T7- and T3-specific RNA polymerases: characterization and mapping of the in vitro transcripts read from T3 DNA.

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CopyrightX)1977 AmericanSociety for Microbiology Printed inU.S.A.

T7-

and T3-Specific

RNA

Polymerases: Characterization and

Mapping of the In Vitro

Transcripts Read from T3 DNA

MIRIAM GOLOMB1 AND MICHAEL J. CHAMBERLIN*

Department of Biochemistry, University ofCalifornia, Berkeley, California 94720

Received for publication 25 June 1976

The late genetic region of the T3 bacteriophage genome is transcribed by a phage-specified RNA polymerase, the product of T3 gene 1. In vitro, purified T3

RNA polymerase acting with T3 DNA template synthesizes six major RNA

products, with molecular weights of 5.5 x 106, 4.2 x 106, 1.7 x 106, 0.87 x 106,

0.52 x 106, and 0.23 x 106. Theseare designated T3 RNA species I through VI, respectively. These RNAs are formed in equimolar amounts, with the exception of T3 species V, which is made in approximately twice this amount. T3 RNA

species I, II, and VI have been mapped by the use of terminally deleted

templates andarefoundtooriginate from promoters located at 56, 67, and 98%

genome length, respectively, and to share a common terminator at approxi-mately 100%. T3 species III, IV, and V must originate from the class III region of the T3 genome between 37.5 and 56%. Although the pattern of transcription by T3 RNA polymerase in vitro closely resembles that of the RNA polymerase from

therelatedphageT7,neitherpolymerase is able to use major promoter sites for theother enzyme at an appreciable rate.

Transcription of the T3 phage chromosome in lymerase recognizes only a single major pro-the bacterial cell proceeds in two stages, cor- motersite on T3 DNA. With T7 DNA, T7 po-responding to the "early" and "late" stages of lymeraseinitiatestranscriptionat sevenmajor infection (5, 13).Theearly region of the genome promoters, distributedthroughout the class III (leftmost 20% on the conventional genetic map) regionof the T7 genome (6). Thus both T3and istranscribed by the Escherichia coli RNA po- T7RNA polymerases are highly selective, yet lymerase (EC 2.7.7.6), whereas the remainder each appears to possess its own unique specific-ofthe chromosome is read by aphage-specific ity. This is remarkable since the two phages

RNApolymerase, theproductof gene 1 (5), as areclosely related andprobablyderived froma

has been shown for the related phage T7 (3). common ancestor (2, 4).

TheT3 RNApolymerase, asingle polypeptide We have continued our comparison of the of molecularweight 100,000, has been isolated transcriptional specificities of T7 and T3 RNA and found to require T3 DNA as a template for polymerases inthehope that this will serveas

RNAsynthesis. ThepurifiedT3 RNApolymer- aguidetoessential biochemicalandbiological

asetranscribed therstrandofT3 DNA exclu- features ofphage transcription maps. In

pre-sively, suggestingthatitishighlyselective in viousstudieswehave located four of themajor

vitro (5). promoter sites readby T7 RNApolymerase on

Although T3 RNA polymerase closely resem- theT7genomeand positioned a common termi-bles its T7 counterpartinstructureand in enzy- nationsignalforthesepromotersneartheright

maticproperties, the two phageenzymesdiffer end of the DNA. Weshowed, inaddition, that markedly in their response to different DNA the remaining major T7 promoters read by T7 templates. T7 RNA polymerasetranscribes T3 RNApolymerasewerealllocatedintheregion

DNA atapproximately 50% ofthe rate at which of the genome coding for so-called class III it transcribes T7 DNA, whereas T3 RNA po- proteins, from 40 to 100% on the T7 physical

lymerase transcribesT7 DNAatonly10to20% map (6). We report here the characterization of theratefound with T3 DNAastemplate(5). andpreliminarymapping of the majorinvitro

Thetranscription product ofT7RNApolymer- transcription products of T3 RNA polymerase ase with T3DNAtemplate has been shownto with T3 DNA.

beasingleRNAspeciescorresponding inmap

location to T7 species IIb; hence T7 RNA po- MATERIALS AND METHODS

1Presentaddress: Department ofBiochemistry,Wash- Procedures employed for the assay of T3 RNA ingtonUniversitySchool ofMedicine, St.Louis,MO 63110. polymerase and thein vitrosynthesis of T3 RNAare

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on November 10, 2019 by guest

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744 GOLOMB AND CHAMBERLIN J. VIROL.

those described previouslyfor T7 RNApolymerase the same technique to theiranalysis. T3 RNA (7), withthe substitution of T3DNAastemplateat a polymerase was allowed to transcribe T3 DNA

final concentrationof 0.3mM. (AllDNAconcentra- template in a standard reaction mixture

con-tions are here given in terms of concentration of

taining

a-[32P]CTP.

At 10

min

after the addi-nucleotide.) T3 RNA polymerasewasisolated froma

late-exponential-phasecultureofDG156, aRNase

tion

ofenzyme, the reaction was stopped by the

1- strain of E. coli B/r (H. Dougan, Ph.D. thesis addition of anequal volume of a solution con-Universityof California, Berkeley, 1973) grownina

taining

50 mMEDTA and 0.5% sodium dodecyl New Brunswick fermentor and harvested 15 min sulfate, and the mixturewasapplieddirectly to afterinfection by T3. Thephage RNApolymerase a1.75%acrylamide-0.5% agarose slab gel (Fig.

waspurified by a modificationof the procedure of 1).

Maitra and Huang (9) and had a final specificactiv- Six major and three minor RNA bands are

ityof 91,000 U/mg. One unit ofT3 RNApolymerase discernible on autoradiograms of such gels,

leadsto a rate of incorporation ofCMPequalto 1 corresponding to at least six discrete size

nmol per h under standard assay conditions. The

purification of T7 RNA polymerase was as previ- classes of RNA. The apparent molecular ouslydescribed (3) its specific activity was 76,000 U! weights of these RNA species were determined

mg. ' , fromtheir

electrophoretic

mobilities relativeto

Two preparations ofE. coli exonuclease III were those ofthe in vitrotranscripts of T7 DNA with used; these were the generous gifts ofI.R. Lehman T7 RNA polymerase. Apparent molecular (Stanford University) and C. C. Richardson (Har- weights of the T7transcripts, relative to a

se-vard University). Aspergillus oryzae S1 nuclease ries of "standard" RNAs, have already been was thegift of P. Berg (Stanford University). Analy- established (7). Table 1 shows the size distribu-sis of RNAsby electrophoresis on acrylamide-aga- tion of the six major T3 in vitro

RNAs,

desig-rosegels (7) andconditions forS1digestionoftem- t . .

plate DNAs (6, 15) have been previously described. nated T3

transcrlpts

I to VIinorder of decreas-Exonuclease III digestions (8) were performed at ing molecular weight. Three quite long (and 37°C, in a reaction mixture containing50 mM Tris- presumably polycistronic) and three compara-hydrochloride buffer (pH 8.0), 0.5mM MgCl2, 10mM tively short RNAs span a molecular weight 2-mercaptoethanol, 1.5 mM T3 DNA, and 20 to 200U rangefrom 5.5 x 106 to 2.2 x 105.

ofexonuclease IIIper ml. The extent ofdigestion This size distribution is highly reminiscent of wasmonitored by following the release of acid-solu- that of the in vitro transcripts of T7 DNA with

ble nucleotides, as measured by UVabsorption at T7 RNApolymerase,shown inTable1for

com-260 nm (6).

arison.

Each

major

T3

transcri

t

can

be

corre-The nomenclature employed previously to de- latith

ma

transcripthang e

rorre-scribe the transcription units read by T7 RNA po- lated with a T7 transcript having a roughly lymerase has been modified for use with the T3 and similar molecular weight, although there are heterologous phage systems. The major RNAs tran- significant differences in detail (the apparent

scribed by T7 or T3 RNA polymerases from their molecular weight of T3 transcript V, for in-homologoustemplates are designated by Romannu- stance, is significantly greater than that of T7 merals inorder of decreasing size (7). The template transcript V).

fromwhich the transcript is read issignified by the Assuming that the apparent molecular designation "T3 transcript" or "T3 species" (e.g., "T3 weights for T3 RNAs correspond

to,

or are

pro-transcriptIII"). Transcripts read by T7polymerase . t .

from T3 DNAor by T3polymerase from T7DNA are

portional

tolarueRrNA

chain

lengths, one can designated by the Roman numeral correspondingto calculate the molar ratios of the six T3 RNAs that ofthehomologous T3 or T7 transcript, andthe made in a standard 10-mm reaction from the natureof thepolymerase is specified, i.e., "T3 tran- relative mass of RNA in each size class (7)

script IIIb read by T7polymerase." The nomencla- (Table 1). Here significant differences emerge ture can be extended to additional (minor) tran- between T3 and T7transcriptionalpatterns.T7 scripts bydesignating these with Roman numerals RNA polymerase synthesizes equimolar beginning with VII. amounts of each of the six T7 RNAs, with the

exception of T7 species III, which has been RESULTS shown to be a mixture oftwo

transcripts,

T7

transcript HIa and T7 transcript IIIb, which

RNAs madeinvitro by T3 RNA polymerase arise from physically distinct transcription

with T3 DNA template. Previous work has units(6). T3 RNA polymerase, in contrast,

syn-shown that the sevenmajor RNAs transcribed thesizes only one molarequivalent of T3 species

byT7RNApolymerasewith T7 DNA template III, the RNA species corresponding in molecu-can readily be resolved by electrophoresis on larweight to T7 species III, which implies that

dilute polyacrylamide-agarosegels (7, 11). An- at least one T7transcription unit (IIIaorIIIb) ticipating that thetranscriptionproductsof T3 has no counterpart read by T3 polymerase on DNA with T3 RNA polymerase would be a the T3 genome. A furtherdifference is that T3

similarly complexmixture of RNAs, we applied RNApolymerase transcribes two molar

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k,-~ 4 TABLE 1. Molecularweightsand molar ratiosof

a

b'

UmajorT3 RNA and T7 RNApolymerasetranscripts

formedin vitroa

Phage Species Mol wt (x10-6) Molar ratio

T3 I 5.5 ± 0.5 1.0

II 4.2 ± 0.3 1.0

III 1.7 ± 0.2 0.9

4 IV 0.87 ± 0.02 0.8

V 0.52 0.02 1.9

VI 0.23 ± 0.01 0.8

T7 I 5.5 ± 0.5 1.1

II 4.5 ± 0.5 1.0

III 2.0 ± 0.2 1.7

IV 0.84 ± 0.04 1.2

V 0.44 ± 0.04 0.9

VI 0.22 ± 0.02 1.1

aApparent molecular weights of T3 RNAs were

s r & j

^obtained

from electrophoretic mobilities on 1.75%

_

[l wpolyacrylamide-0.5%agarosegelsrelativetothe six

T7 RNAs formed in vitro with T7 DNA template (7). Molarratios relative to T3 species II were calculated from the relative amounts of a [32P]CTP incorpo-rated into each species during a 10-min synthetic reaction and their respective molecular weights. Molecular weights and molar ratios of T7 in vitro

mU

*RNAs, shown for comparison, are taken from

Go-lomb and Chamberlin (7).

lents of T3 species V. Hypothetical

arrange-mentsgiving risetosuchadoublet are: (i) two

geographically distincttranscriptionunits

hav-ing

thesame

length (analogous

toT7

transcrip-tion units IIIa and

IlIb),

(ii) a

pair

of

closely

overlapping

transcription

units with

adjacent

promoters andashared terminator, and (iii) a single transcription unit with apromoterthat is

employed

twice as

efficiently

as the other

majorT3 promoters.

Besides the six major RNA species, T3 RNA

polymerase transcribes three minorRNA spe-cies, ofapparent molecular weights 10 x 106, 8.0 x 106, and 6.5 x 106,whicharetentatively

designated T3 species VII, VIII, and IX,

respec-tively. Like their T7 counterparts (M. Golomb,

Ph.D. thesis, University of California,

Berke-ley, 1974; 6), these minor species are tentatively

interpreted as "read-through" derivatives of

majortranscripts (seebelow).

FIG. 1. RNAsformedinvitrobyT3 and T7 RNA polymerases with T3 and T7DNA templates. The RNA made ina10-min syntheticreaction byT3 or

T7 RNA polymerasewasresolvedbyelectrophoresis

on a1.75%polyacrylamide-0.5% agarose gel.

Auto-radiographs show: transcriptionof T3DNAwith(a) T7RNApolymerase and (b) T3 RNA polymerase; transcriptionof T7 DNA with (c) T7 RNA polymer-aseand(d)T3 RNApolymerase. Tracks(b), (c), and (d)arefrom thesamegel; track (a), shown for

com-parison, isfromaseparategel.

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RNAs transcribed in vitro by T3 RNA po- overlapping transcription units that terminate lymerase with T7 DNA template. Under our near the ends of the genome, as with T7. We standard reaction conditions, T7 RNA polymer- therefore carried out a series of limited (2%

asetranscribes T3 DNAastemplateatabout 50 genomelength) and one of more extensive (up

to 70% of the rate at which it transcribes T7 to40%)terminal excisions on T3 DNA tolocate DNAastemplate; theproductof this transcrip- transcription units terminating neartheright

tion is a single RNA species arising from a end of the chromosome.Transcriptionfromthe

transcription unitcongruentwith T7 transcript heterologous (T7RNApolymerase) promoter at IlIb (84 to 100% onthe T7 chromosome) (6, 7). 84% genome length on T3 DNA served as an

T3RNApolymerase transcribes T7 DNA with internal control for thisprocedure.

considerably lower efficiency (5), orabout 10to Limited excision of T3 DNAtemplate and

20% of the rate with T3 DNA under standard mapping of species IV. E. coli exonuclease III conditions. It was therefore of interest to find wasemployedto remove shortsegments (from

outwhich, ifany, transcription signals onthe 0.28% to 1.4%genomelength)from the 3'-OH-T7chromosome arerecognizedby T3 RNApo- terminatedends of T3 DNA, and the resulting lymerase. Figure 1 shows the products of this 5'-P-terminated single-stranded "tails" were

heterologous reaction, resolved on a 1.75% cleaved withsingle-strand-specific Si nuclease acrylamide-0.5% agarose gel. Surprisingly, from Aspergillusorwyzae.Theresulting series of each of the major T7 RNA speciestranscribed truncated DNAswastranscribed with T3 RNA by T7 RNA polymerase appears to be made polymerase, and the RNA products were

re-with T3 polymerase. The relative molar solved by gel electrophoresis. When less than

amountsof eacharesimilartothose found with 1.5%ofthe T3genomeisexcised, T3 species VI T7 RNA polymerase (7). Several conclusions is the only transcript synthesized by T3 RNA

maybe drawnfromthisexperiment. (i) T3 RNA polymerase to besignificantly altered. The

ex-polymerase uses all of the T7 promoters, but tentofremoval of T3 templatestrandDNAand

very poorly. (ii) T3 RNA polymerase uses T7 the decreaseinmolecularweight of T3 species terminators efficiently. This may be inferred VIareplottedas afunctionof incubation time from theappearanceof RNA bandsattheposi- with exonuclease III in Fig. 2. As increasing

tionsof T7 species IV and V; if theinternal T7 amountsof DNAareremoved from the T3

tem-terminatorwerenotrecognized,or werepoorly plate, T3 species VI isprogressively shortened. recognized, these products wouldnotbefound. From thisobservation, andthefact thatall T3 (iii) Asshownpreviously, T7RNApolymerase RNAs are transcribed rightward (5), one can

recognizes a strong promoter at map position conclude that theterminator for T3 speciesVI

84% on the T3 genome (6, 7). However, no mustoccur at orvery neartheright end of the

equivalentstrongpromoterfor T3 RNApolym- T3 genome. Furthermore, since the extent of

erase existsatthis location on the T7 genome RNA shortening equals theextent of

shorten-(seebelow). ingofDNAtemplate strandtowithinan

esti-It should be noted that the poorly utilized mated error of 0.2% genome length, one can promoters readby T3 RNA polymerase on T7 locate the transcription unit for T3 species VI DNAarethe first clear demonstration ofaclass moreprecisely thanhas beenpreviously estab-of "weakpromoters"readbythephage polym- lished for T7 species VI (6). The termination

erases. The existence of such weakpromoters signalfor T3 speciesVI mustlie within0.2%, or

was previously suggested to account for the 80 nucleotides, of the right end of the T3

ge-failure to find major transcripts that contain nome. Fromthe molecular weight of T3species

classIIgeneticsequences amongtheseven ma- VIand its estimated termination at 100% one

jor transcripts formedby T7 RNA polymerase can position its promoter at 98.2%. Thus the

with T7DNA, eventhoughclassII message is promotergoverning T3 transcript VI issituated transcribed invitro (Golomb, Ph.D. thesis; 10, to the right of gene 19 and must overlap the

12). region ofterminal redundancy (4).

Mapping ofT3 invitro transcriptionunits. Transcription of extensively shortened T3

The obvious similarities between thetranscrip- RNAs.AreductioninRNAmolecular weight of

tionalpatternsforT3and T7 RNApolymerase less than 105, although sufficient to map T3

with theirhomologoustemplate encouragedus speciesVI, isdifficulttodetect in the two larg-toattempt to maptheT3 in vitrotranscription estRNAspecies (molecular weights, 5.5 x 106 unitsby the technique usedtomapfour of the and 4.5 x 106,respectively) withpresent meth-sevenmajorT7latetranscriptionunits(6).This ods of resolution. Eventhe failure to detect a

method, the analysis of RNAs made in vitro shortening ofT3species III with short template

from enzymatically shortened templates, is excision does not rule out a terminal location

particularly well adapted to the mapping of for this transcription unit (for instance, one

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° 0.20C immediately to the left of thepromoter for T3

o species VI). Hence, more extensively deleted

x o

templates

mustbe used to locate these RNAs

E 0.15

unambiguously.

With extensive

digestion

of

template,

a

limiting

factorinthe

interpretation

of mapping data is the synchrony of attack by

,0.l0 _ / _ exonuclease

Ill.

A

slight

broadening

inthe

dis-tribution of extent of DNA removed can lead to

E / adramatic loss in resolution of the

correspond-.= 0.05 _ / _

ing

RNA

transcript

on

gels

(6).To obviatethis

problem, very high molecular ratios of

exonu-c I I I I clease III to DNA mustbe used (unpublished

oY₁₀2 ₂₀t ₃₀ ₄₀ ₅₀ ₆₀l

~~~~~observations).

_osrain)

cl0056Using conditions that favor synchronous

at-Time in minutes of Exo m treatment tackonDNA molecules (6),regions of T3DNA varying in extent from 3 to 38% of the 3'-OH-FIG. 2. Decrease in molecular weight of template

terminated

strand of T3 DNA weresuccessively strand T3 DNA and T3 species VI RNA as a function removed with exonuclease III, and the

result-of exonucleolytic digestion result-oftemplate. Exonuclease ing

YPtexonu

clea

ved

th sl

IIIdigestion (8) wasperformedat37°Cin a reaction

ng

5'-P-terminated tails were cleaved with Si

mixture (4.0 ml total volume) containing 50 mM nuclease. These extensively truncated DNAs

Tris-hydrochloride buffer(pH 8.0), 0.5 mM MgCl2, servedastemplates for the in vitrosynthesis of

10 mM 2-mercaptoethanol, 1.5 mM T3 DNA, and 20 RNAsby T3 RNApolymerase.

Mg

of exonuclease III per ml. After the incubation To estimatethe synchronyof excision, these

periods indicated, 0.9-ml portions were combined DNAs were first transcribed with T7 RNA

po-with 0.1 mlof concentratedSi buffer (300 mM so- lymerase, which produces a single, well-defined dium acetate buffer fpH 4.61-5 mM ZnCl2-2 M RNA

species,

T3transcript

lHb,

previously as-NaCl), 1.6 pg ofSi enzyme was added, and the

signed

to a

transcription

unit read from 83 to mixture wasincubatedat50°C for40 min. Release of 97.5%on

the T3

genome.The molecular

weight

acid-soluble nucleotides was measured by adding

of

this

RNA is 2.0 x 106, and it

decreases in

0.1-mlsamples oftheexonucleaseIIIorSi digestion

mixture to2ml of3.5%perchloric acid at0°C.After proportionto theterminal deletion of the

tem-10min, the mixtures werecentrifuged, and the ab- plate. With extensive shortening oftemplate, sorbanceof the supernatantwas read at 260 nm in a the heterogeneity in length of this product in-Zeissspectrophotometer. After digestion with Si nu- creases, providingasensitive measure of lossof clease,thesampleswere twice extracted with phenol synchrony during thecourse of exonucleaseIII andthendialyzed

against

asolution

containing

0.05

digestion

(6).

Figure

3showsa

polyacrylamide-MNaCl,0.01 MTris,and 10-4 M EDTA. Untreated agrsgeofteRAmdbyTRN po

and

exonucleolytically

shortened T3 DNAswereused agarose gel ofthe RNA made by T7 RNA po-astemplatesina 10-minsynthetic transcriptionreac- lymerase With this series ofterminally deleted

tion(7) with a-[32P]CTPaslabeled substrate. Each T3templates. As previouslyreported, the

sin-mixture(0.1 ml total volume) contained 70 U of T3 gle transcriptincreases in electrophoretic

mo-RNApolymerase per ml and 0.3 mMDNA template. bilityand hence decreasesin apparent molecu-Thereaction wasterminated by the addition of 0.2% lar weight with increasing removal of template sodiumdodecyl sulfateand 20 mMEDTA, and por- strand DNA. T3 transcript

IIb

read by T7 po-tions were subjected to electrophoresis in a 1.75%

lymerase

is no longer detectable on a 1.75% polyacrylamide-0.5% agarose gel (7). After autora- polyacrylamide-0.5% agarose gel after 60

min

diography,theapparent molecular weight of T3 spe- ofincubation of the T3 templatewith

exonucle-cies VI transcribed from each template was

deter-minedfromits mobility relative to a set of standard ase III. At this

podit,

2.04 x 106 daltons of RNAs(RNAs madein vitroby T7 RNApolymerase template strand DNA, including the entire

re-withT7 DNA template). Decrease in DNA template gion coding for this RNA, has been degraded strand molecular weight (0) is one-fourth the total (see below). However, during the period of

amountofDNAmadeacidsoluble by digestion with digestion during which T3 transcriptiIlbis still

exonucleaseIII andSi nuclease and isexpressed in discernible on gels (30 min, or 1.08 x 106

dal-molecular weight values. The relative error of this tons of template strand DNA removed), the

determination isestimated at ±+10%, corresponding width of the band corresponding to this RNA

to 016%genome length. Decrease in T3 species VI increases by at most

10%,

indicating a maxi-RNA molecularweight (A)wascalculatedfrom the i.

apparent decrease in molecular weight of this RNA mum

ncrease

in heterogeneity of template species, determined from the increase in its mobility strand length of less than 0.2%. This is better duringelectrophoresis. The relative error of this de- synchrony than previously achieved with this termination is estimated at +3%, corresponding to technique (6) and justifies the use of this

0.05%genome length. method to map thelonger RNAs.

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i

- (In{

j

Fig. 3,

!

tracksatof[see alsoFig. 5],a

sig-nificant proportion ofradioactivelylabeled ma-terial remainsatthe top of thegel. This

mater-ial, which has not been further characterized, has been previously observed inthe transcrip-tion products of DNA templates treated with exonuclease III [6]. As it isespeciallyprominent

0 vi i whenSitreatmentisomitted from the

prepara-tionoftemplate [unpublished observations], it

maybe RNA-DNA

hybrid

or

nonspecific

aggre-gates formed with incompletely degraded 5'-OH-terminated

strands.)

The shortening of T3 transcript IIIbreadby T7 polymerase is plotted as a function of the

extentof exonuclease treatmentin

Fig.

4. It is

apparent

that RNA

shortening

for this

species

equals

DNA

shortening during

the first 30min

of incubation. We had previously mapped the

terminator for this RNA at 98.5% genome length. However, taking into accountthe

esti-matederror inmeasuringthe amountof DNA

removed, these results suggest that the

termi-.j_

natorfor this

species

lies within 0.5% genome length, or about 200 nucleotides, from the right-handedend of the T3 genome. Since this termi-nator must be identical to that governing T3

transcript VI,

the true

position

is within 80

nucleotides ofthe rightend of the genome.The

promoterfor T3 speciesIEDbread by T7 polym-erase can nowbe positioned more accuratelyat

approximately 84.1% genome length, or

slightly to the right of our previous estimate

(83%).

FIG. 3. Electrophoretic analysis of RNA made in vitroby T7RNA polymerase with exonucleolytically

shortenedT3 DNA template. Exonuclease III diges-tionwasperformed and assayed as described in the

legendtoFig.2. The reaction mixture (6.0 ml) con-tained1.53mMT3 DNA and 200 U of exonuclease III per ml.After the incubation periods indicated, 0.9-mlportionswerecombined with0.1ml ofconcen-trated Si buffer,1.6 pgofSi nuclease wasadded, andthemixturewas incubated at 50°C for 40 min. Thesamples wereextractedthree times withphenol and dialyzed. Untreated and truncated T3 DNAs

wereusedastemplatein a10-min synthetic reaction (0.1 ml) with a-[32P]CTP (final concentration, 0.4

mM)aslabeled substrate. Samples were mixed with 0.25% sodium dodecyl sulfate and 25 mM EDTA andsubjectedtoelectrophoresis in a 1.75% polyacryl-amide-0.5% agarose gel. Tracks (a-g): T7 RNA

po-lymerase transcript with T3 DNA template treated with exonuclease IIIfor (a) 150min,(b) 120min,(c)

60min,(d)30min,(e) 20min, (f)10min, and (g) 0

min.Track(h)isthe T7RNApolymerase transcript

withintact T7DNAtemplate.DNAtemplate

concen-trations were:(a)0.4mM,(b)0.4mM, (c)0.5mM,

(d)0.4mM, (e)0.4mM,()0.3mM, (g)0.3mM, and (h)0.3mM.

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2.0 a

b

c

d

e

f

g

h

0

E ° 2.0 _

C)

2D0 40 60

Time in minutes of Exo m treatment

FIG. 4. Shortening of template strand T3 DNA andofT3species IIIbreadbyT7RNApolymeraseas afunction ofduration ofexonuclease IIItreatment.

RNAshortening (A) was calculated from the change

in

mobility

ofT7

species

IIIb in the

gel

shown in

Fig.

nih

3. The relative error in this determination is esti-matedat ±4%(6). DNAtemplatestrand excision (0)

was calculated as one-halfthe total DNA rendered acid soluble during exonuclease III treatment. The relative error in DNA excision is approximately

Figure

5shows the results of transcribingthe sameseriesoftemplateswithT3 RNA

polymer-ase. Threemajor T3 RNAsare affectedbythe

terminaldeletion, whereas threeremain

unal-tered. As expected, species VI (molecular

weight,2.3 x 105) isnotdetectedas a

transcrip-tionproductof any of these deletedtemplates,

sincetheminimumexcisionoftemplatestrand DNA in this experiment is 4.2 x 105 daltons.

The mobilities of T3 species III, IV, and Vare

unaffected byexonucleolyticcleavage of37.5%

ofthegenome.Thus, theirterminatorsmustbe

located tothe left of62% on the genetic map.

The molar abundance of T3 speciesIII, IV, and Visunchanged by the excision of upto 31%of

each strand, indicating that their promoters

must lie to the right of31% genome length.

Upon removal of37.5% ofeach strand, these

three species can still be detected, implying thattheirpromotersoccur totherightof37.5%;

however, the abundance of theseRNAs,aswell

asthe total amount ofRNA

synthesized

from

thishighly truncated DNA, is

considerably

re-duced. Thesimplestexplanationfor this

obser-FIG. 5. Electrophoretic analysis ofRNAsmadein

vitrobyT3 RNApolymerasewithexonucleolytically

shortened T3template. The RNA madein a10-min

syntheticreactionwith70UofT3RNApolymerase

per ml, and the series oftruncated T3 templates

described inthelegendtoFig.3 wasanalyzedona 1.75% polyacrylamide-0.5% agarose gel. T3 DNA

templatewastreated with exonuclease IIIfor:(a)150 polymerasetranscript with T7 DNA template. DNA

min,(b)120min,(c)60min,(d)30min,(e)20min, templateconcentrations are as described in the

leg-(f)10min, and (g)0 min. Track(h) istheT7 RNA endtoFig. 3.

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vation isthatthe promoters for T3 species III, genetic map. Since all the RNA formedbyT3

IV, andV are in theneighborhoodof38%, and RNA polymerase with T3 DNA template is

that their recognition is impaired by proximal complementary to the r strand, all

transcrip-deletion. Alternatively, asynchronous cleavage tion units are read in a rightward direction. during the course of the reaction could have Furthermore,since these speciesareshortened degraded these promoter sites on a fraction of in parallel with truncation of the template

the DNA molecules. strand, they must overlap and sharea common

T3 species I and II are shortened as the tem- terminator near the right end of the T3 ge-plate is truncated(Fig. 5). Table 2 shows this in nome.From the data for T3speciesI, with their

quantitative form. Within the limits of error, high uncertainty, onecannot eliminate the

pos-T3 species I andII are shortened to an extent sibility of a terminator locatedimmediately to

equal to the shortening of the DNA template the left of T3 species VI; however,the data for

strand. Their terminators must therefore lie T3species IIand for the T3transcriptHIIbread near 100% on the genetic nmap, although the by T7 RNApolymerase aremoreaccurateand

uncertainty is greater than for T3 species VI rule outthispossibility. The simplest

arrange-(about 1% genome length for T3 species II and ment consistent with the data isoneof

overlap-2% genome length for I). Nevertheless, it is ping transcription units for T3 speciesI, II, and

evident that T3 species I and II must share a VI, which share asingle termination signalat common terminator with T3 species VI, and or near 100% (see Fig. 4 of the accompanying this must be quite near the right end of the paper [1]). From the apparent molecular

genome. weightsof theseRNAs, theirpromoters can be

In addition to the six major RNA species, positioned at56,67, and 98.2%,respectively. minorRNAs canbe detected inthe transcript The three remaining transcription units for

from native T3DNA. Two ofthese, species VII T3species III, IV, and V must lie in the interior

and VIII, with apparent molecularweights of of theT3genome, to the left of 56% and to the (7.5 + 0.8) x 106 and (6.1 ± 0.5) x 106, respec- right of 38%. Byanalogywith T7 species IIIa,

tively, are evident in Fig. 5. By 120 min of IV, and V (11), it is attractive to suppose that excision (3.9 x 106 daltons of template strand thesespeciesoverlap,with promoters located at

DNA removed), these species have shifted to approximately 43, 49, and 52% and a common

positions correspondingtoapparent molecular terminatorat56%. According tothis hypothe-weights of5.1 x 106 and3.2 x 106,respectively, sis, the two minor T3 RNA species, VII and implying amolecularweightdecrease of2.4 x VIII, whichoriginzate atapproximately 40and 106 forthe larger species and 2.9 x 106for the 50%, respectively, can most easily be inter-smaller. Thus, these minor RNAs must also preted as "read-through" RNAs, arising from terminate at or near the right-handed end of anoccasionalfailure ofT3RNA polymeraseto

the T3 genome. recognizethe termination signalat56%. Their

This information is sufficient to allow ap- molecular weights andmap position are those proximate positioning ofthe major transcrip- expected for RNAs initiating atthepromoters

tion units for T3 species I and II on the T3 for T3speciesIII and IV or Vandterminating

TABLE 2. Reductioninapparentmolecularweight of RNAstranscribed from extensively truncated

T3DNAtemplatea

Durationof Reduction in Reduction in apparent mol wt of RNA(x10-6)

exonuclease template T3species T3species T3species T3

species

T3species T3species treatment strand DNA T3 speciesI T3(4.2 x T3 (1.7

xeiV

T3 87 x V(0.52 x VI

(0.23

IIeT3c

x

(2.s

0

(min) (x10-6) (5.5 x106)b I(4) 107)

1V06)

1065)

10.2)

x 10 )

10 0.42 +0.02 0.1 + 0.4 0.1 ± 0.2 0 0 0 _d 0.45 + 0.02

20 0.77 ±0.04 0.4 + 0.4 0.7 + 0.2 0 0 0 - 0.76 ± 0.03

30 1.08 ± 0.05 0.7 ± 0.4 1.1 ± 0.2 0 0 0 - 1.12 ± 0.04

60 2.0 ± 0.1 1.6 ± 0.4 1.9 ± 0.2 0 0 0 -

-120 3.9 ± 0.2 3.2 ± 0.5 2.7 ± 0.5 0 0 0 -

-150 4.7 ± 0.2 +e + Of Of Of -

-a Reductionintemplatestrand DNA and reduction in apparent molecularweightof RNA are defined as for Fig. 4. Error

in measurement oftemplate strand excision is estimated from the precision of the assay procedure, and error in measurementofreductionin RNAmolecularweight is estimatedfromtheprecisionin mobility of a given RNA species on gels.

bMolecularweightoforiginaltranscript.

cReadbyT7RNA polymerase.

d,Species absentorunidentifiable.

e+,Speciespresent but size notdetermined.

fSpecies reducedinamount.

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at100%. Two minor transcriptional units, cor- inthe specificity ofthephage RNA polymerase,

responding in size and map position to read- points to a strong selective advantage inherent

through extensions ofT7 species IIIa and IV, in such an arrangement. Some advantages of

have previouslybeen identified for T7 (Golomb, such an overlapping transcriptional pattern are

Ph.D. thesis; 6). evident. The entire late region is read much

DISCUSSION more

rapidly

than if

only

a

single transcription

DISCUSSION unit were employed. In addition, there is a

The six major transcriptsformed by T3 RNA three- or fourfold enhancement of the mRNA

polymerasewith T3 DNAhave been character- for gene 10, for

example,

which suggeststhat

ized with respect to size and relative molar the positioning of several

promoters

for one

abundance. Three transcriptional units have genetic region is an effective mechanism of been assigned to definite chromosomal loca- transcriptionalcontrol thatmaywell be used in tions, and the remaining three transcription more complex

organisms

to

amplify

transcrip-units are shown to be situated in the "late" tion ofcertain regions. Finally, the

arrange-region of the genetic map. Noneof the six major mentseemstominimizeterminator

signals

on

primary transcripts stems from the "early" re- the genome, although it is not evident to us

gion, and the data given here make it highly whythis should be of value.

unlikelythat any of them contain information Although the overallpatternof

transcription

for "class II" proteins (from 20 to 42% on the has beenconservedduringthe

divergence

of T3 genetic map [8]). Thistentative positioning is and T7 from their

hypothetical

common

ances-confirmed by studies oftranscriptionof recom- tor, some differences in detail have

appeared.

binant phage DNAsintheaccompanying paper Thus T7 RNApolymerase

recognizes

a

strong

(1). Theoverall patternof transcriptional units promoter on T7 DNA at 84% on the

genetic

for T3phageisquite similar tothat of T7 phage map, but T3 RNApolymerase does not

recog-(6, 10); two clusters of overlappingtranscription nize apromoterat this site onits

homologous

units covertheentire region of the genome that template.That sucha

promoter

onceexistedon

specifies "class III" phage proteins. The pri- T3DNA issuggested bythe abilityof T7RNA

mary transcripts corresponding to "class II" polymerase to

recognize

a

strong

promoter

at genes, for either T3 or T7, remain to be charac- 84% genome length on T3 DNA.

Presumably

terized. thecommon ancestor of T3 and T7

possessed

a

The similarity of thetranscriptionalmaps for promoter sequence at this site andaRNA po-T3 and T7 phages is in accord with the high lymerasecapableofrecognizing it;in thecourse

degree of DNA homology between the two ofevolution the RNA

polymerase

of T3lost the

phages (4) and the colinearity of their genetic ability torecognize thissignal. Itis,

however,

maps (1). However, this resemblance is some- puzzling that T3 would retain a "latent" pro-whatsurprising in light of several striking dif- moter sequence that it is

incapable

of

utilizing.

ferencesbetweenthe two,including: (i) the con- Another difference between the T3 and T7 siderable divergence in gene size between T3 patterns oftranscriptionis the

synthesis

of two and T7 (0. G. Issinger, Ph.D. thesis, Univer- molarequivalents of T3

species

V

by

T3 RNA sity of Freiberg, Freiburg, West Germany, polymerase, ascomparedtooneof T7

species

V 1973), (ii) the low homology of certain genes byT7RNApolymerase. Themapping informa-(e.g., gene10[4]) and, finally, (iii) the failure of tion given here argues

against,

but does not T3 and T7 RNA polymerases to utilize effec- eliminate, thepossibilitythat two

separate

ge-tively promoter sites read by each other. The neticregionsgiverise todistinct RNAs of this latter point isunderscored by the ability of both size. It ismorelikely,however,that T3

species

T7 and T3 RNApolymerases to recognize pro- V stems from a unique chromosomal location moter sites at 98% genome length on their re- andthat thepromoterforthisspeciesisutilized spective DNAs

(Pv1),

although these promoters twice as efficiently as other T3 promoters or

arenotmutuallyrecognizable and the resulting exists in tandemduplication.

transcript spansthe region of terminal redun- ACKNOWLEDGMENTS

dancy, which is virtually norhomologous be- Wewould liketothankGeorgeKassavetesfor his assist-tween the two phages. This specificity of the ance in the experiments shown in Table 2 and Hildburg

phagepolymerasesislikely to bemaintained in Beier forhelpful discussions.

vivo since no recombinant phages have been This researchwassupported by Public HealthService

found thatrequire reading of essentialgenesby grant

GM12010

from the National Institute for General

anonhomologous RNA polymerase (1). Medical

Sciences.

The conservation of the unique transcrip- LITERATURE CITED

tionalpatternofT7andT3, which has survived 1. Beier,H., M. Golomb, and M. Chamberlin. 1976.

Isola-evolutionary changes inpromoter sequence and tion of recombinants between T7 and T3

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752 GOLOMB AND CHAMBERLIN J.

phages and their use in in vitro transcriptional map- Biol. 23:355-363.

ping. J. Virol. 21:753-765. 9. Maitra, U., and H. H.Huang. 1972. Initiation, release, 2. Beier, H., and R. Hausmann. 1973. Genetic map of andreinitiation of RNA chainsby bacteriophage-T3-bacteriophage T3. J. Virol. 12:417-419. inducedpolymerase from T3 DNA Templates. Proc. 3. Chamberlin, M., J. McGrath, and L. Waskell. 1970. Natl.Acad. Sci. U.S.A.69:55-59.

New RNApolymerase from E. coli infected with bac- 10. Niles, E. G., and R. C. Condit. 1975. Translational teriophageT7.Nature (London)228:227-231. mappingofbacteriophage T7 RNAssynthesized in 4. Davis, R. W., and R. W. Hyman. 1971. A study in vitroby purifiedT7 RNApolymerase. J. Mol. Biol.

evolution:the DNA base sequencehomologybetween 98:57-67.

coliphages T7 and T3. J. Mol. Biol. 62:287-301. 11. Niles,E.G., S. W.Conlon,and W. C. Summers.1974. 5. Dunn, J. J., F. A. Bautz, and E. K. F. Bautz. 1971. Purification andphysicalcharacterization of T7 RNA Different template specificities of phage T3 and T7 polymerase fromT7-infected Escherichia coli B+. Bio-RNA polymerases. Nature (London) New Biol. chemistry13:3904-3911.

230:94-96. 12. Pachl, C., and E. T. Young. 1976. Detection of polycis-6. Golomb,M., and M.Chamberlin.1974. Apreliminary tronic and overlapping bacteriophage T7 late tran-mapof themajor transcription unitsread byT7RNA scripts by in vitro translation. Proc. Natl. Acad. Sci. polymeraseontheT7andT3bacteriophage chromo- U.S.A. 73:312-316.

somes.Proc. Natl. Acad.Sci. U.S.A. 71:760-764. 13. Studier, F. W. 1972. Bacteriophage T7. Science 176:367-7. Golomb, M., and M. Chamberlin. 1974. Characteriza- 376.

tion of T7-specific ribonucleic acid polymerase IV. 14. Studier, F. W., and N. R. Movva. 1973. SAMase gene of Resolution of the major in vitro transcripts by gel bacteriophage T3 is responsible for overcoming host electrophoresis.J.Biol. Chem.249:2858-2863. restriction. J. Virol. 19:136-145.

8. MacHattie, L. A., D. A. Ritchie, C. A. Thomas, Jr., and 15. Sutton, W. D. 1972. A crude nuclease preparation suita-C.C. Richardson. 1967.Terminalrepetition in per- ble for use in DNA reassociation experiments. muted T2 bacteriophage DNA molecules. J. Mol. Biochim. Biophys. Acta240:522-531.

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