Vol. 254, No. 2, Issue of January 25, pp. 339-349, 1979 Prrnted in U.S.A.

Construction and Properties of a Cell-free System for Bacteriophage T4 Late RNA Synthesis*

(Received for publication, May 31, 1978)

Dietmar Rabussay and E. Peter Geiduschek

From the Department of Biology, University of California, San Diego, La Jolla, California 92037

A cell-free system for synthesizing bacteriophage T4 late RNA is described. The system, which is based on the “cellophane disc” technique introduced by Schaller and co-workers (Schaller, H., Otto, B., Niisslein, V., Huf, J., Hermann, R., and Bonnhoeffer, F. (1972) J. Mol. Biol. 63,183-200), provides favorable conditions for T4 DNA and RNA synthesis in uitro. Total RNA synthesis can be sustained for more than 1 h at 25°C and initiation of early and late RNA chains occurs in vitro. The capacity to yield cell-free systems which make T4 late RNA in vitro is acquired by virus-infected cells as they make late RNA in ho. The in vitro synthesized RNA is highly asymmetric. The conditions which optimize the in vitro system with respect to several parameters (total extent of T4 transcription, rate of transcription, asymmetry, fraction of T4 late RNA, and sensitivity to inhibition by rifampicin and streptolydigin) are described.

The regulatory mechanisms for the transcription of the late genes of bacteriophage T4 are likely to differ from those that are already known to function in procaryotes. 1) Late genes, as opposed to T4 early and middle genes, normally require T4 DNA replication for their transcription. This “coupling” of T4 DNA replication and late transcription occurs at two levels, via the common use of at least one T4 protein, gene product (gp)’ 45, in the transcription and replication machinery, and via the provision of a “competent” template structure. 2) T4+ DNA contains hmC instead of C. The substitution of hmC by C results in severely defective late transcription which abol- ishes the synthesis of T4 late proteins. The effect of the nucleotide substitution on early and middle transcription has not been thoroughly studied. 3) The RNA polymerase which functions in T4-infected cells during the late period of infec- tion is an extensively modified host enzyme; its (Y subunits are ADP-ribosylated and it contains four T4-specific subunits (gp 33, 55, and two proteins coded by as yet unidentified genes). That these modifications are so much more numerous than any others that are now known is compatible with the spec- ulation that the structure of T4 late promoters differs funda- mentally from the structures of known promoters (for a review of all the above, see Ref. 1).

* This work was supported by grants of the Cancer Research Coordinating Committee, University of California (to D. R.) and the National Institute of General Medical Sciences (to E. P. G.) The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “adver- tisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

’ The abbreviations used are: stl, streptolydigin; rif, rifampicin; CM RNA, chloramphenicol RNA; SDS, sodium dodecyl sulfate; c.e., cell equivalent; hmC, 5-hydroxymethyl cytosine; C, cytosine; Mops, 4- morpholinepropanesulfonic acid; gp, gene product; PA RNA, prean- nealed early and late RNA, SSC, 0.15 M NaCl, 0.015 M sodium citrate.

A suitable in vitro system for the study of T4 late transcrip- tion has not previously been available. Several attempts to transcribe T4 late genes in vitro using purified T4 DNA and RNA polymerase, including templates and enzymes isolated from T4 infected cells, have failed. The search for “factors” which would stimulate T4 late transcription in these systems has not been rewarded with sustained success. Crude lysate systems prepared from T4-infected cells have shown varying amounts of T4 late transcription in vitro. In all cases, the transcription efficiency, the ratio of late to early transcription, or the duration of late transcription in vitro were low and no initiation of late transcription in vitro was demonstrated (1).

We have developed a cell-free system based on the “cello- phane disc” technique introduced by Schaller and co-workers (2). Since T4 DNA replication and late transcription are connected in vivo, it was our intention to provide favorable conditions in vitro for T4 DNA replication as well as tran- scription. In addition, we wanted a system that would allow us to add or remove, activate or inactivate, relevant compo- nents. In this paper, we describe the properties of the system and present proof that this in vitro system resembles very closely the transcription pattern of T4-infected cells in vivo. We also show that the synthesis of late T4 RNA is initiated in vitro. We have recently shown that added, T4-modified RNA polymerase specifically stimulates transcription of T4 late genes in vitro (3). The relation between T4 DNA repli- cation and late transcription in vitro is the subject of a subsequent paper.2

MATERIALS AND METHODS

Section a: Bacteria and Phage

Escherichia coli B” (su-) was the standard host strain. A strepto- lydigin-sensitive (stl‘) mutant of B” (DPR 4) and a DPR 4 mutant with a streptolydigin-resistant RNA polgmerase (DPR 4-10, stl’) were isolated ai described elsewhere.’ E. coli AB105 (RNase I-, RNase III-. met-. (h). Hfr) was from P. H. Hofschneider (Max Planck Institut fiir Bioch&i& Munich, West Germany). CR63 (SU+I) was the per- missive host for T4am mutants.

T4am M41 (gene e, lysozyme) was used as the standard phage; it shows a normal development except that it does not lyse the infected host cell. T4am 292 (gene 55) is deficient in late transcription.

Section b: Chemicals

Streptolydigin was a gift of the Upjohn Co. (Kalamazoo). Rifamp- icin was a gift of Lepetit s.p.a. (Milano). The following substances were purchased from sources noted in parentheses: actinomycin, NAD’, thymidine, and spermidine (Calbiochem, La Jolla); uridine, dithiothreitol, Brij 58, and Mops (4.morpholinepropanesulfonic acid) (Sigma, St. Louis); deoxyribonucleoside and ribonucleoside triphos- phates (P-L Biochemicals, Milwaukee, or Schwarz Bio Research, Orangeburg); phosphoenolpyruvate (Boehringer, Mannheim); egg white lysozyme, DNase I, RNase I and RNase Tl (Worthington, Freehold); various bacterial culture media, Bacto Agar and Noble

’ D. Rabussay and E. P. Geiduschek, manuscript in preparation.

339

by guest on October 28, 2020

http://ww

w.jbc.org/

Dow

nloaded from

340 Cell-free T4 Late RNA Synthesis

Agar (Difco, Detroit); EGTA [ethylenebis(oxyethylenenitrilo)]tetra- acetic acid and various other reagent grade chemicals (J. T. Baker, Phillipsburg); Triton X-100 (Atlas Chemical, San Diego); [“‘Clthy- midine and [“HIUTP (Schwarz/Mann, Orangeburg).

5-Hydroxymethyl-dCMP (hmdCMP) was prepared in two ways which yielded final products with virtually identical properties: (a) unglucosylated T4 DNA was hydrolyzed and the mononucleotides were separated by ion exchange chromatography on Dowex 1 (4); (b) hydroxymethylation of dCMP (5). In each case, hmdCMP was reacted with ATP and crude T4 deoxynucleotide kinase in the presence of pyruvate kinase and phosphoenolpyruvate to yield hmdCTP. The latter was purified on QAE-Sephadex (quaternary aminoethyl Seph- adex) acetate and checked for purity by thin layer chromatography (6).

The following filters and membranes were used: nitrocellulose filters, 24-mm diameter, type HA, 0.45~pm pore size (Millipore, Bed- ford); glass microfiber filters, 24-mm diameter, type GF/C (Whatman, England); cellophane membrane discs, 12-mm diameter, 22-pm thick (Kalle, Wiesbaden Biebrich, West Germany).

Section c: Media, Buffers, and Incorporation Mixtures

M9S medium, which is M9 medium supplemented with 1% casa- mino acids (7), was used for growing phage stocks. Phage were plated on tryptone bottom agar using tryptone top layer agar.

Agar plates A and B were as described (2) except that Noble Agar and 10 mM MgC12 were used. TGNO buffer contains (per liter): 0.5 g of NaCl, 8 g of KCl, 1.1 g of NH&l, 0.2 g of MgCL, 12.2 g of Tris, 0.8 g of pyruvic acid, 0.16 mmol of Na&Od, and 2 mmol of CaCL and the pH is adjusted to 7.4 with HCl. TMA/glycerol buffer contains 25 mM Tris/acetate, pH 7.4, 5 mM magnesium acetate, 10 mru NH&l, 1 mM mercaptoethanol, 50% (v/v) glycerol. TGNO/Brij 58 is a mixture of 4 parts TGNO buffer and 1 part of 5% (w/v) Brij 58. SSC is 0.15 M NaCl, 0.015 M sodium citrate. The compositions of the various incor- poration mixtures for nucleic acid synthesis are given in Table I. Unless otherwise noted, dCTP, rather than hmdCTP, was used. Stop solution is 1% (w/v) SDS, 0.05 M EDTA, 10% saturated Na4P207, 17.5 pg/ml of denatured salmon sperm DNA, pH 7.0.

Section d: Preparation of the in Vitro System

The procedure, which is based on Schaller et al. (2), involves the lysis of a concentrated suspension of bacteria placed on a small disc of cellophane. The lysis takes place in two steps. Spheroplasts are

TABLE I Composition of incorporation mixtures

Mixture

A B C D E

l7lM

ATP 0.1 1.0 CTP 0.1 0.065 GTP 0.1 0.065 [3H]UTP 0.2 0.065

dATP (hm)dCTP” dGTP dTTP

0.02 0.02 (0.02) 0.2 (0.02) 0.2

0.02 0.02 0.02 0.02

Thymidine 0.17 0.17 Uridine 0.2 1.25

Phospho- enolpyru- vate

NAD’

2.5

Dithiothrei- to1

KC1 MgAcz K.Mops

(PH 7.5)

0.1

0.05

100 100 6 5

20 20

Spermidine

1.0 0.2 0.4 0.2 0.1 0.2 0.2 0.1 0.2 0.2 0.2 0.4

0.02 0.02 (0.02) 0.2 0.2

0.02 0.02 0.02 0.02

0.17 0.17 1.25 1.25

2.5 2.5

0.1 0.1

0.1 5

100 200 100 5 10

20 20 20

2.0 1.0

n Concentrations of hmdCTP are given in parentheses.

first formed on an isotonic agar plate, A. The disc supporting the spheroplasts is then transferred to a hypotonic agar plate, B. The resulting osmotic shock lyses the spheroplasts. In detail, the basic protocol (Method A) is as follows. E coli B” were grown in 31 ml of Hershey broth at 37’C in a rotary shaker bath (generation time, 25 min) to AW = 0.3 (1.2 x 10” cells/ml), infected with T4am M41 (10 plaque forming units/cell), and 17 nCi/ml of [2-‘4C]thymidine (53 Ci/mol) was added. Survivors were determined 1 min later (usually less than 0.1%). Eighteen minutes after infection, cells were chilled in ice water, centrifuged, and resuspended in 1 ml of TGNO buffer; the suspension was transferred to an Eppendorf conical tube and centri- fuged in an SS-34 Sorvall rotor accelerated to 10,006 rpm, maintained at that speed for % min, and then turned off. The supernatant was completely removed and the pellet was resuspended in 200 ~1 of TGNO/Brij 58 (about 1.5 x 10”’ cells/ml).

One microliter of 1ySOzyme solution (I mg/ml in 0.1 M pOtaSSiUm Mops, pH 6.5) was spread on a cellophane disc on agar plate A at 5°C and 1 ~1 of the cell suspension was added. After 30 min at 5°C the disc was transferred to agar plate B and air (at about 20°C) was blown over the disc to evaporate all “excess” liquid. This was judged visually. The “drying” time was 6 to 8 min.

Method A has been modified according to the needs of specific experiments with respect to the following parameters: volume of Hershey or Penassay broth, amount of [14C]thymidine (5 to 30 nCi/ml), cell density at time of infection (0.6 to 1.2 x 10R cells/ml) and density of the final cell suspension, and drying procedure. When- ever the specific values of these parameters are considered to be important, they are given in the legends to the tables and figures.

Section e: RNA Synthesis in Vitro

Cellophane discs were removed from agar plate B and placed on a 50-~1 drop of incorporation mixture. Drops of incorporation mixture were placed in a closed plastic Petri dish which was kept on a temperature-controlled brass plate at 25°C. The temperature inside the drops was 23-24’C. Standard incorporation time was 30 min. RNA synthesis was terminated by transferring a disc into 1.5 ml of stop solution and heating in boiling water for 2 min. The sample, including the cellophane disc, was cooled, precipitated with 1.5 ml of 10% Cl&COOH, filtered through a nitrocellulose membrane, washed with 2.5% Cl:&COOH and 70% ethanol, dried, and counted in a toluene-based scintillation liquid. Radioactivity incorporated into RNA was normalized to lo8 cell equivalents, using the internal stand- ardization provided by the 14C-labeled DNA. The internal standardi- zation factor was determined by precipitating a sample of the infected bacterial culture of known optical density immediately after harvest- ing. The background (which is subtracted in the presented data unless otherwise specified) was determined from discs which had been incubated on a drop of incorporation mixture for 2 min in the presence of 40 mrvr EDTA.

Section f: Analysis of RNA

(i) RNA Sire Determination on Sucrose Gradients-RNA synthe- sis was stopped by placing the disc in 0.3 to 0.5 ml of 1% SDS, 1 mM EDTA, pH 7. The sample was heated in boiling water for 2 min, cooled, and a O.l-ml aliquot was layered on top of a sucrose gradient.

The 5 to 20% (w/v) linear sucrose gradients were prepared in nitrocellulose tubes for the Beckman SW 50.1 rotor over a cushion of 0.4 ml of 40% sucrose solution. All sucrose solutions contained 10 mM Tris.HCl, pH 8, 10 UIM NaCl, 1 UIM EDTA, and 0.1% SDS. After centrifugation for 4% h, 48,000 rpm, 18”C, fractions were collected from the tube bottom. To each fraction (approximately 0.2 ml), 0.5 ml of 10% Cl&!COOH was added and 25 samples were simultaneously filtered through a sheet of GF/C glass fiber paper in an apparatus designed by D. Freifelder (8). (The complete processing time for 25 samples with this device was about 10 min.) Precipitates were washed extensively with 2.5% CLCCOOH, 0.1 M Na4P20i, then with 70% (v/v) ethanol in water. Finally, the whole sheet of GF/C paper was rinsed with ethanol/water after the lid of the filtering apparatus had been removed. The paper was dried and cut to separate the individual samples which were counted in toluene-based scintillation fluid. The amount of radioactive macromolecular material layered on each gra- dient was determined by precipitating and filtering a O.l-ml aliquot of the sample as described below for the gradient fractions. We usually recovered greater than 90% of input material in the gradient fractions.

(ii) DNA-RNA Hybridizations-In vitro RNA synthesis was stopped by placing the cellophane disc in 0.5 ml of 0.1 M EDTA, 0.25% SDS, pH 7.0, and boiling for 2 min. The cellophane disc was removed, %o volume of 1 M sodium acetate, pH 5.2, was added and the sample

by guest on October 28, 2020

http://ww

w.jbc.org/

Dow

nloaded from

Cell-free T4 Late RNA Synthesis 341

was repeatedly extracted with equal volumes of water-saturated phenol at 70°C. After dialysis against 0.1 SSC, the [‘H]RNA was hybridized to the separated strands of T4 DNA (9, 10). The input radioactivity was determined by mixing an aliquot of [“H]RNA sample (usually 50 ~1) with 0.2-ml stop solution, precipitating with 0.2 ml of 10% Cl&CdOH, filtering ontoa nitrocellulose membrane filter, wash- ing with 2.5% Cl&COOH and 70% ethanol, drying, and counting in a toluene-based scintillation fluid. The concentration of r3H]RNA never exceeded 10 rig/ml and it was accompanied by RNA from less than 3 x 10’ cells (this would contribute -18 pg/ml of unlabeled RNA if extracted directly from cells rather than from the discs after prepa- ration and incubation). T4 DNA I and r strands were each used at 10 pg/ml, except for the experiment shown in Fig. 8. In the absence of DNA, 1 to 2% of the radioactivity of the [“H]RNA samples bound to filters and this was subtracted as background. The percentage of r- strand-specific [3H]RNA was calculated as (counts per min hybridized to the r strand) x lOO/(sum of counts per min hybridized to the I and r strands).

Percentage of hybridization efficiency was calculated as 100 x (sum of counts per min hybridized to r and 1 strands)/(input counts per min) x 1.13. The factor 1.13 corrects for the different counting efficiencies of the hybridized and acid-precipitated RNA samples on nitrocellulose membrane filters. This factor was determined by hy- bridizing T4 early in viuo [“HIRNA, which contains only labeled T4 RNA, to a saturating excess of T4 DNA 1 strand and dividing the number of counts per min hybridized ( y) by the number of input acid- precipitable counts per min (z) (y/z = 1.13). Comparable experiments with T4 in viuo late RNA, T4 DNA 1 and r strands, yielded the same correction factor.

In routine assays, with single concentrations of different batches of DNA strands, the hybridization efficiency of in viuo RNA varied between 80 and 95%. We therefore normalized the hybridization efficiency obtained in individual sets of experiments with in vitro RNA to the hybridization efficiency obtained with in viuo T4 late [“HIRNA which was used as a standard. We refer to this normalized quantity, i.e. (per cent hybridization efficiency with [“H]RNA sample) x lOO/(per cent hybridization efficiency with the in uivo T4 probe) as the relative T4 hybridization. We take the relative T4 hybridization to be equal to the percentage of total transcription which is T4- specific. Consequently, we calculate T4-specific RNA synthesis in vitro as: (total RNA svnthesis) x (relative T4 hvbridization)/lOO.

Hybridization-competition and hybridization” analyses were done under the same conditions. Unlabeled in viuo T4 RNA samples, used at concentrations up to 2 mg/ml, were prepared as follows: (a) chloramphenicol (CM) RNA was extracted from cells infected with T4 (5 phage/bacterium) for 5 min at 30°C in the presence of 200 pg/ml of chloramphenicol, the latter added 5 min before infection; (b) early RNA was extracted from cells 5 min after infection at 30°C; (c) late RNA was extracted from cells 20 min after infection. Preannealed early and late (PA) RNA was prepared by annealing 6.4 mg of early RNA with 14.9 mg of late RNA for 4 h at 70°C in 3.6 ml of 2 x SK!.

(iii) RNA-RNA Hybridizations-T4 [‘H]RNA was annealed alone or with an excess of unlabeled T4 in viuo RNA in 0.4 M NaCl, 0.08 M

Tris. Cl, pH 7.5,4 mru EDTA for 3 h at 70°C (11). Samples were then digested in 0.12 M NaCl, 0.023 M Tris.Cl, pH 7.5, 10 mM MgCL, 0.8 mM EDTA with 1 pg/ml of Tl RNase, and 10 ag/ml each of pancreatic RNase and DNase, at 37’C for 90 min. Precipitation and counting of the samples were as described (3). Percentage of RNA-RNA duplex formation was calculated as (counts per min in RNA-RNA duplex) x lOO/(input counts per min). Input counts per min were determined by precipitating an equal amount of [“HIRNA which had been sub- jected to the same conditions as the hybridized sample (see above). Percentage of symmetry of transcription was calculated as: 100 x (per cent RNA-RNA duplex formation with 590 pg/ml of PA RNA)/(relative T4 hybridization).

RESULTS”

Section a: General Properties of the System; Time

,’ Portions of this paper (including some of the results, Figs. 2, 3, 4, and 5, and Tables II, III, and IV) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are available from the Journal of Biological Chemistrv. 9650 Rockville Pike. Bethesda. Md. 20014. Request-Document No. ‘78M-877, cite author(s), and include a check or money order for $2.10 per set of photocopies. Number key for reference to major headings: 1, Introduction; 2, Materials and Meth- ods; 3, Results; 4, Discussion.

Course-The cellophane system’s unconventional constitu- tion is associated with certain peculiarities, which it is well to consider at the outset. 1) The cellophane disc which serves as a mechanical support for the concentrated cell lysate, is a semipermeable membrane. This allows rapid changes of com- position of substrates and other low molecular weight com- ponents to be made by simply transferring the disc from one drop of incorporation mixture to another. On the other hand, even relatively low molecular weight substances added to the incorporation mixture do not act as promptly or as efficiently as would be anticipated for homogeneous systems. High mo- lecular weight substances, such as heparin or RNA polymerase must be added directly to the cell lysate, that is, on top of the cellophane disc. To distribute material uniformly on top of a disc requires care.

2) The lysate on top of the cellophane disc is a nonuniform gel of variable thickness. This implies different accessibility of different parts of the gel to substances diffusing in from the incorporation drop and variably restricts lateral and vertical diffusion of small and large molecules within the gel.

Despite these peculiarities of the cellophane system and its laborious preparation, it has been found more suitable for the transcription of late T4 genes in vitro than other systems that were tested. These included a crude lysate system based on the procedure of Wickner and co-workers (12), a system employing toluenized cells based on the procedure of Peterson and co-workers (13), and a system consisting of highly concen- trated “nucleoid” structures prepared according to a modifi- cation4 of the method of Stonington and Pettijohn (14). Such systems synthesized RNA less efficiently and for only approx- imately 10 min.

Before turning to the details of the experiments on the effects of substrate composition, we want to outline their rationale. The main goal was to generate in vitro RNA syn- thesis which resembled T4 RNA synthesis in uiuo in the late period of infection as closely as possible. Accordingly, we had to optimize RNA synthesis for T4 specificity, high proportion of late RNA, low symmetry, high rate, and duration. T4 late transcription in uiuo is related to T4 DNA replication. There- fore, we also maintained favorable conditions for DNA syn- thesis. The conditions of the latter and the relation between T4 DNA replication and transcription in vitro are the subject of a forthcoming publication.’

Our present understanding of the system evolved from numerous experiments, only a fraction of which are presented here. Optimization of the system was done in many steps, over an extensive period of time. Each of the optimizations that is summarized below was ultimately established by confirmatory experiments in which the relevant parameter alone was varied. In developing the system, we used five different incorporation mixtures (Table I), some of which have similar properties. There are two reasons for this variety: (1) the system can be, and has been, separately optimized for different properties; and (2) optimization of the different components was done in several steps. A judgment of the suitability of the different mixes for different purposes will be given at the end of this section.

The time course of RNA synthesis with two different incor- poration mixtures is shown in Fig. 1. The approximately l- min lag in the onset of RNA synthesis probably represents the time required for the diffusion of substrates to sites of transcription. A linear phase of RNA synthesis follows for 40 min in incorporation mixture A (Fig. la) or for 20 min in incorporation mixture B (Fig. lb). (The differences are due to the substrate composition and not to the different rates of drying the discs in the two experiments; longer drying times

4 R. Wu, unpublished results.

by guest on October 28, 2020

http://ww

w.jbc.org/

Dow

nloaded from

Cell-free T4 Late RNA Synthesis 342

Time Cmin)

20 40 60 Time (mid

FIG. 1. Time course of RNA synthesis with two different incorpo- ration mixtures. a, cell-free systems were prepared from T4am M41- infected E. coli B” by Method A but drying on agar plate B was for 45 min at 5°C (2.1 x 10’ c.e./disc, on the average). Incorporation mixture A, but with hmdCTP, 0.1 mM [3H]UTP, no uridine, and 0.2 mM dithiothreitol, was used. Relative T4 hybridization increases with increasing in vitro incubation time from 80% at 10 min to 100% at 60 min. The inset shows the effect of transferring discs at 60 min (arrow) to a drop of fresh incorporation mixture at 25°C (Cl). Control discs remained on the original drop of incorporation mixture (m). b, system preparation by Method A (2.4 x lo7 c.e./disc, on the average) except that drying of the discs was with 5°C air (drying time, approximately 20 min). Incorporation mixture B was used. The relative T4 hybridi- zation efficiency was 97 + 4% for all RNA samples. n , 0, total RNA synthesis; A, Z-strand-specific T4 RNA, 0, r-strand-specific T4 RNA.

shorten the duration of the linear phase of RNA synthesis, data not shown). The subsequent slowing down is not avoided by transferring a disc to a fresh drop of incorporation mixture (inset of Fig. la). We have observed variations of the degree of subsequent slowdown, depending partly on particular batches of components of the incorporation mixture but also depending on steps in the preparation which we cannot yet identify. With an incorporation mixture that has been opti- mized for extensive T4 RNA synthesis (incorporation mixture E), the initial rate of RNA synthesis of Fig. lb can be sustained for more than 60 min (data not shown). The initial rate for all three mixes is about the same. For Mix E it is 4.0 pmol of UMP or about 12 pmol of nucleotide incorporated/min/lO’ cell equivalents. Fig. 1 also shows the time course for T4 DNA r- and Z-strand-specific RNA synthesis. For our purposes, it is satisfactory to regard r-strand-specific RNA as identical with late RNA and Z-strand-specific RNA as identical with early plus middle RNA (compare “Results, Section e”). In Fig. la, about 50% of the T4 RNA is r-strand-specific during the first 40 min of the in vitro reaction, but this fraction decreases to about 40% at 60 min. In Fig. lb r-strand-specific RNA is continuously about 55% of the total RNA synthesized in vitro. The corresponding in uiuo RNA would be approximately 65% r-strand-specific (compare “Results, Sections e and g”).

The values of relative T4 hybridization (defined under “Materials and Methods, Section f”) of all RNA samples with Mix B (Fig. lb) are close to 1. This signifies that the in vitro RNA is almost entirely, or entirely, T4-specific. This is not true for RNA synthesized with Mix A (Fig. la). In this case, the relative T4 hybridization increases steadily from 80% at 10 min to 100% at 60 min, indicating that incorporation of ribonucleotides initially is not exclusively into T4 RNA. Fur- ther analysis (see below) points to polynucleotide phospho- rylase as being responsible for the initial non-T4 RNA syn- thesis in Mix A (polynucleotide phosphorylase activity de-

clines significantly after the first 10 to 20 min of the in vitro reaction; data not shown). The varying relative T4 hybridi- zation was taken into account when the amount of I- and r- strand-specific RNA was calculated for each point in Fig. lb. Accordingly, the amount of r-strand-specific RNA was calcu- lated as: (total RNA synthesis) x (per cent relative T4 hy- bridization) x (per cent r-strand-specific RNA) x 10e4. The amount of Z-strand-specific RNA was calculated analogously.

Section b: Substrate Requirements-This section is pre- sented in the miniprint supplement which follows this paper.

Section c: Preparation of the Cell-free System-This sec- tion is presented in miniprint at the end of the paper.

Section d: Sedimentation Analysis of the in Vitro RNA Product-The cellophane system, which contains all macro- molecular components of the cells from which it has been derived, has a relatively high level of nuclease activity. In order to observe the rate of T4 RNA decay during the prep- aration of the system and during in vitro RNA synthesis, we labeled E. coli BE from 14 to 18 min after T4 infection at 37°C with r3H]uridine and harvested the cells at 18 min. The system was prepared and in vitro RNA synthesis with unlabeled precursors was allowed for 0, 1, 3, 6, 10, 20, or 40 min, respectively. Samples were collected at these times, heat- denatured, layered on top of sucrose gradients, and centri- fuged. Total ribosomal RNA was used as a sedimentation marker in a separate gradient run under identical conditions. The sedimentation profile obtained from an aliquot of cells taken just before preparation of the in vitro system was comparable with previously reported patterns (18) or showed slightly smaller RNA than some previous estimates (19). At zero time, that is, when the cell-free system had been prepared but there had been no in vitro RNA synthesis, the sedimen- tation profile showed only a slight drift to smaller RNA size. Increasing periods of in vitro RNA synthesis shifted the sedimentation pattern more and more to smaller chain length. We calculated that there is, on the average, approximately one break per lo4 nucleotides of RNA per min.5

When the same experiment was repeated with E. coli AB 105 (RNase II, III-), essentially the same result was obtained.

Despite the considerable RNase activity of the system, it is possible to demonstrate the growth of RNA chains in vitro, although it is not possible to determine their growth rate accurately. A pulse-chase experiment yielded the results shown in Fig. 6. A 3-min pulse with [3H]UTP in vitro at 25°C was followed by different periods of RNA synthesis in the presence of excess unlabeled UTP, which shut off further incorporation of label (data not shown). Chase periods of 1 and 3 min show a shift of the labeled RNA to larger size. At 9 min, degradation of that larger RNA is clearly seen.

In the next experiment, we examine the size distribution of the transcripts created during the course of RNA synthesis in vitro. RNA synthesized with Mix A (but containing 0.2 M

instead of 0.1 M KCl) shows the following sedimentation patterns (Fig. 7~): The “background” sample, obtained by a

’ This rough calculation was done as follows: weight average sedi- mentation constants, SW, were calculated for each gradient (not shown). These were then normalized to the sedimentation constant of the control sample (s,,,~). Since s is proportional to M”.“3 in this medium (20), the ratio of weight average chain length of the control sample to all other RNA samples is given as (.~~~,.s~)(~~‘~‘~‘or (s,,/,,)‘.‘. For swc, we calculated a value of 12.2 S, giving a weight average chain length of 1020 nucleotides. The size distribution of all samples is broad. We, therefore, assumed a ratio of weight to number average chain lengths of 2 for all samples and took 510 to be the initial number average chain length, n,. The best line through the time points of sulc,sw gave n,/n = 2.5 at 30 min, corresponding to the introduction of 1.5 breaks/510 nucleotides in 30 min or 1 break/min/104 nucleotides, assuming zero order kinetics of phosphodiester bond cleavages.

by guest on October 28, 2020

http://ww

w.jbc.org/

Dow

nloaded from

Cell- free T4 Late RNA Synthesis 343

II 1

Fmm “under FIIC!lO” “Yrnbll

FIG. 6. Growth and degradation of in vitro labeled RNA. Cello- phane discs were prepared from T4am M41-infected E. coli B” according to Method A. Discs were incubated 3 min on incorporation mixture A containing 0.2 M KC1 and 50 pM r3H]IJTP (10,000 Ci/mol). Then, the underside of each disc was blotted on filter paper and it was transferred to a fresh drop of incorporation mixture which con- tained 200 pM unlabeled UTP instead of [“HIUTP. Incubation on unlabeled mix was for 0 (direct transfer to stop mix), 1, 3, and 9 min

1 a) 30

1 5~0 b)

1-min incubation in the presence of 40 IIIM EDTA, shows a small amount of radioactivity (presumably bound to protein and nucleic acids) sedimenting between 0 and 7 S. The pattern remains the same for in vitro incubations up to 40 s in the absence of EDTA (data not shown). This is consistent iwith a lag of about 1 min in the incorporation of [3H]UTP into RNA (Fig. 1). One-minute in vitro RNA contains RNA chains with sedimentation constants up to 16 S (i.e. 1500 nucleotides long). This supports the notion that a considerable portion of all RNA chains which grow during the first minutes in vitro have already been initiated in uiuo since, in 20 s, one would expect polymerization of no more than 200 to 300 nucleotides corresponding to a rate of 10 to 15 nucleotides/s for T4 RNA synthesis at 25°C. The same argument can be made for the sedimentation profile for 3-min in vitro RNA (i.e. for 2 min and 20 s of incorporation of [3H]UTP into RNA). The largest components of 9-min in vitro RNA are about 23 S and the median size is about 9 S. Thirty-minute in vitro RNA clearly contains a high proportion of degradation products as indi- cated by the high proportion of RNA that is smaller than 7 S. RNA synthesis with 0.1 M instead of 0.2 M KC1 generates essentially the same size distribution of products (data not shown).

The size distribution of RNA that has been pulse-labeled after a 30-min synthesis period in vitro is shown in Fig. 76. The average sedimentation rate, which is greater than that of the 30-min sample in Panel a, reflects the size distribution of growing RNA chains at 30 min in vitro (with, doubtless, some contribution from degradation). The average chain length increases in the 2- and 3-min samples but decreases again in the 9-min sample of Fig. 7b. The average and maximum size of in vitro RNA is larger when synthesis takes place at high ionic strength (0.4 to 1 M KCl). The sedimentation patterns

in Panels a, b, c, and d. Each disc was then processed as described under “Materials and Methods.” The arrows locate the positions of 23, 16, and 5 S RNA.

Fraction number Fraction number Fraction number

Fm. 7. Size of RNA chains growing in vitro under different con- ditions. Cellophane systems were prepared from T4am M41-infected E. coli B” by Method A, except that no [‘%]thymidine was added and the final resuspension of cells was in 300 ~1 of TGNO/Brij 58. Discs were transferred from agar plate B to 50+1 drops of incorpora- tion mixture A containing 0.2 mru UTP. Incubation was at 25’C for 1, 3, 9, or 30 min. One disc was incubated on incorporation mixture A containing 40 mM EDTA for 1 min (“0 min” sample). Each disc was put into 0.3 ml of 0.1% SDS, 1 mM EDTA (pH 7.5), heated to 100°C for 2 min, and kept on ice until a 100~~1 aliquot was layered on a sucrose gradient (run and processed as described under “Materials

and Methods”). a, incorporation mixture .A with 0.2 M KC1 and [JH]UTP (10,000 Ci/mol). b, RNA synthesis was allowed for 30 min on incorporation mixture A with 0.2 M KC1 and unlabeled UTP. Then, discs were transferred to a drop of the same incorporation mixture but containing [3H]UTP (10,000 Ci/mol). The underside of each disc was blotted on filter paper before transfer to the new drop. Incorpo- ration of [3H]UTP was allowed for 1, 2, 3, or 9 min. c, same as a but with 1 M KCl. Symbols indicate the time period of in vitro RNA synthesis: 0, 0 min (EDTA); 0, 1 min; a, 2 min; A, 3 min; 0, 9 min; W, 30 min. The solid line without data points is the sedimentation profile of E. coli total ribosomal [‘%]RNA (23, 16, and 5 S).

by guest on October 28, 2020

http://ww

w.jbc.org/

Dow

nloaded from

344 Cell-free T4 Late RNA Synthesis

for RNA synthesized in 1 M KC1 are shown in Fig. 7~. The appearance of distinct peaks, including one of very low molec- ular weight material, is characteristic for this RNA. The observed differences between high and low ionic strength RNA could reflect effects on synthesis or degradation of RNA, or both.

Section e: Hybridization Analysis of the in Vitro Synthe- sized T4 RNA-We now examine fidelity of T4 transcription in the cellophane system in detail, using three hybridization methods: 1) hybridization of the in vitro synthesized RNA to the separated strands of T4 DNA; 2) hybridization-competi- tion with various T4 in viva RNAs, and 3) RNA-RNA duplex formation between labeled in vitro and excess unlabeled in vivo RNA.

For this analysis, the in vitro RNA was synthesized with incorporation mixture E for 30 min at 25°C. The composition of Mix E is derived from the optimization experiments of Section b and the preparation of the cell-free system is derived from the optimization experiments of Section c (See “Results” in miniprint). Two in uiuo labeled T4 RNA samples serve as reference. One (Sample 2, Table V) has been labeled from 17 to 19 min after T4 infection at 37°C and represents RNA synthesis at the time of harvesting cells for preparation of the in vitro system. The other (Sample 3, Table V) has been labeled 3 to 5 min after infection and represents T4 early RNA synthesis. The result of hybridizing this RNA to an excess of the separated strands of T4 DNA is shown in Table V. The high hybridization efficiency of the in vitro [3H]RNA indicates that it is almost exclusively T4-specific and the proportion of r-strand-specific RNA is high in both Samples 1 and 2. In view of the low and comparable degree of symmetry of all three RNA samples (see below and Table VI), the r transcripts that are made in vitro must come from late genes; this in turn means that the proportion of late RNA synthesis is only 20 to 25% lower in vitro than it is in vivo.

Further analysis of this in vitro RNA by hybridization- competition employs four unlabeled competitor RNA probes (“Materials and Methods, Section f ‘): ( a) “chloramphenicol” (CM) RNA mainly contains RNA sequences proximal to T4 early promoters and contains essentially no anti-message RNA; (b) “early” RNA contains essentially all the sequences which are transcribed from the 1 strand in the early region of the T4 genome, including low concentrations of I-strand-spe- cific anti-late RNA; (c) “late” RNA contains essentially all the late gene messages, many of the early RNA sequences, and low concentrations of anti-late as well as anti-early RNA; (d) “preannealed early and late” (PA) RNA contains essen- tially all early “sense” sequences in single-stranded form but sequesters anti-message in double-stranded RNA. The results of hybridization-competition experiments with limiting amounts of T4 I strand DNA are shown in Fig. 8. The competition patterns are very similar (Panels a and c). It is significant that CM RNA competes with labeled in vivo late RNA and with the in vitro RNA less completely than with

labeled in uivo early RNA (for the last named RNA, we observe a competition plateau at about 15% residual hybridi- zation; data not shown). This difference in competition ability reflects differences in the synthesis of early-promoter-proxi- mal RNA: the Z-strand-specific RNA which is made late in infection, probably is initiated at other than early promoters, i.e. at middle promoters and possibly at a small number of late promoters. This result suggests that the cellophane sys- tem may also be useful for studying T4 middle transcription.

The competition patterns with T4 r strand DNA are also comparable for labeled late in uivo and in vitro RNA (Fig. 8, Panels b and d). However, about 4% of the in vitro RNA is not competed out and must therefore be rarely present in, or altogether absent from, in vivo RNA. In summary, by the criteria of hybridization-competition analysis, our in vitro RNA resembles the corresponding in vivo RNA very closely.

T4 transcription in vivo is not strictly asymmetric. At about 2 min after infection, when middle transcription starts, anti- messenger RNA also first appears. It is this anti-messenger content of T4 transcription in vivo which complicates the use of asymmetry of transcription as an absolute criterion for transcription fidelity. However, by comparing labeled in viuo and in vitro RNA and by using the PA and CM RNA probes in RNA-RNA duplex formation, it is possible to characterize the quality and quantity of anti-message synthesis. CM RNA is almost completely asymmetric but its usefulness as a probe for anti-message synthesis is limited because CM RNA is transcribed from only a part of the early region of the T4 genome. PA RNA is the most sensitive probe for anti-message, since it should contain a complete set of sense sequences but little or no anti-sense sequences in the form of single-stranded RNA.

The results of the RNA-RNA duplex formation analysis are shown in Table VI. A large fraction of the label in Sample 2, the in viuo late RNA, is driven into RNA-RNA duplexes by the anti-late RNA in unlabeled in viuo early RNA (Column 3). In PA RNA, this anti-late RNA has been predriven into RNA-RNA duplexes and does not react with the labeled RNA (Column 4). The residual RNA-RNA duplex formation may be due to late transcription on the anti-sense r strand of early genes (Columns 2 and 4). The in vivo early RNA (Sample 3) shows very low symmetry with all three of these probes. RNA made with E. coli holoenzyme and intact double-helical T4 DNA in vitro (Sample 4) is, of course, highly asymmetric (Column 5) but contains a significant, though low proportion of anti-late sequences (Column 4) due to anti-late RNA syn- thesis along the I strand (11, 21, 22). RNA made with yeast RNA polymerase II and denatured T4 DNA (Sample 5) or with E. coli core polymerase and “nicked” T4 DNA (Sample 6) is almost, if not completely, symmetric: the 40 to 48% r transcription (Column 5) is matched by a very high degree of complementarity to PA RNA (Column 4) and by considerable complementarity to CM RNA (Column 2). Examining the in vitro RNA (Sample 1) in comparison with these other mate-

TABLE V

Hybridization analysis of T4 in vitro and in vivo RNA RNA Sample 1 was synthesized with Mix E at 25°C in a cellophane system prepared according to Method A. The final resuspension of cells

was in TGNO buffer and there were, on the average, 3.4 x 10’ c.e./disc. Labeled in vivo RNAs were prepared as described in Ref. 7.

Sample Period of labeling Specific activity Input Relative T4 hybridi- zation

% )’ transcription

CPdM CPm 1 30 min in vitro 9.4 x 10” 2330 50 2 17 to 19 min in vivo (37°C) 8.8 x IO4 1750 (1:)" 65 3 3 to 5 min in vivo (30°C) 4.2 x 10” 790 (100)” <1

” Value for Sample 1 normalized to Samples 2 and 3 as described under “Materials and Methods, Section f.”

by guest on October 28, 2020

http://ww

w.jbc.org/

Dow

nloaded from

Cell-free T4 Late RNA Synthesis

Unlabelled RNA (mg/ml)

FIG. 8. Hybridization-competition analysis of T4 in vitro and in uiuo RNA. In vitro [“HIRNA (Sample 1 of Table VII) and in vim late [3H]RNA (Sample 2 of Table V) were hybridized to T4 DNA 1 strands ( Panels a and c) or r strands ( Panels b and d) in the absence or presence of different competitor RNAs. T4 DNA strands (1.5 pg/ml) and 2.5 or 100 rig/ml of RNA for Samples 1 and 2, respectively. Concentrations of competitor RNA are indicated on the abscissa. One

rials, emphasizes its similarity with in viva late RNA (Sample 2) and its only slightly greater degree of symmetry than in uiuo late RNA.

Section f: Initiation of Late T4 Transcription Occurs in the Cellophane System-Initiation of late transcription is crucial for the usefulness of this in viva system. We therefore provide evidence for such initiation in two independent ways. First, the time course of RNA synthesis in the absence and presence of the transcription-initiation inhibitor, rifampicin, is examined. We have already noted that the in vitro system is not completely inhibited by rifampicin. Fig. 9 shows that RNA synthesis levels off prematurely if rifampicin is present in the incorporation mixture from the beginning (Curve b, A), while RNA synthesis continues in the control lacking rifampicin (Curve a, n ). The shape of Curve b and the height of its plateau suggests that the system contains many initiated RNA chains when the in vitro incubation starts. This is to be expected since the distribution of nascent RNA chain lengths must be, to a first approximation, random upon harvest of the cells and they should be completed in vitro. Some chains may also be initiated upon transfer of the discs onto the incorpo- ration mixture before the drug efficiently blocks further initi- ation. When discs are transferred after 10 min of [%JUTP incorporation to a second rifampicin-containing, but otherwise identical, drop of incorporation mixture, an intermediate re- sult is obtained (Curve c; 0). Analysis of the RNAs synthe- sized in 60 min under the conditions described for Curves a, b, and c all show the same proportion of r- to l-strand transcription (-0.4:0.6). This result indicates that the differ- ence between the plateaux of Curves b and e is a measure of both early and late RNA chain initiation during the first 10 min of incubation.

345

Unlabelled RNA (mg/ml)

hundred per cent hybridization, in the absence of competitor is 254 and 328 cpm for RNA Sample 1 in Panels a and 6, respectively. The input is 1165 cpm/sample; a background of 9 cpm has been subtracted. For RNA Sample 2 in Panels c and d, 100% hybridization is 230 and 298 cpm, respectively. The input is 875 cpm/sample and a background of 16 cpm has been subtracted. Competitor RNA: 0, CM; 0, early; W, late; A, PA.

Another experiment which provides evidence for initiation of late transcription in vitro is based on the kinetics of T4 RNA synthesis at different ionic strengths (Fig. 4). In the presence of 800 mM KCl, RNA synthesis levels off approxi- mately as it does in the presence of rifampicin (compare Figs. 4 and 9, Curve c). We interpret this to mean that 800 mM KCl, like rifampicin, allows elongation but not initiation of RNA chains. However, the blocking effect of KC1 is completely and rapidly reversible (Fig. 10). RNA synthesis resumes upon transfer of a disc from an incorporation mixture containing 800 mM KC1 (0) to one with 200 InM KC1 (m). The initial rates of RNA synthesis at 800 mM KC1 and after transfer to 200 mM KC1 are comparable. RNA labeled from the time of transfer to low ionic strength until 60 min later had a r: 1 transcription ratio of 0.35:0.65. The corresponding ratio for RNA labeled from 0 to 30 min in 800 mM KC1 in the same experiment was 0.46Zo.54.

That resumption of RNA synthesis after transfer to low ionic strength due to initiation of new RNA chains is demon- strated by its complete inhibition by rifampicin (Fig. 10, 0). Streptolydigin has essentially the same effect as rifampicin (Fig. 10, a). We conclude from the experiments represented in Figs. 8 and 9 that initiation of early as well as late RNA chains does occur with high efficiency in the cellophane sys- tem.

For some time, we were not consistently able to observe rifampicin- and streptolydigin-dependent inhibition of RNA synthesis. Upon further analysis, we found three reasons for this irreproducibility. 1) Systems prepared with Brij 58 are less sensitive to drug action. This may be due to the formation of mixed micelles between the detergent and the drugs. 2) Incorporation mixture A usually requires higher drug concen-

by guest on October 28, 2020

http://ww

w.jbc.org/

Dow

nloaded from

346 Cell-free T4 Late RNA Synthesis

TABLE VI

RNA-RNA duplex formation between drfferentpreparations of T4 in vitro and in vivo RNA

[“H]RNA Samples 1 to 3 are identical with those in Table V. RNA Sample 4 was synthesized in 30 min at 25°C with 0.51 )~g of T4 DNA, 50 ).d of incorporation mixture B and 0.64 ag of holo-RNA polymerase. RNA Sample 5 was synthesized in 400 ~1 of 50 mM Tris, pH 7.5, 6 mM

NaF, 0.7 mM dithiothreitol, 550 PM CTP and GTP each, 930 PM ATP, 32 PM r3H]LJTP (10 Ci/mol), 1.5 mM MgClz, 1.9 mM MnC12, 50 mM (NH&S04 containing 60 pg/ml of heat-denatured T4 DNA and RNA polymerase II from Saccfzaromyces cerevisiae (a gift from S. Fuhr- man, Department of Biology, University of California, San Diego), for 30 min at 30°C. RNA Sample 6 was synthesized in 100 ~1 of incorporation mixture B containing 2.55 pg of T4 DNA, 0.2 pg of pancreatic DNase, 7.55 pg of E. coli core RNA polymerase for 30 min at 30°C. RNA-RNA duplexes were formed with 5, 200, 19,2,2, and 5 rig/ml of [“H]RNA for Samples 1 to 6, respectively. CM, early, late, and PA unlabeled RNA were used at concentrations of 1650, 1570, 1460, and 1600 yg/ml, respectively.

Labeled RNA driven into RNA-RNA duplexes with unlabeled in uiuo RNA: % r-tram

Sample ‘H-labeled RNA scription

1 2 3 4 ~- 5

9j-ecb Timehin)

30 60 90 Time imin)

none CM early

% PA -

1 30 min in vitro 0.6

2 17 to 19 min in

vivo 1 1

5.3 32.6 4.7 50

3.7 28.9 2.9 65

3 3 to 5 min in oivo 0.3

4 In vitro RNA: E. coli holopolymer- 1 2 ase and double ’ helical T4 DNA

0.5 0.7

35.3

47.1

0.9 <I

FIG. 9 (left). Inhibition of RNA synthesis by rifampicin. The in vitro system was prepared according to Method A (0.8 x 10’ c.e./disc, on the average). Discs were transferred directly from the agar plate B (5°C) onto drops of incorporation mixture (25°C) and incubated for the times indicated. Curve a (m), incorporation mixture A, but with 200 mM instead of 100 mM KCI; Curve b (A), incorporation mixture A with 200 mM KC1 and 400 pg/ml of rifampicin; Curve c (O), after a lo-min incubation on incorporation mixture A with 200 IIIM KCl, discs were transferred to the same incorporation mixture which contained, in addition, 400 pg/ml of rifampicin. The underside of each disc was biotted on falter paper before it was placed on the new drop of incorporation mixture. All points represent the average of two experiments.

5 Zn vitro symmet- ric RNA: yeast RNA polymerase 6.0 II and denatured T4 DNA

6 In vitro symmet- ric RNA: E. coli core polymerase 4.8 and nicked T4 DNA

18.4

27.9

4.7 2

40.5 48

36.1 40

trations (especially of streptolydigin) to obtain a given inhib- itory effect. We have not traced the components of Mix A which are responsible for this effect, but it is not explained by the limited but clearly observable polynucleotide phosphoryl- ase activity that is permitted by Mix A. 3) Osmosis has a strong impact on the rate of dispersion of the drugs through the cellophane membrane. For example, in the experiment in Fig. 10, the osmotic effect that accompanies the shift from 800 to 200 mM KC1 accelerates the diffusion of the drugs from the incorporation mixture to the RNA polymerase. If discs are transferred to isotonic or hypertonic drops of incorporation mixture with inhibitor, drug action is delayed (data not shown).

There are two further reasons for concluding that initiation of late T4 RNA synthesis occurs in vitro. One is that the relationship of the size distribution of the in vitro RNA product (Fig. 7), to the time course of RNA synthesis (Fig. 1) requires that new RNA chains be started continuously. The second argument concerns the transcription of late genes by added, purified T4-modified RNA polymerase. In such exper- iments, different exogenous RNA polymerases stimulate tran- scription in a way which is consistent with the regulation of T4 late transcription in uivo (3).

FIG. 10 (right). Reinitiation of RNA synthesis at lower ionic strength and its inhibition by rifampicin or streptolydigin. The in vitro system was prepared according to Method A (1.2 x lo7 c.e./disc, on the average). RNA synthesis was with Mix A with 800 mM KC1 (0). After incubation for 30 min (25”C), six discs were transferred to drops of Mix A containing 200 mrvr KC1 (m). During transfer, the underside of each disc was briefly blotted on filter paper to avoid carryover of incorporation mixture from one drop to the other. Other discs were transferred to drops of Mix A (200 mM KCl) containing 400 pg/ml of rifampicin (0) or streptolydigin (A). Each point repre- sents the average result from two discs.

Section g: Development of the Capacity to Make T4 Late RNA in Vitro-The potentiality for T4 late RNA synthesis in vitro develops after phage infection as shown in Fig. 11. The capacity for total in vitro RNA synthesis reaches a maximum soon after infection and decreases thereafter (m). However, the capacity for late transcription (0) first appears between 5 and 10 min and reaches a maximum about 15 min after infection. Total RNA synthesis in uivo drops immedi- ately after T4 infection, increases again after 1 to 2 min until it reaches a maximum at about 6 min and declines thereafter with a half-time of approximately 10 min. Late transcription starts in viuo about 9 min after infection at 30°C. Thus, the capacity for late transcription in vitro follows late transcrip-

tion in vivo. The capacity for [3H]TTP incorporation in vitro (0) de-

creases immediately after infection, rises to a maximum at about 10 min and then declines gradually. Host DNA synthe- sis in vivo is shut off very soon after phage infection; T4 DNA synthesis starts about 5 min after infection and continues thereafter. The sharp decrease of the in uitro [3H]TTP incor- poration capacity after 10 min does not reflect the in uivo situation and we do not know the reason for this discrepancy. However, the increase in the capacity for [3H]TTP incorpo- ration in vitro precedes the appearance of late transcription capacity in vitro just as T4 DNA replication precedes late transcription in vivo.

by guest on October 28, 2020

http://ww

w.jbc.org/

Dow

nloaded from

Cell-free T4 Late RNA Synthesis 347

vitro system for T4 middle transcription. The two kinds of systems are prepared in quite different ways and a comparison

-I 40 s

I -2

I, ' I'

o-u , I I J 0 IO 20 30

Time after infection (mid

FIG. 11. Development of the potentiality for in vitro RNA and DNA synthesis during T4 infection. E. coli B” were grown in 80 ml of Hershey broth in the presence of 1.5 $i of [14C]uracil (60 Ci/mol) to a Asao = 0.2 and infected with T4am M41 as described under “Mate- rials and Methods.” Aliquots of the culture were removed at 5 min before or 2, 10, 15, 20, and 30 min after infection and immediately chilled by pouring on frozen medium and shaking in ice water. Cell- free systems were prepared according to Method A (1.5 x lo7 c.e./disc, on the average). In vitro incorporation of [3H]UMP and [“H]?MP were measured on separate discs. [3H]TTP had a specific activity of 500 Ci/mol. Incorporation mixture B was used with 100 pM CTP, GTP, and UTP. In vitro incubation was for 10 min at 25°C. Total r- strand-specific UMP incorporation was obtained for each point by multiplying total UMP incorporation by the fraction of r-strand- specific RNA. The relative T4 hybridization of the in vitro [“H]RNAs varied between 90 and 100%. Percentage of symmetry was 4.4, 3.5, 5.4, 6.9, 7.6, 9.6% of total input radioactive label for the samples collected from 2 to 30 min, after infection, respectively. w, total UMP incorporation; M, r-strand-specific UMP incorporation; w total TMP incorporation.

DISCUSSION

The in vitro system we have described exhibits the tran- scription properties of the phage T4-infected cells from which it is derived. It is capable of initiating the synthesis of new RNA chains in vitro, roughly one-half of which are transcribed from the T4 DNA r strand. The rate of total RNA synthesis and initiation in vitro is relatively high, when compared with the in viva rate.

According to several criteria, the fidelity of transcription in vitro is also remarkably high. The relative proportion of r transcription is only 20 to 30% less than it is in uivo and when appropriately optimized, the asymmetry of the in vitro tran- scripts is almost identical with that of in viuo transcripts. The character of the in uitro transcripts, as judged by hybridiza- tion-competition, also closely resembles that of in viuo late RNA. The resemblance involves not only the late transcripts read off the T4 r strand but also the I strand transcripts which are mainly composed of those early and middle RNA species which are already present in cells before viral DNA replication begins. The pattern of I transcription changes in uivo as a result of what we have previously called the “early regulatory event” (1). The resemblance between I strand transcripts that are made in vitro and those that are being made in uiuo at the time of collecting the cells, suggests that the cellophane disc system will also be useful for studying T4 middle transcription. Brody and collaborators (23) have already constructed an in

between them may prove instructive. In the preceding section, we have emphasized the conditions

which optimize the performance of the cellophane disc system. The absence of any single crucial contribution means that some late transcription can be obtained under many condi- tions, but also makes fine tuning more, rather than less, laborious. Moreover, the performance of the in uitro system can be validly judged by different criteria: DNA synthesis, total extent of T4 transcription, initial rate of transcription, asymmetry, fraction of r transcripts, depression of nontran- scriptive polymerization of ribonucleotides, and sensitivity to inhibition by antibiotics. Optimization according to different criteria leads to somewhat different conditions of synthesis. However, conditions which represent a reasonably satisfactory compromise for general experiments can also be specified and have been discussed at the end of “Results, Section b” in miniprint.

RNA is depolymerized by nucleases as it is synthesized in the cellophane disc system. This, and the limited number of cells that are lysed on a cellophane disc, may make it difficult to construct a coupled system for DNA-dependent protein synthesis without extensive modification. However, the sys- tem is adaptable to certain kinds of complementation experi- ments. Those which we have found most successful thus far have involved DNA replication proteins and RNA polymer- ases containing the T4-specific regulatory subunits (3).’

As far as we are able to determine, the cellophane system is strikingly better for T4 late transcription than any of the conventional alternative systems that have previously been tried (24-28). The advantages of the present system may have to do with the high concentrations of all components or with the mechanical requirements of T4 late gene expression (27), or both. A requirement for components at high concentration might reflect participation of assemblies of weakly bound components in T4 late transcription. The E. coli cytoplasm is a densely packed gel of protein and nucleic acid (29) and the cellophane disc comes much closer to achieving such concen- trations of reacting components than does any other in vitro system. If there are mechanical requirements of T4 late tran- scription, they might be due to constraints on the tertiary structure of DNA. The analysis of such requirements could lead us to a better understanding of DNA competence for T4 late gene expression.

Acknowledgments-We thank M. Filip for technical assistance, C. and K. G. Lark for their introduction to the cellophane system, and S. Fuhrman and K. Jacobs for critically reading the manuscript.

REFERENCES

1. Rabussay, D., and Geiduschek, E. P. (1977) in Comprehensive Virology (Fraenkel-Conrat, H., and Wagner, R. R., eds) Vol. 8, pp. 1-196, Plenum Press, New York

2. Schzdler, H., Otto, B., Niisslein, V., Huf, J., Herrmann, R., and Bonhoeffer, F. (1972) J. Mol. Biol. 63. 183-200

3. Rabussay, D.; and Geiduschek, E. P. (19j7) Proc. Natl. Acad. Sci. U. S. A. 74,5305-5309

4. Hurlbert, R. B., and Furlong, N. B. (1967) Methods Enzymol. 12, 193-202

5. Alegria, A. H. (1967) Biochim. Biophys. Acta 149, 317-324 6. Randerath, K., and Randerath, E. (1967) Methods Enzymol. 12,

323-347 7. Bolle, A., Epstein, R. H., Salser, W., and Geiduschek, E. P. (1968)

J. Mol. Biol. 31, 325-348 8. Freifelder, D., and Brown, A. G. (1976) Anal. Biochem. 74,

154-159 9. Guha, A., and Szybalski, W. (1968) Virology 34,608-616

10. Wu, R., and Geiduschek, E. P. (1975) J. Mol. Biol. 96, 513-538 11. Geiduschek, E. P., and Grau, 0. (1970) in RNA Polymerase and

by guest on October 28, 2020

http://ww

w.jbc.org/

Dow

nloaded from

348 Cell-free T4 Late RNA Synthesis

Transcription (Silvestri, L., ed) pp. 190-203, North-Holland Publishing Co., Amsterdam

12. Wickner, W., Brutlag, D., Schekman, R., and Kornberg, A. (1972) Proc. Natl. Acad. Sci. U. S. A. 69,965-969

13. Peterson, R. L., Radcliffe, C. W., and Pace, N. R. (1971) J. Bacterial. 107, 585-588

14. Stonington, 0. G., and Pettijohn, D. E. (1971) Proc. Natl. Acad. Sci. U. S. A. 68, 6-9

15. Fuchs, E., Millette, R. L., ZiIlig, W., and Walter, G. (1967) Eur. J. Biochem. 3, 183-193

16. Imae, Y., and Okazaki, R. (1976) J. Virol. 19,435-445 17. Huang, W.-M., Ngo, D., Olivera, B., and Mikkelsen, D. (1976)

Fed. Proc. 35, 1418 Abstr. 18. Asano, K. (1965) J. Mol. Biol. 14, 71-84 19. Adesnik, M., and Levinthal, C. (1970) J. Mol. Biol. 48, 187-208 20. Boedtker, H. (1968) J. Mol. Biol. 35,61-70

21. Notani, G. W. (1973) J. Mol. Biol. 73,231-249 22. Frederick, R. J., and Snyder, L. (1977) J. Mol. Biol. 114,461-476 23. Thermes, C., Daegelen, P., De Fraciscis, V., and Brody, E. N.

(1976) Proc. Natl. Acad. Sci. U. S. A. 73,2569-2573 24. Hall, B. D., and Crouch, R. (1968) in The Biochemistry of Virus

Replication (Laland, S. G., and Froholm, L. O., eds) pp. 49-59, Universitets Forlaget, Oslo

25. Snyder, L. R., and Geiduschek, E. P. (1968) Proc. Natl. Acad. Sci. U. S. A. 59,459-466

26. Travers, A. A. (1970) Cold Spring Harbor Symp. Quant. Biol. 35,241-252

27. Jdrstad, K., Haarr, L., and Nygaard, A. P. (1973) Ninth Interna- tional Congress of Biochemistry, July 8 to 9, 1973, Stockholm, 194 Abstr., Svenska Kemistaamfundt, Stockholm

28. Maor, G., and Shalitin, C. (1974) Virology 62, 500-511 29. Kennell, D., and Riezman, H. (1977) J. Mol. Biol. 114, l-21

by guest on October 28, 2020

http://ww

w.jbc.org/

Dow

nloaded from

Cell-free T4 Late RNA Synthesis

J

: : 81

P c ::

by guest on October 28, 2020

http://ww

w.jbc.org/

Dow

nloaded from

D Rabussay and E P GeiduschekRNA synthesis.

Construction and properties of a cell-free system for bacteriophage T4 late

1979, 254:339-349.J. Biol. Chem. 

  http://www.jbc.org/content/254/2/339Access the most updated version of this article at

 Alerts:

  When a correction for this article is posted• 

When this article is cited• 

to choose from all of JBC's e-mail alertsClick here

  http://www.jbc.org/content/254/2/339.full.html#ref-list-1

This article cites 0 references, 0 of which can be accessed free at

by guest on October 28, 2020

http://ww

w.jbc.org/

Dow

nloaded from