0022-538X/78/0025-0224$02.00/0 JOURNAL OF VIROLOGY, Jan. 1978, p. 224-237 Copyright © 1978 American Society for Microbiology

Vol. 25, No. 1 Printed in U.S.A.

Transcriptional Specificity of a Multisubunit RNA Polymerase Induced by Bacillus subtilis Bacteriophage PBS2 STEVE CLARK The Biological Laboratories, Harvard University, Cambridge, Massachusetts 02138 Received for publication 15 July 1977

Bacillus subtilis phage PBS2 induced the synthesis of two temporally defined categories of phage-specified transcripts. The transcription of phage "early" genes was induced almost immediately after infection; this RNA synthesis did not require phage protein synthesis. Phage "late" gene transcription, on the other hand, was induced at an intermediate time in the lytic cycle; this RNA synthesis required the production of phage proteins. Both classes of transcription were resistant to the drug rifampin and, therefore, apparently did not require the rifampin-sensitive component of the host RNA polymerase. A rifampin-resistant, DNA-dependent RNA polymerase was purified from bacteria infected with PBS2. The highly purified phage polymerase consisted of five distinct subunits that remained associated during zone centrifugation, isoelectric focusing, and disc gel electrophoresis. The synthesis of each of the five polypeptides was induced at an intermediate time in the phage lytic cycle. As judged by hybridization competition, hybridization to DNA restriction fragments, and RNA-RNA annealing, the phage-induced RNA polymerase preferentially and asymmetrically transcribed PBS2 late genes in vitro. These findings suggest that the PBS2 RNA polymerase controls the expression of phage genes late in the lytic cycle.

Bacillus subtilis phage PBS2, a large DNA virus whose genome (molecular weight = 1.5 x 101 [12]) contains uracil in place of thymine (31), exhibits a novel program of phage gene expression. Unlike all other known DNA phages, PBS2 grows normally in bacteria that have been treated with rifampin, even when the drug is administered before infection (21, 22). This finding is thought to indicate that the onset of phage gene expression is controlled by an as yet-unidentified transcribing activity that is distinct from the rifampin-sensitive, DNA-dependent RNA polymerase of the host bacteria. As shown in this report, two different categories of rifampin-resistant transcription by PBS2 can be distinguished. The transcription of phage early genes, beginning almost immediately after infection, does not require phage protein synthesis. Phage late genes, on the other hand, are first transcribed at about 15 min into the lytic cycle; this RNA synthesis is dependent upon new protein synthesis. PBS2 is known to induce the synthesis of a rifampin-resistant RNA polymerase that specifically copies PBS2 DNA in vitro (5). Here I show that the multisubunit phage RNA polymerase preferentially and asymmetrically transcribes PBS2 late genes. MATERIALS AND METHODS Materials. Unlabeled ribonucleoside triphosphates and rifampin were purchased from Schwartz/Mann.

[3H]UTP, [a-32P]UTP, H13'PO4, Na2'SO4, L-[3H]leucine, Protosol, and Omnifluor were purchased from New England Nuclear. DEAE-Sephadex A-50, agarose, and chloramphenicol were obtained from Sigma. Other materials were purchased from the following sources: polyacrylamide gel reagents from Bio-Rad; DEAE-cellulose (DE52) and Whatman cellulose phosphate Pll from Reeve Angel; pancreatic RNase (5x crystallized) and RNase Ti from Calbiochem; DNase (RNase-free) from Worthington; and nitrocellulose filter paper from Schleicher and Schuell. Buffers and media. TY medium is 0.5% Difco yeast extract, 1% Difco tryptone, and 1% NaCl titrated to pH 7.4 with NaOH. After sterilization, the medium is brought to 0.1% glucose, 0.1 mM MnCl2, and 10 mM MgCl2 by addition from sterile stock solutions (10). 121A medium has been described elsewhere (29). PBS2 dilution buffer is 0.025 M Tris-hydrochloride (pH 7.5), 0.1 M NaCl, 0.001% FeCl3, 0.001% CaCl3, and 0.01% MgSO4. Tris-phosphate-EDTA (TPE) buffer for agarose gels is 0.036 M Tris-hydrochloride (pH 7.8), 0.03 M NaH2PO4, and 1 mM EDTA (9). DNA sample buffer is 0.1x TPE with 0.02% bromophenol blue. 2x SSC is 0.3 M NaCl plus 0.03 M sodium citrate (pH 7.4). Hybridization buffer is a mixture of equal volumes of 2x SSC and phenol-saturated 2x SSC adjusted to pH 7.2. Sample buffer for sodium dodecyl sulfate (SDS)-slab gels is 0.01 M Tris-hydrochloride (pH 7.5), 3% SDS, 0.04% bromophenol blue, and 2% 8-mercaptoethanol. Bacterial growth and preparation of PBS2 stocks. PBS2 in all experiments was grown at 320C, using exponentially dividing B. subtilis 3610. For the preparation of PBS2 stocks, bacteria growing in TY

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medium were infected at a multiplicity of infection of ford and Gesteland (6), except that the purification 0.2. After lysis, PBS2 was harvested by precipitation step was omitted. The incorporation of label was with polyethlyene glycol (1). The concentrated phage stopped by adding the culture to 2.5 ml of crushed were partially purified by a short centrifugation (5 frozen 121 medium with 0.1% sodium azide and 2% min at 7,000 rpm in a Sorvall SS-34 rotor) to remove Casamino Acids. The infected bacteria were harvested cellular debris and then a longer centrifugation (1 h by centrifugation and frozen with dry ice-acetone. For at 12,000 rpm) to pellet the phage. This pellet was the experiment of Table 2, the bacteria were labeled suspended in phage dilution buffer and used for the with 0.25 mCi of L-[3H]leucine for two generations infection of large quantities of bacteria needed for the before infection with PBS2. The uptake of L-[3H]leupreparation of PBS2 RNA polymerase or unlabeled cine was stopped by the addition of 10 lig of unlabeled competitor RNAs. PBS2 was further purified on a leucine per ml for 25 min before infection. The radioactively labeled bacteria (from a 5-ml CsCl step gradient, followed by equilibrium banding in CsCl. This highly purified phage (dialyzed against culture) were mixed with 2 g of unlabeled infected phage dilution buffer) was used in labeling experi- bacteria as carrier, and an extract was prepared and ments and for the preparation of PBS2 DNA (see fractionated by using the procedure summarized in Table 1 (through the phosphocellulose chromatograbelow). Large quantities of infected bacteria for the prepa- phy step) scaled down 10-fold to accommodate the ration of the phage RNA polymerase were grown in smaller quantity of starting material. The 0.55 M KCl TY medium (multiplicity of infection, 2 to 5). In all eluate from the phosphocellulose column was concenother cases, the bacteria were grown in 121A medium trated by precipitation with trichloroacetic acid and (or a modified 121A medium for labeling; see below) analyzed by electrophoresis through a long (24-cm) SDS-slab gel as described below. For the experiment and infected at a multiplicity of 5. Purification of PBS2 RNA polymerase. Frozen of Table 2, the 0.4 M KCl eluate from the phosphocells (60 g) harvested at 30 to 40 min after infection cellulose column (which contained partially purified with PBS2 were disrupted by sonic treatment, and a B. subtilis RNA polymerase) was also analyzed by high-speed supernatant was prepared as described pre- this procedure. SDS-slab gel electrophoresis. SDS-slab gel elecviously (5). The supemnatant was partitioned between phases of polyethylene glycol and dextran as described trophoresis was performed, and the gels were stained by Babinet (2), and PBS2 RNA polymerase was re- as described by Linn et al. (13) by using short (10covered in the 4. M NaCl extraction step. The phase- cm) 8% slab gels or long (24-cm) slab gels with an extracted enzyme was concentrated by ammonium exponential 7 to 12% gradient of acrylamide, as desulfate precipitation, dialyzed against buffer C (0.05 scribed by O'Farrell (17). For analysis of 35S-labeled M Tris-hydrochloride [pH 8.0], 0.1 mM EDTA, 0.1 proteins, the gels were dried and visualized by automM dithiothreitol, and 10% [vol/vol] glycerol) con- radioagraphy (Kodak SB-5 film). For determination taining 0.1 M KCI, and applied to a 30-ml DEAE- of the radioactive isotope content of specific proteins, cellulose column equilibrated with the same buffer. bands were cut from the dried gels, reconstituted with The column was washed with 100 ml of buffer C 50 pl of water, frozen in 0.3 ml of Protosol-water (9:1), containing 0.1 M KCI, and the enzyme activity was and shaken overnight at 55°C. The samples were eluted with buffer C containing 0.24 M KCI. This prepared for counting by addition of 10 ml of 0.4% fraction was applied to an 8-ml phosphocellulose col- Omnifluor in toluene. Before counting, the gel fragumn (prepared as described by Shorenstein and Losick ments were removed by filtration through glass wool. Preparation of pulse-labeled and unlabeled [28]) at a flow rate of 15 ml/h. The column was washed with 50 ml of buffer C containing 0.4 M KCI. PBS2 RNA. For pulse-labeling PBS2 RNA, B. subPBS2 RNA polymerase was then eluted with buffer tilis germinated overnight in 121A was diluted 100C containing 0.55 M KC1. The phosphocellulose-puri- fold in a modified 121 medium containing 0.3 mM fied enzyme was dialyzed against buffer C containing K2HPO0 and lacking Casamino Acids. This culture 0.01 M MgCl2 and 0.1 M KCl and further purified by was grown to approximately 1.2 x 108 celLs per ml, DEAE-Sephadex chromatography and zone centrifu- harvested by centrifugation, suspended in one-half the gation through a linear gradient of 10 to 30% (vol/vol) original volume of a modified 121A medium containing glycerol, as described previously (5). Fractions were 10 ltM K2HPO4 lacking Casamino Acids, and infected stored at -25°C after dialysis against storage buffer with PBS2 in the presence of rfampin (25 ug/ml). (buffer C containing 50% [vol/vol] glycerol and 0.05 Three-milliliter portions in 50-ml Erlenmeyer flasks M KCl). PBS2 RNA polymerase activity was mea- were labeled by the addition of 2 to 4 mCi of carriersured as described previously, with PBS2 DNA (20 free H3PO4 at the indicated times after infection. in the presence of rifampin (50 The infection was stopped by pouring the cells over i&g/ml) as template ug/ml) (5). One unit of enzyme incorporates 1 nmol 1.5 ml of crushed frozen 121 salts solution containing of [3H]UMP in 10 min at 37°C. Protein concentrations 0.1% sodium azide. The cells were harvested by cenwere determined by the method of Lowry et al. (16). trifugation and frozen with dry ice-acetone. Total nuLabeling PBS2 proteins. B. subtilis was grown cleic acid was prepared as described by Segall et al. in medium 121A lacking Casamino Acids to a density (26), and DNA was removed by treatment with DNase of 2.5 x 108 cells per ml. The culture was infected and phenol extraction (24). The final aqueous phase with PBS2 at 5 min after treatment with rifampin (25 was brought to 0.01 M Tris-hydrochloride (pH 7.5)-0.5 ,ug/ml). Five-milliliter portions in 50-ml Erlenmeyer M KC1 by addition of 3 volumes of 0.4 M Tris-hydroflasks were labeled with 0.5 mCi of an Na2iSO4-labeled chloride (pH 8.0)-2 M KCI and passed through a Escherichia coli hydrolysate ([3S]methionine) pre- nitrocellulose ifiter (equilibrated with 0.01 M Trispared according to the procedure described by Craw- hylrochloride [pH 7.5] and 0.5 M KCI and mounted

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in a Millipore syringe filtering device) to remove traces of DNA. The filter was rinsed with an additional 1 ml of 0.01 M Tris-hydrochloride (pH 7.5)-0.5 M KCI. RNA was recovered from the combined filtrates by ethanol precipitation. The final pellet was suspended in hybridization buffer. The early competitor RNA was prepared from 4 liters of bacteria treated 5 min before infection with 150 ,ug of chloramphenicol per ml. The cells for the early competitor were rapidly chilled 10 min after infection by pouring them over 1 liter of crushed frozen 121 medium containing 0.1% sodium azide. The late competitor RNA was prepared from 4 liters of infected bacteria chilled by the same procedure 35 min after infection. Cells were harvested by centrifugation and the RNA was isolated as described by Segall and Losick (24). In vitro RNA synthesis. RNA was synthesized with either 8 yg of fraction 6 PBS2 RNA polymerase or 25 ,Lg of B. subtilis core RNA polymerase (a gift of D. Stinchcomb and R. Tjian). The reaction mix was as described by Pero et al. (18), except that the reactions contained 0.1 M KCI and [a-3P]UTP (500 to 1,000 mCi/mmol) was used to label the RNA. PBS2 DNA (25 ug/ml) was used as the template. Rifampin (50 yg/ml) was routinely included in the reaction mix when PBS2 RNA polymerase was the transcribing enzyme. Excess anti-sigma antibody (a gift of D. Stinchcomb and R. Tjian) was added to reactions with core RNA polymerase to depress activity of residual sigma polypeptide. The RNAs were extracted with phenol and precipitated as described by Pero et al. (19), except that the final pellet was suspended in hydridization buffer. RNA-RNA annealing. RNA-RNA annealing reactions contained 100 p1 of hybridization buffer, 0.15 to 0.3 mg of unlabeled in vivo RNA, and about 20 ng of 3P-labeled RNA (1,000 cpm/ng) synthesized in vitro or about 200 ng of pulse-labeled PBS2 RNA (100 cpm/ng) in a total volume of 200,l. The reactions were incubated for 3.5 h at 55°C and then treated with RNase A (20 Mg) and RNase Ti (20 U) in 1 ml of 2x SSC for 1 h at 350C. RNase-resistant material was precipitated with 5% trichloroacetic acid. Preparation of PBS2 DNA. CsCl-banded PBS2 (250 to 400 Mg of DNA per ml) was extracted with SDS as described by Chessin and Summers (4) except that the final dialysis buffer was 0.01 M Tris-hydrochloride (pH 8.0)-0.01 M KCl-1 mM EDTA. Restriction endonuclease digestions. EcoRI digestions were in 0.1 M Tris-hydrochloride (pH 7.5)-5 mM MgC12-50 mM NaCl with 50 Mg of DNA per ml in a total volume of 1 to 3 ml for 1 h at 37°C, using 20 U of EcoRI (a gift of J. Segall) per jg of PBS2 DNA. (One unit of EcoRI digests 1 jg of phage lambda DNA in 15 min at 37°C.) Reactions were terminated by heating to 70°C for 10 min. The DNA was extracted with phenol three times, phenol was removed by extraction with ether, and the DNA was precipitated with ethanol. Agarose gel electrophoresis. DNA fragments were resolved on horizontal agarose slab gels as described by Segall and Losick (25), except that the TPE buffer of Hayward and Smith (9) was used instead of DNA E buffer. For a preparative gel, approx-

J. VIROL. imately 25 to 30 Mug of EcoRI-digested DNA in DNA sample buffer was applied to a 200-ml 0.85% agarose gel. DNA fragments were transferred to nitrocellulose by using the Southern procedure (30) as described by Segall and Losick (25). Hybridization with DNA strips. Nitrocellulose strips containing an imprint of the EcoRI-generated fragments of PBS2 DNA were wetted with hybridization buffer, wound into coils, and placed in 1-cmdiameter vials. The coils were covered with 1-mm glass beads to reduce the volume of the reactions. 3P2 labeled RNA in hybridization buffer (0.25 ml) was added, completely covering the coil, and the vials were sealed with Saran Wrap. Each reaction contained approximately 2 x 10' cpm of 3P-pulse-labeled PBS2 RNA (100 cpm/ng) or 2 x 105 cpm of 3P-labeled RNA synthesized in vitro (1,000 cpm/ng). An input of 2 x 104 cpm of either type of labeled RNA resulted in the same relative intensities of label in the hybrid bands, but the required time of exposure to film was much longer. The hybridization reactions were incubated with gentle shaking at 55°C for 18 h. Under these conditions, more than 80% of the PBS2 DNA was retained by the nitrocellulose strips. (A temperature of 55°C was used because, at higher temperatures, the efficiency of hybridization was drastically reduced due to poor retention of the DNA by the strips; less than 25% was retained during hybridization at 66°C.) The strips were then washed with 2x SSC for 1 h at 550C, treated with heat-treated RNase A (20 Mg/ml) for 1 h at room temperature, and, finally, washed with 2x SSC for 1 h at room temperature. After this last wash, the strips were flattened, dried in vacuo at 800C, and analyzed by autoradiography with Kodak SB-5 film.

RESULTS Enzyme purification and subunit structure. PBS2 RNA polymerase was purified by a modified version of the previously described procedure (5). In this new protocol, the crude extract of phage-infected bacteria was fractionated by the phase extraction procedure of Babinet (2), followed by chromatography on DEAE-cellulose, phosphocellulose, and DEAE-Sephadex. In the final purification step, the enzyme was subjected to zone centrifugation through a linear gradient of glycerol. As summarized in Table 1, enzyme purified in this manner was enriched about 10,000-fold and was obtained in about a 30% yield. The purified enzyme consisted offive different polypeptide species of 80,000 (80K) (P80), 76K (P76), 58K (P58), 53K (P53), and 48K (P48) daltons. All of these polypeptides were distinct from the subunits of B. subtilis RNA polymerase (Fig. 1). Densitometry of a stained disc gel previously indicated that P80, P76, P58, and P48 were present in approximately equimolar amounts (5). However, P53, a component that was not observed in enzyme isolated by the previously published procedure (5), was present

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TABLE 1. Sunmary ofpurification of PBS2 RNA polymerase Ratio of

AAmto A29oa

F'raction

Total(mg) protein

Total (U)b activity

Sp act (U/mg)

Recovery (%)

108 0.03 0.53 3,500 High-speed supernatant 270 0.43 100 0.57 630 Phase-partitioned enzyme 82 0.71 221 1.4 310 Pooled DEAE-cellulose enzyme 70 8.5 189 22.2 1.5 Pooled phosphocellulose enzyme 58 260 0.6 156 Pooled DEAE-Sephadex enzyme 34 93 390 0.24 Pooled glycerol gradient enzymec aA2w0, Absorbancy at 280 nm. bOne unit of enzyme incorporates 1 umol of [3H]UMP in 10 min at 37°C. c Fraction 6 activity and recovery have been normalized, assuiing that all of the fraction 5 enzyme had been applied to the glycerol gradient (see legend to Fig. 2).

(1) (2) (3) (4) (5) (6)

however, preferentially associated with enzyme at the leading edge of the sedimenting polymerase. These results are consistent with the idea that the PBS2 RNA polymerase can exist as a five-subunit complex of 315,000 daltons and that the previously observed four-subunit complex

_B' 'B

P80_O_w -P765

P5-MN

-o

P53-wm-p P48-48wo-

V

1-

(0 ,-.

C *_%

A

D D 12 16 8 A FIG. 1. SDS-polyacrylamide slab gel electrophoFraction Number resis of PBS2 RNA polymerase and B. subtilis RNA FIG. 2. Zone centifugation of PBS2 RNA polymjpolymerase. PBS2 RNA polymerase (A) (4 pg of fraction 6 enzyme) and B. subtilis holoenzyme (B) (a erase. A sample of 0.5 ml (0.15 mg) of the enzyme mixture of 4 pg of fraction 5 holoenzyme and 0.5 pg purified through step 5 of Table 1 (concentrated of fraction 7 simua, prepared as described by Shor- against Ficoll) was submitted to zone centrsfugatwn enstein and Losick [28]) were subjected to SDS-slab through a linear gradient of 10 to 30% (vol/vol) glycgel electrophoresis usig an 8% 10-cm acrylamide erol as described previously (5). Sedimentation standards, ,B-galactosidase and hemoglobin, were sedigel. mented in paraUel in a duplicate gradient. Fractions in variable and less than stoichiometric amounts of 0.75 ml were collected, and 20 yil was assayed as in Materials and Methods (-). A total of of the PBS2 inindifferent different preparations OI _B;S.2 enzyme. described il of each fraction (except 50 of fraction 11) of As shown in Fig. 2, all five polypeptides cosed- the gradient was precipitated with trichloroacetic imented with the PBS2 polymerase activity at acid and subjected to SDS-polyacrylamide slab gel

enzyme.-200

,1

11S during glycerol gradient centrifugation, the electrophoresis, using an 8% acrylamide 10-cm slab final step in the purification procedure. P53 was, gel (shown in the upper panel).

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results from loss of the P53 cornponent during purification. As further evidence for the as;sociation of the five polymerase subunits, purifiied enzyme was subjected to isoelectric focusinig under nondenaturing conditions in a pH 4 to) pH 8 gradient. Protein fractionated by this proccedure was then subjected directly to electrophorresis in a second dimension through an SDS-slab 1gel, as described by O'Farrell (17). As shown in F'ig. 3, isoelectric focusing resolved two forms of t,he enzyme that contained P80, P76, P58, and P'48, but differed in subunit composition by the Ipresence or absence of P53. In constrast, isoe)lectric focusing under denaturing conditions individually resolved all five polypeptides, a ftnding that indicates that each subunit exhibits a distinct isoelectric point (data not shown). The isoelectric points were estimated from thi s analysis to be 5.0, 5.1, 5.8, 5.9, and 5.2 for P80 P76, P58, P53, and P48, respectively. Purified PBS2 RNA polymeraLse was also subjected to electrophoresis througlh a nondenatur-

ing polyacrylamide disc gel. As shown in Fig. 4, this procedure resolved the protein into four different bands. The subunit compositions of these bands were determined by electrophoresis in a second dimension through an SDS-slab gel. This analysis revealed that the protein in bands 1 and 2 in Fig. 4 contained all five polypeptides (possibly enzyme in one of these bands represented an aggregated form of the 315,000-dalton complex). Band 3 predominantly contained only P80, P76, and P58, while band 4 contained only P48. Thus, nondenaturing gel electrophoresis resolved forms of the RNA polymerase (bands 1 and 2 in Fig. 4) that contained all five polypeptides from dissociated forms of the enzyme complex. Therefore, all five polypeptides of PBS2 RNA polymerase can remain associated in approximately equimolar amounts during zone centrifugation, isoelectric focusing, and disc gel electrophoresis. These findings strongly suggest, then, that the five PBS2 proteins bind to one another to form an enzyme complex of 315K daltons. It has not yet been investigated (e.g., by reconstruction of the enzyme complex) whether all IF five subunits are required for enzyme activity. However, since it is possible to obtain an enzymatically active subassembly of four subunits that lacks P53, at least this polypeptide is not essential for transcription of PBS2 DNA in vitro. C Induction of phage PBS2 RNA polymerCA P80 - ase. As shown in the time course experiment .e. P76 of Fig. 5, PBS2 RNA polymerizing activity can first be detected at an intermediate time in the phage lytic cycle. This activity continues to acP58 cumulate until a late time after infection. If P53 PBS2 polymerase is composed of the five polyP48 peptides described in the previous section, it might be expected that all or some of these ± subunits are phage-induced proteins whose synthesis commences several minutes into the lytic cycle. To investigate this possibility, cells of B. subtilis that had been treated with rifampin to host RNA and protein synthesis were block 5.0 5.6 pH 5.0 pulse-labeled with [3S]methionine at 0 to 5, 5 FIG. 3. Isoelectric focusing of PiBS2 RNA polymto 10, and 10 to 15 min after infection with 6 PBS2. The radioactively labeled cells were erase. PBS2 RNA polymerase (6 Wg of mixed with unlabeled infected bacteria as carenzyme in 25 .ld of storage buffer) was subjected to isoelectric focusing in a disc gel (10 by 0.15 cm) rier, and PBS2 polymerase was partially purified containing 4% acrylamide (from a sitock solution con- from the mixture through the phosphocellulose taining 45% acrylamide and 0.6% bissacrylamide), 10% step of Table 1. Radioactive protein in the parsucrose, and 1.9% ampholines (LKB) O( .5% pH range, tially purified polymerase fractions was dis3 to 10; 0.2% pH range, 9 to 11; 0.1 % pH range, 4 to played by electrophoresis through an SDS-poly6; and 0.1% pH range, 5 to 7) at 0 gel was equilibrated in SDS sampl buffer and ana- acrylamide slab gel. (PBS2 polymerase was suflyzed by SDS-polyacrylamide slabg4el electropawresis ficiently purified after phosphocellulose chroas described by O'FarreU (17), us,eng a 10-cm matography that its subunits could be readily acrylamide slab gel. The panel sh ows the region of identified by SDS-gel electrophoresis.) As shown the stained slab gel containing th,e polypeptides of in the autoradiograph of Fig. 6, radioactivity PBS2 RNA polymerase. from [3S]methionine only appeared in the com*.-,,W

fractiwon

t

o

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+

Non-denaturing 166 1

2

3

4

11

P80 P76 P588 P53P48-

FIG. 4. Disc gel electrophoresis of PBS2 RNA polymerase. PBS2 RNA polymerase (10 pg of fraction 6 enzyme in 40 i1. of storage buffer) was subjected to electrophoresis through a 5% acrylamide (from a stock containing 30% acrylamide and 0.8% bisacrylamide) disc gel (10 by 0.6 cm) at 0 to 4°C, using the procedure described by Krakow (11) (upper gel). A total of 5 ug of the enzyme was electrophoresed in parallel in an acrylamide disc gel (10 by 0.15 cm) of identical composition. This gel was equilibrated in SDS sample buffer and analyzed by SDS-slab gel electrophoresis as described in the legend to Fig. 3. The lower panel shows the stained second-dimension gel. ponents of PBS2 polymerase in cells that had been labeled at 5 to 10 and 10 to 15 min after infection. Further evidence that all of the subunits of the PBS2 enzyme are induced after infection was provided by the following "pulse-chase" experiment. Cells of B. subtilis were labeled before infection with [3H]leucine. The incorporation of 3H was stopped by dilution with excess unlabeled leucine. The 3H-labeled cells were next treated with rifampin for 5 min to inhibit host RNA and protein synthesis and were then infected with PBS2. At 10 min after infection, the 30 40 10 20 bacteria were briefly labeled with [3S]methioTime After Infection (min) nine. The double-labeled cells were mixed with FIG. 5. Time course of appearance of PBS2 RNA polymerase activity. For each time point, 2 liters of unlabeled infected bacteria as carrier, and RNA infected bacteria were chilled rapidly at the indicated polymerase was partially purified through the time by pouring the culture over 0.5 liter of crushed phosphoceliulose chromatography step of Table frozen 121 medium containing 0.1% sodium azide 1. Fractions from the phosphocellulose column and then harvested by centrifugation. An extract was contining either the PBS2 or the B. subtilis prepared and fractionated through step 2 of Table RNA polymerase (see Materials and Methods) 1. Enzyme activity was measured by using pH] UTP were subjected to SDS-polyacrylamide slab gel (500 mCi/mmol) in the presence of rifampin (50 The labeled protein bands were electrophoresis. pg/ml), with PBS2 DNA (25 pg/ml) as template as described in Materials and Methods. Protein concen- cut from the gel, and the amount of 3H and 3S trations were determined by the method of Lowry et in each was determined. As summarized in Table 2, the ratio of radioactivity from 3S (the postal. (16).

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P80

-PP76 -P58 --P53 --P48

A B C FIG. 6. Time of synthesis of the subunits of PBS2 RNA polymerase after phage infection. PBS2-infected bacteria were labeled with [jS]methionine in the presence of rifampin during the time periods indicated below, and PBS2 RNA polymerase was partially purified through the phosphocellulose step of the procedure described in Materials and Methods. For each sample, the same percentage (about 20%) of the total fraction 5 protein was precipitated with trichloracetic acid (10%) and subjected to electrophoresis on a long (24-cm) SDS-slab gel as described in Materials and Methods. (The stained gel confirmed that each sample contained approximately equal amounts of the polymerase from the carrier cells.) The locations of the polypeptides of PBS2 RNA polymerase were determined from their mobilities in a stained sample of the purified enzyme in an adjacent slot. The panel is an autoradiogram of the gel. Fraction 5 enzyme from cells labeled (A) 0 to 5 min after infection (15,000 cpm was applied to the gel), (B) 5 to 10 min after infection (35,000 cpm was applied to the gel), and (C) 10 to 15 min after infection (69,000 cpm was applied to the gel).

infection label) to 3H (the preinfection label) several hundred-fold greater in each of the five subunits of PBS2 RNA polymerase than in was

the ,8 and ,B' subunits of B. subtilis RNA polymerase. Thus, the synthesis of all five subunits of PBS2 RNA polymerase (assuming that leucine is present in these polypeptides) was only detected after PBS2 infection, and this synthesis was not prevented by the presence of rifampin. Program of RNA synthesis during the PBS2 lytic cycle. The production of the PBS2 polymerase at an intermediate time after infection suggested that the phage transcriptase might be responsible for the transcription of genes that are activated at a late time in the phage lytic cycle. To investigate this possibility, it was first necessary to study the temporal control of PBS2 genes in infected bacteria. Hybridization competition experiments indicated that PBS2 induces the synthesis of at least two categories of phage RNA. As shown in Fig. 7, radioactive RNAs from bacteria that had been pulse-labeled at either an early (10 min after infection in the presence of chloramphenicol) or a late (35 min after infection) time in the phage lytic cycle were hybridized to denatured PBS2 DNA in the presence of various amounts of unlabeled RNAs as competitor. Unlabeled RNA from cells that had been infected in the presence of chloramphenicol markedly competed with the hybridization of early pulselabeled RNA (Fig. 7a), but only partially inhibited the hybridization of late-labeled sequences (Fig. 7b). In contrast, unlabeled RNA TABLE2. Synthesis of the subunits of PBS2 RNA polymerase after phage infection TMSb (cpm) 3W (cpm) Ratio of 35S Poly' (p Hccm) to 3Hd peptidea 185 0.08 2,310 ifT 130 1,900 0.07 105 30 80K 3,150 57 50 76K 2,850 60 35 58K 2,100 80 23 53 K 1,820 17 85 48K 1,480 a The polypeptides were identified in an SDS slab gel by comparison of their mobilities with those of purified samples of the two enzymes in adjacent slots. The radioisotopic content of each polypeptide was determined from the gel as described in Materials and Methods. b The bacteria were labeled with [MS]methionine at 10 to 15 min after infection. c The bacteria were labeled with L-[3H]leucine for two generations before infection. d The sample of partially purified B. subtilis RNA polymerase (purified through phosphocellulose chromatography as described in Materials and Methods) contained 22,000 cpm of MS and 25,000 cpm of 3H, whereas the sample of the PBS2 enzyme (purified through the phosphocellulose chromatography step; see Materials and Methods) had 42,000 cpm of 3MS and 25,000 cpm of 3H.

SPECIFICITY OF PHAGE PBS2 RNA POLYMERASE

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50

C

CL50-

0.4

0.6

0.8

Competitor

0.4

RNA

0.6

0.8

(mg)

FIG. 7. Competition of the hybridization of 32P-labeled RNA to denatured PBS2 DNA. Hybridization reactions contained the 32P-labeled RNAs described below, 2.5 pg of denatured PBS2 DNA, and the indicated amounts of unlabeled competitor RNA in a total volume of 200 ,lp of hybridization buffer. Hybridization was performed at 550C for 3.5 h and then treated as described by Pero et al. (18). Hybridization competition of (a) early PBS2 RNA pulse-labeled 5 to 10 min after phage infection in the presence of 25 pg of rifampin per ml and 200upg of chioramphenicol per ml; 25% of the input RNA (22,000 cpm) hybridized in the absence of competitor; (b) late PBS2 RNA pulse-labeled 32 to 35 min after infection in the presence of 25 pg of rifampin per ml; 30% of the input RNA (25,00() cpm) hybridized the absence of competitor; (c) RNA synthesized in vitro by B. subtilis core RNA polymerase in the presence of 0.1 M KCI; 32% of the input RNA (18,000 cpm) hybridized in the absence of competitor; (d) RNA synthesized in vitro by PBS2 RNA polymerase in the presence of 0.1 M KCI; 33% of the input RNA (24,000 cpm) hybridized in the absence of competitor. For each reaction, a background of less than 2% of the input was subtracted from the radioactivity that hybridized. RNAs were labeled and purified as described in Materials and Methods. (0) CAM RNA was purified from bacteria treated with 150 pg of chloramphenicol per ml before infection; (0) late RNA was purified from bacteria 35 min after infection as described in Materials and Methods. CAM, chloramphenicol. in

extracted at a late time in the phage lytic cycle efficiently inhibited the hybridization of both early (Fig. 7a) and late (Fig. 7b) pulse-labeled sequences.

These findings suggest that there are at least two classes of PBS2 genes: an early class whose transcription does not require phage protein synthesis and a late class consisting of sequences actively transcribed at a delayed time after in-

231

fection. The early transcripts appear to persist until late in the lytic cycle, as their hybridization was inhibited by the late RNA competitor. This could either indicate that early genes continue to be transcribed or that the early messages are stable. To investigate the program of PBS2 transcription in greater detaiL endonuclease restriction fragments of PBS2 DNA were used to determine at what times during the lytic cycle different segments of the phage genome are transcribed. The restriction endonuclease EcoRI cleaves PBS2 DNA (despite the presence of uracil in place of thymine) into about 50 fragments with molecular weights ranging from 0.4 x 106 to 8 x 106 (J. Segall, personal communication) (Fig. 8). These restriction fragments were separated by agarose gel electrophoresis and transferred to nitrocellulose by the procedure of Southern (30). Strips of nitrocellulose containing an imprint of the EcoRI-cut phage DNA were then annealed with radioactive RNAs from cells that had been pulse-labeled with [32P]phosphate at various times during the phage lytic cycle. Hybrids were identified by autoradiography of the strips. As shown in Fig. 9 and smmarized schematically in Fig. lOa, three different categories of restriction fragments were identified. Fragments in the first category contained sequences that were most actively transcribed early after infection. The transcription of these early sequences appeared to be greatly reduced or turned off after about 20 mr into the lytic cycle (e.g., fragments 14 and 30-31). The second class contained fragments that hybridized to sequences from all of the pulse-labeled phage RNAs tested regardless of the time of labeling. These fragments could contain both early and late genes or early genes whose transcription persisted at late times in infection. PBS2 RNA synthesized in chloramphenicol-treated bacteria specifically hybridized to all of the fragments of these first two categories (Fig. 9, strip B). The third class consisted of fragments with sequences that were first actively transcribed at an intermediate time in the lytic cycle (e.g., fragment 5-6). This late RNA synthesis, which was prevented by prior treatment of the bacteria with chloramphenicol (Fig. 9, strip C), first began about 14 min after infection, but at least one fragment contains sequences that were not actively transcribed until about 30 min into the lytic cycle (fragment 48). It is not known whether this fragment is located at the distal end of a long transcription unit or if it represents a third temporal class of phage genes. In any event, these findings confirmed that there is at least one early and one late class of temporally controlled PBS2 genes.

232

J. VIROL.

CLARK

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A B FIG. 8. Agarose gel analysis of EcoRI-restricted PBS2 DNA. Electrophoresis was on a 0.85% agarose slab gel. The gel was stained for 1 h in electrophoresis buffer (TPE) containing 1 pg of ethidium bromide per ml and then photographed on a short-wave UV transilluminator (27). (A) EcoRI-digested phage lambda DNA (a gift of J. Segall). Beginning at the top, the molecular weights (x 106) of the lambda fragments are 13.7, 4.74, 3.73, 3.48, 3.02, and 2.13 (32).

Transcriptional specificity of PBS2 RNA polymerase. Three different procedures were used to assess the specificity of gene transcription in vitro by purified PBS2 RNA polymerase. First, as a measure of the asymmetry of transcription, radioactively labeled RNA was copied in vitro from PBS2 DNA and then annealed with an excess of unlabeled early or late phage RNA. The proportion of radioactively labeled product that formed RNA-RNA duplexes (as determined by resistance to RNase) served as a measure of the content of anti-messenger sequences in in vitro-synthesized RNA. As summarized in Table 3, only about 16% of the RNA copied by PBS2 RNA polymerase formed RNARNA duplexes with a mixture of early and late mRNA. Interestingly, the asymmetry of this transcription was influenced by the ionic strength of the reaction mixture; at low ionic strength (i.e., no KCl added to the reaction mixture), PBS2 RNA polymerase generated more anti-mRNA sequences (data not shown). For comparison, B. subtilis core RNA polymerase, an enzyme that is known to exhibit little transcriptional specificity, synthesized greater than 40% anti-mRNA. (Theoretically, 50% antimRNA would be expected for the product of completely random transcription.) As a further comparison, only a small proportion of in vivosynthesized RNA that had been pulse-labeled at an early or a late time in the lytic cycle formed RNase-resistant duplexes with unlabeled early and late mRNA. As a second indication of transcriptional specificity, RNA synthesized in vitro by PBS2 polymerase was hybridized to denatured PBS2 DNA in the presence of unlabeled early or late RNA competitors. As shown in Fig. 7d, the late RNA competitor was significantly more effective than early RNA in inhibiting the hybridization of in vitro-generated sequences. Taken together with the previous demonstration of asymmetric RNA synthesis (Table 3), this finding suggests that the PBS2 transcriptase preferentially copied phage late genes in vitro. For comparison, both the early and late competitors sigificantly inhibited the hybridization of the in vitro product of B. subtilis core RNA polymerase (Fig. 7c). This result is consistent with the finding (Table 3) that core enzyme produces a high proportion of both sense and anti-sense sequences whose hybridization to PBS2 DNA would be inhibited by the competitor RNAs. Finally, as an indication that the PBS2 tran(B) 3 pg of EcoRI-digested PBS2 DNA. The bands are identified by numbers beginning with the largest

fragment at the top of the gel.

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SPECIFICITY OF PHAGE PBS2 RNA POLYMERASE

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FIG. 9. Hybridization ofpulse-labeled 2P-labeled RNAs to nitrocellulose strips containing EcoRI fragments of PBS2 DNA. Filter strips, prepared as described in Materials and Methods, containing about 2 pg of EcoRI fragments of PBS2 DNA, were hybridized with 32P-labeled RNAs (2 x 105 cpm total) labeled during the indicated time periods after infection in the presence of 25 pg of rifampin per ml. RNA from uninfected bacteria was prepared from B. subtilis pulse-labeled for 3 min with ffP]phosphate 5 min after treatment with rifampin. 32P-labeled chloramphenicol RNA (strips B and C) was labeled during the indicated time periods as described in the legend to Fig. 7 and prepared as described in Materials and Methods. No hybridization was detectable to strip A (uninfected RNA). A total of 4 to 8% of the input labeled RNA hybridized to strips B to G. Strips A to G were exposed to Kodak SB-5 film for 2 to 4 days. A total of 10 to 15% of the input RNA hybridized to strips H to L. These strips were exposed to film for 1 to 2 days. The

numbers to the right of the strips identify the DNA fragment (see Fig. 8) to which each hybrid band corresponds. The identification of a band with two numbers (e.g., 5-6) indicates that it is not possible to distinguish between hybridization to either one of two different DNA fragments. Hybridization bands that are not numbered could not be unambiguosly analyzed because of poor resolution (e.g., DNA fragments 2-4 and 7-10) or because of very low levels of hybridization (e.g., fragments 29 and 43-45).

234

J. VIROL.

CLARK

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------l 40 A B 30 10 20 Time After Infection (min) FIG. 10. Schematic summary of the program of Ri'VA synthesis during the PBS2 lytic cycle. The numbe rs on the left-hand side correspond to the hybridiza,tion bands labeled in Fig. 9. The three types of 1t tes are used to indicate changes in the relative anbtount ofhybridization to each band. Thus, a dotted 1- te represents trace or substantially reduced levels of hybridization; a solid line indicates significant hy,bridization; and a heavy solid line indicates in--

creased hybrticzatnon over that represented by a solid line. (a) Program of RNA synthesis in vivo obtained from the hybridization data of Fig. 9. (b) Schematic representation (for comparison to the time course data) of hybridization of in vitro RNA to nitrocellulose strips. These RNAs were synthesized in vitro by (A) PBS2 RNA polymerase and (B) B. subtilis core RNA polymerase. The data are summarized from Fig. 11.

scriptase was preferentially copying phage late sequences, in vitro-generated RNA was annealed to DNA imprints of EcoRI-cut PBS2 DNA. As shown in Fig. 11 and summarized in Fig. l0b, the in vitro-generated RNA hybridized to most of the fragments that had been shown to contain late sequences. This included fragments that hybridized to both early and late pulse-labeled RNA as well as fragments that specifically hybridized only late-labeled sequences. Two exceptions were fragments 42 and 46, which contain sequences that were actively transcribed late in infection but which hybridized little of the in vitro-generated RNA. In contrast to fragments containing late sequences,

EcoRI fragments that contain only early genes hybridized little (e.g., fragments 14 and 22-23) or none (e.g., fragments 30-31 and 33) of the in vitro-synthesized RNA. In a control experiment, B. subtilis core RNA polymerase produced sequences that hybridized to nearly all of the EcoRI fragments. These findings support the conclusion that the PBS2 RNA polymerase selectively transcribes in vitro at least part of the late class of PBS2 genes.

DISCUSSION PBS2 induces at least two temporally defined categories of phage gene transcription. Phage RNA synthesis begins almost immediately after infection; this transcription requires neither the synthesis of new phage proteins nor the function of the normal rifampin-sensitive component (the ,B subunit [14]) of the host cell RNA polymerase. Transcription of some phage genes is activated

between 14 and 25 min into the lytic cycle. This late class of RNA synthesis is dependent on

phage-induced protein synthesis, as transcription is restricted to PBS2 early genes in bacteria that have been treated with chloramphenicol before infection. Like the early phage genes, PBS2 late sequences are copied by a polymerizing activity that is insensitive to rifampin. How is this program of phage gene transcription controlled? Two lines of evidence suggest that the PBS2 RNA polymerase whose isolation is described in this paper determines the expression of phage late genes. First, the synthesis of all five subunits of the PBS2 enzyme is induced at an intermediate time in the lytic cycle, just TABLE 3. Anti-message sequence content of PBS2 RNA synthesized in vitro and phage RNA pulselabeled in vivo

[nP]RNA forming duplexes

with excess unlabeled in vivo RNAs (%) RNA synthesized

In vitro Fraction 6 PBS2 RNA

Early

RNAa

Early

Late RNAb

andlate RNA

10

13

16

16

42

42

polymerase

B. subtils core RNA polymerase In vivo

5 2 3 Early RNA' 7 7 6 Late RNAd aPrepared from bacteria treated with chloramphenicol 5 min before infection and harvested 10 min after infection. bPrepared from infected bacteria harvested 35 mm after infection. Pulse-labeled with H332PO4 5 to 10 min after infection in the presence of chloramphenicol. d Pulse-labeled with H332PO4 32 to 35 min after infection. c

Voi- 25, 1978

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SPECIFICITY OF PHAGE PBS2 RNA POLYMERASE

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FIG. 11. Comparison of the hybridization patterns of in vitro RNAs with the patterns of pulse-labeled in vivo RNAs. Filter strips, prepared as described in Materials and Methods and containing about 2 pg of EcoRI fragments of PBS2 DNA, were hybridized with 32P-labeled RNAs (2 x 105 cpm) synthesized in vitro in the presence of 0.1 M KCI by (A) B. subtilis core RNA polymerase or (C) PBS2 RNA polymerase (see Materials and Methods) or pulse-labeled in vivo (B) 5 to 10 min after infection in the presence of 200 pg of chloramphenicol per ml or (D) 30 to 33 min after infection. A total of 10 to 15% of the input 3Plabeled RNA hybridized to strips A, C and D; these strips were exposed to film for 1 to 2 days. About 5% of the input hybridized to strip B, which was exposed to film for 4 days. The bands are numbered on the right as described in the legend to Fig. 9. The letters on the left identify hybridization bands that are characteristic of RNA synthesized (e) early or (1) late after infection.

235

before the activation of late phage transcription. The phage-inducedpolymerizing activityreaches its maximal accumulation during the period when PBS2 late genes become most actively transcribed. Second, the purified polymerase selectively and asymmetrically copies late sequences from PBS2 DNA in vitro. Proof for this interpretation, however, will require the isolation of phage mutants that are blocked in late gene expression. The PBS2 RNA polymerase, a large enzyme with a sedimentation coefficient of llS, appears to consist of five different subunits having a total molecular weight of 315,000. These five polypeptides remain associated with the PBS2specific transcribing activity during a 10,000-fold purification. The five-subunit structure for the RNA polymerase is supported by the demonstration that all five polypeptides can remain associated during zone centrifugation, isoelectric focusing, and disc gel electrophoresis. Also, in support of the functional relatedness of the subunits, all five polypeptides are phage-induced proteins whose synthesis is induced synchronously during the phage lytic cycle in bacteria that have been treated with rifampin before infection. At least one of the five polypeptides is not, however, required for polymerizing activity in vitro. Polymerase lacking the P53 subunit (which can apparently be lost from the enzyme during purification) actively copies PBS2 DNA in vitro (5), although the possibility that this polypeptide plays a role in the selectivity of transcription is not excluded. An investigation of the function of each of the five subunits requires the reconstruction of the enzyme complex from its individual components. The multisubunit composition of the PBS2 RNA polymerase differs strikingly from the single polypeptide structure of the other known phage-induced RNA polymerases (reviewed in reference 3). This observation, as well as the unusually large size of the PBS2 genome (1.5 x 101 daltons [12], six times as large as T7 DNA [8]), suggests the interesting possibility that PBS2 gene expression might be controlled in a temporally defined program by the sequential interaction of regulatory polypeptides with the PBS2 enzyme complex. (An analogous mechanism of sequential modification of B. subtilis RNA polymerase is known to control transcription by phage SPOl [reviewed in reference 15].) Yet, the experiments described here have demonstrated the existence of only two classes of phage genes. It is possible, however, that other subcategories of temporally regulated phage transcription were not detected in the present study. For instance, different classes of phage genes that were located within the same

236

CLARK

endonuclease restriction fragment would not have been distinguished by the experiment of Fig. 9. A candidate for a subclass of temporally regulated sequences is represented by fragment 48, as this DNA segment was not activated until 30 min into the lytic cycle. It is possible, however, that the late sequences in this fragment are simply located at the promoter-distal end of a long unit of transcription. Although the isolation of a rifampin-resistant polymerase that selectively synthesizes late RNA can account for gene expression late in the lytic cycle, the regulation of PBS2 early genes is not understood. Early RNA is produced in cells that have been treated with rifampin (and/or chloramphenicol) before infection. Therefore, early genes are probably not copied by the unmodified host cell RNA polymerase. Indeed, purified B. subtilis holoenzyme exhibits very little activity with PBS2 DNA as a template (5). Possibly, the phage virion injects either a new polymerase or a protein that modifies the host cell RNA polymerase. The identification of EcoRI fragments that contain only early genes should now provide a highly specific hybridization assay for this novel polymerizing activity. The coliphage N4 has transcriptional properties that are similar to those of PBS2. Although N4 does not grow in the presence of rifampin, phage early RNA synthesis is not blocked in cells that have been treated with both rifampin and chloramphenicol before infection (23, 33). Recently, an RNA polymerase activity has been detected in disrupted N4 virions that could account for the drug resistance of early gene transcription (7, 20). Although this enzyme has not been isolated or characterized, genetic experiments indicate that the virion polymerase is required for early gene expression (7). It will be interesting to determine whether an analogous mechanism accounts for PBS2 early transcription. ACKNOWLEDGMENIS I thank J. Segall, J. Pero, and R. Losick for many helpful discussions, A. Sonenshein for critical reading of the manuscript, and R. Losick for help with the preparation of the

manuscript. Thia work was supported by Public Health Service grant GM 18568 from the National Institute of General Medical Sciences to R. Losick.

LITERATURE CITED 1. Alberts, B. ML, and K. R. Yamamoto. 1970. Rapid bacteriophage sedimentation in the presence of polyethylene glycol and its application to large-scale virus purification. Virology 40:734-744. 2. Babinet, C. 1967. A new method for the purification of RNA-polymerase. Biochem. Biophys. Res. Commun. 26:639-644. 3. Bautz, E. K. F. 1976. Bacteriophage-induced DNA de-

J. VIROL. pendent RNA polymerases, p. 227-246. In R. Losick and M. Chamberlin (ed.), RNA polymerase. Cold Spring Harbor Laboratory, New York. 4. Chessin, H., and W. C. Summers. 1970. Initiation by RNA polymerase on UV or X-ray damaged T7 DNA. Biochem. Biophys. Res. Commun. 38:40-45. 5. Clark, S., R. Losick, and J. Pero. 1974. New RNA polymerase from Bacillus subtilis infected with phage PBS2. Nature 252:21-24. 6. Crawford, L V., and R. F. Gesteland. 1973. Synthesis of polyoma proteins in vitro. J. Mol. Biol. 74:627-634. 7. Falco, S. C., K. Vander Laan, and L B. RothmanDenes. 1977. Virion-associated RNA polymerase required for bacteriophage N4 development. Proc. Natl. Acad. Sci. U.S.A. 74:520-523. 8. Freifelder, P. 1970. Molecular weights of coliphages and coliphage DNA. IV. Molecular weights of DNA from bacteriophages T4, T5 and T7 and the general problem of determination of M. J. Mol. Biol. 54:567-577. 9. Hayward, G. S., and M. G. Smith. 1972. The chromosome of bacteriophage T5. I. Analysis of the singlestranded DNA fragments by agarose gel electrophoresia. J. Mol. Biol. 63:383-395. 10. Katz, G. E., A. R. Price, and M. J. Pomerantz. 1976. Bacteriophage PBS2-induced inhibition of uracil-containing DNA degradation. J. Virol. 20:535-538. 11. Krakow, J. S. 1971. Acrylamide gel electrophoresis as a tool for the study of RNA polymerase and the sigma initiation factor. Methods Enzymol. 21:520-528. 12. Lauer, G., and L C. Klotz. 1976. Molecular weight of bacteriophage PBS2 DNA. J. Virol. 18:1163-1164. 13. Linn, T., A. L. Greenleaf, and R. Losick. 1975. RNA polymerase from sporulating Bacillus subtilis. J. Biol. Chem. 250:9256-9261. 14. Linn, T., R. Losick, and A. L Sonenshein. 1975. Rifampicin resistance mutation of Bacillus subtilis altering the electrophoretic mobility of the beta subunit of ribonucleic acid polymerase. J. Bacteriol. 122:1387-1390. 15. Losick, R., and J. Pero. 1976. Regulatory subunits of RNA polymerase, p. 227-246. In R. Losick and M. Chamberlin (ed.), RNA polymerase. Cold Spring Harbor Laboratory, New York. 16. Lowry, 0. HI, N. J. Rosebrough, A. L Farr, and R. J. Randall. 1951. Protein measurement with the folin phenol reagent. J. Biol. Chem. 193:265-275. 17. O'Farrell, P. H. 1975. High resolution two-dimensional electrophoresis of proteins. J. Biol. Chem. 250:

4007-4021. 18. Pero, J., J. Nelson, and T. D. Fox. 1975. Highly asymmetric transcription by RNA polymerase containing phage-SPOl-induced polypeptides and a new host protein. Proc. Natl. Acad. Sci. U.S.A. 72:1589-1593. 19. Pero, J., J. Nelson, and R. Losick. 1975. In vitro and in vivo transcription by vegetative and sporulating Bacillus subtilis, p. 202-212. In P. Gerhardt, R. N. Costilow, and H. L. Sadoff (ed.), Spores VI. American Society for Microbiology, Washington, D.C. 20. Pesce, A., C. Casoli, and G. C. Schito. 1976. Rifampicinresistant RNA polymerase and NAD transferase activities in coliphage N4 virions. Nature (Lon-

don)262:412-414. 21. Price, A. R., and M. Frabotta. 1972. Resistance of bacteriophage PBS2 infection to rifampicin, an inhibitor of BaciUus subtilis RNA polymerase. Biochem. Biophys. Res. Commun. 48:1578-1585. 22. Rima, B. K., and I. Takahashi. 1973. The synthesis of nucleic acids in Bacillus subtilis infected with phage PBS1. Can J. Biochem. 51:1219-1224. 23. Rothman-Denes, L. B., and G. C. Schito. 1974. Novel transcribing activities in N4-infected Escherichia coli. Virology 60:65-72. 24. Segall, J., and R. Losick. 1975. Effect on asporogenous mutation on rate of bacteriophage Oe transcription in

VOL. 25, 1978

SPECIFICITY OF PHAGE PBS2 RNA POLYMERASE

stationary-phase Bacillus subtilis cells, p. 221-225. In P. Gerhardt, R. N. Costilow, and H. L. Sadoff (ed.), Spores VI. American Society for Microbiology, Washington, D.C. 25. Segall, J., and R. Losick. 1977. Cloned Bacillus subtilis DNA containing a gene that is activated early during sporulation. Cell 11:751-761. 26. Segall, J., R. ITian, J. Pero, and R. Losick. 1974. Chloramphenicol restores sigma factor activity to sporulating Bacillus subtilis. Proc. Natl. Acad. Sci. U.S.A. 71:4860-4863. 27. Sharp, P. A., B. Sugden, and J. Sambrook. 1973. Detection of two restriction endonuclease activities in

29. 30.

31.

32.

Haemophilus parainfluenzae using analytical agaroseethidium bromide electrophoresis. Biochemistry 12: 3055-3063. 28. Shorenstein, R. G., and RK Losick. 1973. Purification and properties of the sigma subunit of ribonucleic acid

33.

237

polymerase from vegetative BaciUus subtilis. J. Biol. Chem. 248:6163-6169. Sonenshein, A. L., and D. Roecoe. 1969. The course of phage 4oe infection in sporulating cells of Bacillus subtilis strain 3610. Virology 39:265-276. Southern, E. M. 1975. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98:503-517. Takahashi, I., and J. Marmur. 1963. Replacement of thymidylic acid by deoxyuridylic acid in the deoxyribonucleic acid of a transducing phage for Bacillus subtilis. Nature (London) 197:794-795. Thomas, M., and R. W. Davis. 1975. Studies on the cleavage of bacteriophage lambda DNA with EcoRI restriction endonuclease. J. Mol. Biol. 91:315-328. Vander Laan, K., S. C. Falco, and L B. RothmanDenes. 1977. The program of RNA synthesis in N4infected Escherichia coli. Virology 76:596-601.