Transcription of denatured T4 DNA

636

BIOCHIMICA

ET BIOPHYSICA

ACTA

BBA 96128

TRANSCRIPTION

JOHN

OF DENATURED

T4 DNA

0. BISHOP

EPigenetics Britain) (Received

Research Group,

Department

of Genetics,

Edinburgh

University,

Edinburgh

(Great

August znd, 1968)

SUMMARY

The synthesis of RNA is described, using denatured T4 DNA and ~ic7ococca4s RNA polymerase. In the reaction mixture resistance of newly synthesised RNA to ribonuclease is initially very high (70-80 %) and falls with time of incubation to reach a steady level in the region of 50 %. A DNA-RNA duplex is synthesised, its RNA/DNA ratio rising to a maximum of about 0.5. Single-stranded RNA and RNA-RNA duplex are also synthesised. Both DNA-RNA and RNA-RNA duplexes show reasonably sharp melting profiles. All molecules of DNA are involved in duplexes with RNA. When the DNA is broken to 6-S fragments, no separation of DNA from DNA-RNA hybrid is observed, showing that each DNA fragment carries RNA. It is proposed that approximately half the DNA between adjacent polymerase attachment sites is transcribed. Measurements by DNA-RNA hybridisation of the homology between messenger RNA and RNA synthesised on the denatured template are in agreement with this. When preformed DNA-RNA hybrid is reincubated with RNA polymerase, RNA is released from the hybrid by a process which is dependent upon the occurrence of RNA synthesis. The RNA released is partly ribonuclease sensitive, partly resistant. On the basis of these experiments a mechanism is proposed to explain the synthesis of RNA-RNA duplex.

lysodeikticus

INTRODUCTION

When the synthesis of RNA in vitro by Escherichia coli RNA polymerase is primed by the single-stranded DNA of bacteriophage yX174, a DNA-RNA duplex is synthesised enzymatically1-3. This duplex, which we shall call ‘enzymatic hybrid’, is resistant to attack by ribonuclease 2- 3. It is made up of approximately equal parts of RNA and DNA and its buoyant density in both CsCl and Cs,SO, density gradients is intermediate between those of RNA and qX174 DNAI-3. The uniquely interesting property of the enzymatic hybrid is its equal content of RNA and DNA. If, as is supposed, these are correctly base-paired, the RNA will contain sequences complementary to all the sequences of the DNA in equal proportion. RNA of this sort provides an extremely useful control material for measurements of homology beBiochim.

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TRANSCRIPTIONOF DENATUREDT4 DNA

637

tween DNA and RNA by techniques such as annealing at high temperature and salt. In the first report of enzymatic DNA-RNA hybrid4, denatured double-stranded DNA was used as primer. The hybrid was shown to be resistant to ribonuclease, and to have a buoyant density in Cs,SO, gradients intermediate between those of RNA and DNA. In the present paper, the synthesis of RNA on a template of denatured T4 DNA is described. The RNA polymerase used is that purified from Micrococcus lysodeikticus5. The properties of this system differ in several important respects from those of the pXI74 DNA-E. coji polymerase system.

METHODSANDMATERIALS Materials

Spray-dried Micrococcus lysodeikticus was obtained from Cambrian Chemicals Ltd., London. Frozen E. coli, strain MRE 600 was kindly supplied by the Microbial Products Division of the Microbiological Research Establishment, Porton. [14C]ATP was from the Radiochemical Centre, Amersham, unlabelled ribonucleotides from Sigma Chem. Co., London, ribonuclease (3 x crystallised) and ribonuclease-free desoxyribonuclease from CalBiochem., London.

RNA polymeyase

Micrococcus RNA polymerase was prepared by the method of NAKAMOTO et al.j, E. coli polymerase by the method of CHAMBERLINAND BERGS, except that 0.01 M

Tris-HCl buffer was substituted for phosphate buffer during elution from DEAEcellulose. Both enzymes were stored at -15’ in 0.05 M Tri-HCl (pH 7.5) containing 50 y0 glycerol. 5 mM dithiothreitol was added to the purified E. coli polymerase. The enzymes were assayed with [14C]ATP as substrate under the conditions described by NAKAMOTOet aL5, except that native T4 DNA was used as primer. Because there is an optimum DNA/enzyme ration with T4 DNA, the same level of enzyme was assayed over a range of DNA concentrations, and activity calculated from the optimum DNA level. The unit of activity is the amount of enzyme catalyzing the incorporation of I nmole of ATP into acid-insoluble form in IO min at 30’. I unit measured with T4 DNA as primer is equivalent to z units measured according to NAKAMOTOet aL5. Following incubation, acid-soluble nucleotides were removed by the filterpaper disc method’, and the radioactivity of the discs measured in a liquid scintillation counter.

DNA

Coliphage T4 was grown on E. cob B and purified by ribonuclease and deoxyribonuclease treatment and differential centrifugation. S2P-labelled T4 was grown on E. coli B in low-phosphate medium and purified by differential centrifugation and banding in 46 y0 CsCl at 35 ooo rev./min in the Spinco S.W. 39 rotor. To label the phage DNA with 14C, bacteria were grown on 3XD (ref. 8) containing 0.5-1 PC [14C]uracil per ml. 14C-labelled phages were also banded in CsCl. DNA was prepared from the purified phage by phenol extraction, the phenol was removed with ether and Biochim.

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the ether with N,, and the DNA was dialysed overnight at o” against 0.01 M TrisHCl (pH 7.5, 0.01 M NaCl). Several preparations examined in the analytical ultracentrifuge were found to have s values between 25 and 30 S, indicating a population of quarter molecule9.

Sydhesis of RNA using denatured DNA T4 DNA was diluted to 50 ,ug/ml in 0.01 M NaCl, 0.01 M Tris-HCl (pH 7.5, 20') and denatured at 98-99” for 7 to 20 min. The denatured DNA was poured into a glass beaker standing in ice. The reaction mixture for the synthesis of RNA (ref. 5) contained, in pmoles/ml: Tris-HCl (pH 7.5 at 30’), IOO; MnCl,, 2.5; spermidine, 1.6; GTP, UTP and CTP, 0.8 each; [i4C]ATP (I C/mole or about 630 counts/min per nmole), 0.4; together with 20 pg/ml heat denatured T4 DNA and RNA polymerase. Incubation was at 30~.

Buoyant density gradient centrifugation Reaction mixtures were adjusted to 0.1 M NaCl and 0.05 y0 sodium lauryl sulphate, held for 2 min at 37’ and added to 2.17 g of Cs,SO, together with 0.1 M Tris-HCl buffer, pH 7.5, at 30°, where necessary, to give a total solution weight of 5 g. The solutions were centrifuged for IO min at 15 ooo rev./min in the S.W. 3g Spinco rotor. The clear solution was removed by piercing the bottom of each tube, leaving behind a floating film which contained the sodium lauryl sulphate. The solutions were placed in a second series of centrifuge tubes, overlaid with liquid paraffin and centrifuged at 33 ooo rev./min for 60 h at approx. 15”. Control experiments with phenol-extracted preparations showed that the buoyant densities of nucleic acids were not affected by components of the reaction mixture in the presence of sodium lauryl sulphate. Samples were collected by piercing the tubes, precipitated with z ml cold 5 o/0 trichloroacetic acid in the presence of 250 pg carrier yeast RNA and after 20 min at o0 collected on 2 cm diameter Oxoid membranes. The membranes were washed with 16 ml 5 o/0trichloroacetic acid, ethanol-ether (I : 2, v/v) and ether, dried, and counted in a liquid scintillation counter. In many cases samples were treated for 30 min at 37” with I ml of 0.3 M NaCl, 0.03 M Tris-HCl (pH 7.5,20”) containing 5 pg/ml ribonuclease, prior to trichloroacetic acid precipitation. In these cases 0.1 ml of 50 o/0 trichloroacetic acid was added, followed after a few seconds by the RNA carrier.

Sucrose

gradients

The sample was adjusted to 0.1 M NaCl and 0.05 y0 sodium lauryl sulphate, mixed with I mg rabbit reticulocyte ribosomes suspended in the same solution, and held at 37’ for 2 min. The total solution (I ml) was layered on a 30 ml linear 15-30 y0 sucrose gradient containing 0.05 o/o sodium lauryl sulphate, 0.1 M NaCl, 0.01 M TrisHCl (pH 7.5, 20’) and centrifuged for 18 h at 23 ooo rev./min in a Spinco S.W. 25 rotor at 20”. 1.5 ml fractions were collected from the bottom of the tube and diluted with z ml H,O. Absorbances were measured at 260 m,u to locate the ribosomal RNA components. The samples were precipitated with 0.4 ml 50 o/otrichloroacetic acid in the Biochim.

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TRANSCRIPTION

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T4 DNA

639

presence of carrier. After IO min at room temp. they were collected branes, and washed and counted as described above.

on Oxoid mem-

Ribonuclease resistance Samples of the reaction mixture were diluted with 4 vol. of 0.35 M NaCl, 0.035 M Tris-HCl (pH 7.5, zoo) and incubated with 0.2-5 pg/ml ribonuclease for 30 min at 37”. RNA was precipitated with trichloroacetic acid, washed, and counted as described above. The ribonuclease was heated to 80” for IO min in 0.15 M NaCl, 0.015 M sodium citrate, pH 5, to destroy desoxyribonuclease activity.

Sonication Aliquots of the reaction mixture were adjusted to 0.1 M NaCl and 0.05 y0 sodium lauryl sulphate and held at 37” for 2 min. z ml portions were chilled in ice and sonicated using a Dawe sonicator set at position 8. 15 second pulses were given, and between pulses the samples were left for 5 min in the ice bath.

DNA-RNA hybridisation The membrane filter method tails of the exact

hybridisation

of GILLESPIE

technique

AND SPIEGELMAN~~

and of the

preparation

was used.

of RNA

De-

are given

elsewhere11,12.

RESULTS

Products synthesised using denatured T4 DNA as primer To investigate the RNA products synthesised using Micrococcus RNA polymerase and denatured T4 DNA, reaction mixtures were resolved by centrifugation to equilibrium in Cs,SO,. The T4 DNA was labelled with 32P and the newly synthesised RNA with [14C]ATP. Fig. I shows a typical experiment of this sort. At all times RNA was found in the region of the DNA band. The amount of this DNA-associated RNA increased with time of incubation, but reached a steady level before being eclipsed by the increasing RNA band. Under the standard conditions of ribonuclease treatment used, about 80 y0 of the DNA-associated RNA forms a hybrid duplex with the DNA, with similar properties to the vX174 DNA-RNA hybrid1-3. The synthesis of RNA using Micrococcus RNA polymerase and denatured T4 DNA differed in two ways from RNA synthesis with E. coli polymerase and vX174 DNA. In the latter case free RNA was not found until the RNA/DNA ratio in the reaction mixture reached about I, and free RNA, once formed, was entirely ribonuclease sensitive3. In the present case, significant amounts of free RNA were found at the earliest times (Fig. I). Furthermore, about 50 o/o of the free RNA was ribonuclease resistant. As synthesis proceeded, the amount of free RNA increased, but its ribonuclease resistance remained proportionally always the same (Fig. I). The melting curve of the ribonuclease-resistant RNA shows that it is a double-stranded RNA-RNA duplex (see below). Biochim.

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0

10 20 30 Fraction No.

Fig. 1. Resolution of reaction products by Cs,SO, gradient centrifugation. x _ - - x, DNA; ?? -@, total RNA; o-0, ribonuclease-resistant RNA. The reaction mixture contained so pg/ml 3aP-labelled T4 DNA, heated to 98.5’ for 20 min and IZ units/ml of Micrococcus RNA polymerase. At 6(a), 15 (b) and 60(c) min 0.3 ml samples were removed and centrifuged in C&SO, as described under METHODS AND MATERIALS. Alternate gradient fractions were treated with ribonuclease. The amount of RNA in each fraction was calculated from the relationship 630 counts/mm = I nmole ATP w I pg RNA. The specific activity of the DNA was 180 counts/min per pg.

Properties of the products of RNA synthesis The DNA and RNA of the q~X174DNA-RNA hybrid duplex could be separated by denaturation with alkali or formamide ly2. To see whether the T4 DNA-RNA hybrid shares this property, double-labelled enzymatic hybrid was synthesised and banded in Cs,SO, to remove free RNA. When the hybrid was recentrifuged in Cs,SO, the greater part of the RNA (80-90 %) again banded with the DNA. After heat denaturation, the RNA and DNA banded separately. When the hybrid was layered on a sucrose gradient and centrifuged, the RNA and DNA sedimented together. After heat denaturation, however, the RNA and DNA sedimented separately (Fig. 2). The mean sedimentation coefficient of the RNA released from the hybrid was approx. 6 S, corresponding to an average molecular weight of about 50 ooo (refs. 13, 14). The mean sedimentation coefficient of the DNA was about 20 S (Fig. za) correspondof the DNA during RNA ing to a molecular weighP of about 10~. Degradation b

0

ic -2cO

c $40. -z :

28 i

16 1

Fraction

No.

Fig. 2. Sedimentation of DNA-RNA hybrid. DNA-RNA hybrid containing 3aP-labelled DNA ( x ) and r4C-labelled RNA( 0) were isolated and centrifuged on sucrose gradients as described under METHODS AND MATERIALS. (a) Untreated; (b) incubated rg min at 37” with 1.7 pg/ml ribonuclease in 0.3 M NaCl prior to the gradient; (c) heated at go”, ro min in 0.1 M NaCl prior to,the gradient. Sedimentation from right to left. The arrows show the positions of 16-S and 28-S rabbit reticulocyte ribosomal RNA markers.

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TRANSCRIPTION

OF DENATURED

T4 DNA

641

synthesis was very little. The observed degradation probably occurred during the preparation and heat denaturation of the DNA. To study its stability at elevated temperatures, enzymatic hybrid was collected from a Cs,SO, gradient and dialysed against 0.1 M NaCI, 0.01 M Tris-HCl. Ribonuclease-resistant RNA was measured after heating samples to different temperatures (Fig. 3). The hybrid melted sharply with a mean temp. of 64”, showing that much of the RNA is correctly base-paired with DNA.

1

Fig. 3. Melting curves of DNA-RNA hybrid (0) and double-stranded RNA (0). In each case the RNA was ‘“C-labelled. The samples were held at the temperature shown for IO min in o. I M NaCl, 0.01 M Tris-HCl (pH 7.5 at 0’) and then chilled. Each was adjusted to 0.3 M NaCl, incubated for 30 min at 30’ with 2.5 pg/ml ribonuclease and then precipitated with trichloroacetic acid. collected on a filter and washed.

The RNA band was collected from the same Cs,SO, gradient and treated in the same way. About half of the total RNA was ribonuclease sensitive, but we are concerned here only with the resistant RNA. The melting curve of this RNA was somewhat sharper than that of the DNA-RNA hybrid, and the mean melting temperature, 74O, was higher (Fig. 3). The sharpness of the melting curve shows that ribonuclease-resistant RNA is correctly base-paired, presumably in the form of an RNA-RNA duplex. GEIDUSCHEK et a1.15 have shown that RNA synthesised in vitro may develop ribonuclease resistance during purification. To test this in the present system, RNA synthesis was carried out with denatured DNA to an RNA/DNA ratio at which 80 y0 of the RNA is not associated with DNA (see below). The reaction mixture was adjusted to 0.3 M NaCl to stabilise RNA hybrids3a6and samples were immediately treated with ribonuclease. Close to 50 “/b of the total RNA was ribonuclease resistant at from 0.2 to 5 pg ribonuclease per ml. Ribonuclease resistance was reduced to less than 5 y0 by heating to 98” for IO min, showing that the resistance was due to RNA duplexes. A sample of the reaction mixture was deproteinised with phenol, and its ribonuclease resistance was unaffected. RNA was purified from a second sample by Biochim.

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centrifugation in Cs,SO,. Again, ribonuclease resistance was close to 50 %. Ribonuclease resistance is evidently developed in the reaction mixture, and is not significantly affected by the purification of the RNA.

Course of RNA synthesis with laative and denatured templates The synthesis of total RNA was followed during the course of a single reaction with denatured T4 DNA. Parallel samples of the reaction mixture were adjusted to 0.3 M NaCl and treated with ribonuclease to measure total ribonuclease-resistant RNA. This is the sum of resistant RNA in both DNA-RNA and RNA-RNA duplexes. Fig. 4 shows that ribonuclease-resistant RNA continued to be formed as long as RNA synthesis continued. The proportion of the total RNA which was resistant was greatest at the shortest incubation times (Fig. 5, upper line), and fell with time of incubation to reach a steady value of close to 50 “/ resistance. When, instead of denatured DNA, native DNA was used as the primer, ribonuclease resistance was very low after short incubation times (Fig. 5, lower line), but increased steadily as incubation continued. However, even after 60 min of incubation with native DNA, ribonuclease resistance had reached only 20 yc. The development of a high level of ribonuclease resistance at early incubation times, falling to a plateau in the region of 50 o/0 resistance, is characteristic of RNA synthesis using denatured DNA. Very similar results were obtained using E. coli RNA polymerase.

0 t, 0

I

20 40 Time(min)

60

Fig. 4. Time course of synthesis of total of incubation were as for Fig. I.

1 Time

(??) and ribonuclease-resistant

(mid

(0) RNA. The conditions

Fig. 5. Relationship of ribonuclease resistance to time of incubation with denatured (0) and native (0) DNA. The data for the denatured DNA are taken from Fig. 4. The incubation with native DNA was carried out in parallel under exactly the same conditions except that the polymerase concentration was 8 units/ml.

The precise relationship between ribonuclease resistance and time varied between several experiments in which the concentrations of DNA and Micrococcus polymerase were varied. However, the level of ribonuclease resistance attained seems to be closely tied to the RNA/DNA (product/primer) ratio (Fig. 6). This suggests that Biochim.

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the course of the reaction is determined by the properties of the template: only when the template has been utilised to a certain extent can the reaction proceed to the next stage. The region of high ribonuclease resistance corresponds to low total RNA synthesis. At this point (Fig. I) the amount of RNA in DNA-RNA hybrid (80 y0 resistant) is greater than the amount of free RNA (50 o/o resistant). The region of 50 T/o ribonuclease resistance corresponds to high total RNA synthesis, where the amount of free RNA is much greater than the amount in DNA-RNA hybrid.

2

RNA/DNA ratio

Fig. 6. Relationship of ribonuclease resistance to RNA/DNA ratio with denatured primer. The conditions were as described for Fig. I, except as shown. __ ____ Expt. No.

Symbol

I 2 3

0

4

:

5

6

0

A A

DNA 20 10 IO 20 20 20

klm~)

Polymerase

(units/ml)

DNA

as

Time (milz) 6, 15. 30, 60 10, 30. 60

12

‘7.7 10.8 8.7 1.5

4, 10, 20 6 15. 45 20 IO

5

Distribution

of RNA in the enzymatic DNA-RNA hybrid The amount of RNA relative to DNA in the enzymatic hybrid was estimated in several experiments after resolution of the hybrid in Cs,SO, (e.g. Fig. I). The specific activity of the s2P-labelled DNA was measured separately, and the specific activity of the RNA was calculated from that of the [r4C]ATP precursor, assuming the G+C content of the RNA to be the same as that of T4 DNA (34 %). The amount of RNA was from 35 to 55 o/o as much as the DNA in different experiments, and of this, close to 80 % was ribonuclease resistant. The DNA-RNA hybrid was not sufficiently well resolved from free RNA to be accurately estimated when the product/ primer ratio was more than 1.5. These figures may therefore underestimate to some extent the maximum proportion of RNA which DNA-RNA hybrid can contain. The underestimate should not be serious, however, because the product/primer ratio rarely exceeds 2.5, and the amount of hybrid reaches an apparent maximum at a product/primer ratio of about I. In a DNA-RNA hybrid containing 50 y. as much RNA as DNA, about half of the total DNA is not hybridised with RNA. To determine whether any DNA molecules were completely free of RNA, enzymatic hybrid was isolated from a reaction Biochim.

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mixture which reached a product/primer ratio of 1.9. The buoyant density of the hybrid in CsCl was measured analytically against a standard of Micrococcus lysodeikticus

DNA. The hybrid

formed

a broad band between

p = I.74 and p = I.& with

a modal point at p = 1.79. No DNA banded at the buoyant tured T4 DNA (p = 1.69x-1.712), with RNA.

The buoyant

density

showing that

density of native or dena-

all DNA molecules

were associated

of the hybrid was closer to that of the DNA than

to that of RNA (p = 1.9). On the assumption that buoyant density in CsCl is roughly proportional to RNA/DNA ratio, this is consistent with an RNA/DNA ratio of less than

I. (The CsCl buoyant

between

the densities

density

of the RNA

of a I : I vxI74 and DNA

DNA-RNA

hybrid

is midway

(ref. z).)

The displacement of the DNA by its inclusion in DNA-RNA hybrid was also measured in preparative Cs,SO, gradients (Fig. 7). Compared with denatured T4 DNA, the DNA in the DNA-RNA corresponding servations1-3

to about

hybrid was displaced

0.01 density

unit.

which show that the buoyant

is not a linear function

This density

by about one half fraction,

is in agreement of DNA-RNA

with hybrid

earlier

ob-

in Cs,SO,

of RNA and DNA content.

Fmction

No.

Fig. 7. Buoyant density distribution of 32P-labelled denatured T4 DNA primer ( x ) and **Clabelled denatured T4 DNA (0). The primer DNA was incubated (20 pg!ml) for 30 min at 30” in a complete reaction mixture containing 10.7 units/ml RNA polymerase and unlabelled nucleotides. After NaCl-sodium lauryl sulphate treatment, 14C-labelleddenatured DNA was added and the mixture was added to the gradient. DNA-RNA hybrid isolated from a parallel incubation with [W]ATP had an RNA/DNA ratio of 0.53 without ribonuclease treatment. Fig. 8. Sedimentatio? of sonicated DNA-RNA hybrid. The reaction mixture contained 20 &ml S$P-labelled DNA and IO units/ml RNA polymerase. Synthesis was allowed to continue for 40 min and the reaction was stopped with NaCl and sodium lauryl sulphate. The sample shown was sonicated for 2.5 min. ( x - - - x ), DNA, specific activity 610 counts/min per ,ug; (O-O), total RNA. The absorbance profile of the ribosomal RNA markers is also shown (0 0 ).

Although not paired with points of hybrid distribution of Biochim.

Biophys.

all DNA molecules are incorporated in DNA-RNA hybrid, they are equal amounts of RNA. This is shown by the slightly different modal RNA and hybrid DNA in Cs,SO, gradients (Fig. I) and by the skewed the hybrid in CsCI. If the unpaired DNA regions are sufficiently Acta,

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long, fragmentation of the hybrid will release free DNA. To investigate the length of the unpaired DNA regions, enzymatic hybrid was severely sonicated. Following sonication the hybrid sedimented with a mean of about 6 S (Fig. 8). Further sonication did not reduce the sedimentation rate of the hybrid. When the duplexed RNA was removed from the sonicated hybrid by heat denaturation, the free DNA sedimented at about 6 S, corresponding to a molecular weight of about IOO ooo (ref. 16) or a length of approx. rium in Cs,SO,. DNA-RNA

300 nucleotides.

The sonicated

On the basis of the results

hybrid

obtained

was centrifuged with 9X174

to equilib-

hybrid1-3,

I :I

hybrid should separate by 0.04 to 0.08 density unit from single-stranded

DNA, corresponding to z. to 4 fractions in the preparative gradient. In fact (Fig. g) the separation of DNA from the RNA of the DNA-RNA hybrid was less than one fraction

and the distribution

Fraction

of hybrid

molecules

remained

heterogeneous.

No

Fig. 9. Cs,SO, centrifugation of sonicated DNA-RNA hybrid. (a) Control; (b) sonicated. The reaction mixture is described in the legend to Fig. 8. The specific activity of the DNA at the time of counting the gradient samples was 430 counts/minperpg. (x - - x ), DNA; (e-a), total RNA.

Evidently the duplexed RNA molecules are distributed over the DNA in such a way that degradation of the DNA to 300 nucleotide lengths leaves most of the DNA molecules still duplexed with RNA. The RNA molecules synthesised on the denatured template are on average about 150 nucleotides in length. This suggests that DNA regions duplexed with individual RNA molecules alternate with non-paired regions of approximately equal length. If so, 3oo-nucleotide stretches will always carry duplexed RNA. Free DNA will be liberated only if fragments containing 150 nucleotides or less are formed.

Hybridisation tured DNA

competition between messenger RNA

and RNA

sylzthesised using dena-

During the development of T4 within the bacterial host two phases of RNA synthesis have been definedr’. During the first 5 min of infection, a special class of RNA known as early messenger is synthesised. At later times a second class known as late messenger is synthesised in addition to the early messenger. When T4 DNA is hybridised with a saturating amount of RNA labelled between 16 and 20 min after Biochim.

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infection, 60 ‘$6 of the hybridising RNA radioactivity is due to early messenger, 40 o/o to late messengerl’. Unlabelled complementary RNA synthesised using denatured T4 DNA was used to compete against the hybridisation with T4 DNA of RNA labelled between o and 4 min and between 16 and 20 min of infection. RNA was isolated from a reaction mixture which reached a product/primer ratio of 1.1, so that the RNA was distributed approximately equally between free RNA and RNA-DNA hybrid. The results of this experiment are shown in Fig. IO. In Fig. Iob they are plotted in such a way that the proportion of hybridising RNA sequences competed against is given by the reciprocal of the slope of the line ll. The slopes of the lines are r.rgfo.oS for the o-4 min messenger and 1.34fo.14 for the 16-20 min messenger. Thus RNA synthesised on the denatured template competed against 84 o/o and 75 oh of the two RNA preparations. RNA synthesised under the same conditions using native T4 DNA, competed against IOO o/o and 80 o/O,respectively, of o-4 min and 16-20 min messenger12.

b

Fig. IO. Competition between labelled messenger RNA and unlabelled complementary RNA for hybridisation with T4 DNA. Each membrane filter carried approx. I pg of S*P-labelled DNA. The messenger RNA was labelled from o-4 min of infection (0-O) and 16-20 min (m-0). (a) Shows a straightforward plot of competition against concentration of unlabelled RNA. (b) Shows a plot of the ratio of unlabelled RNA to proportional competition (g/C) against unlabelled RNA (g).

Synthesis of RNA-RNA hybrid Since a high level of ribonuclease resistance is characteristic of the reaction primed by the denatured template, it seemed possible that RNA-RNA hybrid is generated in some way from the DNA-RNA hybrid. To test this, DNA-RNA hybrid containing 14C-labelledRNA and 32P-labelled DNA was isolated from Cs,SO, gradients and added to the complete reaction mixture containing unlabelled ATP. Samples removed during incubation were centrifuged in Cs,SO, in the usual way (Fig. II). At zero time, some free RNA was found as a contaminant of the DNA-RNA hybrid preparation. During incubation, the prelabelled RNA was progressively released from the DNA-RNA hybrid, appearing as ribonuclease-sensitive and ribonuclease-resistant RNA in the ratio of approx. 2 to I. Release of RNA from the hybrid was entirely dependent on the addition of both precursor nucleotides and RNA polymerase (Fig. 12) showing that the process of RNA release is a result of RNA synthesis. In these experiments, total RNA synthesis de nova was measured in parallel incubations. In the experiment shown in Fig. II the ratio of newly-synthesised RNA to the DNA in the DNA-RNA hybrid primer was 0.45 at IO min and 0.92 at 30 min. DisplaceBiochim.

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ment of RNA from the primer was 57 o/o and 63 %, respectively. Since the RNA/ DNA ratio of the primer was 0.42, synthesis de novo was, respectively, 1.9 and 3.4

Fraction

No.

Fig. II. Displacement of RNA from DNA-RNA hybrid. The isolated hybrid, containing irClabelled RNA (0 ) and S*P-labelled DNA ( x ) was incubated under standard condition with unlabelled nucleotides and 12.5 units/ml RNA polymerase. (a) Control, no incubation, (b) IO min, (c) 30 min at 30’. Alternate fractions were treated with ribonuclease to measure ribonucleaseresistant RNA (0).

Fractmn

No.

Fig. 12. Conditions for displacement of incubated under standard conditions for nucleotides. (a) Control, no incubation, polymerase omitted. x , DNA; 0, total

RNA from DNA-RNA hybrid. The isolated hybrid was IO min with o units/ml RNA polymerase and unlabelled (b) complete system, (c) nucleotides omitted, (d) RNA RNA; 0, ribonuclease-resistant RNA. Biochim.

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times greater than displacement. In this respect, the T4-Micrococcus system resembles thepXr74-E. coli system where a ratio of 2.8 may be calculated from the data of CHAMBERLIN AND BFLR~~.The increase of the ratio with time of incubation is to be expected, since the prelabelled RNA can be released from the template only once. However, the ratio of 1.9 after IO min suggests either that some parts of the template turn over more rapidly than others, or that RNA synthesis is going on by some separate reaction which does not lead to the displacement of RNA from the DNA-RNA hybrid.

Comparison with E. coli RNA polymerase Several experiments were carried out with denatured T4 DNA and E. coli RNA polymerase. The relationship between RNA/DNA ratio and ribonuclease resistance was the same as for the Micrococcus polymerase (Fig. 4). With denatured T4 DNA as primer, the E. coli enzyme synthesised DNA-RNA hybrid and free RNA, about 50 % of which was ribonuclease resistant (Fig. 13). When centrifuged in CsCl in the analytical ultracentrifuge, the DNA-RNA hybrid banded between p = 1.74 and p = 1.81, with a modal point at about p = 1.79. In these respects, and particularly in the synthesis of ribonuclease-resistant RNA, the reaction of E. coli RNA polymerase with denatured T4 DNA resembled the T4-Micrococcus system. b

1 -1.0

Fraction

No.

Fig. I?. Resolution of products of RNA synthesis carried out by E. coli RNA polymerase. CondiGo&of incubation a< for Fig. I except
DISCUSSION

Structure and synthesis of the enzymatic DNA-RNA hybrid Although more molecules of RNA polymerase bind to denatured than to native DNAr8-zO, the number which binds to the denatured template is less than is dictated by the size of the molecule18,1g. This suggests the existence of specific attachment sites on the denatured template, an idea which is supported by the fact that the 5’-nucleotide of the RNA synthesised is predominantly ATP and GTP21-23. The length of singlestranded DNA between polymerase molecules has been estimated as 70 ooo daltons, or about 230 nucleotide+‘. Results of BREMER et a1.20,using denatured T4 DNA, are in agreement with this figure. B&him.

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TRANSCRIPTION

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The results presented in this paper indicate that the en.zymaticTqDNA-RNA hybrid consists of alternating lengths of about 150 DNA nucleotides, every second length duplexed with a single strand of RNA. This suggests that approx. half of the DNA between each pair of adjacent initiation sites is transcribed. The RNA chain grows by the attachment of nucleotides at the 3’-end21,24, and is presumably antipolar to the DNA chain from which it is transcribed. Thus the half of the intersite sequence which lies towards the 3’-end of the DNA strand will be transcribed. This hypothesis satisfactory explains the synthesis of z : I DNA-RNA hybrid. It also explains the observation l2 that transcription of the denatured template is qualitatively limited. A z : I hybrid could result either from the transcription of half of the copies of all DNA sequences, or from the transcription of all copies of half of the sequences. If initiation sites are specific, the first alternative is inconsistent with the hypothesis and the second is consistent with it. If some variation is allowed in the precise point of chain termination, the hypothesis stipulates that transcription of the denatured template should be qualitatively limited to some extent. That this is so is shown by the fact that saturating amounts of native template RNA hybridised with 39 O,&of T4 DNAr2, while denatured template RNA hybridised with only 28 o& (ref. II). The absolute homology between DNA and RNA cannot be estimated in this way, because the availability of the DNA for hybridisation is not known. However, since the homology of the native template RNA cannot be greater than IOO “/b, we can set 70 o/O as the upper limit of the total DNA sequence which may be transcribed from the denatured template. The hypothesis leads to a second prediction. Since duplexed RNA molecules are scattered over the entire template, it follows that they should have much the same proportional homology towards any group of cistrons as towards the molecule as a whole. Two separate groups of cistrons are represented by the ‘early’ and ‘late’ messenger RNA17. The fact that RNA synthesised on the denatured template competes for hybridisation with a similar proportion of pure early messenger and mixed early and late messenger (Fig. IO) is in support of the hypothesis. The quantitative aspects of this experiment are particularly interesting. The messenger RNA in vivo is complementary to only one strand of the DNA within any cistron (Fig. r4a). (For reasons of simplicity the messenger RNA has been drawn complementary to

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-xm

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2.

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Fig. 14. Model of DNA-RNA hybrid formation. DNA is represented by broken lines, RNA by continuous lines, polymerase initiation sites by the symbol xxx. In (a) the complementary strands of a DNA segment are drawn, with RNA in uivo complementary to one of them. (b) Shows the result of RNA synthesis in vitro if attachment sites are in apposition, (c) shows the result if they are distributed randomly in the two strands of the DNA. Biochim.

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one of the DNA strands throughout the length of the segment shown.) RNA syn-

thesised in vitro is complementary to both strands in different places. The RNA which is identical to RNA in vivo will compete against it by hybridising with the same DNA sequences. The RNA which is complementary to RNA in vivo will prevent its hybridisation with DNA by hybridising with it to form an RNA-RNA duplexli. If the initiation sites on the complementary DNA strands are in apposition, since the hypothesis requires the transcription of half the intersite region, the RNA synthesised will be either identical to or complementary to nearly all of the RNA in vitro (Fig. 14b). If the initiation sites on the complementary strands are distributed randomly (Fig. 14~) the RNA synthesised will be identical to or complementary to 75 o/0 of the RNA in vivo. The figure of 75 oh is in reasonably good agreement with the observed competition values of 84 y0 and 75 Oh, and favours the second alternative.

Synthesis of doublestraded RNA The sharp melting curve of the ribonuclease-resistant RNA shows it to be in the form of RNA-RNA duplexes, i.e. double-stranded RNA. The time course of synthesis of double-stranded RNA was completely different when native and denatured templates were used. Under the conditions of these experiments, RNA containing a large ribonuclease-resistant fraction is synthesised only in the presence of denatured template. Since free RNA is available in both reactions, it is concluded that most of the double-stranded RNA is not synthesised by a reaction primed by RNA. On the other hand, it is shown that double-stranded RNA can be produced as one result of displacement of RNA from DNA-RNA hybrid. There is no reason to suppose that RNA would be favoured as a template because of a previous association with DNA. It is therefore reasonable to conclude that this double-stranded RNA is formed in the course of displacement, as a result of RNA synthesis. In the displacement experiments, DNA-RNA hybrid containing labelled RNA was incubated with unlabelled nucleotides. Some two thirds of the radioactivity released was found as single-stranded RNA, and one third was ribonuclease resistant. In experiments in which labelled nucleotides were continuously present, the amounts of resistant and sensitive labelled RNA were approx. equal. This difference can be explained if the labelled ribonuclease-resistant RNA released in the displacement experiments is duplexed with newly-synthesised unlabelled RNA. The synthesis of double-stranded RNA by displacement of RNA from the DNARNA hybrid suggests the following model (Fig. IS). RNA polymerase must have a structural asymmetry to ensure that it moves along the DNA in the correct direction. If polymerase molecules can become attached to the DNA-RNA hybrid in both possible orientations, then in one case the enzyme will transcribe the DNA strand; but in the second it will be forced, if it functions at all, to transcribe the RNA strand. It is proposed that transcription of the DNA strand leads to the displacement of the original RNA strand, so that it is released as single-stranded RNA and replaced with a newly synthesised DNA-RNA hybrid (Fig. 15~); transcription of the RNA strand leads to the formation of an RNA-RNA hybrid between new and old strands, and the separation of the RNA-RNA hybrid from the DNA (Fig. r5d). In each case the newly synthesised RNA forms a duplex with its complement. If this model is Biochim.

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TRANSCRIPTION

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6s

correct, the displacement of twice as much single-stranded as double-stranded RNA from the DNA-RNA hybrid suggests that RNA polymerase transcribes the DNA strand twice as frequently as the RNA strand. It may be asked what mechanism promotes the transcription of the RNA strand. If, as proposed earlier, attachment sites are distributed at random on the complementary strands of the DNA, then in many cases the newly-synthesised RNA complementary to one strand will come to carry a copy of an attachment site on the other strand. This might in some cases act as an initiation site for the transcription of the RNA (Fig. IS). 2 ---__--_--,.-_A a '~~~~~~-~_~_~

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Fig. 15. Model proposed to explain the displacement of single-stranded and ribonuclease-resistant RNA from the DNA-RNA hybrid. DNA is represented by broken lines, and RNA by continuous lines. Hydrogen-bonded regions are joined by dotted lines. Two polymerase attachment sites (xxx) are drawn. (a) Shows the complementary strands of a segment of DNA. (b) Shows DNA-RNA hybrid incorporating the lower strand, synthesised by the attachment of RNA polymerase at Site r. (c) Shows the suggested consequence of the attachment of a second molecule of polymerase at Site r. The old RNA strand is being displaced by a new one. (d) Shows the result of attachment of polymerase at the RNA copy of Site z. RNA duplex is being synthesised. The model has been drawn as though Site z is a site of attachment of polymerase to denatured DNA. This is not a necessary part of the hypothesis.

On the basis of this model, and assuming that newer and older RNA sequences have the same chance of displacement from the DNA-RNA hybrid, the displacement experiments (Fig. II) can be described by R=

where R is the ratio of newly synthesised free RNA to prelabelled RNA released from the DNA-RNA hybrid, and x is the ratio of the released prelabelled RNA to the total prelabelled RNA. (R’ = R+I where R1 is the ratio of total free RNA to prelabelled RNA released from the hybrid). For x = 0.57 the predicted and observed values of R are 1.3 and 1.9. For x = 0.63, the prediction for R is 1.5 and the observed value 3.4. Evidently the model does not describe the situation adequately. Two main possibilities have already been suggested. First, RNA synthesis may occur by a second reaction. Secondly, the assumption of random displacement may be incorrect: instead, some primer sequences may be utilised more readily, and more often than others. Any independently occurring conservative replication process would have to produce equal amounts of double- and single-stranded RNA. For this reason, the second explanation, that some primer sequences are more rapidly turned over, is favoured. Biochim.

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052 ACKNOWLEI)GEMENTS

I am grateful to Mrs. N. ROBERTSON for technical assistance, and to Drs. F. W. ROBERTSON, was provided

M. MELLI and M. BIRNSTIEL by the Medical

Research

for helpful

discussion.

Financial

support

231. Proc. Natl. Acad.

Sci. U.S.,

Council.

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