574.19. Walker, D.H., Jr., and Walker, J.T. (1976). J. Virol. 20, 177-187.20. Stemberg, N. (1979). Virology 96, 129-142.21. Schneider, K., and Beck, C.F. (1986). Gene 42, 37-48.22. Taylor, D.P., and Cohen, S.N. (1979). J. Bacterid. 137, 92-104.23. Miller, J.H. (1972). In Experiments in Molecular Genetics, Cold Spring Harbor Laboratory, Cold Spring

Harbor, N.Y. 352-355.24. Guyer, R.S. (1978). J. Mol. Biol. 126, 347-365.25. Devlin, B.H., Baumstark, B.R., and Scott, J.R. (1982). Virology 120, 360-375.26. Sanger, F., Nicklen, S., and Coulson, A.R. (1977). Proc. Natl. Acad. Sci. USA 74, 5463-5467.27. DeVries, J.K., and Zubay, G. (1967). Proc. Natl. Acad. Sci. USA 57, 1010-1012.28. Heilmann, H., Reeve, J.R., and Punier, A. (1980). Mol. Gen. Genet. 178, 149-154.29. Shine, J., and Dalgarno, L. (1974). Proc. Natl. Acad. Sci. USA 71, 1342-1346.30. Chou, P.Y., and Fasman, G.D. (1978). Adv. Enzymol. 47, 45-148.31. Berg, J.M. (1986). Nature 319, 264-265.32. Landschultz, W.H., Johnson, P.F., and McKnight, S.L. (1988). Science 240, 1759-1764.33. Murre, C , McCaw, P., and Baltimore, D. Cell 56, 777-783.34. Anderson, B.E., Baumstark, B.R., and Bellini, W.J. (1988). J. Bacteriol. 170, 4493-4500.35. Rosenberg, M., and Court, D. (1979). Annu. Rev. Genet. 13, 319-353.36. Ebright, R.H., Cossart, P., Gicquel-Sanzey, B., and Beckwith, J. (1984). Nature 311, 232-235.37. Ptashne, M. (1986). Nature 322, 697-701.38. Stemberg, N., and Hoess, R. (1983). Annu. Rev. Genet. 17, 123-154.39. Scott, J.R. (1980). Curr. Top. Microbiol. Immunol. 90, 49-65 .

This article, submitted on disc, has been automaticallyconverted into this typeset format by the publisher.

7680


the c1 genes of p1 and p7

Documents