Proc. Natl. Acad. Sci. USA Vol. 82, pp. 3134-3138, May 1985 Biochemistry A cIT-dependent promoter is located within the Q gene of bacteriophage X (transcriptional activation/promoter mutation/antisense RNA) BARBARA C. HOOPES AND WILLIAM R. MCCLURE Department of Biological Sciences, Carnegie-Mellon University, 4400 Fifth Avenue, Pittsburgh, PA 15213 Communicated by Allan Campbell, January 11, 1985

ABSTRACT We have found a eII-dependent promoter, nism of this inhibition is not completely understood. Under PaQ, within the Q gene of bacteriophage X. Transcription conditions where cIl is overproduced, such as in a cro- experiments and abortive initiation assays performed in vitro phage, cII-dependent inhibition has been shown to result in showed that the promoter strength and the clI affinity of PaQ severe growth defects for the phage (9, 10). Court et al. (11) were comparable to the other cU-dependent X promoters, PE extended the observations of McMacken et al. (8) to dem- and PI. The location and leftward direction of PaQ suggests a onstrate that a X cI-cy- phage showed a similar early possible role in the delay of X late-gene expression by clI appearance of late proteins as a X c-fcII- phage. They protein, a phenomenon that has been called cM-dependent concluded that cII-dependent inhibition of late protein syn- inhibition. We have constructed a promoter down mutation, thesis resulted from a decrease in Q gene expression through paq-l, by changing a single base pair in the putative clI binding inhibition ofPR transcription by the convergent PE promoter. site of the promoter by oligonucleotide site-directed Indeed, Schmeissner et al. (12) have found a 50% reduction mutagenesis. Thepaq-1 mutant promoter required about 4-fold in PR-specific RNA in a cII+ as opposed to cII- infection. It higher cdi concentrations for maximal activation compared to has been reported, however, that a PE mutation does not the wild-type PaQ. We tested the hypothesis that PaQ is entirely eliminate cII-dependent inhibition (9, 10, 13). The responsible in part for the delay of X late-gene expression by observation (D. Court, cited in ref. 14) that a sequence recombining the paq-1 mutation into a phage showing severe resembling PE exists within the Q gene suggested an ad- cU-dependent inhibition. We found that the paq-l mutation ditional contribution to cII-dependent inhibition. Transcrip- relieved the cIT-dependent growth defect of this phage. The tion from this leftward-transcribing promoter could inhibit paq-1 mutation (in combination with X c1857) resulted in a the synthesis ofthe Q protein, the antiterminator required for clear-plaque phenotype at the permissive temperature of 320C. late gene expression (15), through transcriptional or transla- The role of the PaQ-initiated antisense transcript in the control tional interference. of X development is discussed. We have found that the DNA sequence noted by Court does indeed define a cIT-activated promoter which we call The clI protein of phage X has been called the "critical PaQ (anti-Q promoter). We have tested the hypothesis that switch" in the lysis/lysogeny decision for the phage (1). clI PaQ is responsible in part for cII-dependent inhibition by is required for activation of the PE and PI promoters, which constructing a promoter mutation, paq-1, and by recombin- are responsible for the transcription of the A cI (repressor) ing the mutated promoter into a X phage. We found that the and int (integrase) genes, respectively. Both of these gene recombination of the paq-J mutation with a cro- phage products are required for the establishment of lysogeny. In showing severe cII-dependent inhibition relieved the cII- addition, much of the input to the phage developmental dependent growth defect of the phage. We have also con- program by the host is mediated by effects on cII synthesis structed the X cI857 paq-J phage and found that it displayed or stability (for review, see ref. 2). The activation of PE and a clear-plaque morphology at 32°C. PI by cII has been extensively characterized. Both promoters require cII in addition to RNA polymerase for transcription MATERIALS AND METHODS in vitro and both show poor homologies to prokaryotic promoter consensus sequences (3). Chemical protection, Endonucleases [Ava TI, HindIII, and EcoRI (Bethesda Re- DNase I "footprinting" experiments (4) and in vivo (2) and in search Laboratories); Bgl IT, Rsa I, and Nru I (New England vitro (5, 6) analysis of mutations at the PE promoter have Biolabs); Cla I (Boehringer Mannheim)]; unlabeled shown that the clI site can be defined T-T-G-C- ribonucleoside (ICN) and deoxyribonucleoside triphosphates binding by (P-L Biochemicals); agarose (SeaKem Laboratories, N6-T-T-G-C, where N6 corresponds in position to the -35 Rockland, ME); [a-32P]GTP, [a-32P]ATP, and [a-32P]dATP region hexamer. Interaction of cII with this sequence results (Amersham); acrylamide and N,N'-methylenebisacrylamide in large increases in both the initial binding of RNA poly- (Bio-Rad); cytidylyl(3'-5')adenosine (CpA; Sigma); and merase at the promoter and its subsequent conversion to an 3MM paper (Whatman) were purchased from the sources active complex (ref. 7 and unpublished data). indicated. RNA polymerase was purified from Escherichia The cII protein has an additional role in A lytic develop- coli K-12 by the procedure of Burgess and Jendrisak (16) with ment that has remained obscure. clI acts to inhibit late gene the modifications of Lowe et al. (17); the concentration was expression, a phenomenon which has been called "clI- determined by absorbance at 280 nm, assuming an A 1% of6.2 dependent inhibition." McMacken et al. (8) first observed and Mr = 4.9 x 105. The activity of the RNA polymerase was that aA cII- or A cIII- phage produced late proteins and late 60%, determined as described by Cech and McClure (18). X mRNA earlier than a A cI- phage. They proposed that cIT c1I protein was isolated as described (19) from the over- protein directly inhibited late mRNA synthesis. The mecha- producing strain N6308(pOG7), which was obtained from M. Gottesman (National Institutes of Health) (20); cIT protein The publication costs of this article were defrayed in part by page charge concentration was determined by absorbance at 280 nm, payment. This article must therefore be hereby marked "advertisement" using a molar extinction coefficient of 7.2 x 104 (19), and is in accordance with 18 U.S.C. §1734 solely to indicate this fact. given as nM monomers. X cI protein was a gift of R. T. Sauer 3134 Downloaded by guest on September 30, 2021 Biochemistry: Hoopes and McClure Proc. Natl. Acad. Sci. USA 82 (1985) 3135 (Massachusetts Institute of Technology). The Klenow frag- consensus sequence in Q corresponded to a cII-dependent ment of DNA polymerase was a gift of W. Brown (Carnegie- promoter by in vitro transcription ofX DNA. Phage DNA was Mellon University); it was isolated from the overproducing digested with the restriction endonuclease Ava II to generate strain CJ155 (21) and had a specific activity of 13,000 a run-off transcript from PI (267 bases), Bgl II to generate a units/mg. T4 DNA ligase was isolated as described (22). The run-off transcript from PE (240 bases), and Cla I to generate oligonucleotide primer used for the mutagenesis was ob- a run-offtranscript from the putative PaQ promoter (about 320 tained from the DNA synthesis service at the University of bases). As shown in Fig. 1A, we observed two cII-dependent Pennsylvania. transcripts for each reaction, as well as the self-terminating Solutions. LB, H top agar, and H plates were as described cII-independent transcript from PR (198 bases). One of these by Miller (23). MS buffer is 10 mM Tris Cl, pH 7.5/10 mM cII-dependent transcripts was ofthe size predicted for run-off MgCl2/50 mM NaCl/1 mM dithiothreitol/bovine serum albu- transcription from PI, PE, and PaQ, respectively. The other min (100 gg/ml). Xdil is 10 mM Tris Cl, pH 7.5/10 mM was independent of the restriction endonuclease used and MgSO4. TBE is 90 mM Tris/90 mM, boric acid/2.5 mM was about 220 bases long. As shown in Fig. 1B, this EDTA, pH 8.3. TE is 10 mM Tris Cl, pH 8.0/1 mM EDTA. self-terminated 220-base transcript also originated at PaQ. Assay buffer is 40 mM Tris Cl, pH 8.0/100 mM KCl/10 mM When the plasmid pBH100, which contains an EcoRI frag- MgCl2/1 mM dithiothreitol/bovine serum albumin (100 ment containing the Q gene (see below), was digested with .Ag/ml). EcoRI and transcribed in the presence of cHI, both the Bacterial and Phage Strains and Plasmid Constructions. predicted run-off transcript (320 bases) and the 220-base GM33 [dam 4 (24); from M. Susskind (Univ. of Mas- terminated transcript were observed. The appearance ofboth sachusetts Medical School)], W1485F+:: TnlO-2 [E. coli of these transcripts was dependent upon the addition of clI, K-12 supE42/F+ zzz::TnlO-2 (25); R. Zagursky (National even after long preincubations of the DNA with RNA Cancer Institute)]; C600K- [supE; M. Rosenberg (Smith polymerase or at high RNA polymerase concentrations (data Kline & French Laboratories)]; and XK1500 [AlacUl69- not shown). All three cII-dependent promoters appeared to Sms B1; E. Minkley (Carnegie-Mellon Univ.)] were obtained be of equal strength in transcription experiments, and PaQ from the laboratories cited. GM33F+:: TnJO-2 was con- was active at the same low clI concentrations as were PE and structed for use as a dam-F+ strain. PI. Another cHI consensus sequence occurs near base pair The X phage strains X cI857 Qam73, X cI857 cro20 Qam73, 31,837 in the A DNA sequence (26); this site does not contain X c1857 cro20, and X cI857 cro20 cII68 were the kind gift of a good candidate for a -10 region at an appropriate distance I. Herskowitz (University of California, San Francisco). IR1 from the cHI binding site. We did not observe a transcript from (M13) phage were obtained from R. Zagursky. The X DNA this site under our experimental conditions (data not shown). used for transcription was isolated from purified phage by Design and Construction ofthepaq-1 Mutation. The location using the NaDodSO4/KCl procedure (35). of a cII-dependent promoter within the Q gene (Fig. 2) The plasmid ptacll(XQ), obtained from J. Roberts suggests that PaQ might contribute to the cII-dependent (Cornell), carries X DNA, bordered with EcoRI linkers, from inhibition of X late-gene expression. To test the hypothesis the Cla I site at base pair 43,825 in the X sequence (26) to a that PaQ is responsible in part for cIu-dependent inhibition, we site distal to the carboxyl-terminal end of the Q gene coding designed a promoter mutation, used oligonucleotide site- region at about 44,545, inserted in the EcoRI site of ptacll directed mutagenesis to place the mutation on a plasmid (27). The EcoRI fragment from ptacll(XQ) was isolated and the the mutation from the ligated into EcoRI-cut pZ150 (25) to create pBH100 (see Fig. containing Q gene, recombined 3). Phage Recombination and Analysis. Fresh overnight cul- A B tures (0.1 ml) of C60OK-(pBH100) and C60OK-(pBH110) 1 2 3 4 5 6 1 2 grown in LB with 10 mM MgSO4 and ampicillin (50 ,ug/ml) were infected with -106 of the appropriate phage in 0.1 ml. Phage adsorption took place on ice for 20 min; after a 3-min _ _ incubation at 370C, 3 ml of H top agar was added and the - PaQ mixture was poured onto H plates. After 8 hr at 37°C, 3 ml of -.. - PE Adil was overlayed on each plate and the plates were "' 220 -220 - 220 -220 incubated at 4°C overnight. The resulting lysates were har- . ,,,,,, PR' -PRR vested and tested on C60OK-(supE) for total phage titer and -PR on XK1500 (sup') for the recombinant (Q+) titer. Total phage titers were typically 109-1011 phage per ml and recombination frequencies were 10-4-10-3 with this method. Frequency of reversion of Q- to Q+ was 10-6. Recombinant phage were analyzed by restriction enzyme DNA of Chro- FIG. 1. In vitro activation of X promoters by cII protein. Shown analysis of from plate lysates single plaques. is an autoradiogram of a gel used to separate in vitro transcription mosomal DNA and cellular RNA were first removed by products. Transcription reactions were done in assay buffer with 1 adsorption to an equal volume of DEAE-Sephadex A-25 nM template, 50 nM RNA polymerase, 200 ,uM unlabeled nucleoside equilibrated with Adil (28). Phage DNA then was isolated by triphosphates, and 20 AM [a-32P]GTP (4000 cpm/pmol). Reactions the NaDodSO4/KCl procedure (35). A spermine precipita- were initiated by the addition of RNA polymerase; heparin was tion step (29) was also included before the last ethanol added after 15 min of synthesis. Reactions were stopped 5 min later precipitation to remove agar contaminants. Although the by the addition of 10 mM EDTA and tRNA (160 jig/ml) and were amount of DNA isolated was variable with this method, Nru precipitated with ethanol before electrophoresis in a 5% polyacryl- I digestion was complete and contamination with chromo- amide/7 M urea/TBE gel. (A) X cI857 S7 b2 DNA digested with Ava II (lanes 1 and 2), Bgl II (lanes 3 and 4), or Cla I (lanes 5 and 6) was somal DNA and with RNA was minimal. transcribed in the absence (lanes 1, 3, and 5) or presence (lanes 2, 4, and 6) of 80 nM cII protein. The reactions also contained 300 nM X RESULTS repressor to prevent RNA synthesis from PL and PR. (B) pBH100 DNA digested with EcoRI was transcribed in the absence (lane 1) or A Site Within the X Q Gene Functions as a cII-Dependent presence (lane 2) of 80 nM clI. See text for explanation of bands Promoter in Vitro. We investigated the possibility that the cII observed. Downloaded by guest on September 30, 2021 3136 Biochemistry: Hoopes and McClure Proc. Natl. Acad. Sci. USA 82 (1985)

A',v I r ,-Wl

pp p~ PE PO PaQ

B 44140 44200 AAAGCTTGAAr.GAAT AGGCAAA GTACTGCAA TGCAACATTCGCTTATGCG TTTCGAACTTCCTT LTCCGTTTCCATGA C AJGCGTTGTAAGCGAATACGC -10 35 G C

FIG. 2. The location and DNA sequence ofPaQ. (A) The location FIG. 3. Site-directed mutagenesis of PaQ. Construction of the of the PaQ promoter is shown relative to the X map (26). Genes are plasmid pBH100 is described in Materials and Methods. To prepare drawn to scale. The startpoints and orientation of the other major X the single-stranded template, a 25-ml overnight culture of GM33- promoters are shown above (rightward) and below (leftward) the X F+::TnlO-2(pBH100) was infected with IR1 phage at a multiplicity map. (B) The DNA sequence ofthe PaQ promoter is shown. The -10 of infection of 10 and diluted into 1 liter of LB with ampicillin at 50 and -35 hexamers are boxed and the c1I binding site is underlined. gg/ml. After overnight growth at 370C, cells were removed by The base-pair change corresponding to the paq-1 mutation is shown centrifugation and phage DNA was isolated from the supernatant as beneath the sequence. Numbers above the sequence correspond to described by Zagursky and Berman (25). The single-stranded base pairs in the X sequence. pBH100 comprised about 5% of the total DNA and was not further purified. The large fragment of pBH100, generated by digestion with HindIII and labeled with [a-32P]dATP to a specific activity of 90 plasmid into phage X, and examined the phenotypes of the cpm/4g, was separated from the small fragment by precipitation with mutant phages. 6% polyethylene glycol (33). Forty-five micrograms of the single- Since PaQ is located within the Q coding sequence, the first stranded mixture (plasmid and IR1 DNA) and 20 ,.g offragment were requirement for the design of the mutation was that the Q annealed by boiling the mixture in a water bath for 3 min and then gene must remain functional. The second requirement was cooling slowly. Twenty micrograms ofthis mixture [-2 ;ug (0.5 pmol) that the mutation should affect promoter strength in a of heteroduplex] and 33 pmol of primer were incubated in MS buffer predictable manner. This requirement eliminated many po- at 550C for 5 min and then cooled on ice for 25 min. A 2-hour repair tential changes within the promoter, since PaQ, like the other reaction at 15'C was initiated by the addition of Klenow fragment (13 units), T4 ligase (14 units), and dNTPs (0.25 mM). The repaired DNA cIu-dependent promoters, shows little homology to the pro- was used to transform C600K-, and plasmid DNA from 38 tetr amp' karyotic consensus sequence for promoters (30, 31). How- colonies was analyzed by Nru I digestion. ss, Single-stranded; ds, ever, several changes were possible within the putative clI double-stranded. binding site. These are predicted to result in decreased promoter function based on the study ofmutations at the A PE promoter (2). As a final requirement, we desired a mutation DNA used for transformation was expected to favor changes that could easily be detected by screening plasmid DNA from in the wild-type template strand rather than the newly transformants for an alteration in the pattern of restriction synthesized mutant strand. enzyme cleavages. An alteration fulfilling the above require- After template, HindIll primer, and oligonucleotide were ments is shown in Fig. 2. The mutation is in the third base of annealed, the single-strand gap was filled in (see Fig. 3). The a Q codon; it does not change codon usage significantly (23 DNA mixture was used to transform E. coli C600K-, and to 29%); it corresponds to the mutation cy3078 in X PE; and colonies were selected on plates containing tetracycline and it creates an Nru I cleavage site. ampicillin. Any bacteria transformed with the ligated HindIII The plasmid pBH100 containing the X Q gene was con- fragment were expected to be tetracycline-sensitive due to structed as described in Materials and Methods. When E. the destruction of the tet promoter. Rapid alkaline prepara- coli F+ strains carrying derivatives of this plasmid are tions (34) of plasmid DNA were obtained from 38 groups of infected with M13 [or in this case, IR1 (32), an interference- transformants; halfofthese contained plasmids with a second resistant mutant offi] both single-stranded plasmid DNA and Nru I site at the position expected for plasmids containing the filamentous phage are secreted as particles packaged with mutation. We estimate a mutagenesis frequency of 25-33%. phage coat-protein. This procedure facilitated the prepara- One ofthe cultures was streaked onto tetracycline/ampicillin tion of single-stranded template DNA for oligonucleotide- plates to obtain single colonies. Plasmid DNA from eight directed mutagenesis with a minimum of manipulations. candidate colonies was characterized by Nru I digestion, and To optimize the site-directed mutagenesis, we limited the all eight contained two Nru I sites. One of these candidates amount of in vitro DNA repair by following the scheme was designated pBH110. shown in Fig. 3. Single-stranded circles complementary to The paq-1 Promoter of pBH110 Requires Higher cil Con- the oligonucleotide were prepared by using pBH100 as centrations for Activation. To confirm the expected behavior described in Materials and Methods. The large HindIII of paq-1, we quantified the amount of clI required for fragment of pBH100 was used as an additional primer. After activation of the wild-type and mutant promoters. We used the single-stranded circles were annealed to the denatured the abortive initiation reaction as an assay for-open-complex plasmid backbone and the synthetic oligonucleotide, there formation (35). The synthesis ofthe product CpApA from the remained only -400 bases of single-stranded gap to be dinucleotide CpA and [a-32P]ATP by RNA polymerase in repaired by DNA polymerase (Klenow fragment), thereby reactions with linearized pBI1100 or pBH110 was dependent reducing the chances for the introduction of other mutations on the presence of cII protein. This abortive initiation by incorrect repair synthesis. To further increase the yield of product is consistent with an RNA start appropriate to PaQ plasmids containing the desired mutation, the single-stranded (see Fig. 2). As shown in Fig. 4, the concentration of cIl circles were prepared from a dam- strain of E. coli and the required for half-maximal activation ofPaQ was about 30 nM plasmid used for the preparation ofthe HindI1 fragment was monomers. For the paq-1 promoter on pBH110, the half- prepared from a damn' strain. As a result, in vivo repair ofthe maximal concentration was 70 nM. In addition, the switch- Downloaded by guest on September 30, 2021 Biochemistry: Hoopes and McClure Proc. Natl. Acad. Sci. USA 82 (1985) 3137 Table 1. Rescue of Q+ from paq+ and paq- plasmid strains by X c1857 cro20 Qam73 Growth of Q+ Recombination recombinants Infected strain frequency 370C 420C co 0.6 C600K-(pBH110) (paq-J) 0.0020 26/26 14/26 C600K-(pBH100) (paq+) 0.0024 21/21 1/21 CU -Z 0.4- Plasmid-containing strains were infected with X c1857 cro20 Qam73 to obtain plate lysates, as described in Materials and Methods. The recombination frequency given is the titer of the resulting lysate on 0.2- XK1500 (sup') relative to the titer on C600K- (supE). Q+ plaques were picked from XK1500 to test for growth at 370C and at 420C.

0 40 80 120 160 320 ing the presence of the paq-1 mutation (data not shown). The [cl], nM monomers paq-1 promoter down mutation, then, appears to reverse the increased cIT-dependent inhibition seen in X cI857 cro2O. FIG. 4. The activation Of PaQ and the paq-1 mutant promoter by c~l. The fractional activity of the PaQ (e) and paq-1 (A,) promoters is PaQ also contributes to the lysis/lysogeny decision of shown as a function of c~l concentration. Reaction mixtures (40 sul) wild-type X. This conclusion is based on the following

in assay buffer contained 1 nM plasmid linearized with EcoRI , 50 nM findings. We infected C600K-(pBH110) with X cI857 Qam73 RNA polymerase, 0.5 mM CpA, 20 kLM [a- 12P]ATP (500 cpm/pmol), and found that about half of the Q+ recombinants gave clear and the indicated concentrations of cpq.The amount of CpApA plaques at 32°C. All recombinants from an infection of synthesized was quantified by paper chromatography as described C600K-(pBH100) (paq+) gave turbid plaques at 32°C. Similar (35), using 1.7 M (NH4)2SO4 as solvent. Activities were corrected for results were found at 37°C. At 42°C the recombinants from plasmid background by subtracting the synthesis of CpApA on the pBH110 cross resulted in a mixture of large and small plasmid DNA digested with Rsa I, which cuts within the PaQ promoter. Activities were normalized to the maximum activity clear plaques. Phage from one ofthe large plaques was shown observed in the experiment. This corresponded to 28 CpApA/min to contain the paq-J mutation by using Nru I digestion of per promoter for pBH100 and 38 CpApA/min per promoter for purified DNA. This phage, X cI857 paq-J, also gave clear pBH110. The reactions were initiated by the addition of RNA plaques at 370C and 32°C. We conclude that the wild-type PaQ polymerase and activities were determined at 5 min. promoter is important for the normal lysogenic response ofX. like activation of PaQ with increasing cII protein concentra- DISCUSSION tions was not observed with the paq-1 promoter. Similar profiles of c1I activation were observed with supercoiled We have demonstrated the existence of a cII-dependent templates in abortive initiation assays and with DNA frag- promoter, which we call PaQ, within the Q gene of bacterio- ments in run-off transcription assays (data not shown). phage X. The construction and properties ofa promoter down The Effect of the paq-1 Down Mutation on Growth of X c1857 mutation, paq-J, have provided new information both on the cro2O and X c1857. The paq-1 mutation on pBH110 is interaction of cII with promoters and on the function of PaQ contained within a wild-type Q gene. Infection of suppressor in bacteriophage X development. The paq-J mutation was strains carrying pBH110 with Q- amber-mutant phage should predicted to result in an increase in the concentrations of cII result in Q+ recombinants, some ofwhich should also contain required for activation, based on the definition of the cII the paq-J mutation. However, we did not know whether the binding site (4). We found that paq-J required 2.5-fold more paq-J phage would display a detectable phenotype on plates. cII for half-maximal activation and 4-fold more cII for Therefore, we used X c1857 cro20 Qam73 for our initial saturation in vitro. The effects of the paq-J mutation on recombination experiments. Our strategy was based on the promoter function measured in vitro provide additional properties of this cro mutation (10), which appears to result confirmation for the sequence determinants of cII activation in temperature-sensitive cro function. In combination with proposed by Rosenberg and colleagues (4). This model was the cI857 mutation, the cro20 phage is not viable at 42°C. This based primarily on the effect of many mutations at only one inviability is due to enhanced cIT-dependent inhibition, pre- of three cII-dependent X promoters. The properties ofpaq-J sumably because of cII overproduction, since mutations in phages suggest that PaQ has an important role in X develop- the PE promoter or the cIT protein restore phage viability. We ment. In the following, we argue that these properties are reasoned that if part of cIT-dependent inhibition were due to most simply explained by adopting a model in which PaQ transcription from the PaQ promoter, a X c1857 cro2O paq-J function in vivo is responsible for an inhibitory effect on Q phage should be viable at 42°C. gene expression. The sup' strains C600K-(pBH100) and C600K-(pBH110) The phenotype ofthe phages bearing the paq-J mutation in were infected with X cI857 cro20 Qam73. The resulting combination with the cro2O mutation suggests that PaQ is lysates were used to infect XK1500 (sup°), to obtain Q+ in part cII-dependent inhibition. This conclu- recombinants, and C600K- (supE), to determine the titer of responsible for the lysates. As shown in Table 1, the recombination frequen- sion must be qualified because the growth defects of cro- cies were similar for both crosses. Individual Q+ phage phage are known to be complex. Phage and host replication plaques were picked from both crosses and tested for their are affected (36, 37) and both trans- and cis-acting functions ability to grow at 37°C and 42°C by streaking them over lawns (36) have been implicated in the growth inhibition. For the of XK1500 at each temperature. More than half of the Q+ stringent cro- mutations (e.g., cro27), all of these factors plaques from the pBH110 cross grew at 42°C. Plate lysates probably contribute. For the temperature-sensitive cro2O, were made from single plaques from the pBH110 cross, and however, introduction of a cII- or cy- mutation is sufficient phage DNA from seven of these lysates was analyzed for the to allow growth of the phage at the restrictive temperature. presence of seven rather than six DNA fragments after This result indicates that cII-dependent inhibition is primarily digestion by Nru I. All ofthese c1857 cro2O Q+ recombinants responsible for the growth defect of this phage. Since the which grew at 42°C contained the extra Nru I site, establish- introduction of the paq-J mutation into a X cI857 cro2O phage Downloaded by guest on September 30, 2021 3138 Biochemistry: Hoopes and McClure Proc. Natl. Acad. Sci. USA 82 (1985) also allows growth, we conclude that PaQ is partially respon- 7. Shih, M.-C. & Gussin, G. N. (1983) J. Mol. Biol. 172,489-506. sible for cIT-dependent inhibition. 8. McMacken, R., Mantei, N., Butler, B., Joyner, A. & Echols, The clear-plaque phenotype of a X c1857 paq-J phage H. (1970) J. Mol. Biol. 49, 639-655. showed that PaQ function is important in X development 9. Oppenheim, A., Belfort, M., Katzir, N., Kass, N. & Op- under cro' conditions, where penheim, A. B. (1977) Virology 79, 426-436. extreme cIT-dependent inhibi- 10. Herskowitz, I. (1971) Dissertation (Massachusetts Institute of tion does not occur. An explanation for the clear-plaque Technology, Cambridge, MA). morphology of X cI857 paq-J could be that an increase in the 11. Court, D., Green, L. & Echols, H. (1975) Virology 63, synthesis of the Q protein channels the phage into the lytic 484-491. program. Other mutations that increase Q synthesis have 12. Schmeissner, U., Court, D., Shimatake, H. & Rosenberg, M. been selected and analyzed; one of these (byp) also has a (1980) Proc. Natl. Acad. Sci. USA 77, 3191-3195. clear-plaque morphology (38). PaQ transcription could also 13. Knoll, B. J. (1979) Virology 92, 518-531. interfere with 0 and P expression. However, the inhibitory 14. Friedman, D. I. & Gottesman, M. (1983) in Lambda II, eds. effect of cIl on X DNA replication is not as significant as its Hendrix, R. W., Roberts, J. W., Stahl, F. W. & Weisberg, R. A. (Cold Spring Harbor Laboratory, Cold Spring Harbor, effect on late-gene expression (8). Taken together, these NY), pp. 21-51. results are consistent with the model in which PaQ contributes 15. Roberts, J. W. (1975) Proc. Natl. Acad. Sci. USA 72, to X development by decreasing the expression of Q during 3300-3304. the period of uncommitted phage growth when cII is active. 16. Burgess, R. R. & Jendrisak, J. J. (1975) Biochemistry 14, We do not as yet know the length of the in vivo transcript 4634-4638. originating at PaQ. A sequence containing features of pro- 17. Lowe, P. A., Hager, D. A. & Burgess, R. R. (1979) Bio- karyotic terminators, including a stem and loop, is located chemistry 18, 1344-1352. about 220 bases from the RNA start of PaQ. We observed 18. Cech, C. L. & McClure, W. R. (1980) Biochemistry 19, some termination at this site in vitro. The PaQ transcript may 2440-2447. 19. Ho, Y.-S., Lewis, M. & Rosenberg, M. (1982) J. Biol. Chem. have been detected in vivo by Smith and Hedgpeth (39). They 257, 9128-9134. reported an RNA species =200 nucleotides long that hybrid- 20. Oppenheim, A. B., Gottesman, S. & Gottesman, M. (1982) J. ized to the X 1-strand in the region between base pairs 39,168 Mol. Biol. 158, 327-346. and 44,972. The size of this RNA suggests that the termina- 21. Joyce, C. M. & Grindley, N. D. F. (1983) Proc. Nat!. Acad. tion at +220 observed in our in vitro reactions may occur in Sci. USA 80, 1830-1834. vivo. Processing in vivo of a longer transcript by nucleases 22. Panet, A., van de Sande, J. H., Lowen, P. C., Khorana, H. G., could also have produced the same RNA species. Ho and Raae, A. J., Lillehaug, J. R. & Kleppe, K. (1973) Biochemistry Rosenberg (personal communication) have found that PaQ 12, 5045-5050. functioned in vivo when the appropriate DNA fragment was 23. Miller, J. H. (1972) Experiments in Molecular Genetics (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY), pp. inserted into one of the galK expression vectors and cIl was 433-434. supplied by temperature induction of a defective X prophage. 24. Marinus, M. G. & Morris, N. R. (1974) J. Mol. Biol. 85, Stephenson (personal communication) has also observed 309-322. cII-dependent transcription from PaQ in vivo, using a lacZ 25. Zagursky, R. J. & Berman, M. L. (1984) Gene 27, 183-191. expression plasmid. 26. Daniels, D., Schroeder, J., Szybalski, W., Sanger, F., The mechanism by which PaQ participates in cII-dependent Coulson, A., Hong, G., Hill, D., Petersen, G. & Blattner, F. inhibition is unknown. Transcriptional interference with (1983) in Lambda II, eds. Hendrix, R. W., Roberts, J. W., PR-initiated transcription by the convergent transcription Stahl, F. W. & Weisberg, R. A. (Cold Spring Harbor Labora- from PaQ or translational interference of tory, Cold Spring Harbor, NY), p. 649. by hybridization 27. Amann, E., Brosius, J. & Ptashne, M. (1983) Gene 25, sense and antisense RNAs could both result in decreased Q 167-178. expression. Transcriptional interference with PR by cII- 28. Benson, S. A. & Taylor, R. K. (1984) BloTechniques 2, activated PE has been reported (12). Translational interfer- 126-127. ence by antisense RNAs has been shown to decrease expres- 29. Hoopes, B. C. & McClure, W. R. (1981) Nucleic Acids Res. 9, sion of the TnJO transposase gene (40), ompF (41), and other 5493-5504. genes (42). 30. Rosenberg, M. & Court, D. (1979) Annu. Rev. Genet. 13, 319-353. We are grateful to Ira Herskowitz for several discussions on 31. Hawley, D. K. & McClure, W. R. (1983) Nucleic Acids Res. cII-dependent inhibition and for several phages used in out experi- 11, 2237-2255. ments. We thank J. Roberts, M. Gottesman, and R. Zagursky for 32. Enea, V. & Zinder, N. D. (1982) Virology 122, 222-226. sending us plasmids and strains prior to publication. This research 33. Lis, J. T. & Schleif, R. (1975) Nucleic Acids Res. 2, 383-389. was supported by Grant GM30375 from the National Institutes of 34. Birnboim, H. C. & Doly, J. (1979) Nucleic Acids Res. 7, Health. 1513-1523. 35. McClure, W. R., Cech, C. L. & Johnston, D. E. (1978) J. Biol. 1. 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