Volume 4 Number 5 1977

Nucleic Acids Research

Physicochemical studies on interactions between DNA and RNA polymerase. Unwinding of the DNA helix by Escherichia coli RNA polymerase* James C. Wang, John H. Jacobsent and Jean-Marie Saucierl Dep. Chem., Univ. California, Berkeley, CA 94720, tUniv. Chicago Medical School, Chicago, IL 60637, USA and I Institute Gustave-Roussy, 94800 Villejuif, France

Received January 1977 ++ ABSTRACT +In a medium containing 10 mM Tris, pH 8, 10 mM Mg , 50 mM K and 10 mM NH4, the binding of an E. coli RNA polymerase holoenzyme unwinds the DNA helix by about 2400 at 370C. In this medium the total unwinding of the DNA increases linearly The number of with the molar ratio of polymerase to DNA. binding gites at which unwinding can occur is very large. If the K concentration is increased at 200 mM, the enzyme binds to only a limited number of sites, and the bound and free enzyme molecules do not exchange at an appreciable rate. The unwinding angle of the DNA per bound enzyme in this high salt medium is measured to be 1400 at 37°C. The total unwinding angle for a fixed number of bound polymerase molecules per DNA

is strongly temperature dependent, and decreases with decreasing temperature. INTRODUCT ION It was realized in the early sixties that the transcription of a DNA helix by an RNA polymerase might require the transient disruption of a number of base pairs of the template (for a discussion, cf. Chamberlin and Berg, 1963). The idea has gained support mainly from two types of evidence: that RNA polymerase can copy a single-stranded DNA template and that the transcription process appears to involve a temperature and ionic strength sensitive step which could be attributed to the disruption of DNA base pairs (see the reviews by von Hippel and McGhee, 1972; Chamberlin, 1974). Because of the shortage of physico-chemical techniques capable of detecting a relatively small structural change resulting from the interaction between two very large molecules, there have been few experiments which examined directly the structural change of the DNA helix upon the binding of RNA polymerase molecules. The discovery of covalently closed DNA by Vinograd et al. (1965) led us to the use of the covalent closure of a circular o Information Retrieval Limited 1 Falconberg Court London Wl V 5FG England

1225

Nucleic Acids Research DNA for measuring angular alterations of the DNA helix. The general strategy is to convert two samples of a circular double-stranded DNA containing a few single-chain scissions (nicks) per molecule to the covalently closed form under two different sets of conditions. The difference in the average topological winding number of the two samples can be measured subsequently, which gives directly the total angular alteration of the DNA helix from one set of conditions to the other (Wang, 1969a; 1971). If the presence of single-chain scissions is undesirable, it is sufficient to introduce transient nicks into covalently closed DNA samples by a combination of DNase and ligase, by ligase in the presence of AMP (Modrich et al., 1972), or by other enzymes which can transiently break and join the DNA backbone (Germond et al., 1975; Pulleyblank et al., 1975). In a note published several years ago (Saucier and Wang, 1972), we have reported that the binding of an E. coli RNA polymerase to A DNA is accompanied by a small but definitely measurable unwinding of the DNA. This unwinding does not require triphosphates, is not abolished by rifampicin, and is observed at 37°C but not at 0°C. No such unwinding is detectable with core polymerase without the a subunit. In our earlier work we used zone sedimentation of a mixture of two differently labelled X DNA samples, closed in the presence and absence of RNA polymerase and subsequently deproteinized, to determine the difference in the average topological winding number of the samples. This technique, though highly sensitive, is tedious. A much easier method, gel electrophoresis, has recently been introduced (Keller and Wendel, 1975). The newer technique is ideal for measuring differences in topological winding numbers of small covalently closed circular DNAs. We report here our results on the unwinding of doublestranded coliphage fd DNA by the holoenzyme of E. coli RNA polymerase. EXPERIMENTAL Materials. E. coli RNA polymerase holoenzyme was prepared as described by Hsieh and Wang (1976);E. coli ligase was a generous gift of Dr. Paul Modrich. The double-stranded

1226

Nucleic Acids Research replicative form of coliphage fd was isolated from cells infected with the phage in the absence of chloramphenicol. E. coli strain 993, a F rk mk derivative of K12 (Arber, 1966), was grown in Tryptone broth at 37°C with vigorous aeration. Phage was added at a multiplicity of infection of approximately 5 when the cell density reached about 5 x 108 cells/ml. Aeration was interrupted for 10 min upon the addition of phage to facilitate adsorption, and then resumed for 90 min. After which the culture was chilled, and the covalently closed double-strand replicative form of fd DNA was isolated as described by Wang (1969b). In most of the experiments, double-stranded circular fd DNA containing 1-2 random singlechain scissions per molecule (nicked double-stranded fd DNA) was used. This form of the DNA was obtained from the covalently closed form by the procedure described by Wang (1974a) or by the procedure described by Hsieh and Wang (1975). The extinction coefficient of RNA polymerase is taken as 3.1 x 105 M 1 cm 1 at 280 nm (Richardson, 1966a; Berg and Chamberlin, 1970), and that of double-stranded fd DNA at 260 nm is taken as 6600 M (nucleotides) cm or 7.6 x 10 M 1 (Berkowitz and Day, 1974). These (double-stranded fd DNA) cm values were used in calculating the stoichiometric molar ratios of RNA polymerase to DNA. Methods. Covalent closure of nicked double-stranded fd DNA in the presence or absence of RNA polymerase was carried out by first incubating 100 p1 of the reaction mixture at the desired temperature for several minutes to allow thermal equilibration. The reaction was then initiated by the addition of 2 p1 of a ligase stock (% 80 Lehman-Olivera units per ml in 0.05 M potassium phosphate, pH 6.5, 50% glycerol), via a 10 p1 microsyringe with a fine needle (Hamilton). It is important to avoid perturbing the temperature of the reaction mixture significantly during the addition of the ligase; the microsyringe was chosen for this reason. Mixing was achieved by shaking the tube containing the reaction mixture without removing the tube from the constant temperature bath. The reaction was terminated by the addition of 10 p1 of a solution containing 0.2 M Na3 EDTA and 5% sodium dodecyl sulfate. After 1227

Nucleic Acids Research 3 min at 37°C, the mixture was extracted with 100 p1 of buffer saturated phenol which had been distilled under N2. Analyses of the relative topological winding number of the DNA samples covalently closed under different conditions were done by the gel electrophoresis method first used by Keller and Wendel (1975), as described and interpreted by Depew and Wang (1975). The DNA samples after phenol extraction were mixed with a stock solution containing sucrose and a tracking dye, bromophenol blue, and then loaded on a slab gel and electrophoresed as described. Control experiments showed that the removal of phenol by dialysis prior to electrophoresis had no effect. Boundary sedimentation runs of RNA polymerase and DNA-RNA polymerase complexes were carried out in a Model E analytical ultracentrifuge (Spinco) equipped with a photoelectric scanner and a reflective focusing lens. Scans were taken at a wavelength of 233 mp. This wavelength was selected to give a reasonable ratio of protein absorbance to DNA absorbance. Below 233 nm the signal to noise ratio of our scanner system decreases rapidly. RESULTS

Measuring the Unwinding of the DNA Helix by Gel Electrophoresis. Figure 1 illustrates the electrophoretic patterns of two nicked double-stranded fd DNA samples after treatment with ligase, in the absence and presence of E. coli RNA polymerase holoenzyme. In the absence of the polymerase [Figure 1(a)], a small amount of nicked DNA remained. The rest of the DNA was converted to covalently closed molecules, which migrate as a group of bands enveloped by a Gaussian curve. Two adjacent bands in this distribution differ by one in their topological winding number a, and the distribution in a is a result of thermal fluctuations in DNA configurations at the time of closure by ligase. More detailed interpretations have been presented previously (Depew and Wang, 1975; Pulleyblank et al., 1975). For the particular sample shown in Figure 1(a), the center of the Gaussian distribution is located at 0.3 turns to the right of the most intense band of the distribution.

1228

Nucleic Acids Research

(b)

(a)

Distance migroted

Figure 1. Electrophoretic patterns of double-stranded fd DNA covalently closed by ligase in the absence (a) and presence (b) of E. coli RNA polymerase. Each incubation mixture (100 pi) contained 10 mM Tris-HCl pH 8, 10 mM MgC12, 50 mM KC1, 10 mM NH4Cl, 0.1 mM Na3 EDTA, 10-5 M NAD, 10 mM 2-mercaptoethanol, 50 pg/ml bovine plasma albumin, and 44 pmoles of fd DNA. The sample with RNA polymerase contained in addition 350 pmoles of E. coli holoenzyme. Closure by ligase for the particular samples were carried out at 18WC, in a manner described in the experimental section. Electrophoresis was carried out at room temperature, using 0.8% agarose gel. The electrophoresis buffer was 40 mM Tris*HCl, pH 8, 5 mM Na Acetate, 0.5 mM Na3 EDTA. After electrophoresis the gel was stained with ethidium, photographed, and the negative was traced by a Joyce-Loebl microdensitometer, as described by Depew and Wang (1975). Because of the changes in temperature and ionic environment, the covalently closed DNA became positively twisted during electrophoresis (Depew and Wang, 1975; Wang, 1969a). Unwinding of the DNA helix by RNA polymerase reduces the average topological winding number of the DNA upon closure by ligase, and therefore the DNA was less positively twisted during electrophoresis when compared with the sample closed in the absence of RNA polymerase. The sense and degree of twisting of the DNA samples during electrophoresis can be changed by changing the electrophoresis buffer and the temperature of electrophoresis (Pulleyblank et al., 1975). For samples closed at 37WC, electrophoresis was usually carried out at 4WC in the electrophoresis buffer with 5 mM MgCl2 substituting 5 mM Na Acetate. 1229

Nucleic Acids Research The sample shown in Figure l(b) was covalently closed by ligase under identical conditions as the control sample shown in Figure 1(a), except that approximately 8 polymerase molecules per fd DNA were present at the time of sealing. The presence of the polymerase interferes somewhat with the joining of the single chain scissions by ligase, as indicated by a small increase of the amount of nicked species in the sample. It does not appear likely, however, that RNA polymerase binds strongly to the nicks. The group of covalently closed species form a Gaussian distribution similar to the control sample, except that the center of the Gaussian distribution is displaced to the right by 3.2 turns from the center of the Gaussian of the control sample. In other words, the samples closed in the presence and absence of the polymerase differ by 3.2 turns in their average topological winding numbers. It is experimentally straightforward to show that the sample closed in the presence of the polymerase is deficient in its topological winding number relative to the control sample (cf. Depew and Wang, 1975). Therefore the presence of 8 polymerase molecules per fd DNA unwinds the DNA by a total of 3.2 turns or 1200 degrees at the temperature and in the medium specified in the legend to Figure 1. Theoretically the width of the Gaussian distribution for the sample closed in the presence of the polymerase is expected to be somewhat different from that of the control. This difference arises because of a distribution in the number of bound RNA polymerase molecules per DNA, and possible differences in configurational fluctuations between the DNA and the DNA-RNA polymerase complex. As the unwinding measurements are not affected by the width of the distributions, we will not analyze this aspect in the present communication. Unwinding of the DNA Helix by RNA Polymerase. Figure 2 depicts the total unwinding of the fd DNA as a function of , the stoichiometric molar ratio of RNA polymerase to DNA during the closure of the DNA by ligase. Results from three sets of measurements are shown. The first set of measurements were done at 37WC in a medium containing 10 mM Tris- HC1, pH 8,

1230

Nucleic Acids Research

370C 0 50 mM K* 30000

0

2000 0

3:

1

C

q

Figure 2. Total unwinding of a double-stranded fd DNA as a function of g, the stoichiometric molar ratio of RNA polymerase to DNA. The conditions of covalent closure are described in the text and indicated in the figure. 10 mM MgCl2, 50 mM KCl, 10 mM NH4C2, 0.1 mM Na3 EDTA, 10 M NAD, and 50 ijg/ml of bovine plasma albumin. The second set of measurements were done at 37°C in a medium identical to that of the first set except that the concentration of KCl was 200 mM. The third set of measurements were done at 31°C in a medium identical to that of the second set. As we will present in a later section, in the medium containing 50 mM KCl essentially all the RNA polymerase molecules present were bound to the DNA. The total unwinding of the DNA helix increases linearly with , with a slope of 240 degrees per RNA polymerase. When nicked double-stranded fd DNA is used in such experiments, measurements are difficult at j much greater than 30, as the fraction of DNA which can be converted to the covalently closed form by ligase becomes small, presumably due to the binding of some of the polymerase molecules to regions containing the single-chain scissions. In order to extend measurements to higher values of d, we started with the 1231

Nucleic Acids Research covalently closed replicative form of fd. The ligase cofactor NAD, normally present in the incubation medium was replaced by 0.4 mM of AMP, and the amount of ligase was increased to 15 units/ml. Under these conditions the ligase catalyzed reaction can go both ways and ligase serves both as an AMP dependent endonuclease and a joining enzyme (Modrich et al., 1972). The net result is to allow the covalently closed DNA molecules to assume topological winding numbers representing the lowest free energy state (the "relaxation" of the covalently closed DNA molecules). Such an experiment showed that more than 50 turns per fd DNA could be unwound by RNA polymerase molecules. When the KC1 concentration in the DNA-RNA polymerase incubation mixture is increased from 50 mM to 200 mM, the total drops (Figure 2). Lowering unwinding of the DNA at a given the temperature from 37WC to 31WC decreases further the total unwinding of the DNA at a given g (Figure 2). These results will be discussed after we have presented our results on the fraction of RNA polymerase molecules bound to the DNA in these media. Measuring RNA Polymerase Binding by Boundary Sedimentation. To determine the fraction of polymerase molecules bound to DNA, boundary sedimentation of the complexes was carried out in an analytical ultracentrifuge. A representative tracing for a sample with a a of 17.6 in the high KC1 medium at 37°C, taken at a wavelength of 233 nm, is depicted in Figure 3. Two boundaries are seen. The slower sedimenting boundary contains no DNA, as shown by relative heights of the boundary taken at wavelengths from 233 nm to 280 nm. This boundary moves with a sedimentation coefficient (uncorrected) of 17 S, which is identical to the sedimentation coefficient of RNA polymerase holoenzynme measured under the same conditions. It is therefore concluded that part of the polymerase is not bound to the DNA. By comparing the height of this boundary to that of an RNA polymerase solution of the same total concentration, the fraction of unbound polymerase is calculated to be 0.55. Therefore at a a of 17.6, in the high KC1 medium the average number of bound RNA polymerase per fd DNA is about 8. The faster sedimenting boundary moves with a sedimentation coef-

1232

Nucleic Acids Research

Distance sedimentd

Figure 3. Boundary sedimentation of a solution containing 10 mM Tris*HCl, pH 8, 10 mM MgCl2, 200 mM KC1, 10 mM NH4Cl, 0.1 mM Na3 EDTA, 1 mM 2-mercaptoethanol, 1.08 x 10-8 M fd DNA and 1.9 x 10-7 M E. coli RNA polymerase. A 12 mm double-sector charcoal filled Epon centerpiece was used; the reference solution was the same buffer without DNA and RNA polymerase. An unblackened rotor prewarmed to 37°C was used, and the centrifuge chamber temperature was set at maximum. The cell containing the sample solution was allowed to equilibrate to the rotor temperature for 20 min before the start of the run. The temperature control unit was switched off, upon reaching the running speed of 30,000 rpm, to avoid convection. Temperature drift during the run was about 0.5°C. Photoelectric scans were taken at 233 nm. It was necessary to lower the mercaptoethanol from the usual 10 mM to 1 mM, otherwise it absorbs too much light and the signal to noise ratio suffers greatly. Control experiments showed that the lowering of the mercaptoethanol concentration had no effect on the unwinding

angle.

ficient (uncorrected) of 31.4 S, which is faster than that of the nicked double-stranded circular fd DNA, 23.5 S. It is anticipated that the binding of RNA polymerase molecules should increase the sedimentation coefficient of the DNA, both from theoretical considerations and from previously published results (Richardson, 1966b; Pettijohn and Kamiya, 1967). Results obtained in the high KC1 medium are summarized in Table 1. In the medium containing 50 mM KC1, at j = 7 or 19 only one sedimenting boundary was seen, either at 5°C or 37WC. The possibility that free and bound RNA polynerase molecules are rapidly in equilibrium (relative to the sedimentation rates) in the low KC1 medium is inconsistant with binding results previously reported (Richardson, 1966; Pettijohn and Kamiyon, 1967). Therefore the single boundary observed is interpreted as indicating that all RNA polymerase molecules in the solution were bound to the DNA. 1233

Nucleic Acids Research Table 1.

Unwinding

Sedimen-

tation Angle Total TemperaCoeffiUnwind- Per Bound Bound Fraction ture, cient Bound RNAP/fd °C ing Polymerase 31.4S 1400 11000 7.9 0.45 37 17.6 23.5S 0 26.OS 600 3400 6.2 0.65 9.6 31 28.1S 800 5500 7.0 0.36 19.1 21.3S 0 Temperature Dependence of the Average Unwinding Angle. We have already seen from data in Figure 2 and Table 1 that in the high KC1 medium the unwinding angle is strongly temperature dependent. We have examined this temperature dependence in some detail in the low KC1 medium. The results are depicted in Figure 4. Two sets of data are shown. Samples of the first set (open symbols) were first mixed at 0°C and kept at 0°C for 1 hour. The samples were then incubated at the temperatures indicated for 30 min, after which ligase was added as described in the experimental section. Samples of the second set were first mixed and kept at 0°C for 1 hour as those of the first set. They were then incubated at 370C for 10 min, and then at the temperatures indicated for 2-3 hours prior to the addition of ligase. All measurements were done for a a of 8. The strong temperature dependence is evident. Without prior incubation at 370C, there is no unwinding detectable at 0°C, in agreement with previous results (Saucier and Wang, 1972). At 100C unwinding becomes easily detectable. At a given temperature, samples with and without pre-incubation at 370C do not give the same unwinding angle in the region where the unwinding angle is sensitive to the temperature. To test the time course of the change in the unwinding angle following the temperature shift, an experiment was carried out in which samples of the DNA-RNA polymerase complex, at a q of 8, were first incubated at 370C for 10 min. They were then placed in a 50C constant temperature bath. Ligase was then added to the samples at specified times, and the joining reaction was terminated 30 min after the addition of

1234

Nucleic Acids Research

e 1000

I

I

0~~~~

0 10

20

30

40

Temperature, °C The temperature dependence of the unwinding of fd DNA at a fixed 2 of 8. Open circles: samples closed by ligase at the temperatures indicated without pre-incubation at 37°C. Filled circles: samples pre-incubated at 37WC for 10 min. See text for details. Figure 4.

ligase. The results are shown in Figure 5. To test the time required to close the nicks by ligase under the particular reaction conditions, a separate experiment was carried out and the half time for the conversion of the fd DNA to the covalently closed form was found to be about 10 minutes. DISCUSSION In the low KC1 medium (50 mM KC1, 10 mM Mg at 370C the total unwinding of double-stranded fd DNA increases linearly with qL, the stoichiometric molar ratio of RNA polymerase to DNA. Since in this medium essentially all RNA polymerase molecules are bound, the slope of such a plot gives the unwinding angle per RNA polymerase bound. From data in Figure 2, the slope has been found to be 2400 per polymerase molecule. This value is a lower limit for the angular alteration, as we have counted every polymerase molecule in our preparation as capable of unwinding the DNA helix. Several lines of evidence indicate that at least 50% of the molecules of our preparation are functionally active.- Filter binding assays with phage T7 DNA and an 1100 base pairs long restriction fragment of T7 DNA

+),

1235

Nucleic Acids Research containing the early promoters showed that at a a of 1 60% of the DNA molecules were retained (Hsieh, 1976). The expected value from a Poisson distribution is 63% if each and every polymerase molecule can form a filter-retainable complex with the DNA. With the 1100 base pairs T7 fragment, at a a of 5, more than one half of the polymerase molecules were found by electron microscopy to be bound specifically to the early promoters. These bound polymerase molecules disappeared if the complex was incubated with triphosphates and rifampicin prior to fixation for electron microscopy, indicating that such bound enzyme molecules could synthesize RNA chains (J. Hirsch and R. Schleif, personal communications). Based on these results we feel that it is unlikely that the actual unwinding angle per bound polymerase exceeds 500°. Previously Saucier and Wang (1972) found that each bound RNA polymerase unwinds DNA by an angle 10 times the unwinding angle of ethidium. Taking the ethidium unwinding angle as 260 (Wang, 1974b), the unwinding angle by an RNA polymerase is calculated to be 2600. The agreement between this value and the 2000

!

GP 0

1000 c2

6

C Z

0

20

40

60

80

100

120

time, minutes

Figure 5.

The time course of the change in the unwinding angle Measurements were done with samples with j = 8, and t is the time from the shift in temperature to the addition of ligase. The zero time point was for a sample closed at 370C. Since the ligase reaction has a half time of about 10 min, the actual time course is better represented by displacing all points except the zero time point to the right by approximately 10 min.

following a temperature shift from 37WC to 50C.

1236

Nucleic Acids Research value obtained in the present work is fortuitous, as uncertainties in such measurements are fairly large. In our 1972 work the RNA polymerase preparation used contained a large fraction of inactive molecules, and the unwinding angle was calculated based on the number of bound polymerases measured by filter binding assays, with a somewhat arbitrary assumption that it required two bound polymerase molecules to retain a X DNA on the filter. The unwinding of the DNA helix by RNA polymerase in the low KC1 medium is not limited to binding to the promoter sites. The maximal unwinding angle we have measured is over 50 turns per fd DNA. Taking the unwinding angle as 2400 and the number of base pairs covered per polymerase as 50, the maximal value we measured corresponds to the covering of more than 60% of the DNA. Our results are consistent with the interpretation that each and every polymerase bound to the DNA in the low KC1 medium unwinds the DNA helix. Spectrophotometric measurements on the increase in absorbance of the DNA at 260 nm upon the binding of RNA polymerase molecules also support this interpretation (Hsieh, 1976). A more detailed analysis on this point will be presented elsewhere. When the KC1 concentration of the medium is increased to 200 mM, the situation becomes rather different. The sedimentation results show that not all of the polymerase molecules present are bound to the DNA. At a S of 18, about 8 polymerase molecules are bound per fd DNA. By measuring the retention of restriction fragments of fd DNA on filters, Seeburg and Schaller (1975) found that there were at least 6 RNA polymerase binding sites in a medium similar to our low KC1 medium. These sites were found to be promoters at which chain initiation could occur. Since filter binding assay measures only sites at which polymerase molecules are bound strongly, it is plausible that the binding sites in our high KC1 medium include most or all of the promoter sites they reported. A quantitative comparison of the number of sites detected by different methods is difficult. The boundary sedimentation method we used perturbs the system the least, with the pressure gradient being the only additional factor introduced by cen1237

Nucleic Acids Research trifugation. It should be noted that the free RNA polymerase concentrations of some of the samples used in the boundary sedimentation measurements were fairly high. The free RNA polymerase concentration of the high KC1 sample with a a of 17.6 is readily calculated to be 1.0 x 10 7 M. Thus a site with an intrinsic binding constant significantly higher than 107 would be counted as a site in our measurements, but may escape detection in measurements with a much lower polymerase concentration. Since two boundaries are seen in our sedimentation measurements of the complexes in the high KC1 medium, and the same sedimentation coefficient is observed for the RNA polymerase and the free RNA polymerase in the presence of the complex, it can be concluded that the bound polymerase molecules do not exchange with the free ones at an appreciable rate at 37 or 31WC (see for example, Schachman, 1959). The unwinding angle per bound polymerase in the high KC1 medium, 1400 at 37WC, appears to be significantly lower than the value 2400 in the low KC1 medium at the same temperature. Lowering the temperature to 31°C decreases the unwinding angle further in the high KC1 medium (Table 1 and Figure 2). The strong temperature dependence of the unwinding angle is evident from the results depicted in Figure 4. It should be pointed out that the results shown in Figure 4 were obtained with a fixed ratio of polymerase to DNA. Therefore the curves reflect the temperature dependence of the average unwinding angle. Wle cannot determine whether the corresponding curves for an RNA polymerase bound to a given site would have shapes similar to the ones shown in Figure 4, or whether at certain sites the DNA helix might be fully unwound even down to fairly low temperatures. Though the data for the high KC1 medium shown in Figure 2 is suggestive that the ratio of 'the average unwinding angles at 37 and 310 is insensitive to a, the range for the number of bound polymerase molecules is too small to draw a reliable conclusion. Measurements on the temperature dependence of the efficiency of transcription off different promoters indicate that different promoters have different profiles (Dausse et al., 1976). If one assumes that the tem-

1238

Nucleic Acids Research perature dependence of transcription is correlated with the temperature dependence of the unwinding angle, then the curves shown in Figure 4 represent composites of many such curves for different sites. Qualitatively the curves are likely to reflect the qeneral shape of the temperature dependence, but a quantitative analysis of the curves seems unjustified at this time. Tne type of thermal hysterisis of the average unwinding angle is interesting. The pre-incubation at 37°C might lead to binding to a different set of sites the average unwinding at which differs from that of the set of sites the polymerase molecules occupy without pre-incubation. Data in Figure 5 also indicate that following a temperature shift from 37WC to 5WC the unwinding angle approaches the final value fairly slowly. Dubert and Hirschbein (1969) reported that preincubation of RNA polymerase and phage X DNA at 37WC increased the initial rate of transcription at 0°C. The effect of preincubation became smaller if the initial rate of transcription was measured minutes after the shift to 0°C, but persisted even after two hours following the temperature shift. Hsieh and Wang (1976) observed that pre-incubation of RNA polymerase and a restriction fragment of phage T7 DNA containing the early promoters at 37WC affects the pattern of transcripts at 4WC, and that this effect persisted for at least 1 hour following the temperature shift. These results support the notion that transcription is initiated at sites where the DNA helix is unwound. It does not appear, however, that the formation of an unwound DNA-RNA polymerase comples is sufficient for chain initiation. In the low KC1 medium, the number of sites at which unwinding can occur far exceeds the number of promoter

sites at which RNA chains can initiate. The magnitude of the unwinding of the DNA per bound polymerase is about 1/2 to 1 turn from our measurements. It should be pointed out that a priori there is no reason to believe that chain initiation requires the unwinding of the helix by an exact angle. Our results support the notion that a critical unwinding of the DNA helix is necessary for transcription. The exact magnitude of unwinding, however, might not 1239

Nucleic Acids Research be crucial and might be dependent on the temperature, the ionic environment, and the DNA sequence. ACKNOWLEDGMENT We thank Professor Stuart Linn for providing us with stocks of fd phage and its host, Dr. Tao-shih Hsieh for his help in the preparation of RNA polymerase, Mrs. Rosaline Yu for her help in the preparation of fd DNA, and Professor Michael Chamberlin for stimulating discussions. This work has been supported by a grant (GM 14621) from the U. S. Public Health Service. REFERENCES

*Dedicated to Jerome Vinograd. This paper is the second of a series on DNA-RNA polymerase interactions from this laboratory, the first being Hsieh and Wang (1976). 1 Chamberlin, M. and Berg, P. (1963) Cold Spring Harb. Symp. Quant. Biol. 28, 67 2 von Hippel, P. H. and McGhee, J. D. (1972) Ann. Rev. Biochem. 41, 790 3 Chamberlin, M. (1974) Ann. Rev. Biochem 43, 721 4 Vinograd, J., Lebowitz, J., Radloff, R., Watson, R. and Laipis, P. (1965) Proc. Nat. Acad. Sci. U.S. 53, 1104 5 Wang, J.C. (1969a) J. Mol. Biol. 43, 25 6 Wang, J.C. (1971) in Procedures in Nucleic Acid Research (edited by Cantoni, G. L., and Davies, D. R.) 2, 407 (Harper and Row, New York) 7 Modrich, P., Lehman, I. R., and Wang, J. C. (1972) J. Biol. Chem. 247, 6370 8 Germond, J. E., Hirt, B., Oudet, P., Gross-Bellard, M. and Chambon, P. (1975) Proc. Nat. Acad. Sci. U.S. 72, 1843 9 Pulleyblank, D. E., Shure, M., Tang, D., Vinograd, J. and Vosberg, H.-P. (1975) Proc. Nat. Acad. Sci. U.S. 72, 4280 10 Saucier, J.-M. and Wang, J. C. (1972) Nature New Biol. 239, 167 11 Keller, W. and Wendel, I. (1975) Cold Spring Harbor Symp. Quant. Biol. 39, 199 12 Hsieh, T. S. and Wang, J. C. (1976) Biochemistry, in press

Arber, W. (1966) J. Mol. Biol. 20, 483 Wang, J. C. (1969b) J. Mol. Biol. 43, 263 Wang, J. C. (1974a) J. Mol. Biol. 87, 797 Hsieh, T. S. and Wang, J. C. (1975) Biochemistry 14, 527 Richardson, J. P. (1966a) Proc. Nat. Acad. Sci. U.S. 55, 1616 18 Berkowitz, S. and Day, L. (1974) Biochemistry 13, 4825 19 Depew, R. E. and Wang, J. C. (1975) Proc. Nat. Acad. Sci. U.S. 72, 4275 20 Berg, D. and Chamberlin, M. (1970) Biochemistry 9, 5055 21 Richardson, J. P. (1966b) J. Mol. Biol. 21, 83 22 Pettijohn, D. and Kamiya, T. (1967) J. Mol. Biol. 29, 275 23 Hsieh, T. S. (1976) Ph.D. Thesis, University of California at Berkeley 13 14 15 16 17

1240

Nucleic Acids Research 24 25 26 27 28

Wang, J. C. (1974b) J. Mol. Biol. 89, 783 Seeburg, P. H. and Schaller, H. (1975) J. Mol. Biol. 92,

261 Schachman, H. K. (1959) Ultracentrifugation in Biochemistry, Academic Press, New York Dausse, J.-P., Sentenac, A., and Fromaqeot, P. (1976) Eur. J. Biochem. 65, 387 Dubert, J. M. and Hirschbein, L. (1969) Biochem. Biophys. Res. Comm. 34, 149

1241