Role of DNA in RNA synthesis

(1967) 26, 19-38

J. Mol. Biol.

Role of DNA in RNA Synthesis XI.j- Selective Transcription of X DNA Segments in vitro by RNA Polymerase of Exherichhia coli STANLEVN.COHEN,UMADASMAITRAAND

JERARD HURWITZ

Department of Developmental Biology and Cancer Albert Einstein College of Medicilze Bronx, New York 10461, U.X.A. (Received 12 December 1966) transcription of native DNA isolated from mature bacteriophage /\ was studied using highly puSed preparations of DNA-dependent RNA polymerase isolated from E. coli W. Half-length segments of sheared h DNA were separated by density-gradient oentrifugation, and the RNA polymerase products synthesized on the whole X DNA template and on each of its separated halves were characterized with regard to their nearest-neighbor nucleotide frequencies, base composition, average chain-length, sedimentation velocity, and ability to anneal with specific segments of the template. The priming efficiencies of the h DNA halves were compared, and the influence of certain alterations in the secondary or tertiary structure of the h DNA template on the RNA products formed in vitro was examined. These studies indicate that transcription of native X DNA by the E. coli polymerase in vitro is not random; specific template regions present predominantly on the AT-rich (right) half of linear X DNA are preferentially transcribed throughout the duration of in vitro RNA synthesis. Denaturation of the h DNA template results in elimination of selective copying. Neither free cohesive ends nor linearly intact DNA are essential for the selection mechanism. In vitro

1. Introduction It has been well established that the expression of genetic information in viruses, as well as in more complex organisms, is subject to temporal control; not all genes are expressed simultaneously (Jacob, Fuerst & Wollman, 1957; Luria, 1962; Epstein et al., 1963). “Early” and “late” functioning cistrons have been distinguished in h, T-even and other bacteriophages, and in certain instances genetic maps have shown a clustering of genes the expression of which is temporally related (Jacob et al. 1957 ; Campbell, 1961; Epstein et al., 1963; Eisen et al., 1966). Although the biological mechanism responsible for specific temporal control of gene expression is not known, there is evidence that such regulation may occur at the level of transcription (KanoSueoka & Spiegelman, 1962; Protass & Korn, 1966; Skalka, 1966). Experiments by Bremer, Konrad, Gaines & Stent (1965) and by Maitra & Hurwitz (1965) have indicated that transcription of DNA1 in vitro may also be a specifically t A preliminary report of this work has been presented (Cohen, 1966). The preceding paper in this series is Maitra and Hurwitz (1965). $ Abbreviations used: DNA, deoxyribonucleic acid, concentrations of which are expressed in terms of nucleotide phosphorus; RNA, ribonucleio acid (concentrations are expressed in terms of nucleotide phosphorus); SDS, sodium dodecyl sulfate; SSC, standard saline citrate solution (0.15 M-sodium

chloride

plus 0.015 &I-sodium

citrate

(pH

7); 2 x SSC, 0.30 Ivr-sodium citrate

(pI;I 7); mRNA, messenger RNA; TCA, trichloroacetio acid. 19

20

8. N.

COHEN,

U. MAITRA

AND

J. HURWITZ

regulated process. Initiation of RNA chains in the DNA-dependent RNA polymerase of Escherichia CO& on a variety of DNA templates occurs preferentially with purine nucleoside triphosphates; i.e. pyrimidines constitute the principal class of DNA sit’es at which such chains are started. Recent studies (Berg, Kornberg, Franeher & Dieckmann, 1965; Crawford, Crawford, Richardson & Slayter, 1965; Richardson, 19663; Jones & Berg, 1966) of the binding of RNA polymerase to DNA templates have shown that only certain sites of the template are occupied by enzyme molecules at a given time. However, it has not been clear whether such binding of enzyme to DNA and the in vitro RNA synthesis, which under appropriate conditions is its consequence, takes place randomly along the length of the template or whether it occurs selectively at specific DNA sites. DNA from the bacteriophage X is especially useful for studying the specificity and control of RNA synthesis in vitro. Large segments of this DNA are widely divergent in their base composition (Hershey & Burgi, 1963; Hershey, 1964) permitting the separation of fragments of sheared X DNA by column chromatography (Hogness & Simmons, 1964) or density-gradient centrifugation (Hershey, Burgi & Davern, 1965; Nandi, Wang & Davidson, 1965). Thus, in vitro transcription from selected portions of the X genome can be studied. In the present experiments, X DNA was sheared into half-length molecules which were separated by C&SO, gradient centrifugation. RNA polymerase products were synthesized using whole X DNA and each of its halves as templates, and these products were characterized with regard to their nearest-neighbor nucleotide frequencies, base composition, average chain-length, sedimentation velocity, and ability to hybridize with separated and isolated segments of X DNA. The relative priming efficiencies of the halves were compared, and effects of shearing, sonicating or denaturing the h DNA template on certain properties of the RNA products were examined. Our results indicate that transcription of native X DNA by the DNA-dependent RNA polymerase in vitro does not occur randomly along the DNA template, but rather that the enzyme is directed by the template to copy selectively certain DNA segments. Similar tidings have been recently observed independently by Naono & Gros (1966). The possible influence of the cohesive ends of mature h DNA (Hershey, Burgi & Ingraham, 1963; Ris & Chandler, 1963) on the ability of this template to prime and direct RNA synthesis in vitro was an additional subject for the present investigation. These cohesive ends, which consist of complementary nucleotide base sequences (Hershey & Burgi, 1965), and which are important in determining biological infeetivity of X DNA (Strack & Kaiser, 1965; Kaiser & Inman, 1965), can be joined in vitro with sites on the same molecule to form closed or circular molecules, or with sites on different molecules to form dimers, trimers, etc. (Hershey, Burgi & Ingraham, 1963; Ris $ Chandler, 1963; MaeHattie & Thomas, 1964). Furthermore, “outside-in” reconstituted whole molecules of A DNA can be formed by coherence of the complementary nucleotide sequences at the ends of half-length segments (Wang & Davidson, 1966; Cohen & Hurwitz, unpublished observations). As part of the present investigation, initiation and synthesis of RNA i,n vitro were studied using linear, circular, aggregated and sheared-cohered forms of h DNA as templates. Our observations indicate that these various DNA forms have similar priming abilities. Furthermore, no differences were observed in the RNA products formed on these structurally different templates.

ROLE

OF DNA

1X RSA

SYNTHESIS

21

2. Materials and Methods (a) Preparation and purification of X phage Lysates of X phage were prepared either by infection of E. coli W3110 with Xc,b+ (both obtained from E. Burgi) as described by Burgi (1963), or by induction of E. coli K112(h) thy- (CR34, obtained from Dr H. Eisen) as described by Korn & Weissbach (1962). The media used were peptone broth (Burgi, 1963) for external infection, or M9 medium (Adams, 1959) fortified with 1.5% Casamino acids for thymineless induction. Lysates of tritiated h phage were prepared by induction of E. coli K12(h) thy- using 3H-labeled thymine (New England Nuclear Corp., specific activity 1 u/m-mole) as described by Korn & Weissbach (1962). Initial batches of phage were purified as described by Burgi (1963). Later preparations of X lysates were concentrated by the liquid-polymer phase technique described by Albertsson (1960) as modified by Watanabe & August (1967), and further purified by differential centrifugation followed by two CsCl (Harshaw, optical grade) gradient centrifugations as described by Kaiser & Hogness (1960). (b) Preparation of DNA DNA was isolated from purified phage after treatment with freshly re-distilled phenol (Malinkrodt Chemical Co.) using the procedure described by Kaiser & Hogness (1960), and was examined by either boundary sedimentation (Doty, McGill & Rice, 1958) or zone sedimentation (Vinograd, Bruner, Kent & Weigle, 1963; Burgi & Hershey, 1963) to determine its sedimentation velocity. Portions were also examined by analytical boundary or zone sedimentation in alkali (0.1 M-NaOH, 0.9 M-NaCl, 0.001 M-sodium EDTA) (Studier, 1965) to ascertain the frequency of single-strand breaks in the DNA. Most preparations of unlabeled h DNA were free from detectable single-strand breaks, but such scissions were present in all samples of 3H-labeled r\ DNA examined (specific activity, 8 to 15 pc/ymole of DNA phosphorus). Therefore, only unlabeled (linearly intact) whole X DNA was used as a template for RNA synthesis; use of 3H-labeled h DNA was restricted to loading on nitrocellulose membranes for hybridization experiments. Various forms of h DNA (namely, linear molecules, circles and aggregated dimers and trimers were prepared as described by Hershey et al. (1963). Reconstituted whole molecules of X DNA (SZO,w= 32s) were prepared from sheared molecules by the method used to prepare h circles (Hershey et al., 1963). In both instances, cohesion of the ends of h DNA depends on hydrogen bonding of the terminal complementary nucleotide sequences (Wang & Davidson, 1966). Sonicated DNA was prepared from native linear h DNA using an MSE sonicator (Instrumentation Associates, Inc.). Sonication (manual tuning) at 0°C of 400 mpmoles of DNA dissolved in 10 ml. of SSC for 30 set using a 0.75-in. sonication probe resulted in the conversion of linearly intact DNA to fragments sedimenting at Sz,,, = 9 S. Less than 2% hyperchromicity resulted from this procedure. Single-stranded DNA was prepared by denaturation with 1 i?r-NaOH as described by Gillespie & Spiegelman (1965). (c) Shearing of A DNA and separation. of halves Shearing of whole X DNA into half-length molecules was accomplished with a Fisher steady-speed stirrer fitted with a Vertis stainless-steel macro shaft and blades, and was monitored by gradient centrifugation in CsCl (Hershey, 1964). Quantities of 15 to 25 ml. containing DNA at a concentration of 40 to 50 mpmoles/ml. dissolved in 0.005 M-sodium borate buffer (pH 9.0) containing 0.1 M-Na2S04 were stirred in a Vertis loo-ml. fluted flask at 2100 rev./min for 35 min. The shearing procedure produced less than 2% increase in absorbancy of the DNA. Physical separation of the two halves of h DNA was accomplished by a modification of the procedure described by Wang & Davidson (1966). This procedure utilizes the preferential binding of mercuric ions to the AT-rich half of r\ DNA to increase its buoyant density in Cs.$O, gradients, thereby enabling its ready separation from the GC-rich X DNA half. HgCl, (molar ratio of Hg to DNA phosphorus = 0.4) and CsZS04were added immediately following shearing in most instances, and 4- to S-ml. portions were run in a Spine0 no. 40

22

S. N. COHEN,

U. MAlTRA

AND

J. HURWITZ

fixed-angle rotor for 42 to 60 hr at 38,500 rev./mm at 4’C. When gradient centrifugation delayed, samples of DNA were stored at -10°C; stored samples were heated to ‘75°C for 3 min and then rapidly cooled to 0°C to convert any aggregated molecules to linear DNA before CszSOB and HgClz were added. Following centrifugation, fractions of 6 drops each were collected through a hole pierced in the bottom of the tube, 1 ml. of water was added to each fraction, and the absorbancy was determined at 260 m+ The fractions containing each of the principal peaks (about 4 to 5 fractions per peak) were pooled, and Hg2 + removed by dialysis against four one-liter changes of 0.1 M-EDTA (pH 8), followed by four one-liter changes of SSC. Certain samples of DNA were concentrated subsequently by dialysis against 25% polyethylene glycol (Carbowax 6000, Union Carbide) in O-05 M-N&l, O*OOl M-Tris buffer (pH 7.5), or by slow evaporation under a gentle stream of air. Portions of each of the DNA peaks were further checked in the analytical ultracentrifuge (Spinco model E) in CsCl gradients with an appropriate density marker (DNA from Bacillus subtilis phage 2C) in order to verify the completeness of separation and to ascertain the identity of each peak. The buoyant density of this DNA marker is l-742 g/cm3 when compared with that of E. co& DNA, taken to be 1.710 g/cm3 (Schildkraut, Marmur & Doty, 1962). Samples of the separated halves examined by boundary and/or zone sedimentation had SzO,w values of 24.5 to 26 s. When run under alkaline conditions as described above, a heterogeneous mixture of DNA fragments ranging from 15 to 20 s resulted, indicating that the process of shearing had produced one or more breaks per strand. Certain samples of unsheared whole A DNA subsequently used as template were run in C&SO, gradients with HgCl, and were then subjected to the dialysis procedure described above. No evidence of single-strand breakage was observed when the dialyzed samples were examined by analytical boundary sedimentation under alkaline conditions. W&S

(d) RNA polymerme RNA polymerase was prepared from E. co& W by a modification (Maitra & Hurwitz, manuscript in preparation) of the procedure of Furth, Hurwitz & Anders (1962). The specific activity of preparations used in these studies was 2000 to 3000 units (defined by Furth et al., 1962) per mg protein. Enzyme preparations showed absolute dependency on added DNA for ribonucleotide incorporation into RNA and were free from detectable DNase or RNase activity. No breakdown of cc-3zP-labeled RNA (100 pc/pmole) or 3H-labeled DNA (15 pc/pmole) into acid-soluble material was observed after incubation with 5 to 50 units of enzyme under standard assay conditions for 24 hr. Furthermore, samples of DNA examined by boundary sedimentation in SSC containing 0.1 M-NaOH following incubation with similar amounts of enzyme for 30 mm under standard assay conditions showed no evidence of single-strand breaks. (e) Initiation Initiation @2P-labeled

of RNA nucleoside

(f) Sedimentation Zone sedimentation to 30% linear sucrose 0.2% SDS.

experiments

chains and average RNA triphosphates, as previously analysis

chain-length described

were (Maitra

determined & Hurwitz,

using 1965).

of RNA

of RNA products (Britten & Roberts, 1960) was performed gradients containing 0.05 ivr-Tris buffer (pH 80), 0.1 ~-Nacl

in 15 and

(g) Nearest-neighbor frequency analysis Nearest-neighbor frequency analysis of RNA products was carried out as described previously (Hurwitz, Furth, Anders & Evans, 1962; Skalka, Fowler & Hurwitz, 1966). [32P]Ribonucleoside triphosphates labeled in the x-phosphate positions were purchased from either the International Chemical & Nuclear Corp. or Schwarz BioResearch, and were further purified by column chromatography on Dowex l-Cl (Lehman, Bessman, Sims & Kornberg, 1958). Following alkaline hydrolysis of the RNA products, the 2’ (3’)32P-labeled mononucleotides were separated by high-voltage electrophoresis using a buffer system containing pyridine, glacial acetic acid and water in the proportions 1 : 10 : 89, respectively. Samples were spotted on Whatman no. 3 MM filter paper and were subjected

ROLE

OF DNA

IN RNA

SYNTHESIS

23

to electrophoresis at 6000 v for 90 min using a Savant power supply and tank. The filter paper was cut into l-cm strips which were counted individually in vials containing a standard phosphor solution (16 g 2,5-diphenyloxazole and 4 g 1,4-bis2(5phenyloxazolyl) benzene (Packard) in 3.8 liters toluene). The frequency of dinucleotide pairs was calculated using a Control Data model 160A computer programmed for solution of the simultaneous equations described by Jesse, Kaiser & Kornberg (1961). (We are indebted to Miss Joan Lucas and Dr Josiah Maoy III, of the Albert Einstein College of Medicine, for these computer determinations.) (h) Isolation of RNA products for hybridization RNA was synthesized in our standard RNA polymerase assay (Furth et al., 1962), except that Mg2+ were used in place of Mn2+ for most experiments. Studies were carried out to ascertain that RNA products synthesized with either MnZ + or Mg2 + showed similar patterns of hybridization. Following the designated period of synthesis, DNase (electrophoretically pure, Worthington) was added to a final concentration of 20 pg/ml., and the mixture was incubated for an additional 15 min at 37OC. Control experiments (Cohen & Hurwitz, unpublished data) using 4 to 20 mpmoles of 3H-labeled h DNA (specific activity 10 &pmole) as template indicated virtually quantitative conversion of DNA to acid-soluble material by the procedure. The mixture was then heated at 1OO’C for 3 min and diluted with an equal volume of 2 x SSC containing 0.5% SDS (95% SDS, Matheson, Coleman & Bell). The solution containing the RNA product was equilibrated with an equal amount of freshly distilled phenol saturated with 2 x SSC, the layers were separated by brief oentrifugation, and the aqueous layer was dialyzed for 2 hr each against successive l-liter changes of (a) 2 x SSC containing O*5o/o SDS, (b) repeat of dialysis 1, and (c) 2 x SSC containing 0.05% SDS. The RNA product was removed and assayed for total non-dialyzable radioactive material. Acid-precipitable material was determined by the standard Millipore filter assay after acidification with 5 vol. of cold 5% TCA. Usually, about 85 to 90% of the non-dialyzable radio-active material present in the final product was acid precipitable and alkali sensitive and was defined as RNA. (We have observed that there is either a decrease in counting efficiency or loss of [32P]RNA when Millipore filters are used for absorbing RNA from acid solutions. These observations may account for the apparent loss of [32P]RNA.) (i) Hybridization procedure Hybridization of RNA to DNA was performed as described by Gillespie & Spiegelman (1965) using 3H-labeled h DNA or its isolated halves and 32P-labeled RNA products. Preliminary studies were done to ascertain the effects of temperature, ionic strength, annealing time and non-specific RNA on the hybridization of whole h DNA and each of its halves with their own RNA products. Annealing was done at optimal conditions (as described below) and at DNA levels in excess of the amount required to saturate the RNA used.

3. Results (a) Xeparation and identi$cation of X DNA halves A typical cesium sulfate density-gradient centrifugation of the Hg2+ complex of sheared ;\ DNA is shown in Fig. 1. Two major peaks are evident. The small shoulder on the fist of these peaks was observed with most samples run immediately following shearing, as well as with samples which were stored and were subsequently heated and cooled prior to centrifugation (Materials and Methods). The fractions constituting each of the major peaks and the shoulder were separately pooled, dialyzed, and rebanded in the analytical ultracentrifuge in a CsCl gradient using B. subtilis phage 2C DNA as a density marker (p = 1.742 g/cm3). Figure 2 shows a microdensitometer tracing of the gradient centrifugation analyses of each of the major peaks. The calculated densities of l-7042 g/cm3 and l-7150 g/cm3 correspond to base compositions of 45 and 55% G + C, respectively (Schildkraut et al., 1962). The ultraviolet-absorbing material comprising the shoulder banded at the same density as native whole

24

S. N.

COHEN,

U. MAITRE

5

15

IO

20

AND

25

‘Fraction

FIG. 1. Cs,SO, gradient

centrifugation

30

J. HURWITZ

35

40

45

50

number

of Hga+ complex

of sheared DNA.

Shearing conditions were as described in Materials and Methods. Sheared h DNA (250 mpmoles) M-sodium borate (pH 9.0), was mixed with 1.05 ml. dissolved in 6 ml. of 0.1 M-Na,so,-@005 of 10e4 ivIa-HgCl, at 0°C and Cs,SO+ was added to the solution to a density of p = I.508 at 25%. The mixture was centrifuged at 4% in a no. 40 fixed-angle rotor at 38,000 rev./min for 46 hr in a Spine0 L2 preparative ultracentrifuge. The rotor was allowed to stop without braking, and 6.drop fractions were collected promptly (in the cold) through a hole pierced in the bottom of the tube. 1 ml. of water was added to each fraction, and the absorbancy at 260 rnp was determined using a Zeiss speotrophotometer.

Density

in CsCl

FIG. 2. Microdensitometer (Joyce-Loebl) tracing of photographs taken during analytical ultracentrifugation of the pooled fractions comprising the major peaks shown in Fig. 1. The DNA samples comprising each peak were dialyzed as described in Materials and Methods. Portions containing 1.5 pg of DNA were separately mixed with 2 pg of B. subtilis phage 2C DNA which was used as a reference standard (p = 1.742) and centrifuged in solutions consisting of 0.02 M-Tris (pH 8.5) and CsCl (to p = 1.710) at 44,770 rev./mm at 25% for 20 hr. Standard 12-mm Beckman cells containing Kel-F 4’ sector centerpieces were used for these centrifugations.

h DNA (l-710 g/cm3) and presumably represents a small amount of reconstituted whole X DNA formed by interaction between the cohesive complementary ends of the molecule during centrifugation under conditions of very high salt concentration (CsCl), even at 4°C (cf. Nandi et al., 1966).

ROLE

f

, 20

0

OF DNA

I 40

60

IN

RNA

I 100

I 80

25

SYNTHESIS

I 120

I 140

I 160

160

Time (min) FIG. 3. Kinetics

of RNA

synthesis

primed

by whole X DNA

and isolated

A DNA

halves.

Reaction mixtures (0.5 ml.) contained 25 pmoles of Tris buffer, pH 7.5, 80 mpmoles each of GTP, CTP and UTP; 40 mpmoles of ar-32P-labeled ATP (1.5 x lo5 cts/min/mpmole); 5.0 pmoles ofMgC1,, 0.8 pmole of 2-mercaptoethanol; 2 units of RNA polymerase and 4 mpmoles of whole X DNA or one of its halves. Incubation was carried out at 37’C. Samples (50 ~1.) were removed at intervals shown and were pipetted into 2 ml. cold 5% TCA. The samples were then treated and counted as described previously (Furth et al., 1962). -O-O--, whole h DNA; --A--A--, AT-rich X DNA half; --a---@--, CC-rich X DNA half. (b)

Priming

eficiency

of whole h DNA

a,nd halves of h DNA

product in The kinetics of incorporation of CI-32P-labeled ATP into acid-insoluble RNL4 polymerase reactions primed by similar concentrations of whole X DNA and each of its separated halves are shown in Fig. 3. Both the initial rate of incorporation and the yield at bhe end of 180 minutes were less in reactions primed with the GC-rich (left) half of h DNA than in reactions using either whole h DNA or its AT-rich (right) half as template. The relatively poor priming ability of the GC-rich segment of X DNA is further evident from the results shown in Table 1. The amount of RNA TABLE

Effect of primer

concentration

Template concentration (m~moles/ml.) 4 8 16 32 48

on RNA

1 synthesis primed

by h DNA

templates

RNA synthesis observed with /\ DNA templates Sheared h AT-rich half GC-rich half Whole h (ml*moles/30 min) 0.50 0.95 1.45 1.68 1.97

0.52 o-94 1.51 1.71 2.02

0.50 1.05 1.44 1.69 I,95

0.27 0.53 0.78 0.91 1.06

Reaction mixtures (0.1 ml.) contained 5 pmoles Tris buffer, pH 7.5; 16 mpmoles each of GTP, CTP, and UTP; 12 mpmoles cr-s2P-labeled ATP (2.1 x lo4 ots/min/m~mole); 0.1 pmole MnCI,; 0.5 pmole MgCl,; 0.8 pmole 2-mercaptoethanol; 0.5 unit RNA polymerase; and DNA as indicated. The reaction mixtures were incubated at 37°C for 30 min, and were then treated and counted as described by Furth et al. (1962). Total RNA synthesis was calculated from [,x-~~P]ATP incorporation, using base incorporation factors determined by nearest-neighbor analysis of the RNA products (see below). Values are expressed as incorporation per 0.5 ml. reaction mixture in order to be directly comparable with other data presented.

26

S. N. COHEN,

U. MAITRA

AND

J. HURWITZ

synthesized on this template over a range of primer concentrations from 4 mpmoles to 48 m/rmoles/ml. was 50 to 60% of that synthesized in reactions primed by whole X DNA, sheared X DNA or the AT-rich half of X DNA under similar conditions. (c) Effect of structural alterations in the h DNA template on RNA synthesis in vitro Table 2 shows the effects of certain structural alterations in the X DNA template on initiation of polyribonucleotide chains and on the average size of the product formed in the RNA polymerase reaction. Although the whole X DNA template used in these experiments was free from single-strand breaks, it should be recalled (Materials and Methods) that both of its isolated halves had single-strand nicks as a result of the shearing process. The values shown for both RNA synthesis and initiation are yields, rather than rates, and have been determined at a time (70 minutes) when RNA chain initiation is complete and total ribonucleotide incorporation has virtually reached a plateau. The average length of completely formed RNA chains was determined by dividing total ribonucleotide incorporation by the sum of [y-32P]ATP and [‘-32P]GTP incorporation; virtually no chain initiation was observed with pyrimidine nucleotides under the conditions used. As seen in this Table, no significant differences were observed in RNA chain initiation or synthesis among the products primed by native linear h DNA, h DNA circles, h DNA aggregates, or cohered X DNA halves. In contrast, denaturation of the X DNA template resulted in a sharp rise in total initiation, a decrease in RNA synthesis, and a consequent reduction of average RNA chain-size, as has been observed previously with other DNA templates (Maitra $ Hurwitz, 1965; Bremer, Konrad I% Bruner, 1966). Sonication of the template also TABLE

2

Parameters of RNA synthesis on native and altered X DNA templates DNA template

RNA synthesis

Initiation [Y-~~P]ATP [Y-~~P]GTP (ppmoles incorporated)

Average RNA chain-length

(70 min)

Native linear X AT-rich X half GC-rich X half

1210 1170 780

0.076 0.153 0,077

0.212 0.346 0.500

4250 2350 1350

h circles (hydrogen-bonded) X aggregates Sheared h Sheared-cohered h Sonicated A Denatured h

990 1300 1120 1050 700 550

0.067 0.091 0.120 0.086 0.157 0.270

0.145 0.208 0.200 0.164 0.160 1.450

4150 4350 3500 4100 2200 320

RNA synthesis was carried out in reaction mixtures (0.5 ml.) containing 25 pmoles Tris buffer, pH 8-O; 80 mpmoles each of ATP, CTP and UTP; 40 mpmoles of cr-32P-labeled GTP (3.3 x lo4 cts/min/m~mole); 4 pmoles 2-mercaptoethanol; 0.5 pmole MnCl,; 2.5 pmoles MgCl,; 5 units RNA polymerase; and 4 mpmoles of DNA as indicated. Reaction mixtures were incubated for 70 min and assayed as described above. Initiation experiments were carried out as previously described (Maitra & Hurwitz, 1965), using y-32P-labeled ATP and [y-32P]GTP (activities 6.2 x lo6 cts/min/mpmole and 8 x lo6 cts/min/mpmole), respectively, in different reaction tubes. Incubations were at 37’C for 70 min. Total RNA synthesis was calculated from incorporation of [LX-~~P] GTP using base incorporation factors obtained by nearest-neighbor analysis. The various forms of h DNA were prepared as described in Materials and Methods.

ROLE

OF DNA

IN

RNA

SYNTHESIS

27

resulted in decreased RNA synthesis, but no significant increase in initiation sites resulted from this procedure. Average chain-length was reduced as a result of the decreased rate of synthesis observed in reactions primed with sonicated DNL4. Figure 4 shows the results of sucrose gradient zone centrifugation analysis of RNA products synthesized on whole X DNA and on each of its halves. The RNA products were examined in this case following 30 minutes of synthesis, i.e. during the period of rapid chain growth (Maitra $ Hurwitz, 1965; Bremer et al., 1965). A marked

RNA product

1200-

made from DNA

whole k

2J3s

RNA product 2 F 12003 Y

made from ,4 DNA half

AT-rich

23s

.$ 800.? z g

2

400fd

1200-

I

I

RNA product

I

made from ;\ DNA half

Fraction

CC- rich

number

FIG. 4. Zone sedimentation analysis of RNA products transcribed in vitro from whole h DNA and X DNA halves. Reaction mixtures (0.5 ml.) were prepared as described in the legend to Table 2, except that 4.5 pmoles of MgCI, were substituted for MnCI, and cc-32P-labeled GTP (specific activity of 105 cts/min/m/Lmole) was used as the radioactive label. Following incubation at 37°C for 30 mm, the reactions were stopped by the addition of an equal volume of 0.05 M-Tris buffer, pH 7.5 containing SDS and EDTA (final concentrations 0.5% and 0.025 M, respectively). The mixture was gently layered on 29 ml. of a 15 to 30% sucrose solution gradient containing 0.05 nn-Tris buffer, pH 7.5, 0.1 M-N&I and 0.2% SDS, and centrifuged for I6 hr at 25,000 rev./min at 28°C in an SW 25.1 Spinco rotor. 3H-labeled ribosomal RNA (sedimentation coefficient 23 s) ws,s used as a density marker. Following centrifugation, l-ml. fractions were collected through a hole pierced in the bottom of the tube, and the RNA was precipitated with 3 ml. 5 % cold trichloroacetic acid. The precipitates were collected on Millipore filters, washed with cold 1% TCA, dried at 60% for 30 min and counted in a toluene-base phosphor (see Materials and Methods). A liquidscintillation spectrometer set for double label counting was used to determine 3eP and 3H. The peak of the ribosomal RNA density marker (23 s) is indicated by an arrow.

28

S. N. COHEN,

U. MAITRA

AND

J. HURWITZ

heterogeneity of chain-size in RNA formed after 30 minutes of incubation is evident with all three templates, but three principal peaks consisting of 23 s, 12 s and 6 to 7 s fragments (determined by comparison with a ribosomal RNA marker) are seen with each RNA product. In addition, a broad shoulder in the range of 35 to 45 s is seen in the dripping pattern of the product made on whole X DNA but not with the h halves. While the possibility that this rapidly sedimenting RNA represents aggregates of shorter chains has not at this point been ruled out, this finding suggests that a number of RNA chains approaching 25,000 nucleotides (Kurland, 1960) in length may be synthesized on the intact h DNA template in vitro and would imply that certain RNA polymerase molecules can transcribe about half of the X DNA template as a single polyribonucleotide chain. (d) Nearest-neighbor analysis studies The results of nearest-neighbor analysis of RNA products formed after 70 minutes of incubation using whole h DNA and each of its halves as templates are shown in Table 3. In each instance, the RNA synthesized was greater than the total amount TABLE

Nearest-neighbor

analysis

Dinucleotide

Ratio

of RNA products synthesized with whole h DNA and each h half

AT-rich

pair

3

half

Whole h DNA

GC-rich

half

ApA UpU CpA UpG GpA UpC CpU ApG GpU ApC GpG CpC UP A APU CPG GPG

0.0813 0.0910 0.0683 0.0833 0.0604 0.0619 0.0598 0.0530 0.0573 0.0630 0.0489 0.0420 0.0551 0.0826 0.0443 0.0630

0.0865 0.0759 0.0720 0.0791 0.0598 0.0578 0.0533 0.0551 0.0582 0.0514 0.0525 0.0503 0.0512 0.0765 0.0530 0.0693

0.0489 0.0574 0.0692 0.0774 0.0582 0.0626 0.0556 o-0504 0.0535 0.0620 0.0751 0.0681 0.0306 0.0544 0.0828 0.0913

A U G C A +U/G

0.265 0.291 0.230 0.214 1.252

0.259 0.272 0.240 0.229 1.134

0.207 0.230 0.287 0.276 0.777

+C

The reaction mixtures were as described in Fig. 3, except that in each case, a single s-32P-labeled nucleoside triphosphate (activity 4.5 to 8~ lo4 cts/min/m~mole) and 10 units of purified E. wli RNA polymerase were added. Following incubation for 70 min at 37’C, 1.5 mg of carrier bovine serum albumin was added; the RNA was precipitated with cold 5% TCA containing 0.01 M-sodium pyrophosphate and was washed twice with cold 2% TCA. The precipitates were dissolved in 0.5 ml. of 0.3 N-NaOH and incubated overnight in tightly sealed tubes at 37’C. A mixture of unlabeled marker 2’ (3’)-ribonucleotide markers was added, the digests were neutralized with Dowex 56-H+, and the pH was adjusted to between 6 and 8 with NaOH. The Dowex resin was removed by filtration through glass wool, and the digests were concentrated to dryness in a rotary evaporator at room temperature. They were then dissolved in 0.15 ml. of water, spotted on Whatman no. 3 paper, and subjected to high-voltage eleotrophoresis in pyridine-acetic acid-water buffer (1 : 10 : 89), pH 3.5, at 6000 v for 1.5 hr. Electropherograms were cut into l-cm wide strips which were placed in vials and counted in a Packard Tri-Garb liquid-scintillation spectrometer as described in Materials and Methods. Greater than 90% recovery of the initial acid-precipitable radioactive RNA was observed in each experiment.

ROLE

OF DNA

IN

RNA

SYNTHESIS

“9

of DNA template present in the reaction mixture. As shown in this Table, the products of the two halves can be readily distinguished by differences in the frequencies of individual mononucleotide bases and certain key dinucleotide pairs (e.g. ApA, UpU, ra t ios of 1.252 and O-777 observed in the RNA made from GpC, etc.). (A+U)/(G+C) isolated AT-rich and GC-rich halves (45 and 55% G+C, respectively), reflect the divergent over-all base composition of these templates. As seen in this Table, the frequencies of individual nucleotide bases and key dinucleotide pairs in the RNA formed with whole X DNA as template resembles those formed in the RNA made from the AT-rich half. Furthermore, the (A+U)/(G+C) ratio (l-134) determined for the whole h product suggests that AT-rich regions of the template are preferentially transcribed; random copying would have produced a ratio which is the average of those observed with halves (1.015). It is also evident from the data in Table 3 that the frequencies of complementary mononucleotides (i.e. A and U, G and C) and those of complementary antipolar dinucleotide pairs (e.g. ApA and UpU; GpU and ApC) are unequal in all three of the RNA products. This finding suggests that complementary segments of both strands of the native DNA template may not be copied equally in vitro. In contrast, denaturation of the h DNA template yielded an RNA product the nearest-neighbor analysis of which shows essentially equal values for complementary nucleotides bases and dinucleotide sequences (Cohen & Hurwitz, unpublished observations). Despite these suggestions of strand selectivity by the RNA polymerase in vitro, analysis of the RlSA synthesized on the native X DNA template did not reveal an excess of purines over pyrimidines as has been reported for h-specific RNA synthesized in vivo (Skalka, 1966). (e) Hybridization

experiments

The results of the nearest-neighbor analysis experiments presented above indicated that transcription of intact h DNA ilz vitro did not occur randomly along the length of the genome. The inference that segments of the AT-rich half of the native X DNA template are preferentially copied in the RNA polymerase reaction was substantiated by DNA-RNA hybridization experiments performed on nitrocellulose membranes using the method of Gillespie & Spiegelman (1965). Because of the size differences and the divergent base compositions of the DNA templates and the RNA products, preliminary experiments were performed to ascertain: (a) the degree of retention of each (denatured) h DNA half by nitrocellulose membranes under different salt concentrations; (b) effects of salt concentration on hybridization with each DNARNA combination; (c) effects of the duration and temperature of annealing on the capacity of each type of DNA to hybridize with its own RNA product ; (d) effects of concentrations of the reactants on hybridization; (e) effects of non-specific RNA in competing with the hybridization of X DNA with its template; (f) levels required to saturate homologous sequences with the types of DNA and RNA used; and (g) effects of conditions of the post-annea’ling RNase digestion on the final amount of DNA-RNA hybrid obtained. These control experiments, which will not be presented here in detail, indicated that: (a) Whole X DNA and both of its halves were retained equally by nitrocellulose membranes when loading of 3H-labeled denatured DNA onto membranes was carried out in 6 X SSC. (b) Lowering the salt concentration of annealing solutions below 1.5 x SSC reduced hybridization of all three templates, whereas raising it above 4 x SSC increased non-specific DNA-RNA hybridization

30

S. N.

COHEN,

U. MAITRA

AND

J. HTJRWITZ

significantly; between these concentrations of SSC, the hybridization of each DNA template with its own RNA product was quantitatively similar. (c) Hybridization was roughly proportional to the concentrations of the reactants in annealing mixtures having a volume of O-5 to 1.5 ml. (d) The most satisfactory annealing temperature range in 2 X SSC was 66 to 69°C; temperatures above 73°C resulted in decreased annealing. (e) The amount of hybridization was maximum in each instance by nine hours, and remained unchanged for up to 22 hours of annealing. (f) No interference with h DNA-RNA hybridization resulted from a 150-fold excess of unlabeled RNA isolated from yeast; the specificity of hybridization was further examined in the cross-hybridization experiments with h DNA halves described below. (g) Pancreatic RNase (20 pg/ml.) had no differential effect on the various h DNA-RNA hybrids formed when used as described by Gillespie & Spiegelman (1965). Pigure 5 shows the effect of DNA concentration on hybridization of whole h DNA and each of its halves with their homologous RNA products under appropriate conditions. In each instance, a maximum of about 45 to 50% of the input RNA annealed with its own DNA template, Saturation of 25 ppmoles of input RNA occurred near 1.5 mpmoles of DNA (DNA to RNA ratio N 60) with all three DNA templates. Table 4 shows combined results of five experiments in which whole h DNA and each of the X DNA halves were hybridized with their own RNA products and reciprocally with the products of other X DNA templates. It can be seen from these results that the RNA product prepared with the AT-rich half of X DNA hybridized equally well with whole h DNA and with its own template; about 4% of the input radioactive RNA could be annealed with the GC-rich h DNA half. The RNA synthesized with GC-rich X DNA mirrored this hybridization pattern. When RNA formed on whole X DNA was annealed with each of the halves, a non-random pattern of homology was

FIG. 5. Hybridization of RNA products formed from whole DNA and h DNA halves with their own templates. RNA products were prepared as described in Table 2, except that 25 woks of Tris buffer, pH 7.5 was used and all four ribonucleoside triphosphates employed were labeled with x-~~P (activity of each was 2.5~ lo6 ots/min/m~mole). Following incubation at 37% for 50 min, the reaction mixtures were treated as described in Materials and Methods. 20 to 30 ppmoles of each isolated RNA product was annealed with different amounts of 3H-labeled DNA at 68% for 16 hr in 0.5 ml. of 2 X SSC. Subsequent treatment of nitrocellulose membranes containing DNA-RNA hybrids, and assay of the percentage hybridization were as described in Materials and Methods. -@--•-, Whole X DNA; -O-O-, AT-rich half; -A-A--, CC-rich half.

ROLE

OF DNA

IN

RNA

TABLE

Hybridization

4

of whole X DNA and each of its isolated halves with RNA products synthesized in vitro

RNA synthesized

AT-rich

31

SYNTHESIS

from:

DNA used

Hybridization o/0 input RNA hybridization

X half

Whole h ATX GCA

39 to 43 38 to 45 3 to 6

GC-rich X half

Whole x ATA CC/\

37 to 44 3 to 6 38 to 43

Whole X DNA

Whole h AT/\ GCA

38 to 44 37 to 43 10 to 15

RNA was prepared and hybridizations were performed as described in Materials and Methods and Fig. 5. The values presented are the range obtained in five separate experiments.

observed. This product hybridized equally well with whole h DNA and with the X half, but only 10 to 15% of the input radioactive RNA annealed with the @Z-rich half of the molecule. The high degree of homology of the whole h RNA product with the AT-rich half of h DNA is in keeping with the preferential copying of AT-rich segments of whole X DNA demonstrated by nearest-neighbor analysis and indioates that in vitro transcription of native h DNA is selective. AT-rich

(f) Eflect of duration of in vitro RNA synthesis on selectivity of transcription It was of interest to determine whether the threefold preference of the RNA polymerase for synthesizing RNA from the AT-rich half of X DNA was manifest throughout the duration of in vitro RNA synthesis. In order to determine this, RNA products were isolated and purified following incubation of template with enzyme for different times. Table 5 shows the effect of the duration of enzymic synthesis on

Hybridization

of RNA products synthesized at various incubation times

RNA synthesized on whole X DNA template Duration of mpmoles synthesis synthesized (min) 5 10 20 45 100

0.20 0.38 0.64 1.10 1.46

y0 of input RNA hybridized with: Whole AT-rich GC-rich X DNA half half 18.4 25.6 37.5 40.2 40.8

16.8 27.2 37.4 38.1 42.7

7.1 8.3 11.8 12.4 14.9

RNA synthesis was carried out at 37% for the time period indicated. The RNA polymerase products were isolated and hybridizations were performed as described in lkkterials and Methods and in Fig. 5.

32

S. N.

COHEN,

U. MAITRA

AND

J. HURWITZ

the annealing characteristics of the RNA product made using whole h DNA as template. It can be seen from the Table that the proportion of input RNA showing homology with each of the X DNA halves remained approximately the same throughout the duration of synthesis, although the efficiency of hybridization was reduced in the product formed early during in vitro RNA synthesis. In addition, the RNA products synthesized during various stages of polyribonucleotide chain growth on whole h DNA showed no change on dinucleotide frequencies with increasing RNA synthesis (Cohen & Hurwitz, unpublished observations). These data indioate that when purified E. coli RNA polymerase and linear h DNA are used for RNA synthesis i~z vitro, there is no temporal shift to increased copying of the GC-rich half, as has been reported by Skalka (1966) to occur in vivo. (g) Effect of DNA and enzyme concentrations on selectivity of transcription

It was also of interest to determine whether the ratio of AT-rich half copying to GC-rich half copying could be influenced by the relative amounts of RNA polymerase and DNA template used during in vitro RNA synthesis. A large excess of enzyme might change this ratio by allowing copying of additional (less preferred) template sites; conversely, only the very most preferred sites might be transcribed in the presence of limiting enzyme and excess template. In order to study this, RNA products were prepared using 5 to 300 units of purified E. co& RNA polymerase and 2 to 100 mpmoles of whole h DNA template per incubation tube. Other experiments (Cohen & Hurwitz, unpublished data) had shown that under the assay conditions used, 5 units of RNA polymerase were completely saturated by 30 mpmoles of h DNA (i.e. one enzyme molecule for every 3750 to 8000 DNA nucleotide pairs, based on a molecular weight of 0.4 to 0.9 x lo6 daltons for the purified E. coli RNA polymerase (Crawford et al., 1965; Richardson, 1966a; Maitra & Hurwitz, manuscript in preparation)). TABLE 6

EJjCectof DNA to enzyme ratio on the selectivity of transcription of X DNA in vitro

DNA concentration (m~moles/O.S ml.)

2 4 4 4 20 20

50 100

RNA polymerase (units/O.5 ml.)

300

5 50

100

5 120 5 5

Approximate DNA to enzyme ratio (nucleotide pairs per enzyme molecule) 4-10

500-1000 50-100 25-50 2500-6000 100-200 6250-14,000 12,500-28,000

o/0 input RNA annealing with: AT-rich GC-rich Whole X DNA X DNA half h DNA half

46 46 44 45 44 47 41 47

36 40 43 40 42 42 41 42

22 12 16

18 14 16 12 13

RNA products were prepared as described in Materials and Methods and in Fig. 5. The indicated amounts of whole h DNA template and purified E. coli RNA polymerase were used in each reaction mixture. Nucleotide pairs per enzyme molecule were calculated assuming a molecular weight of 1966a; Maitra & 0.4 to 0.9 x lo6 daltons for the enzyme (Crawford et al., 1965; Richardson Hurwitz, manuscript in preparation) and an average molecular weight of 325 per DNA nucleotide.

ROLE

OF DNA

IN

RNA

33

SYNTHESIS

The results of annealing these RNA products with whole X DNA and its halves are shown in Table 6. These data indicate that transcription of the GC-rich h DNA half was relatively increased when RNA polymerase was present in excess of one enzyme molecule per 25 to 50 nucleotide pairs, but that some preferential copying of sites present predominantly on the AT-rich half was still evident. An increase in the enzyme to DNA ratio to one molecule of enzyme for every 5 to 10 nucleotide pairs (approximately 10,000 enzyme molecules per molecule of DNA based on a molecular weight of 3.2 x lo7 daltons for whole h DNA (Burgi & Hershey, 1963; Caro, 1965; Studier, 1965)) still did not completely abolish selectivity of transcription. In the reciprocal experiment, reduction of the enzyme to DNA ratio to as low as one enzyme molecule per 20,000 DNA nucleotide pairs did not increase AT-half to GC-half copying significantly beyond the three to one selectivity observed in our earlier experiments. (h) Effects of structural alteration in X DNA template on selectivity of transcription The effects of certain alterations in the X DNA template on hybridization of products formed in the RNA polymerase reaction are shown in Table 7. It is evident TABLE

7

Effects of alterations of the A DNA template on hybridization of RNA products oh of input RNA hybridized X DNA template

Native linear Circles Aggregates Sheared Sheared-cohered Sonicated Denatured

Whole A DNA

38

to 44

18.7 28.2

AT-rich half

38

to 42

1m 18.9

with: GC-rich half

10 to 15 8.9 20.0

The various structural forms of X DNA were prepared as described in Materials and Methods. Isolation and hybridization of RNA products were performed as described in Fig. 5 and in Materials and Methods. Incubation was at 37% for 50 mm in each instance.

from this Table that preferential copying of sequences on the AT-rich X half is not affected by altering the free complementary ends present on native linear h DNA by the formation in vitro of X circles, aggregates or cohered halves. Furthermore, shearing or sonicating the template did not eliminate selectivity of transcription by the RNA polymerase. The RNA synthesized on both son&ted X DNA and denatured ;\ DNA showed a generally decreased efficiency of hybridization, an effect which may have been related to a decrease in the size of the RNA products made on these templates. However, only denaturation of the template resulted in elimination of selective copying of the AT-rich half.

4. Discussion At least four principal possibilities exist regarding sites of the h DNA template which may be selected by the RNA polymerase for the formation of RNA chains in

34

8. N.

COHEN,

FIG. 6. Possible

U. MAITRA

schemes for transcription

AND

J. HURWITZ

of DNA

template

in u&o.

vitro (Fig. 6). Such chains may begin and end randomly along the length of the template, as in (a); RNA synthesis may begin at a single site and proceed along the template with newly formed polynucleotide chains terminating at various specific or random sites, as in (b) ; one or more specified segments of the template may be repeatedly copied, as in (c) ; or there may be preferential polymerization of RNA chains on certain portions of the template while other segments are transcribed to a lesser extent, as in (d). The studies reported here indicate that in. vitro transcription of native whole h DNA by purified E. coli RNA polymerase is not random (as depicted in possibility (a)), but rather that the enzyme is directed by the template to copy certain DNA regions preferentially. Possibility (lo) is effectively eliminated by our observation that RNA products formed on the whole h DNA template at various times after initiation of RNA synthesis show similar patterns of hybridization, i.e. sites present predominantly on the AT-rich (right) half of the linear X DNA molecule are preferentially transcribed to the same extent throughout the duration of in vitro RNA synthesis. The model presented as possibility (c) depicts selectivity of transcription as being dependent on the presence of a greater number of initiation sites on certain segments of the DNA template. In possibility (d), initiation of polynucleotide chains is equal on various DNA segments ; but different extents of polymerization of RNA chains are responsible for preferential transcription of certain template regions in vitro. We cannot distinguish with certainty between these last two models, or a combination of them. In evaluating these models, it is important to point out that binding of enzyme to template and initiation of polynucleotide chains along the template are obviously necessary, but not su$icient conditions for RNA synthesis; binding, initiation and polymerization can be separate phenomena (Anthony, Zeszotek & Goldthwait, 1966; Maitra & Hurwitz, manuscript in preparation). Various investigators (Hurwitz et al., 1962; Fox & Weiss, 1964; Bremer et ab., 1966; Jones & Berg, 1966) have shown that denatured DNA is capable of binding larger amounts of RNA polymerase than an identical quantity of the native template, and recent studies (Maitra & Hurwitz, 1965; Bremer et al,, 1966) indicate that initiation of RNA chains is greater on denatured than on native DNA. Yet, chain growth is slower on denatured templates (Maitra & Hurwitz, 1965; Bremer et aZ., 1966) and total RNA synthesis is reduced. Our studies of RNA initiation and synthesis using isolated halves of h DNA as templates provide additional evidence that initiation and polymerization of RNA chains can occur independently. Although initiation was equal on both X DNA halves, total RNA synthesis was reduced on the isolated GC-rich half of the template. Extension of these observations to the RNA products made on intact h DNA must

ROLE

OF DNA

IN

RNA

SYNTHESIS

36

be done cautiously, however, since single-strand breaks (see above) and possibly localized regions of strand separation can occur as a result of the shearing process. (We attempted to determine whether initiation of RNA chains occurred equally on specific segments of unsheared h DNA by using RNA products labeled with [3H]UTP and a mixture of [y-32P]ATP and GTP for annealing with isolated h DNA halves. Unfortunately, the results of these studies have been difllcult to interpret, since hybridization of so speci6c a locus as a single y -32P-labeled nucleoside triphosphate present at an RNA chain terminus has not been reproducible in our hands, presumably because of its susceptibility to RNase attack. It would be expected that “frayed ends” of annealed hybrids would be attacked by RNase used in our standard hybridization procedure.) Large variations in RNA chain initiation that would be expected to result from altering the enzyme to DNA ratio (Richardson, 1966b; Maitra & Hurwitz, manuscript in preparation) over a 25-fold range did not affect preferential transcription of the AT-rich half of X DNA. Reduction of the ratio of AT-rich half copying to GC-rich half copying was observed only when enzyme was present in large enough excess to allow one polymerase molecule for every two to four DNA nucleotide pairs ; and even under these conditions, specificity of transcription was not completely abolished. It is not clear whether reduction of selectivity by such a large excess of enzyme resulted from initiation of new RNA chains at less preferred template sites, or from decreased RNA chain polymerization. Either of these possibilities would be compatible with the decrease in average RNA size always observed at high enzyme to DNA ratios (Maitra & Hurwitz, manuscript in preparation). Denaturation of the X DNA template resulted in elimination of selective in vitro transcription by the E. coli RNA polymerase, an effect which may also be related to either a generalized decrease in the extent of chain elongation or to the uncovering of new non-specific single-stranded sites at which the enzyme can bind (Maitra 8: Hurwitz, 1965; Colvill, Kanuer, Tocchini-Valenti, Sarnat & Geiduschek, 1965). The marked increase in RNA chain initiation which was observed following disruption of the native helical secondary structure of the X DNA template favours the latter possibility. Our preliminary studies (Cohen, 1966) indicated that sonication of X DNA also reduced selectivity of transcription. Subsequently, it became evident that DNA preparations used for these experiments contained variable amounts of denatured material. Later studies performed with X DNA fragments having an intact helical structure indicated that linearly intact h DNA is not essential to the selection mechanism. Apparently, the template sequences determining specificity of transcription are small enough to avoid disruption by sonication of the DNA to fragments having a molecular weight of 600,000 to 800,000 daltons. In addition, the mechanical creation of new DNA termini by this procedure does not influence the capacity of the enzyme to transcribe certain DNA regions preferentially. In vitro cohesion of the ends of linear X DNA extracted from mature phage did not influence either the priming ability of the DNA template or the capacity of the enzyme selectively to copy certain DNA regions. It may also be inferred from our studies of RNA synthesis using structurally altered forms of DNA that in vitro transcription does not require single-stranded DNA termini for the initiation of polynucleotide chains, since hydrogen-bonded circular h DNA molecules showed no detectable differences in template function from linear h DNA. Furthermore, the

36

S. N.

COHEN,

U. MAITRA

AND

J. HURWITZ

selection mechanism was not affected by presenting the enzyme with “outside-in” reconstituted whole h DNA molecules formed in vitro when the halves are made to cohere. Presumably, this mechanism does not depend on a specific linear orientation of the template halves and does not require the presence of specific nucleotide sites at the DNA termini. (The possibility that the RNA polymerase may alter the physical structure of the various X DNA forms added to incubation mixtures must be considered in interpretation of these results. The effects of enzyme-template interactions on the tertiary structure of X DNA were not studied in the present experiments.) It should be emphasized that the DNA used in all of these studies was isolated from mature X bacteriophage, and may not be the structural form of template transcribed in vivo by the RNA polymerase. Earlier investigations (Chamberlin & Berg, 1962 ; Furth et al., 1962 ; Cohen & Hurwitz, unpublished data) have shown that mature X DNA is a relatively poor template for the E. coli RNA polymerase in vitro when compared with other phage DNA preparations, and recent observations (Jones & Berg, 1966) suggest that h DNA contains many fewer binding sites for this enzyme than does phage T7 DNA, for example. It is tempting to speculate that one or more of the recently described intracellular forms of h DNA (i.e. covalent circles, concatenated or “replicative” forms) (Bode & Kaiser, 1965; Smith & Skalka, 1966; Weissbach, Lipton & Lisio, 1966) might show an altered capacity to function as a template with regard to priming efficiency and/or selectivity of transcription in vitro. Recent studies by Skalka (1966) indicate that h-specific messenger RNA made in vivo early after phage infection is transcribed primarily from the AT-rich half of h DNA, a segment which is comprised mostly of “early” cistrons of phage development (Jacob, Fuerst & Wollman, 1957; Campbell, 1961). Thirty minutes later, in vivo transcription extends to the GC-rich half, which contains the “late” functioning cistrons A to J. We have confirmed Skalka’s findings using mRNA pulse-labeled with [3H]uridine 30 to 32 minutes (early) or 60 to 62 minutes (late) after addition of mitomycin C to logarithmically growing cultures of E. coli K12 (A) (Cohen & Hurwitz, unpublished data). In contrast with these in viwo results, our in vitro experiments indicate that transcription of mature h DNA by the purified E. coli polymerase remains restricted to predominantly the AT-rich h half over a wide range of incubation times, and shows no tendency for temporal extension to the GC-rich region. Presumably, a gene product of the AT-rich X DNA half is required in order for a shift to transcription of the (‘late” regions to occur; its absence in our in vitro system results in repeated preferential transcription of segments of h DNA containing “early” functioning cistrons. (Although the polynucleotide sequences transcribed in vitro are from the same template segments as those copied at early times in, vivo, quantitative differences in the ratios of AT-half copying to GC-half copying observed in these two systems mitigate the view that X in vitro RNA can be completely equated with early h mRNA.) Recent evidence that X mutants defective in certain early functions show a pleiotropic defect in late functions (Protass & Korn, 1966; Dove, 1966; Eisen et al., 1966) and a decrease in total late messenger RNA synthesis (Joyner, Isaacs, Echols & Sly, 1966) is consistent with this interpretation. Furthermore, recent studies in our laboratory (Cohen & Hurwitz, unpublished data) indicate that i?z.vivo transcription of h DNA remains restricted to predominantly its AT-rich half when DNA replication is prevented. These experiments were carried out under conditions of continued thymine deprivation following thymineless induction of thymine-requiring E. coli

ROLE

OF DNA

IN

RNA

37

SYNTHESIS

K12(X) (Kern & Weissbach, 1962). Parallel observations have been reported by xaono & Gros (1965), who found that mRNA formed under conditions where DNA was synthesis could not occur was rich in adenine and uracil. When DNA replication permitted, a second class of mRNA rich in guanine and cytosine appeared. Although replication of phage DNA is a necessary condition for the normal synthesis of late h mRNA, it apparently is not a sufficient one; sus Q mutants appear to replicate their DNA normally, but nevertheless show decreased production of late h messenger RNA (Joyner et al., 1966; Dove, 1966; Eisen et al., 1966). During the past few years, workers in several laboratories (Khesin, Gorlinko, Sheyakin, Bass & Prozorov, 1963 ; Luria, 1965 ; Colvill et al., 1965 ; Geiduschek, Snyder, Colvill & Sarnat, 1966) have observed that E. coli RNA polymerase products synthesized in vitro using native DNA from T-even phages as template competed almost exclusively in DNA-RNA hybridization experiments with early messenger RXA made in vivo. Although the intramolecular homogeneity of T-even DNA does not allow ready separation of its segments for subsequent examination of in vitro transcription from specific template regions, the competition experiments with these phages nevertheless indicate that selective transcription of restricted portions of phage DNA template in vitro may be a general phenomenon. We acknowledge the helpful suggestions initial stages of this work. These studies Institutes of Health, the National Science Council of the City of New York. One of us from the American Cancer Society.

of Dr E. Burgi and Dr J. Marmur during the were supported by grants from the National Foundation and the Public Health Research (S. N. C.) acknowledges an award (PF no. 276)

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