In vitro synthesis of late bacteriophage phi 29 RNA.

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JOURNALOFVIROLOGY,June 1983,p.690-702 0022-538X/83/060690-13$02.00/0

Vol. 46, No. 3

In

Vitro

Synthesis of Late Bacteriophage 4)29 RNA

R.DOUGLAS HOLDERANDH. R. WHITELEY*

Departmentof Microbiology and Immunology, UniversityofWashington, Seattle, Washington 98195 Received 14 February 1983/Accepted 4 March 1983

A crude P-100 fraction prepared from Bacillus subtilis 21 min after infection with wild-type phage 4)29 supported the in vitro synthesis of late 4)29 RNA by added RNA polymerase. Synthesis of late RNAwasalso detectedwhen purified

4)29 DNA was transcribed by RNA polymerase in the presence of an S-150

fraction obtained by lysis of 4)29-infected cells in thepresenceof1 MNaCl. Late

4)29RNA was not synthesized when either the P-100 or the S-150 fractionwas

prepared from cultures infected with 4)29 having amutation ingene 4.

Bacillus subtilis phage 4)29 contains linear

double-stranded DNA having a molecular

weight of approximately 12 x 106 (3). The

genome consists of 18 cistrons, and the

func-tions and products ofmostofthe cistrons have

beenidentified(28, 33). Immediately after

infec-tion, thehost RNA polymerase transcribes the

early genes, which account for approximately

40% ofthe coding capacity ofthe genome. As

shown inFig.1,the early RNAisproducedfrom

the"light strand" of429 DNA{i.e., the strand

of lower density in

CsCl-polyuridylic-polygua-nylic acid [poly(UG)] gradients [31]} and is

syn-thesized throughout infection (24, 36, 39). The

lateclassofRNA, whichrepresents the

remain-ing 60% ofthe genome, is transcribedfrom the

"heavy strand" (31, 39). Althoughthe onset of

late transcription occurs at approximately the

sametime thatviral replication begins (36), late

transcription isindependent ofviral DNA

repli-cation (24). Synthesis ofthelate class ofRNA

requires the product ofan early 429 gene,

cis-tron 4 (4, 5, 39); the mechanism by which the gene 4 product regulates transcription is not

known.Thelocations oftheearlyand late genes

have been mappedandcorrelated witha

restric-tion map of thegenome (13, 19, 20, 44).

Transcription ofpurified 4)29 DNAby theB.

subtilis RNA polymerase in vitro yields early RNA (7, 19, 20). The number and location of

early 429promoters have been determined (8),

and the DNA sequences of three early

promot-ers have been compared (32). Synthesis of late

mRNA in vitro has not been reported and the location of the late promoters has not been

ascertained, although two polymerase-binding

siteswerefound in the lateregionof the genome

byelectronmicroscopy ofDNA-RNA polymer-ase complexes (39).

Regulationof viraltranscriptionthrough

mod-ification ofB.subtilisRNApolymerasehas been studied extensively during the development of

bacteriophages SP82 (23, 42) and SPO1 (13).

These phages code for several small polypep-tides which bind to and alterthetranscriptional specificity of the host RNApolymerase. Earlier

investigations (17) of RNApolymerase

extract-edfrom 4)29-infected cells showed that approxi-mately 10% ofthe host polymerase containeda

30,000-dalton (30K) peptide which was not

found inpolymerase from uninfectedcells. The

transcriptional specificity ofthe 30K-containing

polymerase with respect to 4)29 DNA was not

determined. It is known that at least the 3

subunit ofthe host RNApolymerase is required

for viral transcription since synthesis of late

mRNA is inhibited by the addition of rifampin

(24,

37).

In the present paper, we report that late 4)29

RNAwassynthesized invitrobypurified

rifam-pin-resistant RNA polymerase from uninfected

B. subtilis in the presence ofcrude P-100

frac-tionsfrom cellsinfected with wild-type4)29and

treated with rifampin toinhibit the endogenous

polymerase.Wealso report thatsynthesis oflate

mRNA was observed when reaction mixtures

containing purified 429DNAandpurified RNA

polymerase were supplementedwitha

superna-tant fraction (S-150) obtained by lysing

4)29-infected cells in the presence of a high salt

concentration. In both instances, i.e., in the presence of eitheraP-100orthe high-saltS-150

fraction, late mRNA was not produced if the

cellswereinfected withphage havingamutation in gene4.

MATERIALSANDMETHODS

Strains andgrowthofphage-infected B. subtilis. B. subtilis SR22, anasporogenous mutantof B. subtilis

168, obtained fromJ.Spizizen(Departmentof

Micro-biology, UniversityofArizona, Tucson),was usedas

the hostforwild-type 429 and as the nonpermissive host for4)29 sus4. Thesuppressor strain, B. subtilis

Su+44, and the sus4(56) mutant were obtained from B. E. Reilly(SchoolofDentistry, Universityof Min-690

on November 10, 2019 by guest

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nesota, Minneapolis). B. subtilisOSB420, a rifampin-resistant mutant of B. subtilis 168, was obtained from L. Brown(Departmentof Microbiology, Oregon State University,Corvallis).

Phage-infected B. subtilis was grown in 750-ml to 10-liter batches of M medium (1% tryptone, 0.5% yeast extract, 1% NaCI). After sterilization, this medium wassupplemented with 0.4 mMMnC12, 5 mM MgSO4, and0.1% glucose added from sterile stock solutions. Cultures were grownat37°C to a concentration of 2 x

108 cells per ml and infected with 5 to 104)29particles percell. After 21 min, sodium azide was added to a 0.01 M final concentration, the temperature was re-duced to 0 to 5°C by the addition of ice, and the cells were harvested bycentrifugation, frozen in a dry ice-ethanolbath, and storedat-70°C. When infected cells were treated with rifampin before harvesting, the inhibitor was added at a final concentration of 10 ,ug/ml

at 18minafter infection, incubation was continued to

21 min, and the cells were collected as described above. Lysates of4)29wereprepared using B. subtilis SR22for wild-type phage and B. subtilisSu+"forthe sus4 mutant, using the M medium described above. Lysis occurred at 36 to 39 min, yielding approximately 101l phage per ml.

Preparation of the P-100 fractionandthe high-salt

S-150fraction.+29-infectedcells which had been treated withrifampin andharvestedat21 minafterinfectionas

described above were usedfor the isolation of both fractions. Except when indicatedotherwise, the fol-lowingoperationswerecarried out at0°C.Toobtain the P-100fraction, frozen cells (ca. 0.2 g [wetweight]) were suspended in 4 ml of buffer A (0.05 M Tris-hydrochloride [pH 7.5], 0.1 M NaCl, 1 mM EDTA, 0.01M2-mercaptoethanol,200 ,ugof

phenylmethylsul-fonyl fluoride perml) supplementedwith30%o

glycer-ol,lysozymewasadded(0.5mlofa5-mg/ml solution),

and thesuspensionwasincubatedfor 25to30minat

100C.Thepreparationwasthencentrifugedfor 20 min

at100,000 xginaBeckman type 40rotor at4°C,the

supernatantwasremovedcarefully, and bufferAwas addedtothesedimenttogiveatotalvolume of 1.5 ml. Thethin, yellowish-brownsediment withaclear

gelat-inouslayer abovewasresuspended by gentle homog-enization with fourorfive strokes ofalooselyfitted

Douncehomogenizerand usedimmediatelyasa

tem-plate for RNA synthesis. In some experiments, the

preparationswerecentrifugedfor10minat10,000 rpm in a Sorvall centrifuge to remove any intact cells before high-speed centrifugation. In most

experi-ments, this preliminary centrifugation was omitted, andif any intact cells were detected after the

high-speedcentrifugation (i.e.,ifagrey-whiteopaque cen-ter of any size could be seen in the yellowish sedi-ment), the preparation was discarded. "Successful lysis" yielded P-100fractions which had anegligible number ofunlysed cells. Direct counts, bymeansofa

Petroff-Hauser counter, showed that there were 2 x 106to 5x10'cells per ml(mostly swollen and atypical

inappearance) inourusualP-100preparationsand ca. 2x 104 viable cellsasdeterminedby plate counts;the original4ml ofsuspensionwascalculatedtohave ca. 5

x 109 cells per ml.

To testforpossibledegradation ofthe DNAin the P-100fraction, 0.5-mlamounts weredilutedwith 2 mlof proteinaseKsolution(50,ug/ml in0.01 M Tris-hydro-chloride [pH7.51-0.01 M EDTA-0.5% sodium

dode-cyl sulfate[SDS])and incubated for 2 hat30°C. The samples were then extracted with phenol, dialyzed

against 100 volumesof 0.1 MTris-hydrochloride(pH

7.5)-imMEDTA for24hat4°C,treated with1 ILgof

RNaseApermlfor 60 minat22°C,andanalyzed by

electrophoresis through vertical agarose slab gels,

using TBE buffer(0.089 M Tris base,0.089 M boric acid, 0.0025MEDTA).

A high-salt S-150fraction was obtained byoneof

two methods. In thefirst, theprocedure givenabove for isolation of the P-100 fractionwasfollowedexcept thatthetreatmentwithlysozymewasreducedto12to

15 min at 10°C, NaClwas added from a 5 M stock solutiontogiveafinal concentrationof 1 M, and the preparationwasthoroughlymixed, cooledto0°C,and centrifuged for 30 minat 150,000 x g in aBeckman type 40 rotorat 4°C. The supernatant was removed and desalted by passage throughacolumn of Sepha-dexG-25 equilibrated with buffer A, and the fraction having the highest absorbency at 280 nm ("high-salt S-150 fraction") was tested for its ability to stimulate latetranscription fromi029DNA. Inthesecond meth-od, two or more P-100fractions isolated in the

pres-enceof 0.1MNaClweresupplementedwith 5 MNaCl

togiveafinal concentration of 1 M, and the prepara-tions were left on ice for 10min, centrifuged,

com-bined, andprocessedasdescribedfor the high-salt S-150 fraction. The second method also yielded preparations which stimulated the synthesis of late RNA, but they were considerably less effective and less stable than thehigh-salt S-150 fractionobtained by thefirst method.

PreparationofphageDNA.Phagewereprecipitated from 10-liter lysatesby the addition of polyethylene glycol (43), dialyzed for 24 to 48 h against buffer B

(0.01 M Tris-hydrochloride [pH 7.5], 0.1 M NaCl, 0.005 M MgCI2),centrifuged through cesium chloride (31), and dialyzed for 12 to 18 h against buffer B

containingEDTAandSDS atfinal concentrations of 0.01 M and1%, respectively.Afterheatingat60°Cfor 2min,anequal volume ofproteinaseK(100 Lg/rnlin 0.01 M Tris-hydrochloride [pH 7.51-0.01 M EDTA) wasadded,and thepreparations were incubated for4 to6 h at37°C and then extracted with phenol. Phage DNAwasprecipitatedwithethanol, dissolved in 0.01 M Tris-hydrochloride (pH 7.5)-i mM EDTA, and dialyzed against the latter solution for24h.

Digestionof DNAwithrestrictionenzymesand elec-trophoresis. 4)29 DNA was digested with EcoRI or HindIll obtained fromNewEngland Biolabs (Beverly, Mass.)accordingtomethods describedby the

manu-facturer. Fragmentswereseparatedby electrophoresis throughavertical 0.7to1.4% agarose slab gel (12 by

14by 0.6 cm),asindicated, using TBE buffer, at100 V. Staining of gels with ethidium bromide, visualiza-tion under UV light, and photography have been describedpreviously (1).

StrandseparationofEcoRI fragmentsof+29DNA. DNA was digested with EcoRl andcomplexed with poly(UG) byamodification ofthemethodof Goldbach

etal.(14). Specifically,10p.gofdigested DNAin0.1 ml of 0.1 mM EDTA was mixed with 0.06 ml of partially hydrolyzed poly(UG)at aconcentration ofI

mg/ml, and themixturewasthen heatedfor3 min at

100°Ctodenature the DNAandquickly cooledto0°C. Acriticalstepin thisprocedurewasthepreparation of partially hydrolyzed poly(UG). Thiswasachievedby

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692 HOLDER AND WHITELEY

mixing 1 volume of a3-mg/ml solution of the polymer (Miles Laboratories, Inc., Elkhart, Ind.) with 0.1 volume of 0.5 M Na2CO3, incubating for 15 min at 67°C, and then cooling and adding 1 volume of 0.05 M HCl and 1 volume of 0.08 M Tris-hydrochloride (pH 7.7).After the addition of Ficoll and bromphenol blue, samples containing denatured fragments complexed with poly(UG) were subjected to electrophoresis on horizontal0.45%agaroseslab gels, using 0.04 M Tris-acetate (pH 7.7)-i mM EDTA as the buffer (14). Electrophoresis was performed at 4°C at a constant current of45 mAfor 18 to 20h, and the gels were stained withethidium bromide.

Invitro synthesis of RNA. When preparations of the P-100 fraction were used as the template, reaction mixtures (total volume, 0.24 ml) contained 60 ,ul of the P-100fraction, 10 to 20pgofB.subtilis OSB420 RNA polymerase, 0.04 M Tris-hydrochloride (pH 7.9), 0.1 M NaCl, 0.015 MMgCl2, 0.01 M 2-mercaptoethanol, 0.5mgof bovine serum albumin per ml, and 10 ,ug of rifampin per ml. The preparations were maintained at 0°C and mixed thoroughly, and an aliquot of 0.1 ml was withdrawn and added to a 4-,ul aliquot of a nucleotide solution containing 8 mM ATP, GTP, and CTP and4 mMUTPplus1 to2,uCi of[a-32P]UTP(30 to 50mCi/mmol; New England Nuclear Corp., Bos-ton, Mass.).The reaction mixtures were incubated for

10 min at 30°C or at the indicated temperature, and RNA was extracted for hybridization as described previously (41). When purifiedDNAwas the template, the same procedure was used except that the P-100 fraction was replaced with 20 to 30 ,ug of4)29DNA per ml.Methods used to assay theactivity ofRNA

poly-merasehave been described previously (1).

Labeling RNA in vivo. RNA was pulse-labeled at intervals during infection by the addition of4,uCi of [3H]uridine (41.5Ci/mmol; ICN, Irvine, Calif.) per ml and extractedaccordingtopreviouslydescribed meth-ods (41). Alternatively, phage-infected cultures were

grown in a low-phosphate medium (1) and pulse-labeled with 100 ,uCi of 32PO4 (carrier-free; New

England Nuclear) per ml.

Hybridizationof[32PJRNAtorestrictionfragmentsof 4o29 DNA. Fragments weretransferred to nitrocellu-lose filters by the methods of Ketner and Kelly (21), and the nitrocellulose was cut into 3 to 4-mm-wide strips. Eachstripwasincubated with[32P]RNAin3to

4mlof buffer C (0.025MTris-hydrochloride[pH 7.5],

0.825 M NaCl) containing 0.1% SDS in a sealed

polyethylenebag for 18to20 hat67°C.Thestripswere

washedinbuffer C for 20min at60°C, treated with10

pugof RNaseAper ml in 0.3MNaCl-0.03 M sodium citrate for20min at 37°C,and washedat 60°C fora totalof 60 minwiththree changes of buffer C contain-ing 0.1% SDS,0.01 Msodium PP,,0.004 M disodium phosphate, and 1 to 2,g ofATP per ml. Thefilters were dried and exposed to Kodak X-Omat film with an intensifyingscreen at -75°C.

RNA-DNA competition-hybridization. Anti-mRNAs may be synthesized by in vitro transcription of4)29

DNA(19).These RNAsmayself-anneal or hybridize with added -competitor RNAs or both, thus leading to spurious results in competition-hybridization

reac-tions. To minimize these effects, the competition-hybridization procedure (22) was modified to permit presaturation with competitor RNAs (26). In brief, nitrocellulose filters (5-mmdiameter) containing2 to 3

,ug of denatured 4)29 DNA were incubated with 0 to 0.75 mg of unlabeled competitor RNA in 0.3 ml of hybridization buffer (0.01 M Tris-hydrochloride [pH 7.5], 0.33 M NaCl, 0.1% SDS) at 67°C for 16 to 20 h. The filters were washed to remove unhybridized RNA,and labeled RNA was added and incubated with the filters as described above. After washing, the filters were treated for 60min at 20°C with a mixture of

RNases AandTI,washed, and dried, and the radioac-tivity was determined in a scintillation counter, using a toluene-based scintillant.

Miscellaneous methods. RNA polymerase was puri-fiedasdescribedby Achberger and Whiteley (1). The procedure of Roberts (34) was used to purify rho factor from Escherichia coli MRE600; rho activity was as-sayed according to Lowery-Goldhammer and

Richard-son (25). Protein concentrations were determined by the methodof Bradford (6), using gamma globulin as thestandard.

RESULTS

Transcriptional map. As indicated in Fig. 1,

early RNA is transcribed primarily from the

EcoRI A andC fragments. One earlytranscript

extendsslightly into theEcoRI Dfragment,and

hybridization to fragment D has been reported

by some investigators (20, 39)but notbyothers

(19). RNAextracted from4)29-infected cells late

ininfectionhybridizestoEcoRIfragmentsB, D,

and E in addition tofragments A and C.

Sym-metricaltranscriptionof boththe B andD

frag-ments can occur(39).The in vitrotranscription

of 4)29 DNA by B. subtilis RNA polymerase

yields several RNA species which hybridize

predominantly to theEcoRI A and Cfragments

(19,20, 39); in vitrotranscriptionmay alsoyield

significant amountsof anti-late mRNA (19).

Strand separation. To study the in vitro

syn-thesis oflate4)29 mRNA, we have determined

the strand specificity of transcripts produced

from a region of the genome containing late

genes-i.e., the EcoRI A and B fragments.

When EcoRI restriction fragmentsof4)29DNA

weredenatured,complexedwithpoly(UG),and

subjected to electrophoresis, the

complemen-tary strands of the two largest fragments each

yielded two ratherdiffuse, but separable,bands

(Fig. 2, lanes a to d). The complementary

strands ofundigested 4)29DNAand thestrands of the EcoRI Cfragmentwere notseparable by

this method, and theDand Efragments usually

comigratedas asingle largeband.

Figure 3 compares autoradiograms obtained

by hybridizing RNAs synthesized during

infec-tion to EcoRI and HindIlI fragments and to

strand-separated EcoRIfragmentsafter

electro-phoresisand transfer ofthefragmentsto

nitro-cellulose membrane filters.Controlexperiments

(Fig. 3, lanes a,b,g, andh)indicated thatearly

and late RNAs hybridized to the EcoRI and HindIII fragments in accordance with the map shown in Fig. 1. Early RNA hybridized to the J. VIROL.

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IN RNA Genetic 12 3 45 6 7 8 &5 9 10 11 12 13 14 15 16 17

1a a Ia a a I I Ia I I I

Map

. , .. -. . . >

early genes A

Hind IllL

(L) 5'. (H)

3'-late genes

B

B G K HMI E J D

a I I ...a .I a

early RNA

late genes earlygene

E D C

A N F C L

early RNA

late RNA

[image:4.488.81.414.68.239.2]

0 25 50 75

FIG. 1. Genetic,physical, and transcriptionalmapsof the4)29genome.

EcoRI C fragment and to the slow-migrating band of the A fragment (Fig. 3, lane d). The latter has been designated "AL," since early RNA istranscribed from the light strand of4)29

DNA (31). The parallel experiment with late RNA(Fig. 3, lane e) demonstrated that in addi-tion to AL, the fast-migrating band of the A fragment and the slow-migrating band of the B fragment were transcribed at late times after infection. These bands were designated "AH"

and "BH," respectively, since late RNA is complementary to the heavy strand of 4)29 DNA. These findings and strand assignments

agreewiththose ofSogoetal. (39), who

report-ed that the fast-moving band of all EcoRI frag-ments of 4)29 DNA, except the A fragment, corresponded to the light strand and the

slow-moving bandcorresponded totheheavy strand. Longer exposures of the autoradiograms

pre-sented inFig. 3 also showedhybridization oflate

mRNAtothe EcoRI D/Efragment region (data notshown). Occasionally, RNA extracted from cultures early in infection showed a small amountofhybridization in the BH region (faint-ly visible in lane d of Fig. 3 and in lane b of Fig. 5), butnohybridizationwasfoundtothe EcoRI Bfragment if the strands of the DNA were not

complexed withpoly(UG) and separated before transfer to nitrocellulose (Fig. 3, lane a).

Be-cause of this occasional spurious hybridization ofearly in vivo RNA to BH, hybridization to both AHandBHwasconsideredtobethemost reliable indicator of the presence of late RNA.

The decreased hybridization of late RNA rela-tive to the hybridization of early RNA to the EcoRI C fragment(Fig. 3, lanes a, b, d, and e)

and tothe HindlIl C fragment (Fig. 3, lanes g

andh) will be discussed separately (R. D.

Hold-erand H. R. Whiteley, manuscriptin

prepara-tion).

In vitrosynthesis oflate RNA.P-100fractions

wereprepared from 429-infected cultures 21 min

afterinfection as described above and used as

templates for the synthesis of RNA. Since DNA replication beginsat8 to10min after infection, thepresence oflarge amountsof 4)29 DNAwas

expected. Electrophoresis of complexes after digestion with proteinase K showed two bands

on agarose gels. One band corresponded in

molecularweight to mature 429 DNA, and the other,

havinf

anapproximate molecular weight

of 30 x 10, probably contained sheared B. subtilis DNA. The samplesalso contained some

high-molecular-weight DNA which didnot enter thegel. Therewasnoevidenceofdegradationof either the hostorphage DNA during the

incuba-tion of thecomplexes with nucleotides and add-edpolymeraseasjudged by electrophoresis. The

FIG. 2. Strand separation of EcoRI fragments of 4)29 DNA by gel electrophoresis (photograph of a 0.45%agarosegel stainedwithethidiumbromide). (a) EcoRI-digested4)29 DNA; (b) heat-denatured EcoRI-digested +29 DNA; (c) EcoRI fragments of DNA which were heat denatured and complexed with

po-ly(UG) before electrophoresisasdescribedin thetext; (d) purified EcoRI A fragment denatured and com-plexedwithpoly(UG) before electrophoresis; (e) puri-fied EcoRI Bfragment denaturedandcomplexedwith poly(UG)beforeelectrophoresis.

100

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[image:4.488.294.395.433.580.2]

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a

bc

d

ef

g

hi

'g-~A

:i -AL

*wA

X

-

-AH

X

BH

DI

BL

0t0:Ujx;0| 0;

D/E

l

_

Z

~~~~~~~~~K

L

FIG. 3. Autoradiograms showing the hybridization of early and late in vivo [32P]RNAs to restriction fragments of4)29DNA.4)29-infectedcells were labeled with32PO4,and RNAs were extracted andhybridized to EcoRI- and Hindlll-digested 429DNA asdescribed in the text. (a) Early RNA(pulse-labeled for 2 to 7min) hybridized toEcoRIfragments; (b) late RNA(pulse-labeled for 25 to 30min) hybridized to EcoRl fragments; (c) photograph of ethidium bromide-stained 0.7% agarose gel ofEcoRI fragments; (d) early RNAhybridized to separated strands of EcoRI fragments; (e) late RNA hybridized to separated strands ofEcoRI fragments; (f) photograph of ethidium bromide-stained 0.45%agarose gel of separated strands ofEcoRI fragments; (g) early RNAhybridized toHindlI fragments; (h) late RNA hybridized to HindII fragments; (i) photograph of ethidium bromide-stained 1.2% agarose gel ofHindlIl fragments.

P-100 fractions also contained many proteins,

including RNA polymerase and probably

ribo-somes, since ribosomesweredetected in earlier

experiments with similar preparations(30).

Sedi-mentationofthe P-100preparations in10to30%

glycerol gradients showed thatRNApolymerase

and additional peptides cosedimented with

DNA, suggesting that the preparations

con-tainednucleoprotein complexes.

Because the P-100fractions contained endog-enouspolymerase, transcription wasperformed in the presence ofrifampin by using

rifampin-resistantpolymeraseisolatedfrom uninfected B.

subtilisOSB420.To ensure thatRNAsynthesis

by preparations containing the P-100 fraction

resulted from de novo initiation by the added

rifampin-resistantpolymerase and did not result

merely from elongationoftranscriptsinitiated in

vivo, cultures were treated with rifampin for 3

minbefore harvestingthecells. The data in Fig.

4show thatthistreatmenttotally inhibited RNA

synthesis by the endogenous polymerase pres-entin the P-100fractions.Itshouldbenoted that

all in vitro transcriptions were performed using

RNA polymerase preparations containing the

deltapeptide (2).

Two typesof P-100 fractionswerecompared:

thoseobtained from cells infected with wild-type

4)29 and those prepared from nonpermissive

cells infected with 4)29 carrying a nonsense

mutation in cistron 4. Mutations in this cistron

havepreviously been shown toprevent

expres-sion of late genes in vivo (4, 5, 39). RNAs

synthesized from such complexes by

exoge-nously added rifampin-resistant polymerase

were analyzed by hybridization to blots of

strand-separated EcoRI fragments of4)29DNA

asinFig. 3. HybridizationtofragmentsAH and BH was defined as indicating the synthesis of

late mRNA.

Transcription of the P-100 fraction derived

from cells infected with wild-type 4)29 yielded

RNAs which hybridized strongly to fragments

AL, AH, and BH and weakly to fragment C

(Fig. 5, lane a). The same fragments were

hy-bridizedby RNAs synthesized in vivo 25 to 30 J. VIROL.

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20

-E

1.5-0

0101.0

0.

1-0.

0~

0.-0 2 4 6 8 10 12 Time

(min)

FIG. 4. RNA synthesis from the P-100 fraction in the presenceand absenceof added RNA polymerase. Reaction mixturescontained 150 ,ul of P-100 fraction, 10 g of rifampin per ml, 50 ,ug of rifampin-resistant RNApolymerase(O or A) or no added polymerase(0 orA),buffer, and nucleotides including[32P]UTPin a total volumeof 0.75. The reactions were incubated at

30°C,and atthe indicated times, S0-pl samples were withdrawn and the incorporation of radioactivity into trichloroacetic acid-insoluble material was measured.

(0, 0)P-100fraction isolated from4)29-infectedcells

which had not been treated with rifampin before

harvesting; (A, A)P-100fraction isolated from o029-infected cells which had been treated with rifampin beforeharvesting.

min afterinfection (Fig. 3, lane e) except that

there was a relatively weaker hybridization to

ALin the latterexperiments.Transcription of

P-100obtained fromcellsinfectedwith thegene 4 mutantdid notyield late RNA (Fig. 5, lane b). Hybridization wasfoundpredominantly to

frag-ment AL and more weakly to fragment BH.

SincehybridizationtofragmentAHcouldnotbe

detected and therewas nohybridizationtothe B

fragment if the poly(UG) complexing step was

omitted (data not shown), the hybridization to

BH was considered to be an artifact. The

ab-senceofhybridizationtotheEcoRI Cfragment

in lane b ofFig. 5 will be discussed elsewhere

(HolderandWhiteley, in preparation). For

com-parison,lane cofFig. 5presents the resultsofa

separateexperiment in which purified4)29DNA was used as the template inplace ofthe P-100

fraction. Late RNA was not synthesized from

this template, as evidenced by the absence of

hybridizationtoAHandBH. Hybridizationwas

observedtofragments AL, C,andD/E,

indicat-ing the synthesis of earlyRNA(19, 20).

Hybrid-izationwasalso found tofragment BL,

indicat-ing the synthesis of anti-late RNA. This

observationis discussed in a later section.

Paral-lel experiments in which the S-100fraction ob-tained from cells infected with wild-type 4)29

was used for the synthesis of RNA by rifampin-resistant polymerase yielded only trace amounts

of RNA, probably because the S-100 fraction

contained relatively low concentrations of

DNA. The hybridization of this RNA was not

investigated.

Confirmation that the P-100 fraction from cells

infected with wild-type 4)29 supported the

syn-thesis of late RNA in vitro came from

RNA-DNA competition-hybridization experiments

(Fig. 6). Thedifferences in hybridization in the

presence of early and late competitor RNAs

were similar to those obtained with

pulse-la-beled in vivo late RNA. In contrast, the data

obtained with RNAsproduced from purified4)29

DNAindicate that only earlyRNA was

synthe-sized; the ability of late RNAto competein this

hybridizationwasduetothefact that early RNA

is produced throughout infection (7, 24, 37).

Hybridization ofRNA synthesized from P-100

tosaturatingamountsof 4)29 DNA indicated that

approximately 25% of the transcripts were429

specific (datanotshown),and therelative

inten-sities of the bands in lane a of Fig. 5 and

a

b

c

AL_

---AL

AHd

~-AH

BL-

*-BL

C-'-C

D/E

D,-);E

FIG. 5. Autoradiogram showing the hybridization ofseparated strands ofEcoRIfragments of4)29DNA with RNAs synthesized in vitro. RNA was synthe-sizedas described inthe text during a 10-min incuba-tion at 30°C, using rifampin-resistant RNA polymer-ase, rifampin, and: (a) a P-100 fraction isolated from cells infected with wild-type429,(b)aP-100 fraction isolated from cells infected with a gene 4mutant of

4)29,and(c)purified4)29DNA.Note that two sets of filterswereused in thisexperiment: one set forlanes a andbandthe other setfor lane c.

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6% HOLDER AND WHITELEY

L..

o

20

-0

0

100

0

2 80

IL A

60

40

20-0

0 0.5 1.0 1.5 2.0 2.5

Competitor RNA

(mg/ml)

FIG. 6. Competition-hybridization experiments in which RNAs synthesized in vivo and in vitro were

used. (A) RNA extractedfrom4)29-infected cells la-beled with 32PO4 at 2 to 7 min after infection and competed with 7-min unlabeled RNA (0) or 30-min unlabeled RNA(0);RNAextracted from4)29-infected cells labeled with32P04at25to30 minafter infection andcompetedwith 7-min unlabeled RNA (A) or 30-minunlabeled RNA(A).(B)[32P]RNAsynthesizedin vitro as described in thetextfrompurified 4)29DNA andcompetedwith 7-min unlabeled RNA(0) or 30-min unlabeled RNA (0); [32P]RNA synthesized in vitro as described in the text from aP-100 fraction isolated from+29-infectedcellsandcompetedwith

7-minunlabeledRNA(A)or30-min unlabeledRNA(A\).

subsequent figures indicate that significant

amountsoflate RNA were synthesized.

Effects oftemperature,dilution,andNaCi.The

capacity oftheP-100fractionisolated from cells

infected with wild-type 4)29 to support the in

vitro synthesis of late RNA was highly

depen-dent upon the temperature at which transcrip-tion was performed. Figure 7 presents dataon the hybridization between strand-separated

EcoRI fragments of4)29 DNA and RNAs

syn-thesized by preparations containing P-100 prepa-rations at 20, 25, 30, 37, and 42°C. Two overall trends were apparent from these

autoradio-grams. First, late RNA was not synthesized at

the two higher temperatures asjudged by hy-bridization to EcoRIfragmentsAH and BH(Fig. 7, lanes d and e), and synthesis of anti-late RNA

was observed (hybridization to fragment BL).

Temperature shift experiments (data not shown) indicated that thefactor orfactorsrequired for late transcription were irreversibly inactivated

in vitro at temperatures higher than 30°C. The

second trend evident inFig.7 was aprogressive

inhibition in the synthesis of early RNA as the temperature was decreased. RNA complemen-tary to the EcoRI C fragment was not synthe-sized at 25°C (Fig. 7, lane b), and at 20°C there

was little detectable transcription from the

EcoRI A fragment (Fig. 7, lane a). Thus, at

lowertemperatures,virtuallyalltranscription by

reaction mixtures containing the P-100 fraction

came from late sequences. Since these assays

were performed with crude preparations, it is

not known whether the apparent temperature

lability of the factor(s) is dueto degradation by

proteases or to some othermechanism.

The efficient synthesis of late RNA in vitro

required relatively high concentrations ofthe

P-100 fraction (50 to 100 ,ul per 240-,ul reaction

mixture; the reaction mixtures also contained

0.5 mg of bovine serumalbumin perml). When

low concentrations wereused(30

RI

per 240-,ul

reaction mixture), therewaslittleor no

hybrid-izationto AH andBH, andhybridizationtoBL

was detected, indicating the synthesis of

anti-a

b

c

d

_e

-AH

w || X -BH

~:

.dl-BL

-D/E

FIG. 7. Autoradiogram showing the hybridization ofseparated strandsofEcoRI fragments of4)29DNA

withRNAssynthesized at different temperatures from

aP-100fraction.[32P]RNAswereextractedfrom

reac-tion mixtures containing rifampin-resistant

polymer-ase and a P-100 fraction isolated from 4)29-infected

cellsafter10min of incubationat:(a)20,(b) 25, (c) 30, (d) 37,and(e)42°C.

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(8)

SYNTHESIS 429

late RNA. It maybe speculated that the gene4

product was presentinlimitingamounts orthat

itwasreadily inactivated by dilution orboth.

The addition of0.05 to 0.15 M NaCl had no

effect on the synthesis of late RNA, although

some inhibitionwasnoted at aconcentration of

0.2MNaCI. Thisfinding is of interest because of

the effect of NaCI in limiting the formation of

nonspecific complexes between RNA

polymer-aseand DNA. Detailed studiesonthebinding of

B.subtilisRNApolymerase to phage SP82DNA

(1)have shownthat in thepresenceof0.1 to0.2

M NaCl, B. subtilis RNA polymerase

prepara-tionswhichcontainthedeltapeptideform

com-plexes onlywithstrongearly phage

promoters-i.e., there is little or no nonspecific complex

formation. The formation of complexesby

SP82-modified forms of RNA polymerase with middle

and late SP82 promoters was even more

sensi-tive to increasing concentrations of NaCl than

the interaction of unmodified polymerase with

earlySP82promoters. If theinteractionof

poly-merase with late 4)29 promoters resembles the

interaction of thisenzymewith SP82promoters,

then the observation that late 4)29 RNA was

produced in the presence of 0.15 M NaCl

sug-gests that this synthesis did not result from a

nonspecificinteraction betweenpolymerase and 4)29DNA.

Location of the promoter(s) for late

transcrip-tion.Asnotedabove,thelocation and number of

late promoters on 4)29 DNA have not been

determined. Tolocalize the site(s)atwhich late

transcription was initiated in vitro in the

pres-ence of the P-100 fraction, transcription was

terminated after increasing intervals of

incuba-tion and theRNAs werethenhybridizedtoblots

of HindIII restriction fragments of 4)29 DNA

(Fig. 8, lanes a to f). Lanes g and h ofFig. 3

show, respectively, the hybridization of RNAs

pulse-labeledatearly and late times of infection.

RNAs synthesized in vivo 2 to 8 min after

infectionhybridized toHindlllfragments B,C,

G, H, and K, whereas RNAs produce 25 to30

minafter infectionhybridized tothe same

frag-ments asearly RNAplusHindIIIfragments A,

D, E, F, I, and J(Fig. 3, lane g).Hybridization

to the two smallest fragments, HindIII-L and

-M, was not detected in any experiments; a similarlackofhybridization to smallrestriction

fragmentswasnotedoriginally bySouthern(40).

RNAssynthesized in vitro duringthefirst30 s

of incubation in the presence ofthe P-100

frac-tion were complementary to the Hindlll B, C,

H,and Ifragments(Fig. 8, lanea).With

increas-ing times of incubation (Fig.8, lanes b to f),

additional regions of the genome were

tran-scribed ina sequence which is consistent with

the transcriptional map shown inFig. 1, ifone

allows for the fact that hybridization to

frag-a

b

c

d

e

f

-A

& w -B -D

* 0*

*-E

-F

*

4*

* *

*-G

-

I--J

L-FIG. 8. Autoradiogram showing the hybridization

of HindIIIfragmentsof+29DNAwith RNAs

synthe-sized afterincreasingintervalsofincubation froma P-100fraction.[32P]RNAswereextractedfrom reaction mixturescontainingrifampin-resistant polymeraseand

aP-100fraction isolated from429-infected cells and incubated at30°Cfor: (a) 30 s,(b)60 s,(c)90 s,(d)2 min, (e)4min, and (f)8min.

mentsLandM was notdetected. The results of

the experiment presented in Fig. 8 suggest that

early RNA synthesis in vitro is initiated on the

HindlIl B, C, H, and I fragmentsandproceeds

from rightto lefton the physical map. In vitro

transcription of the late regions was evidently

initiated in theregionnearorwithin the

HindlIl-H-I region, and the sequential appearance of RNAcomplementary tofragments E,J(faintly hybridized), D, A, and F indicates that the

direction oftranscriptionwasfromlefttoright.

Although the HindlIl I fragment was the first

exclusivelylatefragmenttowhichhybridization

was detected, the exact position of the late

promoter could not be determined since the H

and Ifragmentsareseparated by theMfragment

which didnothybridize. Itisevident, however,

fromFig.8 thattranscription comparabletothat

ofthe H-Iregionwas notinitiated inthemiddle

of the lateregionoratthe Bi and B2sites(39).

The latter two sites, at positions 59.4 and 79.3

fromtheleft end ofthe genome, wereidentified

asbinding sites forB. subtilisRNApolymerase

by electron microscopic examination of

poly-merase-DNAcomplexes.

Elution of thefactor(s)from the P-100 fraction. Treatmentof the P-100fraction with 1 MNaCl,

followed by centrifugation or lysis of

phage-VOL. 1983

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(9)

698 HOLDER AND WHITELEY infected cells in the presen

apparently released the fact

transcription of late sequence

sedimentedby high-speed cei

a to e of Fig. 9 indicate th

increasing amounts ofthe hi

tion (after decreasingthe con

to 0.1 M) to transcription r

purified

4)29

DNAand purifie

wereemployednot onlystimu

of late RNA (hybridization ti

alsoprogressivelyreducedth

RNA (hybridizationto AL ai

RNA (hybridization to

BL)

discussed below, RNAs corr

EcoRI D and E fragments

producedbyreadthrough fro

in EcoRI-C. The P-150 fract

the high-salt treatment was

effective in stimulating late

was a P-100fraction (data not

same experiments were per1

infected with4)29 carrying a

in cistron 4, the high-salt '

(Fig. 9, lane g) did not stimul;

late sequences in vitro, and :

complementaryto theCand

not detected. The lack ofsyl

fragment in this experiment:

a

bcde

S

..:

**

*

p

FIG. 9. Autoradiogram showi ofseparatedstrands of EcoRI fra withRNAssynthesizedinthepr S-150fraction isolated from cellh type4)29 andfrom cellsinfectedi

of4)29.[32P]RNAsweresynthesiz

as described in the text in reac

volume,0.1ml)containing3 ,ug( rifampin-resistantRNApolymera perml,buffer, nucleotides,and ti of thehigh-salt S-150fractionfro] wild-type 429:(a)none,(b)6 ,u1,(

32 p1, (f) none, and (g) 32 ,t1 fraction isolated from cells infe mutant of 4)29.

ice of 1 M NaCl, obtained in two earlier experiments (Fig. 3,

tor(s) required for lanesb, e,and h,andFig. 5, laneb).

:s from the material Sincethe4)29DNA used in theabove

experi-ntrifugation. Lanes ments was purified using proteinase K

treat-iat the addition of ment,thetemplate lacked the

4)29

gene3protein

igh-salt S-150 frac- (16, 35). Synthesis oflateRNA in vitro,

there-icentrationof NaCl fore, doesnotdepend on thisprotein. Previous

reactions in which studies have demonstrated that the gene 3

prod-d RNA polymerase uct doesnotaffect either RNApolymerase

bind-ildated the synthesis ing (39) or in vitro synthesis of early RNA (8,

o AH and BH) but 20).

esynthesisof early The followingpreliminary observations have

ndC) and anti-late beenmade withrespecttotheproperties of the

). Presumably, as high-salt S-150 preparation. (i) Electrophoresis

iplementary to the onSDS-polyacrylamide gelsand onagarosegels

and to BL were showedthat thesefractionscontained acomplex

imaninitiation site mixtureofpeptides and small amountsof

nucle-tion resulting from ic acids (some rRNA and trace amounts of

significantly less DNA). (ii) As stated earlier, theS-150 fractions

transcription than prepared by either of two methods did not

shown). When the provide a template for added RNA

polymer-formed using cells ase-i.e., the addition ofpurified429 DNAwas

nonsense mutation required for RNA synthesis and late

transcrip-S-150 preparations tion. (iii)Norifampin-resistant incorporation of

atetranscription of [3H]UTP into RNA could be detected in the

synthesis ofRNAs presence of4)29 DNA when the high-salt S-150

D/E fragmentswas fraction was used as the source of RNA

poly-nthesis from the C merase in the standard RNA assay procedure

agrees with results (the concentration of NaCi in the eluate was

reducedto 0.1 Mbefore assay).(iv)Theaddition

of the high-salt S-150 fraction obtained from

cells infected with wild-type 4)29 or with the

t

g^

cistron 4 mutant resulted in a two- to threefold

t ~ stimulation in the activity ofthe

rifampin-resis--AL tant RNA polymerase. This increase wasnoted

-A H inassaysinwhich two DNA

templates

(4)29

and

-BH

SP82

DNAs) were employed and probably can

be attributedtothe release ofthe sigma subunit

-BL from endogenous RNA polymerase. (v)

Treat-mentwith 1 MNaClwasrequiredtoreleasethe

active factor(s) from P-100 fractions. (vi) The

-C

cfactor(s)

responsible

for

synthesis

of late RNA

wasrapidlyinactivated bydilution andexposure

totemperaturesgreaterthan 30°C. (vii)To date,

-

D/E

attemptstofractionate the factor(s) by

sedimen-tation in glycerol gradients orby

chromatogra-ing the hybridization phy at 40C on DEAE-cellulose,

phosphocellu-Lgments of+29 DNA

lose,

Biorex

70,

and DNA-cellulose have been

esence of a high-salt unsuccessful.

s infected with wild- In view of the earlier observation (17) that a with a gene 4 mutant small part of the RNApolymerase isolated from

red at 30'C for10min 429-infected cells contained a 30K peptide, it

Ation mixtures (total was ofinteresttodetermine whetherthe

synthe-of

429DNA,4

pLg

of sisoflate

4)29

RNAcouldbecorrelated withthe

efollowginof

armompnts

presence of this

peptide

in the

high-salt

S-150

m cells infected with

preparation.

Electrophoretic comparisons of

'c) 121Ll,(d) 20p1,(e) such fractions obtained fromcells infected with

of a high-salt S-150 wild-type and gene 4 mutantphages showed no

cted with a gene 4 significant differences in theamountsof peptides

in the 25,000- to 35,000-dalton range (R. D. J. VIROL.

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(10)

IN VITRO SYNTHESIS OF LATE 4)29 RNA 699

Holder, Ph.D.thesis,University of Washington,

Seattle, 1981). Furthermore, when extracts of

cells infected with wild-type 4)29 were

precip-itated with antibodytothe coresubunits ofRNA

polymerase and the antibody complexes were

analyzed by electrophoresis on

SDS-polyacryl-amide gels, no additional peptides were found

associated with the core subunits. However,

when thesameantibody preparationswereused

in parallel experiments with extracts of

SP82-infected cells, the SP82-coded peptides were

found in association with thecoresubunits(D. L.

Linemeyer, Ph.D. thesis, University of

Wash-ington, Seattle 1977). These observations,

cou-pledwith thefact thatareproducible isolation of

a 30K-containing form ofRNApolymerase has

notbeenachieved, suggestthat the 30Kpeptide

is not involved in transcription of late genes.

Alternatively, the 30K peptide may havea low

affinity for thecoresubunits and therefore isnot

precipitatedby antibodiestothecore orretained

with the core subunits duringpurification.

In vitrosynthesisofanti-lateRNA. Inspection

of the restriction map of 4)29 DNA (Fig. 1)

indicates that the in vitro synthesis of anti-late

RNA(RNAwhichhybridizestoBL;Fig.5, lane

c and Fig. 7, lanes d and e) results from read

through of transcription beyond a termination

site near the junction of the EcoRI C and D

fragments. Davison et al. (8) have shown that

although RNA polymerase will terminate

tran-scription atthis site invitro, termination is not

efficient and RNA ofhigh molecular weight is

produced by read through beyond this site.

Perhaps such readthroughmay occurinvivoas

well, since hybridization of early RNA to the

EcoRI D fragment has been demonstrated (20,

39). Inciarteetal. (19) reported that the addition

ofacrude extractofB. subtilis decreased

tran-scription through this site and suggested that a

termination factor may be involved. We

at-tempted to isolate a B. subtilis rho factor by

using methods described for the isolation (34)

andassay(25) ofE.coli rho but didnotsucceed (Holder, Ph.D. thesis); subsequently, Hwang

and Doi(18)isolatedarho factor fromB.subtilis

by usingother methods.

However,wefound that the addition of theE.

coli rho factor decreased the incorporation of

[3H]UTP

into RNAbyB.subtilisRNA

polymer-asebyapproximately 30%when

4)29

DNA was

the template and that E. coli rho had noeffect

when polydeoxyadenylic

acid-polydeoxythymi-dylic acid was the template (data not shown).

Figure 10presents dataon the hybridization of

RNAs synthesized in the presence and absence

of E. coli rho. The addition of this protein

inhibited the synthesis of anti-late RNA-i.e.,

RNAthathybridizedto BL(Fig.10,lanesaand

b). Lanes c and d ofFig. 10 provide evidence

a

b

I

.-AL

.. w:

-AH-i-BH

-BL

Q

-c d

S-AA

-B

-L. ._.-c

_w _ -Ew

[image:10.488.274.426.77.224.2]

A

D/-D

E

FIG. 10. Autoradiograms showing the hybridiza-tion of EcoRI fragments of +29 DNA with RNAs synthesized from +29 DNAin the presence and

ab-sence ofEcoRI rho factor. (32P]RNAs were synthe-sized asdescribed in the legendtoFig. 8 in the absence (lanesaand c) or presence(lanes b and d) of 3 Fgof

EcoRIrhofactor. Lanes aand b show the hybridiza-tion to separated strands of EcoRI fragments, and lanes c and d show the hybridization toEcoRI frag-mentsof4)29DNA.

that rho prevented synthesis from the E

frag-ment aswellasfrom theBfragment.E.coli

rho-induced termination occurred, therefore, near

thejunction of the EcoRI D and E fragments.

However,hybridizationofearlyand late invivo

RNAs(Fig. 3) demonstrated thatearly

transcrip-tion did notextendinto the EcoRI-Dregion; in

our experiments, the B, D, and E fragments

represent exclusively late regions. Therefore, although the E. coli rho factor was able to

terminate in vitrotranscription of4)29 DNA by

B. subtilis RNA polymerase and prevent the

synthesisofatleastapartof the anti-lateRNA, it seemsthatthis factor doesnot recognizethe

termination site utilized in vivoby4)29-infected

B. subtilis. It should be noted also that the

pattern ofhybridization in lane c ofFig. 10 is

identicaltothatobserved with late in vivo RNA

(Fig. 3, lane b). Use ofseparated strands,

how-ever, allows discrimination between late and

anti-lateRNAandclearlyshows that

hybridiza-tion of the B fragment in lane c of Fig. 10

represents anti-late transcription.

DISCUSSION

The present investigation demonstrated that

bothearlyand late

4)29

RNAsweresynthesized

invitrobyRNApolymerase fromDNA

provid-edbyacrudeP-100 fraction obtained from

4)29-infected B. subtilis at a late stage ofinfection.

Rifampin was added tothe culturesjust before

extraction oftheP-100 fractiontoinhibit

endog-46, 1983

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(11)

enouspolymerase; transcription was performed

in the presence of rifampin, using a purified

rifampin-resistant RNA polymerase. The

pat-ternofRNAsynthesis bythis system, as

detect-ed by hybridization to restriction fragments of

4)29DNA,was verysimilartothat obtained with

RNA synthesized in vivo late in infection. A

similar in vitro systememployinga crude lysate

of T4-infectedE.coli has recentlybeen reported

to synthesize middle T4RNA (9, 10). Asinthe

present studies, rifampin was used to block

transcription by endogenous polymerase, and

RNA synthesis was catalyzed by added

rifam-pin-resistant E. coli RNA polymerase in the

presence of this inhibitor. It was proposed that

the binding of rifampin-inhibitedpolymerase to

immediate early promoters prevented chain

elongation to the middlegenes, thereby

permit-ting detection of middlegene expression under

the control of the motgene product. It is not

known what effect, ifany, thebinding of

rifam-pin-inhibited polymerasetoearlypromotershad

onthe4)29in vitrosystem.Possibly, such

bind-ingmayhave decreasedtranscription from early

promoters, thus allowing amore active

synthe-sisof late RNA.

Underoptimal conditions oftemperature and

concentration of the P-100 fraction,there was no

synthesis of anti-late 4)29mRNAinourin vitro

reactions. Read through of a termination site

nearthe EcoRI C and Dfragments to produce

anti-late mRNA was observed when purified

4)29DNAwasusedas atemplate in placeof the

P-100 fraction. Addition of theE. coli rhofactor

prevented synthesis of some of the anti-late

RNA, butterminationdidnot occur atprecisely

the same site astermination in 4)29-infectedB.

subtilis. Possibly, a B. subtilis rho factor (18)

functions both in vivo and in the in vitrosystem

toprevent read through.

Previous studies have shown that theproduct

ofcistron 4 isrequired for the synthesis of the

lateclassof429RNAin vivo(4, 5, 13, 39). We

found that late RNAwasnotsynthesizedin vitro

when theP-100fractionwasderivedfromasus4

mutant,suggestingthatlatetranscriptionin vitro

requiresanactivegene 4protein. Earlier studies

(5) suggested that peptide LM 3B may be the

gene 4product;morerecently(11,29)gene 4 has

been cloned and sequenced, and a peptide of

12,500daltons hasbeen identifiedasitsproduct.

BystoppingtranscriptionfromtheP-100

frac-tion afterincreasingintervals of incubation and

then hybridizing the RNAs to blots of HindIII

fragments of+29DNA, welocalizedtheregion

from whichin vitro latetranscriptionwas

initiat-ed. Theseexperiments indicated that late RNA

synthesis beginsintheHindIII-H-Iregion ofthe

genomeand thattranscription proceeds

sequen-tially from left to right, consistent with the in

vivo transcriptional map presented in Fig. 1.

Electronmicroscopy of polymerase-DNA

com-plexes (39) revealed thata polymerase binding

site (A3)waslocated in this region and that there

were twoadditionalbinding sites(B1 and B2)in

the HindIII A and F fragments, respectively.

Although the results of the present studies do

notruleoutthepossibility that therewas

ineffi-cientorweak initiation from the B1 and B2 sites

concomitantly with stronger or more efficient

initiation from the HindIII-H-I region, the

sim-plestexplanation forourresults isinitiationnear

theA3 binding site followed by rightward

tran-scription.

BytreatingtheP-100fraction with1 M NaCl

orbylysingphage-infected cells in thepresence

of 1 M NaCl, the factor(s) required for late

transcriptionwasreleased into thesupernatant.

In vitro synthesis of late RNA was observed

whensuchsupernatantfractions (high-salt S-150

fraction)werepreparedfrom cells infected with

wild-type 429 and added to transcription

reac-tions containing purified 4)29 DNA and RNA

polymerase from uninfected cells. When the

high-salt S-150 fractionwasobtained from

non-permissive cells infected with 4)29 carrying a

nonsense mutation in cistron 4, late transcrip-tionwas notdetected.

Several possible mechanisms could be

pro-posedto accountfor theeffect ofthehigh-salt

S-150fractions in promoting the synthesis of late

RNA by unmodified RNA polymerase from

pu-rified 429 DNA. One possibility is that these

fractions contain thegene 4product and that this

protein is similar to the phage-coded peptides

which replacesigma in themodification ofRNA

polymeraseinSP82-orSPOt-infectedB.subtilis

(13,23, 42).Thus, it could be imaginedthat the

gene 4 product binds to thefree core subunits

whicharepresent in mostpolymerase

prepara-tionsorthat itdisplaces thesigmasubunit from

theholoenzyme, thereby providingthe

specific-ityrequired fortherecognition of late promoter

sequences. However, if this mechanism

oper-atesin thetranscriptionof4)29 DNA, the in vitro

properties ofthefactor(s)aresignificantly

differ-ent from those of the SP82-coded

specificity-determining peptides. The latterbindsufficiently

tightly to the core subunits to be retained

throughoutthepurificationofRNApolymerase,

theyareprecipitated withthecoreby antibodies

tothe core,andthey donotdisplayany unusual

sensitivitytotemperatureordilution(12, 13, 23,

42).

Anotherpossibility isthat the gene4product

functions in antitermination. An antiterminator

mechanismfor latetranscriptionof

4)29

hasbeen

suggested (39) based on the observation that

when large amounts of early RNA and late

chloramphenicolRNAwereused,hybridization

J. VIROL.

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(12)

wasdetectedtothe heavy strand of the EcoRIA

fragment but not to the heavy strand of the

EcoRIBfragment. Sogoetal. (39)proposedthat the EcoRI A fragment may contain a late pro-moter and thattranscription ofthe B fragment

requires an antiterminator protein which is not

producedearly in infection or in the presence of

chloramphenicol.

The lability of the factor(s) which stimulates

the in vitro synthesis of late 4)29 RNA is of

particularinterest. Wefound that thecapacityof

the P-100fractiontosupportthesynthesis of late

RNA decreased as the temperature of

incuba-tion of thereaction mixtures wasincreased and

thatattemperaturesabove30°C, littleor nolate

RNA was produced. Thefactor(s) required for

late in vitrosynthesiswasirreversibly

inactivat-ed at moderate temperatures and by dilution.

Moreover, asthe temperature ofsynthesis was

decreased, synthesis of earlyRNA wasinhibited

so that at 20°C virtually all RNA synthesized

from the P-100 fraction represented

transcrip-tion from late sequences. It appears, therefore,

thatatlow temperatures, B. subtilis RNA

poly-merase may initiate synthesis in vitro at a late

promoter(s)but is unable todo so at early )29

promoters. If the same factor(s) is involved in

late transcription in vivo, it must either be

stabilizedinsomewayorbesynthesized contin-uously, since raising the temperature to 42 to

45°C decreases the burst size ofwild-type 4)29

by only 50% (38). It is known that the

antiter-minator N protein is unstable in vivo at 37°C

(15). Apparently, continuous synthesis is

re-quiredtomaintain Ninlambda-infectedE.coli, since in the presence of chloramphenicol the proteinhasahalf-life ofonly2min (15).

Mangel andChamberlin (27) have shown that

theinteraction betweenE.coliRNApolymerase

and phage T7promoters is temperature

depen-dentand thatpolymerase is unabletoformopen

promotercomplexesattemperatureslower than

15 to20°C. Similar observations have been made

withB.subtilisRNApolymerase andearly SP82

promoters except that the delta-less form of

RNApolymerasewasabletoinitiatesomeRNA

synthesis at temperatures below 20°C (2). The finding that delta-containing B. subtilis RNA

polymerase is unable to transcribe early 4)29

sequences at20°C probablyreflects the inherent

inability of polymerase to separate the DNA

strands atlow temperatures. Since polymerase

was able to initiate synthesis of late RNA at

20°C, however,possiblythe,29 gene4product,

a basic protein (11, 29), can act as a DNA

"melting protein" and assist in separating the

DNA strands to form open complexes at late

promotersites. Asimilar rolehas beenproposed

forthe mot geneproduct, which controls middle

T4RNAsynthesis in vitro (9,10).Alternatively,

it may be speculated that the gene 4 product

interacts with RNApolymerase to permit

bind-ingatlate promoter sites.

ACKNOWLEDGMENTS

This research was supported by Public Health Service grants GM-20784 andGM-26100 from the National Institute of GeneralMedical Sciences. H.R.W. is a recipient of Research Career Award K6-GM-442 from the National Institute of General Medical Sciences.

LITERATURE CITED

1. Achberger, E. C., and H. R.Whiteley. 1980. The interac-tion of Escherichia coli core RNA polymerase with speci-ficity-determining subunits derived from unmodified and SP82-modified Bacillussubtilis RNApolymerase. J. Biol. Chem. 255:11957-11964.

2. Achberger, E. C., and H. R. Whiteley. 1981.The role of the delta peptide of theBacillus subtilis RNA polymerase in promoterselection. J. Biol. Chem. 256:7424-7432. 3. Anderson, D. L., D. D. Hickman, and B. E.Reilly. 1966.

Structure of Bacillus subtilisbacteriophage +29and the length of +29 deoxyribonucleic acid. J. Bacteriol. 91:2081-2089.

4. Anderson, D. L., and B. E. Reilly. 1974. Analysis of bacteriophage +29 gene function: protein synthesis in suppressor-sensitivemutantinfection of Bacillus subtilis. J.Virol. 13:211-221.

5. Anderson, D. L., and B. E.Reilly.1976.Analysis ofgene function in Bacillus subtilis+29,p. 254-274. In D. Schles-singer (ed.),Microbiology-1976. AmericanSociety for Microbiology,Washington,D.C.

6. Bradford, M. M. 1976. Arapid and sensitive method for quantitation ofmicrogram quantities of proteinutilizing the principle of protein dye binding. Anal. Biochem. 72:245-254.

7. Carrascosa, J. L., F. Jimenez,E.Vinuela,andM.Salas. 1975. Synthesis in vitro of429-specific early proteins directed by phage DNA. Eur. J.Biochem.51:587-591. 8. Davison,B.L., C.L.Murray,andJ. C. Rabinowitz. 1980.

Specificityof promoter site utilizationin vitroby bacterial RNApolymerases onBacillus phage429DNA: transcrip-tion mapping with exonuclease III. J. Biol. Chem. 255:8819-8830.

9. deFranciscis, V., and E. Brody. 1982. Invitro system for middle T4 RNA. I. Studies with Escherichia coli RNA polymerase.J.Biol. Chem. 257:4087-4096.

10. deFranciscis, V., R.Favre, M. Uzan, J. Leautey, and E. Brody. 1982. In vitro system for middle T4 RNA. II. Studies with T4-modified RNA polymerase. J. Biol. Chem. 257:4097-4101.

11. Escarnds, C.,and M. Salas.1982.Nucleotide sequence of the early genes 3 and 4of bacteriophage 4)29. Nucleic Acids Res. 10:5785-5798.

12. Fox,T.D.,andJ. Pero. 1974.NewphageSPOl-induced polypeptidesassociated with BacillussubtilisRNA

poly-merase.Proc. Natl. Acad.Sci.U.S.A.71:2761-2765. 13. Geiduschek,E.P., and J. Ito. 1982.Regulatory

mecha-nisms in thedevelopment oflytic bacteriophagesin Bacil-lus subtilis,p. 203-245. In D. Dubnau(ed.), Molecular biologyof bacillus. Academic Press, Inc.,NewYork. 14. Goldbach, R. W., R. F. Evers, and P. Borst. 1978.

Electrophoretic strand separation of long DNAs with poly (U,G)inagarosegels. Nucleic AcidsRes.5:2743-2754. 15. Greenblatt, J.1973.Regulation of the expression of theN

gene of bacteriophage lambda. Proc. Natl. Acad. Sci. U.S.A.70:421-424.

16. Harding,N.E., J.Ito, and G. S. David. 1978. Identifica-tionoftheproteinfirmlybound to the endsof bacterio-phage4)29DNA.Virology84:279-292.

17. Holland, M.,andH. R.Whiteley.1973. RNApolymerase fromBacillusamyloliquifaciens infected with+29 bacte-riophage.Proc. Natl. Acad.Sci. U.S.A. 70:2234-2237.

http://jvi.asm.org/

(13)

18. Hwang, J., and R. H. Doi. 1980.Transcription termination factor rho from Bacillus subtilis. Eur. J. Biochem. 104:313-320.

19. Inciarte, M. R., E.Vifiuela, and M. Salas. 1976. Transcrip-tion in vitro of+29DNA and EcoRI fragments by Bacillus subtilis RNA polymerase. Eur. J. Biochem. 71:77-83. 20. Kawamura, F., and J. Ito. 1977. Transcription of the

genome ofbacteriophage+29: isolation and mapping of the major early mRNAsynthesized in vivo and in vitro. J. Virol.23:562-577.

21. Ketner, G.,and T. J. Kelly. 1976. Integrated simian virus 40 sequences in transformed cell DNA: analysis using restrictionendonucleases. Proc. Natl. Acad. Sci. U.S.A. 73:1102-1106.

22. Lawrie, J. M., G. B. Spiegelman, and H. R. Whiteley. 1976. DNA strandspecificity oftemporal RNA classes producedduring infection of Bacillus subtilisbySP82. J. Virol. 19:359-373.

23. Lawrie, J. M., G. B. Spiegelman, and H. R. Whiteley. 1977.Localization oftranscripts producedin vivoandin vitro on phageSP82 genome.Gene 2:251-262. 24. Loskutoff, D. J., J. J. Pene, and D. P. Andrews. 1973.

Geneexpressionduringthedevelopmentof Bacillus subti-lis bacteriophage +29. I.Analysis ofviral-specific

tran-scriptionbydeoxyribonucleic acid-ribonucleic acid

com-petitionhybridization. J.Virol. 11:78-86.

25. Lowery-Goldhammer, C., and J. Richardson. 1979. An

RNA-dependent nucleoside triphosphate phosphorylase (ATP-ase)associated with rho termination factor. Proc. Natl. Acad.Sci. U.S.A. 71:2003-2007.

26. Lucas, J., and H. Ginsberg. 1971. Synthesis of virus-specific ribonucleic acid in KB cells infected with type 2 adenovirus. J. Virol. 8:203-213.

27. Mangel,W. F.,andM.J. Chamberlin. 1974. Studies of ribonucleic acid chain initiationbyEscherichiacoli ribo-nucleic acid polymerase bound to T7deoxyribonucleic acid.I.Anassayfor therateandextentofribonucleic acid chain initiation. J. Biol. Chem. 249:2995-3001. 28. Mellado, R. P., F. Moreno, E.Vinuela, M. Salas,B.E.

Reilly, and D. L. Anderson. 1976. Genetic analysis of bacteriophage +29 ofBacillus subtilis: integration and mapping ofreferencemutantsoftwocollections. J. Virol. 19:495-500.

29. Mellado,R.P.,and M. Salas.1982.Highlevelsynthesisin Escherichia coli of the Bacillus subtilisphage 4)29proteins p3 and p4 under the control ofphagelambdaPLpromoter. Nucleic Acids Res.10:5773-5784.

30. Mizuno,S.,andH.R.Whiteley.1968.Nuclear fraction of Bacillussubtilisas atemplatefor ribonucleic acid synthe-sis. J. Bacteriol. 95:1221-1237.

31. Mosharrafa, E., C. Schachtele, B.E. Reilly, and D. L.

Anderson. 1970. Complementary strands of bacteriophage +29 deoxyribonucleic acid: preparative separation and transcriptionstudies. J. Virol. 6:855-864.

32. Murray,C. L., and J. C. Rabinowitz. 1982. Nucleotide sequences of transcription and translation initiation re-gions in Bacillus phage4)29early genes. J. Biol. Chem. 257:1053-1062.

33. Reilly,B. E., R. A. Nelson, and D. L. Anderson. 1977. Morphogenesisof bacteriophage4)29of Bacillus subtilis: mapping and functional analysis of the head fiber gene. J. Virol. 24:363-377.

34. Roberts, J. W.1969.Termination factor for RNA synthe-sis. Nature(London) 224:1168-1175.

35. Salas, M., R. P. Mellado, E. Vinluela, and J. M. Sogo. 1978.Characterization of a protein covalently linked to the 5'termini of the DNA of Bacillus subtilis phage4)29.J. Mol. Biol. 119:269-291.

36. Schachtele, C. F., C. V. DeSain, and D. L. Anderson. 1973. Transcription during thedevelopment of bacteriophage 4)29: definition of "early" and "late" 4)29 ribonucleic acid. J. Virol. 11:9-16.

37. Schachtele, C. F., C. V. DeSain, L. A. Hawley, and D. L. Anderson. 1972.Transcription during the development of bacteriophage4)29:production of host- and 4)29-specific ribonucleic acid. J. Virol. 10:1170-1178.

38. Schachtele, C. F., R. W.Oman,and D. L.Anderson. 1970. Effect of elevated temperature on deoxyribonucleic acid synthesisinbacteriophage +29-infected Bacillus amyloli-quifaciens. J. Virol. 6:430-437.

39. Sogo, J. M., M. R. Inciarte, J. Corral, E.Vinuela,and M. Salas. 1979.RNApolymerase binding sites and transcrip-tion map of the DNA of Bacillus subtilis phage429.J. Mol.Biol. 127:411-436.

40. Southern, E. M. 1975. Detection ofspecific sequences among DNAfragments separated by gel electrophoresis. J. Mol.Biol. 98:503-517.

41. Spiegelman, G. B., and H. R. Whiteley. 1974. In vivo and in vitro transcription by RNA polymerase from SP82-infected Bacillus subtilis. J.Biol. Chem. 249:1483-1489. 42. Splegelman,G.B., and H. R. Whiteley. 1978.

Bacterio-phage SP82 induced modifications of Bacillus subtilis RNApolymerase result in the recognition of additional RNAsynthesis initiation sites on phage DNA. Biochem. Biophys. Res. Commun. 81:1058-1065.

43. Yamamoto, K., and B. Alberts. 1970.Rapid bacteriophage sedimentation in the presence ofpolyethylene glycol and itsapplicationtolarge-scale viruspurification. Virology 40:732-744.

44. Yoshikawa,H., and J. Ito. 1982. Nucleotide sequence of themajor early region ofbacteriophage429.Gene 17:323-335.

J. VIROL.

http://jvi.asm.org/