Novel transcribing activities in N4-infected Escherichia coli

VIROLOGY

60,

Novel

65-72 (1974)

Transcribing

Activities

in N4hfected

LUCIA B. ROTHMAN-DENES

AND

Department of Biophysice

and Theoretical Biology, and Institute of Microbiology,

Escherichia

co/i

GIAN CARLO SCHITO

University of Chicago, Chicago, Illinois University of Parma, Italy

Accepted February

60657,

21, 1974

N4 is a small virus containing double-stranded 1)NA of molecular weight 40 X 106, active on Escherichia coli K12 strains. Analysis of N4 transcription in infected cells by pulse labeling and hybridization has revealed the presence of two new RNA polymerizing activities. The first N4 activity is unprecedented. It appears in cells treated prior to infection with both rifampicin and chloramphenicol and, therefore, requires neither transcription nor translation of the phage chromosome. This transcribing activity may be due to a previously undescribed host enzyme. The second activity, maximal at 6 min after infection at 37’, is also rifampicin resistant, but its appearance requires the expression and function of at least two N4 genes. 1NTROI)UCTION

Several host functions are known to be needed by bacterial viruses during their development. Prominent among these is the continuous requirement for at least a portion of the bacterial DNA-dependent RNApolymcrase (Geiduschck and Sklar, 1969; Haselkorn et aZ., 1969). Growth of a number of phagcs is inhibited by rifampicin (Riva and Silvestri, 1972), an antibiotic that blocks initiation of RNA synthesis by interaction with the beta subunit of the host enzyme (Zillig et al., 1970). Growth of bacteriophages T3 and T7 becomes resistant to the drug early after infection (Summers and Siegel, 1969). This resistant phase, however, is prcceded by a short period of rifampicin-sensitive RNA synthesis required to transcribe an early region of the phage chromosome which specifies a new, rifampicin-resistant, RNA polymerase (Chamberlin et al., 1970; Maitra, 1971; Dunn et al., 1971). In rifampicin-pretreated cells, therefore, all known DNA coliphage chromosomes remain genetically silent. We have found that a more complex model of virus-ccl1 interaction may be provided by the development of bacteriophage N4 in Escherichia coli. Growth of this DNA-containing virus does not substantially affect 65 Copyright 0 1974 by Academic Preae, Inc. All rights of reproduction in any form reserved.

cellular RNA and protein synthcscs early in infection (Rothman-Denes et aE., 1972). In fact, although host DNA and cataboliterepressible enzyme syntheses are shut off within a few minutes after infection, stable RNA and other bacterial proteins continue to bc produced (Rothman-Denes et al., 1972). WC have found not only that N4 induces a new rifampicin-resistant RNApolymerizing activity, but that transcription leading to this early expression of the phage chromosome is itself resistant to rifampicin. MATERIALS

ANI) METHODS

Bacterial and phqe strains. HFR 3300 was the nonpermissivc strain used in all cxpcriments. N4 wild type and N4 amber mutants were grown as previously described (Schito, 1973). Aleasurement of RNA synthesis. Bacterial cultures were grown in minimal salt medium (Bollc et al., 1968) supplemented with 0.5 % glucose, 1% casamino acids, at 37°C to 5 X 1Oscolony-forming units/ml. Before infection, cells were centrifuged and resuspended in fresh medium. Phago was added at a multiplicity of infection of 10. At diffrrcnt times after infection 0.2-ml aliquots wrre removed and incubated for 2 min with 3Huridine (Schwarz 25 pCi/ml, 28 Ci/mmolc).

66

ROTHMAN-DENES

At the end of the pulse 0.05-ml samples were treated as previously described (RothmanDenes et al., 1973). Measurement of DNA synthesis. Infected cell samples (0.2 ml) were removed and incubated with [methyl-3H]-thymidinc (Schwarz, 10 &i/ml, 3 Ci/mmole). After 2 min, 0.05-ml aliquots were treated as described for RNA in the preceding paragraph. RNA-DNA hybridization was performed using DNA bound to nitrocellulose filters as described previously (Rothman-Denes et al., 1972), except that labeling was for 3 min with 3H-uridine (Schware, 12.5 &i/ml, 28 Ci/ml). Filters cont’aining 50 pg E. coEi DNA or 2 pg N4 DNA were added to 2 ml of 4 X SSC, 1% SDS containing 1 pg/ml of labeled RNA. Polyacrylamide gel electrophoresis and autoradiography. HFR 3300 was grown as above except that the medium contained 0.05 mM magnesium sulphate. Cells were incubated with 200 pg/ml of rifampicin. After 5 min cells were infected and 2 min later 36S042(ICN, 50 $X/ml) was added. After incubation for 30 min, cells were collected in the presence of 50 mM azide. Samples containing an equal number of cells were prepared and run on 20 % polyacrylamide slab gels, using the procedure and apparatus described by Studier (1973). The gel composition is described by Blattler et al., (1972). Autoradiograms were analyzed with a Joyce-Leobl microdensitometer. RESULTS

Appearance of Rijampicin-Resistant scription after N4 Injection

Tran-

We have previously shown that synthesis of host messenger and ribosomal RNA remains largely unaffected after infection of E. coli cells with bacteriophage N4 (RothmanDenes et al., 1972). Addition of rifampicin 2 min before the labeling of RNA, however, reveals a burst of drug-resistant RNA synthesis which reaches its maximum 4-6 min after phage infection (Fig. 1). The ability of wild-type N4 to induce a rifampicin-resistant RNA synthetic activity is shared by one class of DO amber mutants of N4 which include am12 and am25 (Fig. 2). A second class of DO amber mutants ex-

AND SCHITO

OOW

, min time after infectum

FIG. 1. Rate of 3H-uridine incorporation during N4 infection. Pulses were prepared as described in Materials and Methods in the absence (0-O) or in the presence (O-O) of 200 pg/ml of rifampicin, added 2 min before the labeling period. Values are plotted as percentage of uninfected cells in the absence of rifampicin, at the middle of the 2-min pulse period. 100 on ordinate = 15,500 cpm

time after infection, mm

FIG. 2. Rate of 3H uridine incorporation during infection with N4 amber mutants. am12 (O-O), am25 (O-O), am15 (A-A), am23 (A-A). In all cases rifampicin (200 rg/ml) was added 2 min before the labeling period. Values are plotted as percentage of uninfected cells in the absence of rifampicin at the middle of the 2-min pulse period. 100 on ordinate = 15,500 cpm.

emplified by am15 and am23, failed to induce this drug-resistant RNA synthesis. Therefore, appearance of the rifampicinresistant RNA synthetic activity requires the function of the N4 genes defective in am15 and am23, but is independent of the replication of N4 DNA. Five minutes after wild-type N4 infection, the rifampicin-resistant activity accounts for about 80 % of the total uridine incorporation into RNA in the infected cells (Fig. 1). Moreover, approximately that fraction of the hybridizable labeled RNA is N4 specific

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ACTIVITIES

(Table 1, line 1). This is an unexpected result if we consider that E. coli RNA synthesis is not shut off early after N4 infection (Rothman-Denes et al., 1972). However, analysis of the fractions of hybridizable labeled RNA that arc N4 or E. coli specific (Table 1, line 1) does not allow for a quantitative calculation of the amounts of host and phage RNA made due to differences in hybridization cfhciencies. In addition, the presence of rifampitin could introduce changes in the transcription machinery or specific activities of the RNAs made. Hybridization comprtition cxperimcnts (in progress) will shed light onto this discrepancy. Preliminary cxperimcnts indicate that all early N4 transcription is rifampicin resistant. By tho tenth minute, however, the rifampicin-resistant activity accounts for only 20 % of the uridine incorporated, while some 60 % of the hybridizable RNA labeled from 10 to 13 min after infection is NCspecific (data not shown). These results suggest that a rifampicin-sensitive activity is responsible for part, or possibly all, of late N4 transcription. When N4 DNA replication is blocked by the mutations in am25 or am12, the proportion of uridinc incorporation resistant to rifampicin remains high for an extended period (Fig. 2). Rijampicin-Resistant RNA Synthesis in S4 Injection Appears in the Absence of E. coli RNA-Polymerizing Activity In order to analyze the kinetics of appearance of the rifampicin-resistant activity in greater detail, the drug was added prior to, with, or at different times after addition of the phage. The effect of adding rifampicin 5 min before infection is shown in Fig. 3. It can be seen that synthesis of some RNA species takes place after N4 infection under conditions in which the host RNA polymcrasc is inactive. The lcvrl of incorporation at the peak in Fig. 3 is comparable to the maximum rifampicin-resistant activity in Fig. 1. If it is assumed that enzyme, rather than template, determines the level of incorporation, then we can suggest that most, perhaps all, of the transcription required for the appearance of tht new rifampicin-resistant activity is itself resistant to the drug. The patterns of uridinc incorporation cx-

IN N4 INFECTED TABLE

HOST

67

E. COLI 1

AND EARLY VIRAL RNA SYNTHIGSIS IN N4-INFISCTICD CELLS IN THY: PRESENClC OF I)RUQS

Additions before infectiona CAM, Rif, loo 206 w/ml aghl -+ + + +

RNA labeling time after infection (min)

4-7 4-7 2-5 2-5

RNA specific activity (cpdcrd

% cpm

hybridized N4 DNA

-2Q,ooO 19.1 17,060 42.7 23,799 0.64 1,550 21.5

13.coli DNA 5.5 0.08 6.8 0.6

0 Chloramphenicol was added 5 min and rifampicin 3 min prior to infection. Other conditions as in Materials and Methods.

lime ofler infectIon , min

Fro. 3. Effect of rifampicin added prior to N4 infection on the rate of aH-uridine incorporation. Bifampicin at a concentration of 206 rg/ml was added 5 min prior to infection with N4 wild type. Labeling conditions as in Materials and Methods.

hibited by DO amber mutants of N4 (Fig. 2) are not changed by the addition of rifampicin prior to infection (data not shown). To determine further the characteristics of the RNA synthesized after N4 infection, RNA labeled with 3H-uridine under different conditions was hybridized to E. coli or N4 DNA. The results of this cxpcriment arc given in Table 1. As expected, under normal conditions of infection, a large proportion of the RNA synthesized early in infection is host specific (lint 1). However, when rifampitin is added before infection, the RNA synthesized hybridizes exclusively to phage DNA (line 2). In the prcscncc of chloramphcnicol alone, the synthesis of phage mRNA species is drastically limited (line 3). Addition of

68

ROTHMAN-DENES

AND

SCHITO

RNAs arc translated ill vivo into phagc>specific polypeptidcs. The patterns obtained after SDS polyacrylamidc clcctrophorcsis of 35S-labcled lysatcs of rifampicin-prrtrcatcd N4-infected cells are shown in Fig. 4. As control and reference, patterns of labeled uninfected cells and labeled virions have bmn included. A small numbrr of proteins arc’ lightly labeled in rifampicin-treated uninfected cells. N4+, am12 or am25 infected bacteria, under the same conditions, synthrsizc at least 13 different new polypeptide chains. Only very few phage-specific bands are prcscnt in the autoradiograph of lysatcs of am15 and am23 infcctrd ~11s. It can bc observed that few, perhaps nonck,of the virion structural polypcptides are made in any of The Products of Rijampicin-Resistant Tranthe rifampicin-prctreatcd cells (comparct scription are Translated am12, am25, N4 wild-type with virions). Transcription and translation of N4 gmcs The mRNA species produced in the prescncc of rifampicin arc functional, bccausc the in the prc?cnce of rifampicin also gives rise>to rifampicin as well as chloramphenicol before infection produces a dramatic decreasein the specific activity of the RNA obtained (line 4). In these conditions, the contribution of the host RNA polymerase is eliminated, as seenby the disappearance of RNA hybridizable to E. coli DNA (line 4). It further confirms the fact that some N4 RNA speciesarc synthesized in the presence of both inhibitors. These data clearly indicate that, under conditions in which the host machinery is unable to synthesize host RNA, phagcspecific RNA can be made. They rule out the possibility that cells become impermcablc or resistant to the antibiotic after infection.

Fxo. 4. Protein synthesis in cells infected with N4, in the presence of rifampicin. Acrylamide gel electrophoresis and autoradiography are described in Materials and Methods. Each sample corresponds to 3 X lo* cells. Samples contained the following amount of radioactivity: am12, 98,000 cpm; am25, 178,OCKlcpm; am15, 137,000 cpm; am23 105,000 cpm; N4 wild type, 108,000 cpm; uninfected cells, 64,000 cpm; virions, 250,000 cpm. Electrophoresis was from left to right. Letters a-m designate N4-directed nonvirion proteins; numbers l-10 refer to virion structural proteins (Giraldi et al., 1973).

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functior~al proteins since shut off of host DNA replication, a phagc-coded function, is normal in the presence of the drug (Fig. 5, N4f). Although the onset of phage DNA replication is delayed, phage DNA synthesis does occur, with a 2-fold reduction in the rate of isotope incorporation (Fig. 5, N4+). The fact that on infection with am25, thr arrest of host DNA replication is not followed by a phase of radioactive thymidine incorporation into DNA supports the conclusion that some phage DNA is synthesized in the presence of the drug in N4+ infection (compare Fig. 5, N4f and am25). Addition

IN N4 INFECTED

of chloramphenicol prior to N4+ infection shows that N4 gene expression is required for the shutoff of host DNA replication (Fig. 5, 64+). Other Properties of the Rifampicin-Resistant RNA Synthetic Activities in NQ Infection

To further analyze the characteristics of the rifampicin-resistant synthesizing activities we have studied the action of other inhibitors of transcription after N4 infection. Addition of streptolydigin (an inhibitor of E. coli RNA polymerase at the chain elongation step) before infection, does not prevent the appearance of N4 coded rifampicin-resistant activity (Fig. 6). Therefore, both new transcribing activities are resistant to this inhibitor as well as to rifampicin.

lime after infection , min

Fro. 5. Rate of 3H-thymidine incorporation in N4-infected cells. Chloramphenicol (CAM) at 196 pg/ml or rifampicin (RIF) at UW)pg/ml were added 2 min before infection. Other conditions as in Materials and Methods. 166 on ordinate = 3699 cpm. O-0, Control; A--A, CAM; O-O, RIF.

,’l&5i2xA 0

2

4

6

IO

on N4 rifampiFIG. 7. Effect of AF/ABDPcis tin-resistant “H uridine incorporation. Rifampicin (299 fig/ml) was added 5 min prior to N4 wild-type infection of EDTA-treated cells. When present (0-O) AF/ABDPcis (396 ag/ml) was added 3 min before the labeling period. Culture without AF/ABDPcis (0-O). TABLE

6. Effect of streptolydigin added prior to N4 infection on the rate of aH-uridine incorporation. EDTA-treated HFR 3399 (Leive, 1968) was incubated with 296 pg/ml streptolydigin for 5 min prior to infection with N4 wild type. Incorporation in uninfected cells in the absence of streptolydigin = 14,666 cpm.

8

time ofler infeclh,min.

INHIBITION SYNTHESIS

FIG.

69

E. COLI

2

OF EARLY N4 RNA BY AF/ABDPcis

Additions

before infection

CAM

Rif

199 166 100

169 166 196

bg/rnBa AF/ABDP 20 loo

RNA specific activity (cpmhd 397 12 14

a Chloramphenicol was added 10 min, rifampicin and AF/ABDPcti 5 min prior to infection. EDTA-treated cultures were labeled from 2 to 5 min after infection.

70

ROTHMAN-I)ENES

We have also tested a derivative of rifampicin, AF/ABDP, known to be an inhibitor of rifampicin-resistant RNA polymerases (T7, E. coli rifr) (Chambcrlin and Ring, 1972). As shown in Fig. 7, no rifampitin-resistant N4-coded RNA synthesis is detected if AF/ABDP is added just before the labeling periods. AF/ABDP also inhibits the rifampicin-resistant activity first (Table 2). DISCUSSION

Transcription of the bacteriophage N4 chromosome in infected cells employs at least two new RNA polymerizing activities. The second of these activities, which is maximal at 6 min after infection at 37”, is conventional in the sense t’hat its appearance requires early viral protein synthesis and the proper function of two N4 genes, cistrons 3 and 4 (am23 and am15). The situation is somewhat similar to T3 and T7 infection, in which the product of a single early gene is a rifampicin-resistant RNA polymerase that transcribes the entire late region of the chromosome. N4 infection differs from T3 and T7 infection, however, in that at least two early N4 gene products (cistrons 3 and 4) are rcquired, and the resulting rifampicin-resistant RNA polymerizing activity does not satisfy all subsequent requirements for N4 DNA replication and phage production. Moreover, it is not yet known whether cistrons 3 and 4 code for a new polymerase, modify an existing enzyme, or regulate other undiscovered genes. The first N4 transcribing activity is unprecedented in E’. coli (but see Price and Frabotta, 1972, discussed below). The activity itself is rifampicin-resistant. Since it appears in cells treated prior to infection with both rifampicin and chloramphenicol, this new activity requires neither transcription nor translation of the N4 chromosome. In cells treated with rifampicin, the new activity is responsible for transcription of at least three N4 genes: cistrons 3 and 4, and the N4 gene(s) whose product(s) shut off host DNA synthesis. What is the source of this new transcribing activity? One possibility is that N4 virions carry, and inject together with the viral DNA, either a new RNA polymerase or

ANI)

SCHITO’

factor(s) capable of modifying the host RNA polymerasc. In the latter case, which wc consider extremely unlikely, the factor-induced modification would have to render the host RKA polymerasc resistant to rifampicin only when transcribing N4 DNA, since transcription of host DNA remains rifampicinsensitive after N4 infection. Alternatively, N4 DNA might “capture” the rifampicinresistant RNA polymerase that normally initiates Okazaki DNA fragment synthesis (Sugino et al., 1972), either as a conscquencc of the mode of N4 DNA injection or because N4 DNA cont’ains high affinity sites for that polymerase. Initiation of h’. coli DNA fragment synthcsis is resistant to rifampicin (Sugino et al., 1972). Initiation of the conversion of +X174 single-stranded DNA to double-stranded replicativc form is resistant to both rifampitin and streptolydigin in vitro (Schekman et al., 1972). These properties, ascribed to an RNA polymerizing activity distinct from the known DNA-dependent RNA polymerase, are shared by bot’h early N4 t’ranscribing activities. The program of protein synthesis in x4infected cells, as it is understood to date, contains three classes of proteins (RothmanDenes et al., 1972). Class I (pre-early) includes the products of the genes mutated in am15 and am23. Mutations in these genes prevent the appearance of subsequent classes of N4 proteins. Since these mutations also prevent the appearance of the second rifampicin-resistant t,ranscribing activity (Fig. 2), it is likely that the latter activity is required to transcribe mRNA for N4 class II proteins. The N4 class II proteins (early) include the products of the genes mutated in am12 and am25. These are required for N4 DNA replication, which in turn is a necessary condition for N4 class III (late structural) protein synthesis. Thus the failure to synthesize N4 structural proteins in cells infected with am12, aml5, am23, or am25 can be attributed to either direct or indirect effects of the mutations on N4 DNA replication. The mutations in am12 and am25 permit the synthesis of class II proteins, although N4 DNA replication is blocked. In these cases, rifampicin-resistant RNA synthesis is

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IN N4 INFECTED

E. COLI

71

extended with respect to wild type infection. the National Science Foundation (NSF GB 37557) and the American Cancer Society (ACS BC-139), The presence of this rifampicin-resistant RNA synthesis can be explained by postulat- and in part by funds from the Ministry of Health ing control at two levels: activity of the of Italy (Cap. 1722) granted to the Institute of of the University of Parma. Rifamtranscription machinery or template specific- Microbiology picin and AF/ABDPcis were generously provided ity. In the first, we can assume that the by Dr. L. Silvestri (Lepetit), and streptolydigin rifampicin-resistant activity requires the by Dr. G. B. Whitfield, Jr. (Upjohn Co.). continuous synthesis of labile early N4 products whose synthesis, in the absence of REFERENCES DNA replication, is extended. Alternatively, a late (Class III) protein, not made in the BLATTLISR, 1). P., GARNER, F., VAN SLYKE, K., and BRADLEY, A. (1972). Quantitative electrophoabsenceof N4 DNA replication, may be norresis in polyacrylamide gels of 240y0. J. Chromally required to shut down the rifampicinmatogr. 64, 147-155. resistant transcription. In the second, we BOLLE, A., EPSTEIN, R., S.~LSER, W., and GEIDUassume that only prereplicative N4 DNA is during T4 SCHEK, E. P. (1968). Transcription the template for the rifampicin-resistant development synthesis and relative stability of activity. In this case, failure of N4 DNA early and late RNA. J. Mol. Biol. 31, 325-348. replication would lead directly to the exCHAMBERLIN, M., and RING, J. (1972). Charactended rifampicin-resistant transcription. terization of T7 specific RNA polymerase III. Inhibition by derivatives of Rifamycin SV. Why are class111proteins not made in cells Biochem. Biophys. Res. Commun. 49, 1129-1136. infected with wild-type N4 in the presenceof rifampicin? Functional class I proteins are CHAMBISRLIN, M., MCGRATH, J., and WASKELL, H. (1970). New RNA polymerase from E. coli inmade, functional class II proteins are made, fected with bacteriophage T7. Nature (London) and N4 DNA is replicated under these condi228, 227-231. tions (Figs. 3-5). The answer must be that DUNN, J. J., B~IJTZ, F. A., and BAUTZ, E. K. F. transcription of the late N4 genes requires (1971). Different template specificities of phage the normal, rifampicin-sensitive host RNA T3 and T7 RNA polymerases. Nature (London) polymerase. Consistent with this requireNew Biol. 230, 94-96. ment is our observation that, in cells carrying GEIDUSCHISK, E. P., and SKLAR, J. (1969). Continual requirement for a host RNA polymerase a mutation to rifampicin-resistance in the component in a bacteriophage development. DNA-dependent RNA polymerase, producNature (London) 221, 833-836. tion of N4 phage is completely resistant to GIRSLDI, M., TONI, M., and SCHITO, G. C. (1973). the drug. Structural proteins of coliphage N4. Virology 55, Infection of Bacillus subtilis by PBSB, a 476482. uracil-containing phage, has been reported to HASELKORN, R., VOGXL, M., and BROWN, R. D. be resistant to rifampicin and streptolydigin (1969). Conservation of the rifamycin sensitivity (Price and Frabotta, 1972). In this case, of transcription during T4 development. Nature however, all viral proteins are made, because (London) 221, 836-838. the phage yield is normal in cells to which the LICIVE, L. (1968). Studies on the permeability change produced in coliform bacteria by ethyldrugs are added prior to infection. Unlike enedeaminetretaacetate. J. Biol. Chem. 243, N4-infected E. coli, the proportion of uridine 2373-2380. incorporation resistant to rifampicin in PBS2-infected B. subtilis increases steadily MAITRA, U. (1971). Induction of a new RNA polymerase in E. coli infected with bacteriophage until lysis; Price and Frabotta suggest that T3. Biochem. Biophys. Res. Commun. 43, 443PBS2 virions carry a drug-resistant RNA 450. polymerase. PRICE, A. R., and FRABOTTA, M. (1972). Resistance ACKNOWLEDGMENTS We thank Dr. Robert Haselkorn for helpful discussion and support during the course of this work. We also wish to thank Dr. M. Toni and Ms. K. VanderLaan for help with some of the experiments. This work was supported by grants from

of bacteriophage PBS2 infection to rifampicin, an inhibitor of B. subtilis RNA synthesis. Biothem. Biophys. Res. Commun. 48, 1578-1585. R~va, S., and SILVESTRI, L. G. (1972). Rifamycins, a general view. Annu. Rev. Biochem. 26,199-224. ROTHMAN-DENES, L. B., HASELKORN, R., and SCHITO, G. C. (1972). Selective shut-off of ca-

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tabolite-sensitive host syntheses phage N4. Virology 56, 95-102. ROTHMAN-DILNPX, It., HASKLKOBN,

L.

B.,

by bacterio-

MUTHUKRISHNAN,

S.

and STLJDIKR, W. (1973). A T7 gene function required for shut-off of host and early T7 transcription. “Virus Research” (C. F. FOX and W. S. Robinson, eds.), pp. 227-239. Academic Press, New York. SCHEKMAN, It., WICKNICH, W., WI~STI~XUUAHD, O., BRIJTLAG, I>., GEIDER, K., BETSCH, L. L., and KORNHERG, A. (1972). Initiation of DNA synthesis of 0X174 replicative form requires RNA synthesis resistant to rifampicin. Proc. Nat. Acad. sci. U.S. 69, 2691-2695. SCHITO, G. C. (1973). The genetics and physiology of coliphage N4. Virology 55, 254265. SUGINO, A., HIROSI, S., and OKASAKI, R. (1972).

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SCHITO

RNA-linked nascent I)NA fragments in Escherischia co&. Proc. Nat. Acad. Sci. U.S. 69, 18631867. STUuIKIt, F. W. (1973). Analysis of bacteriophage T7 early RNAs and proteins on slab gels. J. Mol. Biol. 79, 237-248. SUMMERS, W. C., and SIEGEL, 1~. G. (1969). Control of template specificity of E. coli RNA polymerase by a phage-coded protein. 1Valure (London) 223, 1111-1113. ZILLIG, w., ZKCHEL, K., ItABIJSSAY, I)., SCHACHNICR, M., SICTHI, V. S., PALM, P., HIEIL, A., and SEIFERT, W. (1970). On the role of different subunits of DNA dependent RNA polymerase from h’. coli in the transcription process. Cold Spring Harbor Symp. Quant. Biol. 35, 47-58.