Studies of the binding of Escherichia coli RNA polymerase to DNA

J. Mol. Biol. (1972) 70, 197-207

Studies of the Binding of Escherichia coli RNA Polymerase to DNA III. Tight Binding of RNA Polymerase Holoenzyme to Siugle-strand Breaks in T7 DNA DAVID C.~TINK.LE~,JANET RIIW AND MICHAEL J. CHAMBERLIN Departments of Biochemistry and Molecular Biology and the Virus Laboratory University of California, Berkeley, Calif. 94720, U.S.A. (Received 21 September 1971, and in revised form 26 April 1972) Single-strand breaks in T7 DNA enhance transcription by Escherichia cdi core RNA polymerase, probably by providing new sites for RNA chain initiation. Significant enhancement is obtained with a small number of breaks, hence it seems likely that a large proportion of all breaks can serve as RNA chain initiation sites for core polymerase. Single-strand breaks in T7 DNA inhibit transcription by E. co&i RNA polymerase holoenzyme. The inhibition is due to a decrease in the fraction of RNA polymerase holoenzyme molecules that can initiate a T? RNA chain when DNA containing single-strand breaks serves as template. This decrease is probably accounted for by the finding that single-strand breaks serve as tight binding sites for RNA polymerase holoenzyme but that few such sites can serve as RNA chain initiation sites. It is concluded that the structural requirements for a “tight” binding site on DNA for RNA polymerase are less stringent than the requirements for an RNA chain initiation site. Thus, the fidelity of initiation of T7 RNA synthesis is governed not only through regulation of the sites on T7 DNA at which binding of RNA polymerase can occur, but also by what appear to be rigid structural requirements for RNA chain initiation by RNA polymerase once it has been bound.

1. Introduction Transcription of T7 DNA in vitro by bacterial RNA polymerase is restricted primarily to one of the two DNA strands and gives rise to messenger RNA sequences found early in phage infection. This selective and restricted transcription is due in part to efficient promoter site selection during RNA chain initiation (Summers Q Siegel, 1969 ; Goff & Minkley, 1970 ; Chamberlin, 1970) and depends on the presence of sigma subunit on the RNA polymerase molecule (Goff & Minkley, 1970). Measurements of the binding of RNA polymerase holoenzyme to T7 DNA (Hinkle & Chamberlin, 1970,1972a,6) indicate that site selection occurs during binding of RNA polymerase to T7 DNA and involves the formation of a highly stable holoenzyme-T7 DNA complex at or near the T7 promoter site. Formation of this complex requires the sigma subunit of RNA polymerase (Burgess, Travers, Dunn & Bautz, 1969; Berg, Barrett, Hi&e, McGrath $ Chamberlin, 1969) and probably involves the opening of DNA base pairs near the promoter site (Hinkle & Chamberlin, 1970). In contrast, efficient transcription by core polymerase requires a template which is t Present address: Mass. 02115, U.S.A.

Department

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Chemistry,

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single-stranded or contains single-strand breaks (Vogt, 1969; Burgess et al., 1969; Berg, Barrett & Chamberlin, 1971; Iahihama, Murakami, Fukuda, Matsukage & Kameyama, 1971). Furthermore, attachment of core polymerase to intact T7 DNA does not lead to formation of a highly stable complex (Hinkle & Chamber& 1972a). Thus, it appears that sigma subunit is required for the opening of the DNA strands near the promoter site. This suggested that new binding and initiation sites might be introduced into the DNA through introduction of single-strand breaks in the helix. Site-specific breaks are known to be present in some phage DNA’s as they are found in the virion (Thomas & McHattie, 1967) and have been implicated in the regulation of transcription during vegetative growth of other phages (Cascino, Riva $ Geiduschek, 1970). In this communication we report the effect of such breaks on the transcription of T7 DNA in vitro and on the formation of highly stable complexes between RNA polymerase holoenzyme and T7 DNA.

2. Materials and Methods Except where noted, materials and techniques used to measure binding of RNA polymersm to T7 DNA using the nitrocellnlose filter technique have been described in the preceding papers (Hinkle & Chamberlin 1972a$). DNA concentrations are given in mnoles of DNA nucleotide. Single-strand breaks were introduced into T7 DNA using bovine pancreatic deoxyribonuclease (DNase I: Worthington electrophoreticelly purified DPFF). Samples of T7 DNA (300 nmoles) were incubated for 16 min at 37°C in 06 ml. of binding buffer (10 mMTris (pH 80), 10 mM-MgClz, 60 m&r-N&l, 10 mm-2-mercaptoethol, 1 rmr-EDTA) with varying amounts of pancreatic DNase I. DNase was inactivated by incubating the solution at 65°C for 15 min and 0.06 ml. of 0.1 M-EDTA was then added to each reaction mixture. The number of 5’-termini present in each DNA preparation was determined by the method of Weiss, Live & Riohardson (1968). Samples containing 10 to 30 nmoles of T7 DNA were denatured in 60 ~1. of O-1 N-NaOH. After 10 min at O”C, reactions were rapidly adjusted to pH 7.9 with 10 ~1. of O-6 M-Tris base plus 0.75 N-HCl. Bacterial alkaline phosphatase (10 ~1. of 10 units/ml.) was added and reactions were incubated for 30 min at 37°C. Reactions were chilled to O”C, 20 pl. of a solution containing 50 mM-MgClz, 86 m&r-2-mercaptoethanol, 5 mM-potassium phosphate (pH 7*5), and 85 ~M-[~-~~P]ATP (3700 cts/min/pmole) and 10 pl. of T4 polynucleotide kinase (600 units/ml.) were added and incubation was continued for 30 min at 37°C. Acid-insoluble radioactivity was then determined, ss in the standard RNA polymerase assay, by precipitation of the labeled DNA with ice-cold perchloric acid containing 0.1 M-pyrophosphate, followed by 6ltrtEtion through Whatman GF/C glass filters (Berg et al., 1971). Filters from control reactions with no polynucleotide kinase contsined only 0.03 pmole of [Y-~~P]ATP. The number of 6’termini per genome was calculated assuming a molecular weight of 25.6 x IO* daltons for T7 DNA (Freifelder, 1970). We are gmteful to Dr Stuart Lhm for providing the polynucleotide kinaae used for this analysis. Transcription was assayed in O-10-ml. reactions containing assay buffer (40 mM-Tris (pH 8), 10 mm-MgCl,, 10 m&r-2-mercaptoethanol), 40 nmoles each of GTP, UTP, CTP and [w~~P]ATP (7900 cts/min/nmole), 10 nmoles of untreated or DNase-treated T7 DNA as indicated, and 0.2 pg of either RNA polymerase holoenzyme (fraction 6 Berg et al. (1971) ; 14,000 d(A-T) units/mg) or core polymersse (16,000 d(A-T) units/mg). Incubation was for 10 min at 37°C. The incorporation of AMP into an acid-insoluble form was determined as desoribed elsewhere (Berg et al., 1971). Measurement of T7 RNA chain initiation was carried out using [ys2P]ATP or GTP as described elsewhere (Chamberlin & Ring, 1972).

3. Results (a) Effect of single-strand breaks on 7’7 RNA synthesis When single-strand interruptions are introduced into helical DNA the ability of the DNA to serve as a template for core polymerase-directed RNA synthesis is consider-

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Deoxyribonuckose I kg 1

FIQ. 1. Effect of single-strand breeks in T7 DNA on the rate of transcription by RNA polymer* holoenzyme and core polymeraee. T7 DNA (10 nmoles) w8a preinoubeted for 16 min et 37°C with the indicated amounts of pancreatio DNase I in 60 ~1. of sassy buffer plus 60 maa-Necl. The DNase w8a then irmotivated by incubation 8t tV?‘G for 15 min. An assay mixture (100 4.) containing 50 nmolea eaoh GTP, UTP, CTP snd [w~~P]ATP (5200 cts/min/nmole) plus either 0.6 re; RNA polymersse holoenzyme (fraction 6, 20,000 d(A-T) units/m& or 1.2 pg core polymerese (16,000 d(A-T) units/m@ in 86~8~ buffer plus 60 mu-N&l was then edded to each reaction and the incubation wes continued for 10 mm at 37°C. Incorporation of AMP into an acid-insoluble form was determined aa described in Materiala and Methods. when pancreatio DNase I wes omitted from the pre-inoubetion, RNA polymer88e holoenzyme incorporeted 873 pmoles of AMP and oore polymereee inoorporated 72 pmoles of AMP. -O-O-, RNA polymer&se holoenzyme; -e-e--, core polymeram.

ably enhanced (Vogt, 1969). Figure 1 shows the effect of incubating T7 DNA with increasing amounts of pancreatic DNase before its utilization as a template for RNA synthesis by E. wli RNA polymerase holoenzyme or core polymerase. As found for #SO phage DNA (Vogt, 1969), there is a marked increase in the activity of core poly. merase with the DNase-treated T7 DNA. In contrast, transcription of DNase-treated DNA by RNA polymerese holoenzyme is considerably reduced. To determine the relation between the number of single-strand breaks introduced into T7 DNA and the effects on transcription, samples of T7 DNA were prepared by treatment with increasing amounts of DNase. The number of single-strand breaks was measured using polynucleotide kin~se (Weiss et al., 1968) and the template activity of the samples was compared with RNA polymerase holoenzyme and with core polymerase (Table 1). Untreated T7 DNA contained 26 5’-termini per genome after alkaline phosphatase treatment, indicating that in addition to the two 5’-tern&i of the DNA strands there was a maximum of one single-strand break per 3.3 strands. Estimates of the number of unbroken DNA strands in this preparation were also obtained by zonal sedimentation of the strands in alkali (Studier, 1966). By this latter criterion, over 70% of the strands were intact. After treatment of 300 nmoles of T7 DNA with 1 x 10m3 H of DNase, about 20 single-strand breaks have been introduced into the T7 genome.

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1

Effect of single-strand breaks on transcription of T7 DNA Pre-incubation (pg DNase) Number of V-termini per genome Transcription by core enzyme Transcription by holoenzyme

0 2.6 = 100 ilO

1 x 10-e 20 180 54

1 x 10-Z 145 100 20

5x10-’ 396 25 5

Single-strand breaks were introduced into 300 nmoles of T7 DNA by incubation with the amounts of DNase I shown; the exact procedures employed and the determination of the number of 6’-termini per genome are described in Materials and Methods. Samples of treated T7 DNA (10 nmoles) were assayed as templates for RNA polymerase holoenzyme (0.2 pg) or core polymerase (0.2 pg) in a standard RNA polymerase assay. With untreated T7 DNA, RNA polymerase holoenzyme incorporated 95 pmoles of AMP and core polymerase incorporated 19 pmoles of AMP, in a lo-mm reaction.

This leads to a l&fold increase in the activity of core polymerase and about a l&fold decrease in the activity of RNA polymerase holoenzyme. Further cleavage of the DNA leads to a decrease in template activity with both forms of enzyme. The stimulatory effects of single-strand breaks on transcription by core polymerase are most easily explained by the hypothesis that these breaks serve as new initiation sites for core polymerase (Vogt, 1969). Core polymerase is probably unable to initiate RNA chains with an intact helical template (Ishihama et al., 1971). Thus, one might imagine that sigma subunit is required to open the strands of the DNA template prior to chain initiation (Hinkle & Chamberlin, 1970). Since a single-strand break provides a point at which the helix is opened over a few base pairs, it is plausible that such a site might allow core polymerase to initiate an RNA chain in the absence of sigma. A relatively small number of such breaks are required to give good initiation by core polymerase. The data in Figure 1 suggest that maximum stimulation of transcription by the core polymerase is obtained using an amount of DNase suflicient to inhibit transcription by the holoenzyme by about 40% or enough to introduce slightly less than 20 breaks per T7 genome. However, transcription by the core polymerase is significantly stimulated using tenfold less DNase. We assume that the number of single-strand breaks introduced into a sample of T7 DNA is directly proportional to the ratio of DNase I added to T7 DNA substrate. Thus, stimulation of core polymerase occurs with an average of only one to two breaks per genome. This suggests that essentially any break in the DNA can serve as the initiation site for core polymerase. The reason for the inhibition of synthesis by RNA polymerase holoenzyme remains to be explained. Two possibilities are considered: (1) a single-strand break in a DNA strand may block the growth of RNA chains beyond that point; (2) single-strand breaks may serve as points at which tight binding of RNA polymerase can occur but at which chain initiation cannot occur. Qualitatively, (1) would explain the level of inhibition seen in Table 1; at 1 x 10e3 pg DNase there were about 18 breaks per genome or about 1.4 breaks per 2 x lo6 daltons of DNA. Since the transcription by RNA polymerase holoenzyme is restricted to a region of about 2 x log daltons (Millette, Trotter, Herrlich & Schweiger, 1970; Maitra, Lockwood, Dubnoff & Guha, 1970), only breaks in this region would be significant. The probability of no breaks in this region is given by the Poisson distribution as P,,, = e-le4 or about O-25; hence, about 75% of these regions would contain a nick, while transcription is found to be

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inhibited about 50%. These numbers are roughly comparable, but this result does not rule out the second possibility proposed above?. (b) Effect of single-strand breaks on RNA chain initiation To test directly whether breaks in T7 DNA block RNA chain initiation, we have measured the effect of such breaks on the incorporation of [Y-~~P]ATP and GTP into T7 RNA. Experiments were carried out by forming an RNA polymeraseT7 DNA complex at a ratio of RNA polymerase to DNA (r) of about 5 ; nucleoside triphosphates were then added to initiate RNA synthesis snd the reaction was terminated after two minutes. Under these conditions, we assume that RNA polymerase molecules are all bound to class A or tight binding sites on T7 DNA and that because of the very slow rate of dissociation from such sites, exchange between sites does not occur during the reaction. During the short reaction time involved, each active RNA polymerase molecule is able to initiate an RNA chain if it is bound at a site which permits initiation (Chamberlin & Ring, 1972); however, RNA chain termination and repeated initiation is minimized. Thus, the amount of y- 32P-labeled nucleotide incorporated directly reflects the number of active RNA polymerase molecules in the reaction, and by inference, the fraction of the tight binding sites at which chain initiation is permitted. With intact T7 DNA we have argued that essentially all class A binding sites permit chain initiation (Chamberlin & Ring; 1972) however, only about 70% of the enzyme added to the reaction is enzymically active. The results (Fig. 2) show that the introduction of single-strand breaks decreases the amount of [Y-~~P]ATP incorporation markedly but has little effect on the incorporation of [Y-~~P]GTP. The first conclusion is that the total number of RNA chains initiated does decrease with the introduction of single-strand breaks and that the

Deoxyribonuclease

I (pg)

Fro. 2. Effect of single-strand breaks in T7 DNA on the initiation of T7 RNA chains by [Y-~~P]ATP and GTP. Samples (100 nmoles each) of T7 DNA were treated with the amounts of pencreatio DNase I shown for 16 min at 37°C 8s in Fig. 1, and the resulting DNA containing single-strand breeks was ass8yed for its ability to dire& T7 RNA synthesis using [‘W]ATP as substrate, 8s in Fig. 1 (-- l -- l --) or for its ability to initiate T7 RNA chains using [Y-~~P]ATP (-O-O-) or [Y-~~P]GTP (-n-u-) as substrate in 8n identioal reaction. In the latter instance, RNA ohain initiation ~8s measured after 2 min at 37% to prealude re-initi8tion of RNA chains by enzyme whioh had oompleted a round of tmnsoription (Chamberlin & Ring, 1972). In each reaotion 3.4 pg of E. coli RNA polymerase holoenzyme was used (Y = 5). t The results described below, whioh show that single-strand breaks do serve 8s tight binding sites for holoenzyme, suggest in feet that a break does not appreciably deorease transcription by RNA polymerase. This is most simply explained if transoription oan pass through a break or, elternatively, if oh8in termin tion with release of free RNA polymerase oaours 8t such a site. In either case the amount of transcription would not be reduced appreciably.

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decrease in total RNA synthesis approximately parallels the decrease in RNA chain initiation. This result does not rule out the possibility that single-strand breaks in the r-strand of T7 DNA do retard some chain elongation. However, it indicates that the major cause of the decrease in T7 RNA synthesis brought about by single-strand breaks is due to a decrease in RNA chain initiation. This decrease in RNA chain initiation is most easily visualized as being due to the binding of RNA polymerase molecules at tight binding sites, at which they cannot initiate an RNA chain or at which initiation is extremely slow. Direct evidence that single-strand breaks function as new tight binding sites is presented below. The differential effect of single-strand breaks on incorporation of [y-32P]GTP is quite interesting. It has been suggested that T7 messenger RNA chains initiated at the “early” T7 promoter site may be initiated only with ATP (ChamberlinJ970) and this is supported by preliminary annealing studies, which suggest that all of the [Y-~~P]ATP chains are r-strand specific, while the [Y-~~P]GTP chains are not (Ring 6 Chamberlin, unpublished observations). Thus, it may be that all [y-32P]GTP-init,iated T7 RNA chains are initiated at non-specific sites or at single-strand breaks. The fact that the amount of [Y~~P]GTP incorporation does not change appreciably with the introduction of many breaks suggests that a constant fraction of these breaks permit initiation of an RNA chain with GTP. In contrast, the initiation of an RNA chain with ATP at such sites occurs seldom, if at all. (c) Effect of sin&-strand

breaks on the jidelity of T7 RNA synthesis

Since it is believed that in viva all T7 messenger RNA is transcribed from the r-strand of the DNA (Summers & Szybalski, 1968), an estimate of the amount of correct transcription in vitro can be obtained by comparing the amount of T7 RNA annealing to the separated l- and r-strands (Goff t Minkley, 1970). It is usually assumed that all I-strand-specific T7 RNA is due to missense initiation; however, the relation between this value and the total amount of incorrect initiation is not easily obtained. First, since missense initiation can presumrtbly take place on either strand, the amount of I-strand RNA must represent only part of the incorrectly initiated RNA. If we assume that m&sense initiation takes place randomly, then an amount of r-strand RNA equal to the amount of l-strand RNA is incorrectly initiated. Second, when randomly initiated l- and r-strand RNA’s are present together in the hybridization assays, formation of RNA-RNA duplexes takes place (Brady, Diggelmann & Geiduschek, 1970) and these are not retained on nitrocellulose filters. This can lead to a preferential loss of missense RNA, since it is precisely these RNA species that are able to form duplexes+. Thus, the efficiency of annealing relative to the total input of T7 RNA must also be considered in judging the extent of incorrect initiation. Table 2 shows the annealing properties of T7 RNA fractions transcribed by both t Goff & Minkley (1970) have reported that a variable fraction of T7 RNA synthesized by core polymerase is RNase resistant and have suggested that this RNA is present in a DNA-RNA hybrid. Suah a hybrid would not be detected in the filter annealing experiments we describe and might also contribute to a low efficienoy of hybridization. We have seen no evidence for such a hybrid. A small fraction (8 to 10%) of the RNA formed by core polymerase is insensitive to RNase; however, this RNA is not sensitive to a mixture of RNase and DNase I, which destroys over 98% of an authentio DNA-RNA hybrid (Chamberlin & Berg, 1964). To preclude contamination by DNA-RNA hybrids, all RNA products in our experiments were isolated after digestion with DNaae I. In addition, duplicate snnealing studies carried out with denatured T7 DNA (containing both r- and l-strands) on the filters revealed from 92 to 100% of the labeled RNA annealing to the filter in all instances in Table 2.

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TABLE 2

Effect of single-atrand breaks on r-strand-apeci$c T7 RNA aynthesia by RNA polymerase Ellzym0

RNA polymerase core polymerase

pmzzol

ZNA

r-strand

l-strand

Template

holoenzyme

% input RNA (rhybridized

l)/(r+l)

T7 DNA DNase’d T7 DNA

211 182

21 26

87 79

82 75

T7 DNA DNase’d T7 DNA

68 49

37 45

69 48

29 2

3H-labeled T7 RNA was prepared in a standard RNA polymerase assay mixture (0.15 ml.) oontaining 46 nmoles of T7 DNA, [sH]ATP (103 ots/min/pmole) as labeled nucleoside triphosphate and 5 or 8 pg of RNA polymerase holoenzyme (fraotion 6) or core polymerase, respectively. Incubation was for 10 min at 37°C. The reaotion was stopped and DNA degraded by adding 10 H of panareatio DNase. After 10 min at 37”C, the produots were extraoted onoe with phenol. With RNA polymerase holoenzyme, 24.0 and 18.9 mnoles of T7 RNA were formed with intact T7 DNA and DNase-treated T7 DNA, respectively. With aore polymerase, 3.1 and 6.7 nmoles of T7 RNA were formed with intact T7 DNA and DNase-treated T7 DNA, respectively. Samples of the phenol-extraoted produots were used directly for determination of the amount of labeled T7 RNA and for annealing to nitrocellulose filters containing a large excess (2.6 nmoles per filter) of separated r- or l-strands of T7 DNA (Hinkle & Chamberlin, 1970). DNase-treated T7 DNA was prepared as described in Materials and Methods; 45 mnoles of T7 DNA was treated with lo-’ H of DNase I for 16 min at 37°C; this is calculated to introduce about 12 single-strand breaks per T7 genome.

RNA polymerase holoenzyme and core polymerase using intact T7 DNA and DNasetreated T7 DNA (about 12 breaks per genome) as templates. Hybridization was carried out using the nitrocellulose filter technique of Gillespie & Spiegelman (1965). The extent of correct initiation is estimated from the fraction (r - l)/(r + l), which reflects the amount of T7 RNA preferentially initiated on the T7 r-strand. It is clear that over 80% of the RNA sequences transcribed by holoenzyme with intact T7 DNA as template are preferentially initiated on the r-strand. The introduction of about 12 breaks per genome lowers the Sdelity of transcription but not dramatically. If each break were able to serve as an active initiation site, the fidelity of transcription would be expected to decrease precipitously with the introduction of a few such breaks. We therefore conclude that most single-strand breaks are not able to serve as e&ient sites for RNA chain initiation by RNA polymerase holoenzyme, a conclusion consistent with the studies of RNA chain initiation we have described above. Transcription by core polymerase gives rise to variable amounts of r- and l-strandspecific RNA. In this experiment there is a slight excess of r-strand-specific RNA; however, the results of duplicate experiments do not support the preferential synthesis of even a small excess of r-strand RNA. As expected, transcription of the nicked DNA by core polymerase shows no indication of strand selection. It should be noted that as the fraction of T7 RNA preferentially annealing to the r-strand decreases, the efficiency of hybridization also decreases. This is attributed to formation of RNARNA duplexes in the hybridization assay, which leads to a preferential loss of symmetrical RNA as discussed above. (d) Effect of single-strand breaks on the binding of RNA polymerase to T7 DNA To test the effect of single-strand breaks on the binding of RNA polymerase to T7 DNA, we have used the filter binding procedure (Hinkle & Chamberlin, 1970). In

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particular, we wanted to measure the number of tight binding sites for RNA polymerase holoenzyme on T7 DNA, before and after the introduction of single-strand breaks. This measurement can be carried out by direct titration of the number of enzyme molecules bound per DNA (method 2 of Hinkle b Chamberlin, 1972a). However, we have chosen a simpler although more indirect method to obtain this information. If 3H-labeled T7 DNA, is mixed with increasing amounts of an unlabeled DNA, and RNA polymerase (E) is added, then the fraction of enzyme bound to r3H]DNA, is given as E-[3H]DNA1 E-DNA,

K3HlDV1 KIN, =

(1)

[DNA,] K,N,

where K, is the equilibrium constant for binding of E to [3H]DNA1 and N, and N, are the number of binding sites on [3H]DNA, and DNA,, respectively. For large values of K, such that enzyme is quantitatively bound to DNA, E-[3H]DNA1

Et

E-[3H]DNA, = E-r3H]DNA, + E-DNA,

=

WWNAJ N,& H3HlDN&1N& + IDN&I N,K, (2)

where E, is the total amount of enzyme added to the reaction. Under these conditions the amount of E-[3H]DNA1 complex is a direct reflection of the ratio N,K,/N2K,. E-[3H]DNA1 Et

[DNA,] N,K, -l = ( ’ + [[3H]DNA,] N,K, >

(3)

To measure the number of tight binding sites on DNase-treated T7 DNA, increasing amounts of unlabeled, intact or DNase-treated T7 DNA were mixed with 3H-labeled T7 DNA. RNA polymerase was then added and the fraction of 3H-labeled T7 DNA in enzyme-DNA complexes was measured after 30 minutes (Fig. 3). From the amount of 3H-labeled T7 DNA complexed, the fraction of enzyme bound to 3H-labeled T7 DNA can be calculated from the relation R = 1 - expeeU (Hi&e & Chamberlin, 1972a) where E is the efficiency of the assay (about 0.4 in this instance), u is the ratio of enzyme moleoules to DNA molecules and R is the fraction of DNA retained on the filter. Because of the very high values of K for binding of the first 8 holoenzyme molecules to T7 DNA, binding at low ratios of E : DNA (below 3 to 4) can be considered irreversible, and if we assume that the rate constants for binding to the two DNA’s are equal (Hinkle & Chamberlin, 19723) then E-r3H]DNA1 Et

=

[DNA,] N, ’ + [[3H]DNA1] N,

-I -

(4)

From this, it can be seen that if the reciprocal of the fraction of the enzyme bound to 3H-labeled T7 DNA in such an experiment is plotted as a function of the ratio of unlabeled DNA to 3H-labeled T7 DNA (DNAJ3H]DNA,), a linear relationship will result, with a slope equal to N,/N,. When the results (Fig. 3) are presented in this manner, a linear relation results which gives values of N2/N, = 1.0, 3.6 and 28 for competition with unlabeled T7 DNA containing 2 O-6, 20 and 145 breaks per genome, respectively (Fig. 4). Since there are about 8 tight binding sites on T7 DNA for RNA polymerase holoenzyme (N, = S), we conclude that the introduction of 20 and 145 breaks per genome leads to the appearance of 20 and 215 new tight binding sites, respectively, or about

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Unlabeled DNA (n-moles) Fm. 3. Effeot of single-strand breaks on tight binding of RNA polymerase to T7 DNA. Binding of RNA polymerase to 3H-labeled T7 DNA was determined using the nitrooellulose filter assay described previously (Hiie & Chamberlin, 1972a). Reaotions (0.1 ml,) contained O-1 pg RNA polymerase holoensyme (fraction 6, 14,000 d(A-T) units/mg), 6 nmoles of intact 3H-lab-eled T7 DNA(lO,OOOota/min) andtheindioatedamountsof unlabeledT7DNAoontainii 146(-A-A-), 20 (-m--m-) or 5 06 (-O--e--) breaks per molecule. Inaubation was for 30 mm at 37°C. In the absenoe of unlabeled T7 DNA, 60% of the 3H-labeled T7 DNA was bound to RNA polymerase in a non-filterable complex. A blank (4%) representing the amount of sH-labeled T7 DNA retained by the 6lter in the absence of RNA polymerase has been subtracted from eaoh point.

0 Ratio Unlabeled DNA %-labeled DNA

FIG. 4. Analysis of the relative number of tight binding sites for binding of RNA polymerese to T7 DNA oontaining single-strand breaks. The fractions of RNA polymerase bound to 3H-l&&d T7 DNA were oaloulatad from the data in Fig. 3 using the relation R = 1 - exp-fY (Hinkle & Chamberlin, 1972a) where R is the fraation of 3H-labeled T7 DNA retained by the flltar, u is the average number of enzymes bound per molecule of 3H-labeled T7 DNA and c is the apparent effioienoy of the assay, and assuming that in the absence of unlabeled T7 DNA, enzyme is quantitatively bound to sH-labeled T7 DNA. Thus, the fraction of enzyme bound to aH-labeled T7 DNA in the presence of unlabeled DNA is: u/u0 = m1.z~~ = In (1 - R)/ln (1 - R,), where R and R,, are, respeotively, the amount of sH-labeled T7 DNA retained in the presence and in the absenoe of unlabeled DNA. DNA samples oontained 146 (-A-A-), 20 (-W-m-) or 2 06 (-a-a--) single-strand breaks per genome, respeotively. 14

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CHAMBERLIN

1 to l-5 tight binding sites per single-strand break. We conclude that, on average, each single-strand break can serve as a tight binding site for between one and two RNA polymerase holoenzyme molecules. 4. Discussion The introduction of a limited number of single-strand breaks into T7 DNA leads to an appreciable alteration of its template properties in enzymio transcription. There is an increase in transcription by the core polymerase, which is in accord with the notion that this form of RNA polymerase initiates RNA chains predominantly at singlestrand breaks or at the ends of DNA molecules (Vogt, 1969; Hinkle & Chamberlin, 1970; Ishihama et al., 1971). Essentially any single-strand break appears to serve as an initiation point for core polymerase. At the same time, there is a corresponding decrease in the rate of RNA synthesis, RNA chain initiation and the correct initiation of RNA chains by the RNA polymerase holoenzyme. These effects are probably explained by the finding that single-strand breaks can serve as sites at which tight binding of RNA polymerase holoenzyme can occur. Between one and two holoenzyme molecules can bind tightly to each single-strand break. Some of these sites appear to serve as initiation points for non-specific RNA chains, as is shown by the slight decrease in correct initiation measured by hybridization experiments. However, even in the presence of 12 single-strand breaks per T7 genome, RNA polymerase holoenzyme initiates primarily r-strand-specific RNA chains and this result, taken with the reduction in the amount of RNA chain initiation brought about by single-strand breaks, indicates that most single-strand breaks cannot serve as initiation sites for RNA polymerase holoenzyme. The differential ability of RNA polymerase holoenzyme and core polymerase to initiate RNA chains at single-strand breaks suggests that the binding of sigma subunit depresses the ability of core polymerase to initiate a nonspecific chain at a single-strand break. Thus, in addition to facilitating the location of the “promoter” sites for correct chain initiation on T7 DNA, sigma subunit appears to act in some way to depress possible incorrect initiation by holoenzyme molecules tightly bound at single-strand breaks. These results, taken with previous studies of the binding of RNA polymerase to T7 DNA, indicate that there are four possible fates that may befall an RNA polymerase holoenzyme molecule upon binding to T7 DNA; (1) binding to the DNA helix at an unbroken region, which leads to transient (weak) binding and release (Hinkle & Chamberlin, 1972a,b) ; (2) binding near the promoter region, which leads to opening of the DNA strands and the initiation of a specific RNA chain; (3) binding to a singlestrand break or end of the molecule, which leads to initiation of an incorrect RNA chain; (4) binding to a single-strand break or end of the moleoule, at which no chain initiation can occur. Possibility (4) recognizea that while the opening of the DNA strands is a necessary step in RNA synthesis, it is not sticient to ensure RNA chain initiation. This leads us to conclude that the nature of the nucleotide sequence in the vioinity of the region where the strands have opened must have an important role in allowing selective RNA chain initiation. This investigation GM12010 from the

was supported by U.S. Public Health Service research grant no. Institute of General Medical Sciences. One of us (D. C. H.) WRZJ the recipient of a predootoral fellowship (GM40468) from the National Institute of General Medid Scienoea.

BINDING

OF RNA

POLYMERASE

TO SINGLE-STRAND

BREAKS

207

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