THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 248, No. 15, Issue of August 10, pp. 5512-5519, 1973

Printed in U.S.A.

Transcription and Translation of Prereplicative Bacteriophage T4 Genes in Vitro*

(Received for publication, December 22, 1972)

PATRICIA 2. O’FARRELL AND LAWRENCE M. GOLD

From the Department of Molecular, Cellular, and Developmental Biology, University of Colorado, Boulder, Colorado 80506

SUMMARY

Bacteriophage T4 DNA was used to direct transcription and translation in vitro in extracts prepared from uninfected Escherichia coli. The radioactive protein products of the cell-free reactions were examined on sodium dodecyl sulfate acrylamide gels.

We conclude that the cell-free system prepared from un- infected E. coli has the capacity to synthesize most T4 pre- replicative RNAs and proteins. Furthermore, synthesis of those RNAs and proteins occurs in the same temporal order as occurs in uiuo during the first minutes after T4 infection. The relative molar yields of early proteins in vitro are similar to the relative yields obtained in vivo. We found no evi- dence in support of the subdivision of early RNAs into im- mediate early and delayed early species; promoter recogni- tion in vitro is followed by the elaboration of polycistronic mRNAs with no constraints against promoter distal tran- scription.

A small class of prereplicative proteins was not synthe- sized efficiently in vitro. Those proteins are derived from the special class of prereplicative cistrons called quasi-lates; these genes in vivo are under the control of promoters first recognized after a delay of about 1% min. The components required for quasi-late promoter recognition in vivo appear to be missing or nonfunctional in cell-free extracts of un- infected E. coli.

The data in the preceding paper (1) have led us to believe that most prereplicative bacteriophage T4 RNAs and proteins are synthesized under the control of two classes of promoters. The early transcription units may be designated as PE --f Tn, where PE is an early promoter and TE is a termination site (l-3). In addition to early transcription units we described a second prcreplicative class, called quasi-lates, whose boundaries are designated Po ---f To (1, 4, 5); the quasi-late transcription units are poorly expressed if initiations of RNA synthesis are limited by rifampicin to a short time after infection (1). This class of transcripts (also called the postreplicative earlies (5)) is not

* This research was supported by Grant E-624 from the Ameri- can Cancer Society and Grant GB-30517 from the National Science Foundation.

identical with the delayed early RNAs, although the first ap- pearance of quasi-late RNAs coincides with some promoter distal delayed early species (1, 4). No direct experiments have been reported in which recognition of quasi-late promoters was studied in vitro; thus the enzyme responsible for transcription of the quasi-late genes has not been identified.

In the experiments reported below we have asked which pre- replicative cistrons are expressed in a cell-free system derived from uninfected E. co& and directed by T4 DNA (6-10). The cell-free system contains all essential components for the tran- scription and translation of bacteriophage DNAs (7, 9, 11). Our data suggest that the proteins encoded within early transcription units (designated PE -+ TE (1)) are synthesized in vitro, whereas those proteins encoded primarily within quasi-late transcription units are not efficiently synthesized (1). The early transcription units commonly are thought to contain p-mediated termination sites (12) approximately 2000 base pairs from the early promoters (2-4) ; these boundaries have been used to distinguish between promoter proximal (immediate early) genes and promoter distal (delayed early) genes. However, we were unable to find condi- tions under which the cell-free system restrictively transcribed and translated only immediate early cistrons.

MATERIALS AND METHODS

Coupled Cell-free &stem-We have described previously in some detail the methodologies employed in preparing and assay- ing extracts from E. coli (6-10). The emphasis here will be on those parameters which we have found to alter the fidelity of in vitro transcription and translation.

1. The cells which we have used come from an E. coli K-12 strain 514 (13). We have used as well E. co& l3 and several other strains of E. coli K-12. The cells grown in Hershey broth (14) at 37” in a New Brunswick fermentor which provides vigor- ous aeration and a generation time of about 21 min. When the cells reach a concentration of 1 x lo8 per ml they are harvested by pouring quickly onto ice. The cells are centrifuged using a Sorvall continuous flow attachment, washed once in buffer (10 mM Tris-HCI, pH 7.5 + 10 mM magnesium acetate), and frozen quickly in a Dry Ice-acetone slurry. The frozen cells are stored either at -70” or in liquid nitrogen. Cells must be harvested in early log phase. We have studied extensively the behavior of extracts prepared from cells harvested at various cell densities.’ The amount of enzyme synthesis obtained in

1 L. M. Gold, unpublished observations.

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vitro falls drastically in extracts prepared from cells approaching LO9 per ml, although amino acid incorporation in such extracts is unimpaired. Other authors have reported a similar phenom- enon (15, 16).

2. The cells must be broken in a Sorvall Omni-Mixer as described previously (7). We have been unable to obtain highly active cell-free systems by any other method of breaking the cells (including grinding with alumina, sonication, or extrusion of suspended cells through a French press).

3. After separation of the ribosomes and supernatant proteins by centrifugation, the supernatant proteins are freed of con- taminating nucleic acids by adsorption to and passage through a DEAE-cellulose column (7). We use a specific DEAE-cellu- lose (Cellex D, from Bio-Rad). During the low salt elution of the supernatant proteins large amounts of presumably non- essential proteins are lost. Most of these proteins are not lost during the low salt elution if high quality DEAE-cellulose (What- man, DE-52) is used; extracts prepared using DE-52 arc rela- tively inactive.

4. The incubation conditions are the same as described previ- ously except for the addition of exogenous E. coli RNApolymerase (17). The requirement for exogenous RNA polymerase varies somewhat from extract to extract; some extracts are not stimu- lated at all. During incubation the tubes are slowly shaken; the shaking can increase enzyme synthesis 2- to 3-fold. With each new preparation of ribosomes and supernatant protein we systematically vary the concentrations of magnesium acetate, ribosomes, supernatant protein, exogenously added transfer RNA, and exogenously added RNA polymerase (a kind gift from Dr. R. R. Burgess; prepared from E. coli AS19 (18)). The optimum magnesium ion concentration for the synthesis of most enzymes is 11 to 12 mM (11, 19). Typical data for amino acid incorporation in response to T7 DNA as a function of the con- centrations of the biological materials are given in Fig. 1 (which is discussed as well under “Results”). The conditions which we use as standard for T4 DNA-directed transcription and trans- lation include magnesium ion at 12 mM, ribosomes at 3 mg per ml, supernatant protein at about 3 mg per ml, transfer RNA at about 1 mg per ml, and exogenous RNA polymerase at 30 pg per ml. We have found these conditions to be optimal for the synthesis of T4 P-glucosyltransferase. Under these conditions we can drive from 30% to 70% of the ribosomes into polysomes during protein synthesis in vitro.l

The data given for amino acid incorporation in the figures and tables are counts per min per 0.010 ml of the cell-free incuba- tions (as hot trichloroacetic acid-precipitable material (6)). Thus, we routinely obtain during a 30-min incubation about 2 to 10 X lo6 cpm of protein in response to T4 or T7 DNA per ml of reaction. The %-amino acids are present at 50 PCi per ml. The endogenous level of incorporation is usually 5% of the template-directed rate. Thus the maximum amount of amino acid incorporation is about 15% of the input amino acids, which are present at 0.2 mM each. This corresponds to about 70 pg of newly synthesized bacteriophage proteins per ml.

SD%Polyacrylamide Gels-Slab gels are prepared and run as described by others and in the accompanying papers (1, 20- 22). The reaction mixtures from cell-free incubations are not fractionated in any way. After appropriate incubation for transcription and translation (in the presence of 14C-amino acids), t’he reaction mixtures are chilled in an ice water slurry. To each 50.~1 reaction mixture is added 1 ~1 of a solution containing

2 The abbreviation used is: SDS, sodium dodecyl sulfate.

DNase and RNase at 1 mg per ml each. The reaction mixtures are then reincubated at 37” for 1 min, chilled again, and to them are added 200 ~1 of the SDS sample buffer described by Laemmli (21). These mixtures are then heated at 90” for 1 min to solu- bilize all of the material. Samples of 25 to 50 ~1 are run on SDS gels. After running, the gels are fixed in 50% trichloroacetic acid for a few hours, stained in 0.1% Coomassie blue in 50% trichloroacetic acid for 30 min, and destained in 7% acetic acid. The fixing and destaining are essential for gels containing cell- free reaction mixtures. This processing of the acrylamide gel al- lows the complete deacylation of any charged transfer RNA which survived the brief RNase treatment and migrated into the gel. Furthermore, any free 14C-amino acids left in the gel diffuse out during the destaining. The dried gels are then subjected to autoradiographic analysis (21-23).

All other materials and method have been described (1, 4, B-10, 24). The T4 mutants used are described in the accompa- nying paper (20). p factor was purified from E. coli 514 (12,13).

RESULTS

Control Experiments with I’7 DNA-We have performed sev- eral controls for the fidelity of the in vitro system using DNA from the bacteriophage T7 as a template. The early pattern of T7-specific protein synthesis in viuo has been well established by other investigators (22); this early pattern is less complex than the prereplicative T4 pattern. The major early T7-specific proteins synthesized in vivo include one low molecular weight protein (8,700), a DNA ligasc (40,000), and the T7-specific RNA polymerase encoded for by the T7 gene 1 (107,000). In our experiments we varied the concentration of ribosomes, superna- tant proteins, exogenously added transfer RNA, and exogenously added RNA polymerase. We measured amino acid incorpora- tion and asked as well which T7 proteins were synthesized under these various conditions (Fig. 1). We arbitrarily defined the optimum conditions for cell-free RNA and protein synthesis as those conditions which gave the highest yields of T7 ligase and T7 RNA polymerase relative to the amount of protein (or peptide) which accumulated at the dye front during electro- phorcsis. The rationale is that there are a large number of arti- facts inherent to cell-free systems which can bring about in- efficient synthesis of high molecular weight proteins (such as the T7 RNA polymerase). These data (Fig. 1, a to d) suggest that the molar outputs of specific genes in vitro can be sensitive to constraints other than those involved in the determination of gross amino acid incorporation. A typical autoradiogram resulting from a reaction run under optimal conditions is shown in Fig. le; the amounts of specific T7 early proteins resemble early pulses of T7-infected bacteria (22). Densitometer scans of such autoradiograms (as in Fig. lf) suggest that little protein accumulates which is not an authentic T7 early protein. How- ever, we must note that T7 DNA directs the synthesis of an enzymatically active lysozymc during cell-free transcription and translation (9,26). Genetic data show that the T7 lysozyme gene is very far removed from the early T7 region (22). How- ever, it is clear from our gels that relatively little transcription and translation occur in vitro from T7 genes located downstream from the transcriptional termination site located on the promoter distal side of the ligase cistron (22). Thus, the large amount of lysozyme activity resulting from cell-free synthesis (9, 26) reflects a relatively small molar amount of protein.

Gene Assignments for T/t Proteins Synthesized in Vitro-Our previous experiments demonstrated that at least seven T4-spe- cific proteins could be synthesized in vilro: enzymatically active

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I 04 OB 12 24 32 10 3050 100 Ix)

mp/td Cla/ml

FIG. 1. A test for fidelity in vitro with T7 DNA. Cell-free incubations were carried out for 30 min using various concentra- tions of ribosomes (a), supernatant protein (b), transfer RNA (c), or exogenous RNA polymerase (d). T7 DNA was added to a final concentration of 30 pg per ml. As each ingredient was varied, the concentrations of the fixed components were 3 mg per ml of ribo- somes, 3.2 mg per ml of supernatant protein, 1.2 mg per ml of transfer RNA, and 30 rg per ml of RNA polymerase. The amino acid incorporation for each incubation is given in a to d ( l - l ). The ‘4C-amino acids were present at 25 PCi per ml. The products of each incubation were analyzed on 7.5yn SDS-acrylamide gels. The resulting autoradiograms (as, for example, e) were scanned using a Joyce-Loebl microdensitometer (as inf). For each incuba-

cr- and /3-glucosyltransferase, dCMP deaminase, lysozyme, and immunologically competent internal proteins I, II, and III (7, 9). This catalogue may be expanded by observing the products of T4 DNA-directed transcription and translation on SDS-ac- rylamide gels. Such patterns, directed by template DNAs extracted from a variety of T4 mutants (27-29) are shown in Fig. 2. Without exception those genes which we assigned to specific proteins in our in vivo experiments (1, 20) yielded the same gene products during cell-free RNA and protein synthesis. There are large variations in the quantities of each protein syn- thesized; we believe that these quantitative differences reflect the capacity of the coupled cell-free system to mimic accu- rately the earliest events of T4 infection.

Quasi-late Promoters Are Not Recognized in Vitro-We com- pared the proteins synthesized in vitro in response to T4 DNA with those proteins synthesized in vivo. Two types of pre- replicative profiles from T4-infected cells may be obtained; total prereplicative proteins or prereplicative proteins synthesized during infections to which rifampicin is added after 1 min (1). Those proteins synthesized after such rifampicin treatment are encoded within early transcription units (Pn + TE (1)). The products of cell-freeRNA and protein synthesis are quantitatively similar to the proteins synthesized in vivo in infected cultures treated with rifampicin (Fig. 3). Thus, there is relatively little synthesis in vitro of the proteins encoded by genes 43, 46, 32, and rIIB. These data may be supplemented by the data in

tion a fidelity factor was calculated from the scanned patterns. This factor was defined as:

Peak heightii,,,, + peak height,,,, 1 peak heightd,, front

The data obtained are plotted in a to cl (O- - -0). The percent- ages shown in f for the three major proteins are averages of a large number of experiments with T7 DNA in vitro; the numbers are the percentage of the total radioactivity on the gel exclusive of the dye front. We note that positive identifications of the products of the genes 0.3, 1, 3, 4, and 1.3 have been made for these cell-free reactions (22, 25).

Fig. 2; the gene 45 protein, a major prereplicative protein in vivo (defined as under the control of a quasi-late promoter (1, 20)) is a minor fraction of the total cell-free products.

We may use the molar ratio of rIIB to rIIA production as a monitor for the failure to recognize Po sites; in vivo, if initia- tions are restricted to l’n sites with rifampicin, rIIB accumulates at about a 2-fold molar excess over the rIIA protein (1). During coupled transcription and translation, rIIB is synthesized in 2- to 3-fold excess over rIIA3; this may be t.aken as suggestive evidence for restrictive initiations of transcription at PE sites (1; also 3, 5). Thus the elements responsible for quasi-late gene expression in vivo are missing or nonfunctional in the cell-free system.

All Transcripts Generated in Vitro Result from Rapid Promoter Recognition-We now focus again on the early transcription units bounded Pn + Tn. Previously, we studied promoter recognition of five T4-specific cistrons: internal proteins I, II, and III, fi-glu- cosyltransferase, and lysozyme (9). For those markers we were able to show that delays in the generation of specific transcripts reflected the distances between the specific genes and promoters which regulated them (9, 10). We have generalized those ex- periments by analyzing on SDS gels all of the T4 proteins syn- thesized in vitro. In these experiments rifampicin was added at different times after the incubations had started. Amino acid incorporation and SDS-gel analyses (Fig. 4) suggest un-

3 I?. Z. O’Farrell, unpublished observations.

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a) 7.5 % Gel

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Mutant 8271 r638 r-1993 HI8 1252 293 E949 Nll6 N82

Gene 46 rlIB rlIA 32 43 (?I 52 39 44

b) 12.5% Gel

Mutant El0 E51 N55 eG506 eG326

Gene 45 56 42 - -

FIG. 2. Cell-free synthesis of specific T4 proteins. Cell-free incubations were directed by DNA isolated from various T4 non-

added RNA polymerase which was obtained from a rifampicin-

sense and deletion mutants. The products were analyzed on SDS- resistant strain of Escherichia coli; the patterns obtained are slightly different (quantitatively) than all others reported in this

acrylamide gels. These incubations contained an exogenously paper. The dots show the missing prot’eins from each reaction.

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in v/fro

Gene

- 43

I

/ - 46

,

I 9c

i 32 = rlIB

in v/v0

Rifampicin Control FIG. 3. A comparison of T4 proteins synthesized in vitro or

in vivo. Standard incubations were performed for cell-free RNA and protein synlhesis in response to T4 DNA. The radioactive proteins synthesized in vitro were analyzed on SDS-acrylamide gels of 7.5% acrylamide (20,21). For comparison, proteins labeled in vivo for the first 12 min of infection were also analyzed. These proteins were either from control infections or from infections to which rifampicin was added 1 min after the bacteriophage (1). The control infections display the total prereplicative catalogue of T4 proteins, whereas the rifampicin-treated culture displays only the proteins encoded by early transcription units (PE --f TE) (1).

ambiguously that within the 1st min of in vitro incubation all early T4 transcripts had been initiated. We know from our previous experiments that the in vitro system does not transcribe all of the early T4 RNAs until the 5th or 6th min of incubation (8, 9). Thus, all of the early transcripts arise in vitro via rapid promoter recognition and subsequent RNA chain growth. Fur- thermore, these promoters are the same promoters as those rec- ognized immediately after infection (designated PE (1, 5)).

Sequential Appearance of Early T.J Proteins in Vitro-T4 DNA- direct.ed protein synthesis was allowed to occur for short time intervals in the presence of high specific activity 14C-amino acids. The kinetics of protein synthesis, SDS gels of aliquots removed after various times, and the kinetics of specific early protein synthesis are shown in Fig. 5. These data may be compared with similar data for T4 infections carried out in viva during infections restricted to PE recognition (Fig. 2C of Reference 1). The in vivo order and the in vitro order appear to be identical.

Does p Factor Cause Transcriptional Terminations in the Cou- pled &&em?-In order to see whether p factor (12) could restrict the coupled system t.o the immediate early (promoter proximal) subset of early genes (2-4), we added p factor to the basic in vitro system. Thus, p is added in addition to the amount of p present endogenously in the cell-free system. We performed

a>

I I I I I I I I

012345678

Time of Rlfomplcln Addltlon (minutes)

i

Time of Rifampicin O

Addition 012468 -- (minutes)

Template - l-T4 DNA-1 - T4DNA

FIG. 4. Promoter recognition in vitro. Cell-free mixtures were prepared at 0”. The tubes were shifted to a shaking 37“ incubator, and rifampicin was added to the incubations at various times. The final concentration of rifampicin was 100 pg per ml. T4 DNA, when present, was at a final concentration of 30 pg per ml. All in- cubations were shaken for a total of 25 min. The amino acid in- corporation is given (a) for reactions with no exogenous template (O---O) or reactions primed by T4 DNA (0-O). The acrylamide gel analyses are shown in b. The ‘G-amino acids were present at 50 pCi per ml.

these experiments with X and T4 DNA, as X DNA has been shown to have genuine p-mediated terminator sites (12). The data for these experiments are shown in Table I. Clearly X transcription and translation are sensitive to p factor under our cell-free conditions, whereas T4 DNA-directed protein syn- thesis is not. These conclusions are confirmed by SDS-gel anal- yses of proteins synthesized in vitro with or without added p factor (Fig. 6). Thus, in contrast to the experiments in which p factor causes termination of early T4 transcripts in purified systems (3, 30, 31), p factor has no effect on T4 DNA-directed transcription and translation. We note two facts concerning the specific p preparation used in these experiments. First, the preparation was greater than 95yo pure, as judged by analysis on SDS gels, and second, this preparation does inhibit T4 DNA transcription by purified RNA po1ymerase.l However, our

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TABLE I E$ect of p on cell-free RNA and protein synthesis

Cell-free reactions were mixed containing various combinations of AS19 RNA polymerase (150 pg per ml), p factor (20 pg per ml) (12), and T4 DNA (30 pg per ml). The incubations were carried out at 37” for 30 min. The incorporation is given for 0.005 ml of reaction mixture. The apparent amino acid incorporation in the absence of template was 1860 f 50 cpm (this “incorporation” in- cludes both true endogenous protein synthesis and the radio- activity nonspecifically adsorbed to the filters during washing); this background incorporation was subtracted from all values.

Tempfate Exogenous Amino acid p Iactor

1 I incorporation

IL&L&h

Qm

<IO0

<lOO

2,080 170

3,861 3,267

17,829 17,217

.-

-

%

92

15

3

X DNA

T4 DNA

- - + + - - + +

b)

-

p factor inhibits X DNA transcription far more efficiently than it inhibits transcription of T4 DNA.1

No T&pecijk Protein Is Needed to Allow Delayed Early Tran- scription in the Coupled System-Although the coupled cell-free system has been prepared from uninfected E. co&i, one could argue that at an early time during the cell-free incubation a TFspecific protein accumulates which allows the transcription of promoter- distal genes. This notion seems unlikely to us; however, we measured, in collaboration with Dr. Kaye Fields, the effects of either chloramphenicol or amino acid deprivation in vitro on the elaboration of delayed early RNAs. The coupled cell- free system was stimulated by T4 DNA in the presence of [aHI- UTP under various conditions. The RNA synthesized in vitro

was purified and assayed by DNA-RNA hybridization-competi- tion techniques for the presence of delayed early RNA. The data we obtained (Table II) showed that under no conditions was RNA synthesis restricted to the promoter-proximal (im- mediate early) genes. These data also demonstrate that early transcription in vitro is asymmetric; since most early transcrip- tion units (Px + Tx) are recognized, this must be the case as well in viva (24, 32). Previously Black and Gold argued that restriction to immediate-early transcription in viva by chloram- phenicol was a reflection of polarity (9, 33) ; if so, we have failed to preserve in the coupled cell-free system those conditions needed to obtain polarity, since chloramphenicol was without effect on promoter-distal transcription in vitro. Other authors have com- mented on the loss of polarity in vitro (34).

Incubation Time I23456789 IO 15

(minutes)

-1 I 5 I6 I5 Time of Synthesis

(minutes)

FIG. 5. Temporal appearance of T4 proteins synthesized in vitro. In order to visualize proteins synthesized during short intervals a special radioactive mixture was prepared. The 20 unlabeled amino acids were used at a final concentration of 0.06 mM each (down 3-fold from our standard conditions) and the 14C- amino acids were present at 100 &i per ml (up a-fold). The reac- tion mixtures were prepared at 0” and shifted to 37” at zero time. Incubations were terminated by adding 0.1 volume of a mixture containing 100 fig per ml of DNase, 100 pg per ml of RNase, 500 pg per ml of chloramphenicol, and 1 mM of each of 20 amino acids. After an additional 30 s of incubation at 37”, the reactions were cooled to 0” to await further processing. The amino acid accumu- lation into protein is given (a) for incubations with no template (C---O) and incubations directed by T4 DNA (0-O). These data are given for 0.001 ml of reaction mixture. The SDS- acrylamide gels of these samples are presented in b. We have at- tempted to diagram the time at which specific bands first may be distinguished from the background (c).

DISCUSSION

Fidelity of Coupled Cell-free System-The data in Fig. 1 suggest that the coupled cell-free system may be used with confidence to study early bacteriophage DNA transcription and translation. In response to T7 DNA, phage-specific transcripts and proteins are synthesized in amounts which mirror the first minutes after T7 infection (22). The early region of the T7 genetic map has been described by Studier (22). The major early proteins syn- thesized in vivo are encoded by the 0.3 gene, the gene 1 (RNA polymerase), and the gene 1.3 (ligase) ; these are the same pro- teins as those synthesized in largest amounts in the coupled cell-

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Added r ho (pg/mI I 1

Template

- 20

XDNA

FIG. 6. The effects of p on transcription and translation in vitro. p factor was purified from Escherichiu coli 514 according to the met.hod of Roberts (12). Cell-free incubations were directed by either T4 DNA or X DNA (a kind gift of Drs. F. Blattner and W. Szybalski) in the presence of increasing amounts of p. RNA polymerase was added to 30 pg per ml. The autoradiograms for the incubations without template or with x DNA as a template

free system.4 These data, in combination with those of other investigators, suggest that extracts of uninfected E. coli (con- taining RNA polymerase) can efficiently transcribe a restricted region of a bacteriophage genome corresponding to those cistrons recognized immediately after phage infection (5; also see 35).

Coupled Cell-free System Transcribes and Translates Most Early T4 Genes-T4 DNA directs the synthesis of most early transcripts and early proteins (corresponding to Pn ---f Tn (1)). The mech- anism by which early RNAs and proteins are generated is quite simple; a small number of promoters are recognized by the host RNA polymerase and long transcription units are read to give polycistronic RNAs (2, 5, 9, 10). Thus, this portion of the transcriptional development of T4 is not different from a random collection of tryptophan-like operons linked together in the early region of a bacteriophage genome. We emphasize that im- mediate early and delayed early genes are transcribed and trans- lated with high efficiencies. The rIIA gene is a delayed early gene (4, 36, 37); that gene is expressed in large amounts in vitro (Figs. 2 and 3). Furthermore, by hybridization-competition analyses, the delayed early RNAs are synthesized (as a class) with high efficiency (Table II). The similarity between the cell-free system and infections in vivo (Fig. 3) plays a large role in our interpretation that the transcription units PE --) Tn are

4 R. Condit, personal communication.

- 2 4 IO 20

T4 DNA

were exposed for about 10 times as long as the autoradiograms derived from the T4 DNA-directed products. The X DNA-di- rected reaction incorporated 906 cpm above the endogenous con- trol (1,900 cpm), whereas the incorporation with added p was reduced to near that of background. The T4 DNA-directed incu- bations each contained about 45,000 cpm of newly synthesized proteins, independent of the added p.

expressed during T4 development without the mediation of T4-induced proteins (I, 9). It seems unlikely that a T4 protein synthesized in vitro could rapidly modify the cell-free system and thereby loosen any restriction against delayed early tran- scription and translation. The data in Table II support this position.

We have also investigated the ability of the termination factor p to mediate terminations within these early transcription units. Using conditions under which p factor works with high efficiency for X DNA transcription we have been unable to detect any effects on T4 DNA-directed RNA and protein synthesis (Table I, Fig. 6). We have used p concentrations as high as 40 pg per ml and observed no alterations to the T4 DNA-directed patterns of RNA and protein synthesis. Thus we must make at least the quantitative statement that p-mediated termination sites on X DNA have markedly higher sensitivity to p than puta- tive equivalent sites on T4 DNA.

Lastly, we have described a subset of the prereplicative pro- teins which is not synthesized efficiently in the coupled cell-free system. This subset, called quasi-late. accumulates in small amounts in vivo in cultures to which rifampicin was added shortly after infection (1). Thus transcription of the type PQ + To does not occur in cell-free systems derived from uninfected E. coli, as though some essential components are missing. The

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TABLE II

Hybridization-competition analysis of TQ RNAs synthesized in vilro

The conditions for cell-free RNA and protein synthesis were modified to exclude radioactive amino acids and include tritiated UTP at 100 &i per pmole. The incubations were carried out for 8 min at 37” in the presence of T4 DNA under standard conditions (Line 1) or under conditions which might affect promoter-distal transcription (Lines 2 through 4); chloramphenical was present at 100 fig per ml, and p, when added, was present at 30 pg per ml. No exogenously added RNA polymerase was present. The radio- active RN9 was purified (4, 9) and annealed to the separated strands of T4 DNA (24) using standard conditions for DNA- excess hybridization (4, 24). The efficiencies of hybridization without competitor RNAs were 55yo to 75yo (for 1 strand DNA). Hybridizat,ion-competition experiments were performed with either 2 mg per ml of RNA extracted from E. coli infected for 7 min by T4 in the presence of chloramphenicol (immediate early competitor (4)) or 2 mg per ml of RNA extracted 5 to 6 min after a normal infection (total prereplicative competitor (4)); these concentrations of competitor RNA were determined to be high enough to give essentially plateau competitions. For each labeled RNA the background radioactivity (retained by filters after incu- bation without added T4 DNA) was 15 cpm; that value was sub- tracted from all data. All of the points were obtained by dupli- cate analyses. The relative delayed early RNA is the fraction of the total hybridizable RNA which cannot be competed by the immediate early competitor. These hybridization experiments were performed by Dr. Kaye Fields, to whom we are grateful.

The rates of RNA and protein synthesis for the four conditions tested are given in nanomoles of UTP per ml per min and nano- moles of total amino acid per ml per min. (The amino acid incor- poration data were obtained using separate reactions.) These data suggest that the average codon (as a triplet) in vifro gives rise to 2 to 3 amino acid residues of protein.

Cell-free conditions for RNA synthesis

Control incuba- tion..

Plus chloram- phenicol.

Minus 20 amino acids..

Plus p -

I 1 Counts per h;$nsDNA-RNA

Rate of Rate Of RNA protein

synthe- synthesis sis 1 strand ’ syti;a’

Total with im- 1

mediate total,P”-

strand early rephca- competi- tive

tor competi- tor

5.4 13.0 1707 787 74 3 0.46

5.1 <.2 1666 684 101 3 0.41

1.9 1840 720 91 0 0.39 4.4 11.7 1205 532 67 3 0.44

Rela- tive

Total “z;;cd r

strand

cell-free system as now constructed can thus serve as an assay system for those factors in T4-infected cells responsible for the initiation of transcription at quasi-late promoters. It is possible that the previously described modifications to the RNA polym- erase which occur after T4 infection play some role in quasi-late

promoter selection (38-40).

Acknowledgments-We thank Dr. David Hirsh for helpful discussions, and Mr. Tom Hill for much needed assistance. One of us (L.M.G.) thanks Dr. Fritz Lipmann in whose laboratory the work with cell-free systems was begun. We thank Dr. Rich- ard Epstein for his frequent communications concerning these experiments. We also thank Dr. Joyce Silver for a critical read- ing of this manuscript.

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Patricia Z. O'Farrell and Lawrence M. Goldin VitroTranscription and Translation of Prereplicative Bacteriophage T4 Genes

1973, 248:5512-5519.J. Biol. Chem. 

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