THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 257, No. 8, Issue of April 25, pp. 4087-4096, 1982 Printed in U.S.A.

In Vitro System for Middle T4 RNA I. STUDIES WITH ESCHERICHIA COLI RNA POLYMERASE*

(Received for publication, May 5, 1981, and in revised form, October 16, 1981)

Vittorio de Franciscis$ and Edward Brody From the Institut de Biologie Physico-Chimique, 75005 Paris, France

We describe crude in vitro systems from T4-infected cells which reflect in vivo T4 regulation. Lysates from cells which had been infected in the presence of chlor- amphenicol manifest the same polarity of RNA synthe- sis as did the infected cells. Next, we describe a comple- mentation system between lysates which have no RNA synthetic capacity and purified RNA polymerase; in this system, delayed early RNA synthesis in vitro de- pends on the presence of an active mot gene product. Mot activity controls middle mode gene expression in vivo. In vitro, mot activity in the lysate directs RNA polymerase to initiate on regions of DNA that are oth- erwise inaccessible. This mot-dependent delayed early RNA synthesis in vitro is seen at 0.1 and 0.2 M KC1, but not at 0.05 M KCl. We present a model in which mot is a DNA melting protein necessary for recognition of middle promoters by either E s c h e ~ c ~ i u coli or T4-mod- ified RNA polymerase which contains E. coZi u subunit.

Immediately after phage T4 infects Escherichia coli, the host RNA polymerase recognizes early T4 promoters and starts transcription of long polycistronic early messenger RNA (1). In the absence of protein synthesis, p induces premature termination of these early RNA molecules (2-4). The pro- moter proximal regions of such early transcription units, tran- scribed even when p acts, are called immediate early regions; those which are distal to the p site or sites are called delayed early (5). Another class of promoters was f i s t recognized because they were not used immediately after infection (6,7). Subsequently, it was found that their utilization depends on the presence of a T4 function, mot (8,9). These are the middle promoters. Early and middle transcription units overlap to a great extent, making RNA analyses difficult. Early mode transcription refers to RNA synthesis which starts at IE’

* This work was supported by the following grants: Centre National de la Recherche Scientifique (Groupe de Recherche 18, A.T.P. “Biologie Moleculaire du Gene” et “Microbiologie 1979”), Delegation Generale a la Recherche Scientifique et Technique (Convention 8O.E.0872), Ligue Nationale contre le Cancer (Comite de la Seine), and Commissariat a 1’Energie Atomique. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. * European Molecular Biology Organization Postdoctoral Fellow, 1977-1978. Fellow of the Commission of the European Communities, 1979. NATO Postdoctoral Fellow, 1980. Present address, Laboratorio di Embriologia Molecolare, via Toiano 2, Arc0 Felice, Napoli, Italy 80072.

The abbreviations used are: IE, immediate early; DE, delayed early; r i p , rifampicin resistant; am, amber; ts, temperature sensitive; A650, absorbance at 650 nm; m.o.i., multiplicity of infection; CAM, chloramphenicol; rip, rifampicin sensitive; stl, streptolydigin; SDS, sodium dodecyl sulfate; 2xSSC, 0.30 M NaCl plus 0.03 M sodium citrate, pH 7.

promoters; middle mode transcription refers to RNA synthesis which starts at promoters which are mot-dependent in vivo (see also Fig. 5).

Although the early mode of T4 transcription seems to be reproducible in vitro merely by allowing the interaction of host E. coli RNA polymerase and purified T4 DNA (1, 5, 6, lo), middle mode transcription does not seem to take place in this way (6, 11, 12). In contrast, Travers (7) reported that a ribosomal supernatant of T4-infected cells directed host core RNA polymerase to initiate RNA synthesis at sites in the DE region. Trimble and Maley (13) reported that E. coli holoen- zyme transcribed T2 DNA in a middle mode only at low ionic strength. In order to study the way mot controls middle mode transcription, our laboratory has developed an in vitro system in which RNA synthesis in DE regions depends on mot activity (14).

This system depends on the artificial blockage of transcrip- tion of the early mode, so that chain elongation into DE from IE regions is greatly diminished. In this and the following article, we report on the properties of this in vitro system, on the mot dependence of middle mode RNA synthesis, on mot interaction with E. coli a factor, on the ionic strength depend- ence of middle mode RNA synthesis in vitro, and on the partial purification of the template that is competent for middle mode RNA synthesis. A preliminary account of these results has been rendered (15).

EXPERIMENTAL PROCEDURES

Materials-Streptolydigin was a gift of The Upjohn Co. Rifampicin was purchased from Lepetit s.p.a. (Milano, Italy). The sodium salt of heparin (batch RH 818) was purchased from Gipep (Reuil-Malmaison, France). Nitrocellulose filters, 24-mm diameter, type HA 0.45 pm pore size, were from Millipore (Bedford, England). We used Whatman GF/ C glass microfiber filters 25-mm diameter for collecting material precipitated by CLCCOOH. Ribonucleoside triphosphates were pur- chased from Sigma. [3H]UTP of the highest specific activity available was purchased from Amersham, England. All other chemicals were reagent grade.

Media-M9S (16) is our standard medium for bacteriological ex- periments. One liter of M9S contains: Na2HPO4.2H20, 7 g; KHIPOI (anhydrous), 3 g; NaCl, 0.5 g; 1 m~ MgS04; 0.1 m~ CaC12; glucose, 4 g; casamino acids, 10 g. Petri plates contained tryptone bottom agar at 1.5%; top agar was also tryptone agar at 0.7%.

Bacteria and Bacteriophages-E. coli B“ (su-) was the standard host for T4D (wild type) infection. The rif” strain B”-rzf4 was selected as a spontaneous mutant resistant to 150 p g / d of rifampicin on minimal medium agar plates (17). This rif“ strain grows normally in liquid cultures with 200 pg/ml of rifampicin. CR63 or €340 (SUI+) were used to propagate am mutants of T4.

Bacteriophage am BL 292 (gene 55-) has an am mutation in a gene coding for a peptide which binds strongly to the RNA polymerase of the host and which controls late transcription. T4 am B22 (gene 43-) and am E645 (gene 42-) are am mutants, respectively, in the T4 DNA polymerase and in the dCMP hydroxymethylase. T4 ts G1 (gene mot”) is a temperature-sensitive mutant in the gene which controls middle mode expression (8, 9).

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T4 am G1 is an am mutant in the mot gene (9). It is leaky on E. coli BE but is a lethal mutation on the E. coli mutant Tab G23 (18). T4 ts G1-am B22 and am GI-am E645 are double mutants isolated in our laboratory by standard phage crosses.

T4-infected Cells-Bacteria were grown at 30 "C in M9S in a rotary shaker bath to an & = 0.7 (5 X 10' cells/&), a t which time they were infected at m.0.i. of 5-10 with T4 bacteriophage or an appropri- ate mutant. If infection was to be carried out at 42.5 "C the culture was shifted to this temperature when the &XI was at 0.4. L-tryptophan was added 1 min before infection to 20 pg/ml. Surviving bacteria were measured 4 min after infection and were usually fewer than 0.1%. As we have described previously, CAM lysates were prepared by

adding 200 p g / d CAM to cells 5 min before infection (14). Cells were infected at zero time and isolated for lysate preparation 5 min after infection at 30 "C. "Exhausted" lysates have also been described previously (14). These lysates were made from T4-infected rif" bac- teria to which rifampicin has been added. The antibiotic was added to 200 pg/ml either 5 min after infection at 30 "C or 3.5 min after infection at 42.5 "C. The infection was continued for another 15-20 min, after which the infected, rifampicin-treated cells were chilled, centrifuged 10 min at 4000 X g, then resuspended in %o the original volume of 10 m~ Tris-HC1, pH 7.5, and 100 m~ NaCl (in some cases 10 mM MgClz was added). The infected cells were recentrifuged, then resuspended in '/io0 the original volume of 10 m~ Tris-HC1, pH 7.5,lO mM NaC1, and in some cases 0.1 mM EDTA. When indicated, the detergent Brij 58 at 0.5% (w/v) was added as well. The cells were then rapidly frozen. Frozen cells were kept at -20 "C until they were

was retained even if the cells had been kept a t -20 "C for 2 months. to be used for lysate preparation. Transcription specificity in lysates

For the streptolydigin system (19, 20), we isolated E. coli infected with T4, 3 min after infection st 42.5 "C. Infected cells were centri- fuged at 4000 X g, resuspended in 1/10 the original volume of 10 mM Tris-HC1, pH 7.5, 100 m~ NaC1, and then recentrifuged. Cells were then resuspended in %W the original volume of 10 m~ Tris-HC1, pH 7.5, 10 m~ NaCI, and 0.1 mv EDTA, and rapidly frozen. Frozen cells were stored at -20 "C until they were to be used for lysate formation.

Lysate Formation and in Vitro RNA Synthesis-Frozen infected cells at 5 X 10"/ml were thawed and mixed with 0.8 volumes of lysozyme at 1 mg/ml in 10 m~ Tris-HC1, pH 7.5 (14). In some cases, freezing and thawing was repeated twice before lysozyme addition. Lysates were then incubated 5 min at 37 "C and subsequently kept at 0 "C (for up to 1 h) until they were diluted for in vitro RNA synthesis. The standard incorporation mixture for in vitro RNA synthesis included either 20 mM 3-(N-morpholino)propanesdfonic acid, pH 7.5, or 20 mM Tris-HC1, pH 7.5, 10 m~ MgCl,, 1 mM ATP, 0.2 mM GTP, 0.2 m~ CTP, 0.02 m~ C3H]UTP (at a specific activity of 1000-5000 pCi/pmol), and variable KC1 concentrations. When the KC1 concentration is not specified, it was 100 m~ (14). Early experi- ments included CAM at 160 pg/ml, although we found that this affected neither the RNA synthesis capacity nor its specificity. Rif- ampicin, when present, was at 34 pg/ml; streptolydigin, when present, was at 30 pg/ml. Sometimes 1 m~ unlabeled uridine was included in the reactions; this never diminished in uitro [3H]UTP incorporation.

Between 100 and 150 pl of lysate (corresponding to between 2.7 and 4.1 X lo9 infected cell equivalents) was added/ml of incubation mixture. After 10 min of preincubation at 38 OC, RNA synthesis was started by adding RNA polymerase to the reaction mixture. The RNA polymerase was ordinarily resistant to the antibiotic used in the lysate, and was usually between 10 and 25 pg/ml. The incubation, unless otherwise specified, was for 30 min at 38 "C. The reaction was stopped by adding SDS to 1%. At this time, 5 pl of reaction mixture was precipitated with 5 ml of 10% (w/v) cold CLCCOOH, filtered through a GF/C Whatman glass filter, washed with 15 ml of 5% CI&COOH, and then 2 ml of 70% ethanol. Filters were then dried, and radioactivity measured in a toluene-based scintillation liquid. To the rest of each reaction mixture, we added 2 volumes of 10 m~ Tris- HC1, pH 7.5, 10 m~ MgC12, and 50 m~ NaC1. Then NaOAc, pH 5.2, was added to 100 m ~ . Next, a volume equal to the total aqueous volume of water-saturated phenol was added. The samples were then either frozen at -20 "C or directly extracted with hot phenol.

RNA Extraction-The samples were extracted at least 3X with water-saturated phenol at 68 "C. Details of phenol extraction and ethanol precipitation of this RNA have been published (16). We recovered between 7540% of the [3H]RNA after this purification.

Analysis of RNA Synthesized in Vitro-Phenol-extracted [3H] RNA was hybridized with the separated 1 and r strands of T4 DNA (21). Liquid annealings were done in 2xSSC for 16-18 h at 60 'C with 4 pg/ml of either strand. The concentration of [3H]RNA usually did

not exceed 50 ng/ml; the unlabeled RNA derived from the lysate was the equivalent of about 25 pg/ml (4 X lo7 cell equivalents/ml). If no degradation had occurred, this would be the equivalent of about 1.2 pg/ml of T4 specific RNA (T4 RNA comprises about 5% of the total RNA extracted from T4 infected cells (10)). In fact, the rifampicin "exhaustion" lowers the amount of unlabeled T4 RNA contributed by the lysates to much lower levels than this. Hybridization compe- tition was carried out using purified l strand T4 DNA and unlabeled competitor RNA from cells to which 200 pg/ml CAM had been added 5 min before infection (CAM RNA) or to which no antibiotic had been added (early RNA). In each case, E. coli BE was infected at a m.0.i. = 10 with T4D' and the reaction stopped 5 min after infection at 30 OC (14). The unlabeled competitor RNA was present in increas- ing quantities up to 2 mg/ml. DNA-RNA hybrids were then treated with 20 pg/& of pancreatic RNase A 20 min at 37 "C and diluted and filtered through nitrocellulose filters as previously described (IO).

Background counts/min (already subtracted in the data presented) were determined by incubating ['HIRNA without T4 DNA, either in the presence or absence of competitor RNA. Background counts/min were always low, never exceeding 1% of the input r3H]RNA retained on nitrocellulose filters. The fraction of DE RNA was determined by dividing the counts/min annealed to the 1 strand of T4 DNA in the presence of 2 m g / d unlabeled CAM RNA by the counts/min an- nealed in the absence of competitor. Occasionally, complete compe- tition curves were done; even in these cases, only plateau values (2 mg/ml) for competition are given. Although the data are not shown, we always showed that when transcription was 1 strand specific, those counts/min not competed by unlabeled CAM RNA were competed by unlabeled total early (5') RNA (1,3).

Early transcription is almost completely I strand specific in vivo (22). Therefore, in vitro early and middle r strand RNA synthesis is taken as a measure of incorrect initiation. Symmetric transcription

rvas measured as: z, where r represents the counts/min hybridized

to 4 p g / d r strand of T4 DNA and 1 represents the counts/min hybridized to 4 p g / m l l strand of T4 DNA.

Hybridizations were carried out as liquid annealings. Hybridization efficiencies are measured as the counts/min hybridizing to 4 pg/ml 1 strand T4 DNA x 100/cpm precipitated by CLCCOOH. No correction was made for the counting efficiency difference of 3H in the two types of assay (20). Hybridization efficiencies varied between 20 and 50'36, and were slightly lower than corresponding in vivo RNA preparations. They were, nonetheless, always in a linear range of input for the r3H] in vitro RNA and thus always in a range of DNA excess (16). The counts/min annealed to T4 1 strand in the absence of unlabeled competitor RNA was never less than 800.

RNA Polymerase Preparations-E. coli rifR RNA polymerase was purified from strain BE-rif-4 (see above), by a modification of the method of Burgess (23). After the (NH4)ZS04 fractionation step, the enzyme was passed over a high salt agarose column, eluted with buffer A (10 m~ Tris-HC1, pH 7.9, 10 mM MgC12,O.l mM EDTA, 0.1 mM dithiothreitol, 5% (v/v) glycerol) plus 1 M KCI. The eluted enzyme was then precipitated with 50% saturated (NH,),SO,, layered onto a DEAE-Sephadex column, and then eluted with a linear gradient of 0.25 to 0.7 M KC1 in buffer A. Fractions containing enzymatic activity were pooled and concentrated by (NHJzS04 precipitation. This en- zyme was used for many experiments. It has a specific activity of 270 -01 of UTP incorporated/mg of protein in a 10-min reaction at 0.15 M KC1, using T4 DNA as a template. This enzyme was 50% resistant to 50 pg/ml of rifampicin.

Part of this enzyme preparation was resuspended in buffer C (50 rn Tris-HC1, pH 7.9,O.l mM EDTA, 0.1 m~ dithiothreitol, 5% (v/v) glycerol) plus 50 m~ KC1. The enzyme was applied to a Whatman P11 phosphocellulose column equilibrated with the same buffer and then eluted with a linear gradient from 50 to 500 mM KC1 in buffer C. In these conditions, a subunit is found in the flow through and core enzyme is eluted at 300 mM KC1. This core shows only b, P' , and (I bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This core enzyme transcribes purified T4 DNA about 5% as well as it transcribes calf thymus DNA (24). Core RNA polymerase was also 50% resistant to 50 pg/ml of rifampicin.

RNA polymerase was stored at -20 'C in 10 mM Tris-HCl, pH 7.9, 10 II~M MgC12, 100 m~ KCl, 0.1 m~ EDTA, 0.1 mM dithiothreitol, 50% (v/v) glycerol. Streptolydigin-resistant RNA polymerase was the generous gift of Dietmar Rabussay (Bethesda Research Laboratories, Gaithersburg, MD). It was isolated from a streptolydigin-resistant mutant of a strain that had been selected for its sensitivity to streptolydigin. The original strain was E. coli BE and the stlR enzyme

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Middle T4 RNA Synthesis in Vitro 4089

was isolated from the derivative DPR4-10. This enzyme was purified according to published methods and its properties have been de- scribed by Rabussay and Geiduschek (19,20).

Another set of rifampicin-resistant RNA polymerases was used in this and the accmpanying article. They were the .ifR E. coli and T4- modified enzymes isolated and generously provided by Reinhard Mailhammer and Wolfram Zillig (Max Planck Institut, Martinsried bei, Muenchen). The E. coli r i p K12 strain, AJ7-riP 57, was the source of these enzymes purified by the method of Zillig et al. (25). T4-modified RNA polymerase was prepared 15 min at 37 “C after infection with a T4e- (lysozyme-) mutant. Transcription properties of this “4-modified RNA polymerase in a different system are given in Mailhammer et al. (26). We found that the E. coli enzyme incor- porated 156 nmol of UTP into RNA/mg of protein, when T4 DNA was used as a template and the KC1 concentration was 0.1 M. This is for a reaction time of 10 min at 37 “C. The T4-modified RNA polymerase incorporated 56 nmol of UTP into RNA under the same reaction conditions. Mailhammer had found2 that 10 pg of the E. coli enzyme was not further stimulated by adding 6 pg of purified u subunit to the reaction. The same quantity of T4-modified enzyme was stimulated 2.3-fold by 6 pg of u subunit.

The E. coli enzyme showed f i , /3’, a, and u bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. One faint contaminating band was visible between fi and u. The T4-modified enzyme at the same concentration has a few more contaminating bands, the 15,000- dalton polypeptide which binds to RNA polymerase after T4 infection was also visible. The a, /3, and p’ bands were as intensely stained as they had been in the gel of the E. coli enzyme. The u subunit was clearly visible, but at a lower concentration than in the enzyme from uninfected cells.

RESULTS

We have described a crude in vitro system that reproduces CAM-induced polarity in T4-infected cells (14). We had also found that lysates of infected cells that were capable of synthesizing middle mode T4 RNA in uiuo seem to retain this capacity in vitro. When such lysates were prepared from cells whose endogenous RNA polymerase had been inactivated in vivo, exogenous E. coli RNA polymerase synthesized middle mode RNA from the endogenous T4 DNA in the lysate. Lysates derived from E. coli infected by mot- T4 bacterio- phage did not show middle mode RNA synthesis in vitro. They were only, or p r imgy , capable of giving IE transcripts when complemented with E. coli RNA polymerase.

We have carried out extensive analyses of these lysates which we report here. They relate to three aspects of RNA synthesis in T4-infected cells: 1) chloramphenicol-induced polarity; 2) the synthesis of DE RNA in the middle mode in vitro; 3) the role of the T4 mot function in middle mode synthesis.

In Vitro Polarity-When T4 infects E. coli in the presence of CAM, only IE RNA is synthesized. We have shown this to be due to p-mediated transcription termination (2). This p- induced polarity persists in lysates derived from such cells (14). We have investigated several properties of such CAM lysates to see whether polarity depended on some special property not found in lysates of cells infected by T4 without antibiotic (14).

CAM lysates have a higher viscosity than lysates not show- ing polarity, since CAM treatment of host cells prevents T4- induced degradation of host DNA. We sheared CAM lysates through a syringe with a 21-gauge needle until they had the same viscosity (as measured by flow in a 1-ml pipette) as early lysates. This has no effect on the restriction to IE synthesis in these lysates (Fig. 1).

We also tested whether supercoiling of the DNA in such lysates might affect their capacity to show polarity, since any structure analogous to the host “chromoid” would be unaf- fected by our lysing procedure. Using the absorbance at 260 nm as a measure of nucleic acid in these lysates, we calculate

* R. Mailhammer, personal communication.

1 J 0 0.5 1 u ‘1

ma perm1 CAM RNA

FIG. 1. Properties of CAM lysates. CAM lysates were prepared from CAM pre-treated T4‘ infected E. coli BE in our standard way (see “Experimental Procedures”). A parallel lysate preparation was made from cells not pre-treated with CAM. The CAM lysate was treated in various ways, and then 200 pl of each lysate was used per ml of reaction mixture for in vitro RNA synthesis. After phenol extraction, [3H]RNA was used for hybridization competition analysis with unlabeled CAM RNA A, RNA made from control, early lysate; 0, RNA made from control CAM lysate; ., RNA made from a CAM lysate that had been passed 15 times through a 21 gauge needle; V, RNA made from a CAM lysate that had been incubated 10 min at 37 “C with 30 pg/ml of ethidium bromide; 0, as above, but with 100 pg/ml of ethidium bromide; V, as above but with 200 p g / d ethidium bromide; e, the CAM lysate was supplemented with 50 p g / d of purified E. coli RNA polymerase. This stimulated RNA synthesis about 3-fold in the in vitro reaction. All in vitro syntheses were for 30 min at 37 “C.

that 100 pg/ml of ethidium bromide would relax all supercoils in a DNA “chromoid-like” structure, and that twice this concentration would put positive super-coils into the DNA. Incubation of CAM lysates with these concentrations of ethid- ium bromide (and with a lower amount as well) does not affect the in vitro polarity (Fig. 1). Ethidium bromide does decrease the total amount of RNA synthesized, as would be expected from the experiments of Richardson (27).

The polarity in CAM lysates is seen even when chains are initiated in vitro. When we add 50 pg of purified E. coli RNA polymerase to a CAM lysate (200 pl, which is the equivalent of 10” cells) in 1 ml of reaction mixture, we stimulate 3-fold the amount of RNA synthesized. The polar effect remains constant (Fig. 1).

Complementation of Lysates-We have previously de- scribed a system of complementing lysates which allows the production of DE RNA. One lysate shows polarity in vitro (thus no or little DE RNA) and has a rifampicin-resistant RNA polymerase. The other lysate has no RNA synthetic capacity in vitro, because its RNA polymerase has been inactivated by rifampicin in vivo (14). These exhausted lysates are prepared by adding 200 pg/ml of rifampicin to a T4- infected rif” E. coli culture and allowing the rif“ RNA polym- erase to run off to the ends of transcription units. We have made these lysates at different temperatures depending on the particular experiment. The time of addition of the anti- biotic is chosen as the time when most of the T4 early proteins are being synthesized, but the late period for phage develop- ment has not yet begun. We isolate the cells by rapidly chilling them in ice and concentrating them 100 times; the time of isolation is such that the in vivo RNA synthetic capacity is

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TABLE I Effect of rifampicin addition to T4-infected E. coli B E on lysates of these cells

E. coli BE were infected with T4 am BL 292 (557 at a m.0.i. of 5. cells were isolated. Lysates were prepared from these two types of One ml of infected cells was pipetted (at the times indicated) into 3 infection (see “Experimental Procedures”) and 15 pl of lysate was pCi of [3H]uridine, and 1 min later, the pulse was terminated by used for in vitro RNA synthesis in a total volume of 100 pl. Incubation addition of cold 5% CLCCOOH plus 100 pg/ml unlabeled uridine. was carried out for 30 min at 37 “C, after which time the samples Cl&COOH insoluble counts/min were measured after filtration were brought to 5% CLCCOOH, then filtered through GF/C What- through GF/C Whatman glass filters. Cells that were infected with man glass filters. RNA synthesis in vitro employed either: no addi- T4 am BL 292 were either isolated 5 min after infection (for normal tional antibiotics (although there was residual rifampicin in exhausted lysates) or else were treated with 200 p g / d of rifampicin at this time lysates); incubation in 34 pg/ml of rifampicin; incubation in 34 pg/ml and further incubated until 15 min after infection, after which the of rifampicin plus 200 p g / d of CAM.

I n vivo RNA synthesis In vitro RNA synthesis

Pulse time insoluble C13CCOOH Additions CLCCOOH

insoluble

5’ Infected cells cpm

~~

cpm 4“5’ 3.6 X 104 None 4.4 X lo4

Rifampicin 3.3 X lo4 Chloramphenicol and rifampicin 3.1 X lo4

Infected cells incubated in rifampicin 5-15 min 4’-5’ 2.7 X lo4 None 2.1 x lo3

14’-15‘ 4.3 x lo2 Rifampicin 2.0 X lo3 Chloramphenicol and rifampicin 2.6 X lo3

after infection (exhausted lysate) 9-10‘ 2.7 X 103

TABLE I1 Zn vitro DE RNA synthesis from exhausted lysates depends on the presence of rifampicin-resistant RNA polymerase

The lysates came from E. coli BE (rifampicin-sensitive) or E . coli in a total volume of 1 ml: 20 m~ 3-(N-morpholino)propanesulfoNc BE rif-4 (rifampicin-resistant) infected cells. CAM lysates came from acid at pH 7.5; 10 m~ MgC12; 1 mM ATP; 0.2 mM G T P 0.2 m~ C T P cells which had been treated with 200 pg/ml of CAM 5 min before 0.02 m~ [3H]UTP 0.1 M KC1; 160 pg/ml of CAM; 34 pg/ml of infection at 30 “C as described under “Experimental Procedures.” In rifampicin, 150 pl of each indicated lysate. RNA synthesis was carried line 5, the RNA polymerase purified from E. coli E rif-4 uninfected out a t 38 “C for 30 min. [3H]RNA was extracted with hot phenol and cells was added at a concentration of 25 pg/ml. The exhausted lysates hybridized to the 1 strand of T4 DNA (at 4 pg/ml) in the presence of were prepared from E. coli BE infected with T4 am BL 292 (55-) at increasing amounts of unlabeled CAM RNA for 18 h at 60 “C in 2 X 30 “C; rifampicin (200 pg/ml) was added 5 min after infection as SSC. DE RNA represents the percentage of [3H]RNA hybridizing to described under “Experimental Procedures.” The detergent Brij 58 1 strand T4 DNA in the presence of 2 mg/ml of unlabeled CAM RNA, was included at a concentration of 0.5% (w/v) in the buffer used for compared to hybridization without unlabeled competitor RNA. the last resuspension of the lysates. Each reaction mixture contained,

Lysate from CAM-treated infected cells Lysate from normally infected BE cells

Cells Phage Phage Treatment DE ?6

1) BE am BL 292 (55-) No lysate 7 2) BE 15-4 am BL 292 ( 5 5 ) No lysate 10 3) BE am BL 292 (55-) am BL 292 (557 exhausted 8 4) BE I5-4 am BL 292 (55-) am BL 292 (55-) exhausted 35 5) no lysate; purified BE rif-4 RNA am BL 292 (557 exhausted 30

thesized RNA In vitro syn-

polymerase

less than 5% of what it was at the time of rifampicin addition (Table I).

In Table 11, we show data for some of the fundamental properties of this complementation system. Lines 1 and 2 show that cells which exhibited polarity in vivo continue to synthesize predominantly IE species in vitro (14). The muta- tion to rifampicin resistance in the RNA polymerase has no effect on this in vitro polarity (line 2). In lines 3 and 4 we show that the complementation of lysates is unidirectional. When there is a rifampicin-resistant RNA polymerase in the CAM lysate, complementation and synthesis in vitro of DE RNA takes place; if the RNA polymerase in the CAM lysate is sensitive to rifampicin, it cannot reinitiate chains in the complementation assay, and the only RNA synthesized is that which has been initiated in vivo. This is predominantly IE RNA apparently the exhausted lysate does not contain a diffusible substance capable of relieving p-mediated in vitro polarity. The complementation must take place by the diffu- sion of the rifampicin-resistant RNA polymerase from the CAM lysate to the DNA of the early lysate which is capable of giving middle mode RNA. Line 5 shows that the CAM

lysate can be replaced by purified rifampicin-resistant E. coli RNA polymerase (14).

The use of rifampicin in this lysate complementation system leads to two difficulties of interpretation. First of all, the exhausted lysate contains rifampicin-inactivated RNA polym- erase, which is still capable of binding to promoter sites, and which might induce premature termination of RNA chains initiated by the rifampicin-resistant RNA polymerase (28). Secondly, a p subunit which is rifampicin-resistant may have interactions with cell components that a rifampicin-sensitive p subunit does not (29). Lysates prepared from mot+- and mot--infected cells without rifampicin treatment show no mot dependence of DE RNA synthesis in This and other results (see accompanying article) lead us to believe that the rifampicin-inactivated RNA polymerase plays an essential role in our finding mot-dependent DE RNA synthesis in exhausted lysates complemented with rifampicin-resistant RNA polymerase (see Fig. 5). We can show that the mutation to rifampicin resistance in the p subunit is not absolutely

V. de Franciscis and E. Brody, unpublished results.

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Middle T4 RNA

required to obtain mot dependence of DE RNA synthesis in vitro. A streptolydigin-resistant RNA polymerase can, in some cases, substitute for a rifampicin-resistant RNA polymerase (19, 20, 30).

In the streptolydigin system, cells used to make early lysates were isolated at the time at which we would ordinarily add rifampicin. The lysates were incubated with streptolydigin in the presence of streptolydidin-resistant RNA polymerase. Fig. 2 shows the incorporation of UTP into RNA as a function of the concentration of streptolydigin in the lysate. Since strep- tolydigin blocks both initiation and elongation of RNA polym- erase (30), the streptolydigin-inactivated RNA polymerase from the early lysate should be distributed over entire early transcription units and not just at promoter sites as is the case for the rifampicin-inactivated RNA polymerase. Although the streptolydigin-resistant RNA polymerase is partially inhibited by the antibiotic, complementation is taking place. Such a system has been used by Rabussay and Geiduschek (19, 20) for in vitro late T4 RNA synthesis. We find that RNA syn- thesized in vitro in this system contains more DE RNA when the early lysate derives from a T4 mot+ infection than when it derives from a mot- infection (Table IIIB, lines 2 and 4). In this respect, the results parallel those of the rifampicin com- plementation system (Table IIIA, lines 2 and 4). The strep- tolydigin lysates show some mot dependence of DE RNA synthesis in vitro. We presume that this result also depends on the presence of streptolydigin-inactivated RNA polymer- ase on the template DNA. Nevertheless, we find important differences between DE in vitro RNA synthesis in the strep- tolydigin lysate system and that seen in the rifampicin system. One difference has to do with the preparation of early lysates (Table 111, A and B) . In preparing either CAM lysates or early exhausted lysates, we originally used Brij 58 detergent in the lysis procedure. This detergent is capable of partially inacti- vating the anti-a activity found in a 10,000-dalton polypeptide induced in T4-infected cells (31). Rabussay and Geiduschek

I I 10 30 s o l

CONCENTRATION OF STREPTOLYDIGIN

2. Effect of streptolydigin on in vitro RNA synthesis in a crude system. Lysates were prepared from E. coli BE Gected with T4 am BL 292 (gene 55) as is described under “Experimental Proce- dures.” Either no addition (0) or addition of 23 pg/ml of stlR DPR 410 purified RNA polymerase (0) was made to each lysate. Each reaction mixture contained the concentration (in micrograms/ml) of streptolydigin indicated in the abscissa, and 100 pl of lysate/ml. The KC1 concentration was 0.1 M. The in vitro reaction time was 20 min at a temperature of 38 “C. The 100% point is represented by the [3H] UTP incorporation without antibiotic or exogenous enzyme added. The total incorporation of UTP into RNA in the 100% point sample after 20 min of incubation was 2790 pmol/ml, that is, 66.9 pmol/lOs cell equivalents.

~”

Synthesis in Vitro 4091

TABLE I11 Effect of Brij 58 addition on in vitrc DE RNA synthesis and on the

symmetry of transcription (A) In vitro RNA synthesis was carried out at 38 “C for 30 min

using 10 pg/ml of purified rifampicin-resistant BE rif-4 RNA polym- erase. Conditions for the in vitro reaction are as given in the legend to Table 11. The reaction mixtures contained rifampicin at 34 pg/ml, KC1 at 0.1 M, and 150 p1 of the exhausted lysate, in 1 ml of reaction mixture. The Brij 58 when present, was used at a concentration of 0.5% (w/v) in the buffer used for the last resuspension of the lysates and thus was at 0.07% in the in vitro reaction mixture. The exhausted lysates were prepared as described under “Experimental Procedures,” from E. coli BE infected at 42.5 “C either with T4 am BL 292 (55-) or with T4 ts GI (mot-). Rifampicin at 200 pg/ml was added 3.5 min after the infection, and the infection was allowed to continue another 20 min. The percentage of DE RNA synthesized in vitro was deter- mined as described in the legend to Table 11. The symmetry of

transcription (indicated as r ) was calculated as the percentage of

counts/min hybridizing to the r strand of T4 DNA (at 4 pg/ml) divided by the counts/min hybridizing to the 1 strand T4 DNA (at 4 pg/ml) plus the counts/min hybridizing to the r strand (at 4 pg/ml) of T4 DNA. (B) In vitro synthesis was carried out at 38 “C for 20 min in the presence of 23 pg/ml of purified streptolydigin-resistant E. coli DPR 410 RNA polymerase. RNA synthesis conditions are as given in the legend to Table 11. The reaction mixture (see “Experimental Procedures”) included streptolydigin at 30 pg/ml, 0.1 M KC1, and 100 pl of the lysate, but no rifampicin. The Brij 58, when present, was used at a concentration of 0.5% (w/v) in the buffer used for the last resuspension of the lysates and thus was at a concentration of 0.05% in the in vitro reaction. The lysates were prepared, as described under “Experimental Procedures,” from E. coli BE infected at 42.5 “C either with am BL 292 (55-) or with ts G1 (mot-) and isolated 3 min after infection by rapidly chilling the cells. No antibiotic was added to these cells before lysis. DE RNA percentages were calculated as given in the legend to Table 11. Symmetry of RNA synthesis was determined as in (A).

l + r

% %

A) Lysate from cells infected by: 1) am BL 292 (55-) Exhausted + 32.5 7 2) am BL 292 (55-) Exhausted - 29 1.5 3) ts G1 (mot-) Exhausted + 20 3 4) ts G1 (mot-) Exhausted - 14.5 3

B) Lysate from cells infected by: 1) am BL 292 (55-) None + 24 16 2) am BL 292 (55-1 None - 27 4 3) ts G1 (mot-) None + 25 12 4) ts G1 (mot-) None - 17 3

(20) found that lysing cells with this detergent reduced in vitro synthesis of late T4 mRNA. When we complement rifampicin-resistant RNA polymerase with rifampicin-ex- hausted early lysates, the use of Brij 58 in the lysis mixture neither effects the mot dependence of in vitro DE RNA synthesis, nor the symmetry of transcription (as measured by I strand specificity). Early lysates, which are not treated in vitro with antibiotics, and which are then complemented with streptolydigin-resistant RNA polymerase in the presence of streptolydigin, show these same properties only when Brij 58 is absent from the lysing medium (Table IIIB). We do not know if this Brij sensitivity in the early lysates is due to inhibition of the anti-a activity, but we guess that some other target is involved, since Brij 58 decreases specificity of tran- scription in these lysates (increased r strand transcription) whereas inhibiting anti-o activity would be expected to have the opposite effect.

Next, we present the salt dependence of DE RNA synthesis in vitro in the rifampicin lysate system. A number of param- eters of T4 transcription are influenced by salt concentration.

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4092 Middle T4 RNA Synthesis in Vitro

/*

KC1 (MOLARITY)

.I- : P at c--- """ """-""""""", X

i - I I I 0.05 0.1 0 0.20 -

KC1 (MOLARITY)

FIG. 3. Salt dependence of in vitro RNA synthesis with mot+ and mot- lysates. A, C3H]UTP incorporation into RNA is shown as a function of the KC1 molarity in the reaction mixture. Each reaction mixture is 1 ml and the data are shown as [3H]counts/min incorpo- rated into RNA in 10 pl of reaction mixture, which is the equivalent of 2.7 X lo7 infected cells. The RNA polymerase used was either from rifampicin-resistant uninfected E. coli AJ7 or from E. coli AJ7 isolated 15 min after T4 infection at 37 "C (25,26). The E. coli RNA polymerase was used at 18 pg/ml (A, A); the T4-modified enzyme was used at 20 pg/ml (0, 0). Each reaction contained 100 pl of the appropriate lysate; rifampicin was present a t 34 pg/ml. The rifampicin exhausted lysates were made from E. coli BE infected at 42.5 "C with either am B22 (mot'-43-; open symbols) or ts GI-am B22 (mot--43-; closed symbols). Infected cells were treated with rifampicin (200 pg/ ml) 3.5 min after infection (see "Experimental Procedures"). Brij 58 was not used in any step of the lysate preparation or in vitro RNA synthesis. The in vitro reactions were 30 min at 38 "C at which time aliquots were taken for C13CCOOH precipitation. B, the effect of heparin on salt-dependent in uitro RNA synthesis. Lysates are either from mot+ (A, A) or mot- (0, W) infected cells as in A. Conditions were as in A except that only uninfected E. coli AJ7 rifampicin- resistant RNA polymerase has been used. Here we test the C3H]UTP incorporation into RNA of either the same components as in A, (open symbols) or after, in addition, having added heparin to a concentration of 100 pg/ml 1 min after addition of the E. coli RNA polymerase (closed symbols). We show only incorporation after 30 min at 38 "C, although RNA synthesis was also measured at 0,2 ,3 ,4 ,5 ,10 ,15 , and 20 min after enzyme addition in the four cases.

1) IJsing purified T4 DNA and E. coli RNA polymerase, reinitiation of RNA chains requires high (0.2 M KC1) salt concentrations (32); 2) also using a purified transcription system, Richardson (4 ) showed that E. coli termination factor p did not function in T4 RNA synthesis at 0.2 M KC1 (Table

IV); 3) the anti-u 10,000-dalton polypeptide induced in T4- infected cells functions poorly at 0.05 M KCI, and has full activity only at higher salt concentration (0.2 M KC1) (31). We therefore thought that an analysis of salt dependence would clarify the mechanism of DE RNA induction in the comple- mentation system.

We thus examined in vitro RNA synthesis in mot+ and mot- early exhausted lysates complemented with r i p E. coli or T4-modSied RNA polymerase, as a function of salt concen- tration. Using 30-min reaction times, we found (Fig. 3) a large salt dependence of RNA synthesis in these lysates. With complementing RNA polymerase from uninfected E. coli, there is an almost %fold stimulation of RNA synthesis when the salt concentration goes from 0.05 M KC1 to 0.20 M KC1. This stimulation is not seen, however, when the lysate derives from a mot- infection. We think that this stimulation is due to initiation at middle promoters in a u and mot-dependent reaction which apparently cannot take place in the mot- lysate. When T4-modified r i p enzyme is used to complement early exhausted lysates, there is again less synthetic capacity from mot- than from mot+ lysates, but now the capacity for

0" 10 I 20 I - RNA POLYMERASE in pg per ml

FIG. 4. RNA synthesis a8 a function of E. coli RNA polym- erase concentration in mot+ and mot- lysates. The RNA polym- erase used in the in vitro reactions was from the rifampicin-resistant E. coli strain AJ7. In each 1 ml of in vitro reaction was included rifampicin at 34 pg/ml, KC1 at 0.2 M, and 150 pl of the lysate. The exhausted lysates were prepared from E. coli BE infected at 42.5 "C either with urn B22 (mot+&-; A) or with t s GI-am B22 (mot--43-; 0). Rifampicin at 200 pg/ml was added 3.5 min after infection (see "Experimental Procedures"). The abscissa shows the concentration in micrograms/ml of RNA polymerase present in the mixture. The in vitro reaction was carried out at 37 "C for 10 min. At that time 100- pl aliquots were precipitated with 10% cold CLCCOOH, fdtered onto GF/C Whatman glass filters, and counted in a toluene-based scintil- lation liquid. The incorporation in 100 pl of reaction mixture is shown in the ordinate. This corresponds to about 4.1 X 10' cell equivalents. We point out that the calculation of picomoles of UTP incorporated might be too low because we have ignored the unlabeled UTP in the lysate in this calculation.

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Middle T4 RNA Synthesis in Vitro 4093

TABLE IV Mot dependence of DE RNA synthesis in vitro as a function of KC1 concentration

The RNA polymerase from the rifampicin-resistant E. coli AJ7 which time the 3H-RNA was extracted and analyzed as above. The was used at a concentration of 23 pg/ml. The exhausted lysates were streptolydigin-resistant RNA polymerase purified from E . coli DPR prepared from E. coli BE infected at 42.5 “C either with am B22 (437 410 was added to the reaction mixture at a concentration of 23 p g / d or with ts GI-am B22 (mot--43-). Rifampicin (at 200 pg/ml) was The lysates used to complement this enzyme were prepared from E. added 3.5 min after infection (see also the legend to Table I1 and coli BE infected at 42.5 “C with am BL 292 (55-) or ts GI (mot-) and “Experimental Procedures”). Lysates were also prepared using E. coli isolated 3 min after infection (see “Experimental Procedures”). No Tab G23 (rifampicin-sensitive) infected at 30 “C with either am E645 antibiotic was added to these cells before lysis. Brij 58 was not (42-) or with am GI-am E645 (mot--42-). Rifampicin (at 200 pg/ml) included in any step of the lysate preparation or in vitro synthesis. was added 5 min after infection (see “Experimental Procedures”). One ml of reaction mixture included streptolydigin at 30 pg/ml (but Brij 58 was not used in any step of the lysate preparation or in vitro no rifampicin), and 100 pl of the indicated lysate. The KC1 concentra- synthesis. One ml reactions were carried out. Reaction mixtures tion is indicated in the table. The in vitro reactions were carried out included rifampicin at 34 pg/ml, and 100 pl of the indicated lysate; a t 38 OC for 20 min. DE RNA synthesized in vitro was determined as the KC1 concentration is indicated in the table. The reactions were described in the legend to Table 11. Next to each number showing the carried out a t 38 “C for 30 min. [3H]RNA was then extracted and percentage of DE RNA is a number in brackets. The number(s) in analyzed for DE RNA content and symmetry (see Tables I1 and 111). brackets is a measure of the symmetry for each 3H-RNA preparation. The rifampicin-resistant RNA polymerase purified from E. coli BE- This is calculated as the percentage of counts/min hybridizing to the rif-4 was used at a concentration of 10 pg/ml. The exhausted lysates r strand of T4 DNA (at 4 pg/ml) divided by the counts/min hybrid- used for this complementation were prepared from E. coli BE infected iziig to the 1 strand of T4 DNA (at 4 pg/ml) plus the counts/min at 42.5 “C with either am BL 292 (55-) or ts GI (mot-). Rifampicin hybridizing to the r strand of T4 DNA (at 4 pg/ml). The data taken (at 200 pg/ml) was added 3.5 min after infection (see also the legend from Richardson (4) concern experiments using an in vitro transcrip- to Table I1 and “Experimental Procedures”). Brij 58 was not used in tion system with purified T4 DNA and E . coli RNA Polymerase. p any step of the lysate preparation or in vitro synthesis. One ml of protein, when present, was added at a concentration.of 4 pg/ml. In reaction mixture included rifampicin at 34 pg/ml and 150 pl of the these experiments the percentage of DE RNA synthesized in vitro indicated lysate. The KC1 concentration is indicated in the table. was dekrmined by hybridization competition analysis of each RNA, These in vitro syntheses were carried out at 38 “C for 20 min, after using total denatured T4 DNA instead of purified 1 strand.

Purified RNA uolvmerase used for transcriution

In vitro purified Rifampicin-resistant

BE rif-4 Streptolydigin-resistant

DPR 410 system (data

from Richard- son, Ref. 4)

Rifampicin-resistant E . coli AJ7

Bacteriophage used to infect cells for lysates

KC1 concen- 43- tration mot--43- 42- mot--42- 55- mot- 55- mot- No ad- p

dition added % D E (s) W DE (s) % DE (s) % D E (s) S DE

1) 0.05 M 30 (6) 29.5 (9) 30 (IO) 26 (17) 30 3 2) 0.1 M 44 (5) 19.8 (5.5) 23 (8) 29 (1.5) 14.5 (3) 27.4 (4) 17 (3) 26 9 3) 0.2 M 21 (5) 10 (1) 13 (7) 6 (7) 25 24

both decreases as one passes from 0.05 M KC1 to 0.20 M KC1. This is what would be expected from the fact that the anti-a activity in the T4-modifled RNA polymerase is salt-dependent (31). Thus, the salt stimulation seen in mot+ lysate comple- mentation seems to be dependent on E. coli a function. Since reinitiation by E. coli RNA polymerase on T4 DNA in a purified system is also salt-dependent and a-dependent, is this mot+/mot- difference due to the capacity of these lysates to recycle E. coli RNA polymerase at the same early promoters? If this were the case, we would expect that limiting initiation in the complementation reaction to a brief period would eliminate the mot+/mot- difference at high salt. This was tested by the use of heparin, a polyanion that inactivates RNA polymerase not strongly bound to DNA (33). E. coli .ifR RNA polymerase was added to mot+ and mot- rifampicin exhausted lysates, and 1 min later, 100 pg/ml of heparin was added to each transcription mixture. Kinetics of RNA synthe- sis was followed in each case and the results are shown in Fig. 3B. With heparin addition limiting initiation to 1 min at 38 “C, the mot+/mot- lysate difference at high salt is maintained. Thus we conclude that this difference is not only a mot- dependent recycling of RNA polymerase. It is likely that mot functions in vitro to direct RNA polymerase to middle pro- moters just as it is presumed to do in uiuo. Other support that this is so comes from saturation curves of mot+ and mot- lysates with E. coli RNA polymerase. Fig. 4 shows the initial rates of RNA synthesis in mot+ and mot- lysates after com- plementation with increasing amounts of r i p E. coli RNA polymerase. Saturation of the DNA template is seen in mot- lysates whereas mot+ lysates seem to have many more DNA

sites available at the same concentration of RNA polymerase. We presume that this difference is due to the availability of middle promoters in the mot+ but not in the mot- lysates. We next examined the specificity of synthesis as a function of salt concentration. As we had shown previously, when E. coli RNA polymerase is complemented with mot+ early exhausted lysates at 0.1 M KC1, DE RNA is synthesized in vitro. Mot- lysates under these conditions show much less ability to transcribe DE regions (Ref. 14 and Table IV). We show four different pairs of mot+ and mot- lysates (Table IV) at 0.1 and 0.2 M KC1 where this dependence is maintained. When the KC1 concentration is lowered to 0.05 M, the mot+ and mot- lysates give the same percentage of DE RNA when comple- mented with E. coli RNA polymerase. We always look at differences in DE content between in vitro RNA made from mot+ and mot- lysates. It is always found that the absolute levels of DE RNA decrease in going from 0.1 to 0.2 M KC1. The about 2-fold difference in DE content between RNA made from mot+ and mot- lysates does not, however, change (Table IV).

DISCUSSION

In Vitro Polarity-E. coli infected by bacteriophage T4 in the presence of CAM synthesize only IE RNA (3). This effect of CAM is due to p-induced polarity (2); lack of protein synthesis at the time of T4 infection allows p to prematurely terminate early RNA chains. A recent model of polarity has p competing with ribosomes for sites on nascent RNA. p recognizes RNA sites and then starts to move down the RNA chain toward the RNA polymerase in an ATP-dependent

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4094 Middle T4 RNA Syn&hesis in Vitro

reaction (34,35). This movement seems to be inhibited when the nascent RNA is being translated (34). If p arrives at an RNA polymerase molecule pausing on its DNA template, it provokes enzyme and RNA release (34, 35). This complex series of reactions seems to be taking place in lysates from cells infected by T4 in the presence of CAM, even when the RNA chains are initiated in vitro (Fig. 1).

Although we see p-induced polarity in CAM lysates, it is not seen in coupled transcription translation systems using T4 DNA (11) nor is it seen if CAM is added after lysis of cells infected with T4 in the absence of antibi~tic.~ Fractionation of CAM lysates should provide insight into the mechanism of p-induced polarity.

About 4 min after infection, T4-infected E . coli become insensitive to the ability of CAM to induce polarity (36). Recent work from our laboratory4 has shown that this "anti- polarity" is the result of two early regulatory events (37). First of all, mot dependent middle mode RNA synthesis allows initiation on genes that are distal on early transcription units. Even when mot is not functional, however, a second anti- polarity effect renders early transcription units insensitive to the ability of CAM to induce polarity! Our in viuo experi- ments show that the anti-polarity effect has different kinetics in different transcription units, and we suggest that some cis- effect is responsible for this behavior. The in vitro experiments reported here are consistent with such an interpretation. Complementation of early exhausted lysates with CAM ly- sates allows RNA polymerase from the CAM lysate to induce middle mode RNA synthesis in the exhausted lysate, but no substance from the exhausted lysate allows the RNA polym- erase in the CAM lysate to read through p termination sites (Ref. 14 and Table 11). Although T4 proteins are undoubtedly involved in this anti-polarity, we postulate that some cis acting function is necessary as well.

Middle Mode RNA Synthesis in Vitro-Lysates from rif" T4-infected cells have almost no RNA synthetic capacity if the cells had been treated with rifampicin before lysis. The rifampicin treatment in viuo is done in such a way that rifampicin-inactivated RNA polymerase should be largely bound to T4 promoters. It has previously been shown in T7 transcription in vitro that such inactivated, bound RNA po- lymerase can act as a termination signal when transcription units overlap (28). Rifampicin exhausted lysates can be com- plemented with CAM lysates from rif" cells, or with .ifR E. coli RNA polymerase to yield DE RNA, but only if the exhausted lysate was made from infected cells in which the T4 mot gene product had been active.

DE RNA is synthesized in vivo either by chain extension of IE transcripts in the early mode or by mot-dependent middle mode transcription. Nonetheless, DE RNA synthesis is mot- dependent in our in vitro system; we have concluded that we see primarily middle mode (mot-dependent) synthesis of DE RNA because chain elongation in the early mode has been blocked by bound, inactive RNA polymerase. A schematic representation of this in vitro situation is given in Fig. 5.

The results presented here suggest that the mot gene prod- uct is not functioning as an anti-terminator in these lysates. Table IV shows that DE RNA synthesis depends on mot at medium and high ionic strengths, but that. at low ionic strength (0.05 M KCl), DE RNA synthesis becomes mot-in- dependent. If mot were necessarily functioning as a p antago- nist for DE RNA expression, the opposite ionic strength dependence would be expected. p functions well as a termi- nation factor for in vitro T4 transcription only at low ionic strength at 0.02 M KC1, p does not terminate T4 transcripts

C. Themes and E. Brody, manuscript in preparation.

A ) m - lysate r h o site

A

'9 n n 'I 'M

U' U' [I 1

B)mt+ lysate rho lite mnt

'?

tion with R f R RNA polymerase. A, the DNA template in the mot- FIG. 5. A schematic representation of lysate complementa-

rifampicin exhausted lysates is represented by one early transcription unit on T4 DNA. There is an IE and a DE region and a p termination site. We show two early promoters, PE, in the IE region, and one middle promoter, PM, in the DE region. Filled burs ( ) represent rifampicin-inactivated RNA polymerases bound to promoters. These bound, inactive RNA polymerase molecules can act as termination signals (28). We represent them here as being 7540% efficient for termination. RifR RNA polymerase molecules, represented by open bars ( 0 ) can compete for, or initiate at, PE promoters. Such tran- scripts are usually terminated at the next bound RNA polymerase. No competition or initiation can take place at PM because of the lack of the mot gene product. The resulting transcripts are represented by the arrows. B, the same symbols are used. There is now active mot gene product, represented as i, on the DNA at PM. The .ifR RNA polymerase can now compete for (when there is a bound RNA polymerase) or initiate at (when there is no bound polymerase) the middle promoter, giving the distal transcripts. If PM are distributed about equally over early transcription units, it is clear that this leads to a mot-dependent enrichment of DE RNA in this in oitro system.

(4). If mot were necessary to remove p for propagation of RNA into DE regions, we would expect a mot dependence of DE RNA synthesis at low, but not at high ionic strengths.

The kinetics of appearance of rIIB RNA had already sug- gested that the mot-dependent DE RNA expression in this in vitro system is due to middle mode RNA synthesis, and not to an anti-p action in early transcription units (14). In this paper, we have shown that mot activity allows initiation of in vitro RNA synthesis in regions of T4 DNA not readily acces- sible when mot is inactive (Fig. 3, A and B, and Fig. 4). We have recently gathered further evidence for this conclusion; RNA synthesized in vitro after complementation of exhausted lysates with rif" E. coli RNA polymerase contains equd proportions of gene 39 specific RNA when the lysates are derived from mot+ or mot- infections. Gene 39 is expressed in uiuo as an IE function (38). Gene 43 RNA synthesis, on the other hand, is very mot-dependent during the early period of T4 We detect gene 43 RNA only after comple- mentation of mot+, not mot-, lysates.

We have also made exhausted lysates from Tab C 5521 infected with either mot+ or mot- T4 phage. Tab C 5521 is a strain of E. coli with a mutant p which renders it insensitive

T. Mattson, private communication.

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Middle T4 RNA Synthesis in Vitro 4095

to the normal T4-induced anti-polarity functions (39). T4 infections of Tab C strains allow mot-dependent middle mode expression, but are blocked for DE RNA expression in the early mode, because of the Tab C “super”-p. Exhausted lysates from Tab C bacteria show the same mot dependence of DE RNA synthesis as do lysates from Tab’ infected c e k 3 This again suggests that mot is not functioning in these lysates as a p antagonist for early transcription units.

If p is not blocking the extension of IE transcripts in lysates from mot--infected cells, then something else must be; oth- erwise, we would have the same control for DE RNA synthesis that is seen in vivo (6,36). Also, in uitro, T4 DNA and E. coli RNA polymerase give DE RNA by chain extension of IE transcripts (1, 5, 10, 11). A number of results show that the mot dependence of DE RNA synthesis that we have found depends itself on the presence of rifampicin-inactivated RNA polymerase on the template DNA. We have already men- tioned that lysates prepared from mot+- and mot--infected cells without rifampicin treatment show no mot dependence of DE RNA synthesis in uitro. In the accompanying article, we present an experiment in which we have transcribed par- tially purified T4 DNA-protein complexes isolated after mot+ or mot- infection. These complexes show the mot dependence of exhausted lysates only if they are fwst incubated with rifampicin.

Why does DE RNA synthesis become mot-independent at 0.05 M KCl? Could it be that the blocking RNA polymerase- rifampicin complexes dissociate from T4 promoters at low ionic strength? A priori, this is unlikely since RNA polymer- ase-DNA interactions are stronger, not weaker, at low ionic strength (40). Experimental evidence related to this problem is given in the accompanying paper, where we show that the mot dependence of DE RNA synthesis is maintained at low ionic strength when complementations are carried out using rif‘ T4-modified RNA polymerase instead of E. coli enzyme. Thus, the E. coli enzyme really recognizes middle promoters at low ionic strength without mot function. We shall discuss this result below, but we want to point out here that Trimble and Maley (13) had already suggested that E. coli RNA polymerase could recognize middle promoters on purified T2 DNA at low ionic strength, but not at high ionic strength.

We suggest, then, that mot is a T4 protein that interacts with T4 DNA. It is the equivalent of a melting protein or gyrase, at least for middle promoter regions, where it loosens DNA-DNA duplex interactions and allows RNA polymerase to initiate RNA synthesis. At 0.1 or 0.2 M KC1, middle pro- moter recognition depends on mot function when E. coli holoenzyme is used for lysate complementation. At 0.05 M KC1, DNA melting is facilitated by the lower ionic strength so that E. coli holoenzyme can initiate at middle promoters even without mot. That this is not really the physiological situation is suggested by the fact that r strand specific transcripts are always slightly higher at low ionic strength than they are at 0.1 or 0.2 M KCl. This can be seen in Table IV; when E. coli holoenzyme shows mot-independent DE RNA synthesis (0.05 M KCl), there is always a slight increase in the amounts of r strand specific RNA synthesis. This r strand specific RNA synthesis is primarily a reflection of incorrect or nonphysio- logical in uitro RNA synthesis (41, 42).

In the accompanying paper, we show that middle promoter recognition remains mot-dependent even at low ionic strength when T4-modified RNA polymerase is used for complemen- tation. T4-modified RNA polymerase has weakened core-u interactions (43), and recognizes poorly one of the two T4 tRNA early promoters (44). It also has the polypeptides necessary for late promoter recognition in uitro. We think that the mot dependence at low ionic strength of the T4

enzyme is a reflection of weakened core-E. coli u interactions. Thus we suggest that mot interaction with middle promoter DNA allows promoter melting, and that it acts synergistically with the E. coli u subunit. Promoter melting becomes mot- independent at low ionic strength because E. coli holoenzyme has sufficient u subunit to bypass the mot function. Because of the weakened core-u interactions in T4 modified RNA polymerase, middle promoter recognition stays mot-depend- ent with this enzyme even at low ionic strength.

Recently, the cII protein of bacteriophage X has been shown to act by facilitating initiation at certain promoters on XDNA after interaction with this DNA (45). We are presently trying to purify mot protein to see to what extent mot and cII action are similar. We think that they might not be precisely the same; in uiuo, mot seems also to play some role in the T4 anti- polarity effect: although we think it is not the cis acting function we have described above. We had postulated that mot may diminish RNA polymerase pausing during transcrip- tion by a genera! weakening of DNA-DNA duplex interactions (2). Finally, our data suggest that the T4 u hypothesis ( 7 ) is unnecessary. Mot protein and E. coli u suffice to explain middle mode RNA synthesis.

Acknowledgments-We should like to thank Dietmar Rabussay, E. P. Geiduschek, Reinhard Mailhammer, and Wolfram Ziuig for sending us materials used in these experiments. We thank Josette Leautey, Renee Favre, and Marc Uzan for their aid with various parts of the work and for helpful discussions, and E. P. Geiduschek for a critical reading of this and the accompanying article. We acknowledge the participation of Patrick Daegelen and Claude Thermes in the early stages of the development of this in vitro system. We thank M. Grunberg-Manago for her support and encouragement during the course of this work.

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3. Salser, W., Bolle, A,, and Epstein, R. (1970) J. Mol. Biol. 49,

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6. O’Farrell, P. Z., and Gold, L. M. (1973) J. B i d . Chern. 248,

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V de Franciscis and E Brodypolymerase.

In vitro system for middle T4 RNA. I. Studies with Escherichia coli RNA

1982, 257:4087-4096.J. Biol. Chem. 

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