J. Mol. Biol. (1978) 124, 73-85
Transcription II.? Transcription
of Polyoma Virus DNA in Vitro of Superhelical and Linear Polyoma DNA by RNA Polymerase II
BERNARD LESCURE, ANNICK CHESTIER AND MOSHE YANIV Department of Molecular Bioloyy Institut Pasteur 25 rue du Dr Roux 75015 Paris, Prance (Received 3 January
1978)
Several steps in the transcription of double-stranded polyoma DNA by calf thymus RNA polymerase II(B) were analysed. Electron microscopy observations show that, as found for the Eecherichia coli enzyme, the eucaryotic RNA polymerase exhibits a higher affinity for superhelical DNA as compared to linear DNA. The half-life time of complexes formed at 37°C between the enzyme and superhelical DNA is relatively short (30 to 60 s), compared with 30 h for the E. coli enzyme under identical conditions. Binary complexes formed with linear DNA have a faster dissociation rate. The same results were obtained by using the nitrocellulose binding assay or by measuring the decay of heparinresistant complexes. Only ternary complexes, enzyme-DNA-RNA, obtained after the formation of the first phosphodiester bond, are resistant to polyanions. These results suggest that, as for the procaryotic enzyme, RNA polymerase II has to melt locally the double-stranded DNA during initiation, but that it lacks a sigma type factor necessary to stabilise the open initiation complexes. RNA polymerase II synthesizes shorter RNA chains than E. coli RNA polymerase. The formation of RNase I resistant RNA-DNA hybrids during transcription may explain the reduced rate of chain elongation.
1. Introduction The circular double-stranded DNA of the papovaviruses (simian virus 40, polyoma, etc.) is transcribed in vivo by RNA polymerase II (B) as suggested by the sensitivity of viral RNA synthesis to a-amanitin (Price & Penman, 1972; Hildebrandt & Sugden, 1972). The small size of these DNAs, the abundant information about physical and functional maps of SV40 and polyoma (Acheson, 1976), and sequence information (Dhar et aE., 1977 ; Contreras et al., 1977 ; Griffin, personal communication), make t,his template a useful tool for the study of enzyme-DNA interactions. Several steps have been defined in the process of recognition of double-stranded DNA by procaryotic RNA polymerase (Hinkle & Chamberlin, 1971: Dunn & Studier, 1973) : entry, formation of closed complexes, formation of rapid start open complexes and initiation. The signals for the recognition of the DNA by this enzyme were characterized on a t Paper I in this series is Lescure et al. (1976). 73 0022-2836/78/250073-13 $02.00/O
0 1978 Academic Press Inc. (London) Ltd.
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large number of bacterial promoters (Pribnow, 1975; Sekiya et al., 1975). The fact that the formation of stable initiation complexes requires the opening of the doublestranded DNA (Saucier & Wang, 1972) explains the increased affinity of Escherichia coZi RNA polymerase for superhelical DNA (Richardson, 1975 ; Lescure et al., 1976). In the present studies, we set out to characterize better the interactions between the eucaryotic RNA polymerase II and the double-stranded viral DNA. The ability to form stable initiation complexes and to transcribe polyoma virus DNA was studied. We show that RNA polymerase II exhibits a higher affinity for superhelical DNA than for relaxed DNA, and that stable initiation complexes can be obtained only after initiation with a dinucleotide and a single ribonucleoside triphosphate. The elongation with E. coli RNA polymerase, with RNA polymerase II is less than that obtained possibly because of RNA-DNA hybrids formed during transcription with the former enzyme.
2. Materials and Methods (a) DNA
and enzyme preparations
Superhelical and linear polyoma virus DNA were purified as previously described (Lescure et al., 1976). E. coli RNA polymerase was purified from E. coli MRE 600 cells as described by Humphries et al. (1973). The enzyme had a spec. act. of 400 units/mg (1 unit equals the incorporation of 1 nmol UTP in 10 min at 37°C). Calf thymus RNA polymerase B (II) was either a gift from Dr P. Chambon or purified according to the method described by Kedinger C Chambon (1972) applying the following steps: DEAE-cellulose and phosphocellulose columns followed by glycerol gradient centrifugation. The enzyme obtained was free of DNase and RNase contamination: no conversion of superhelical to relaxed polyoma DNA was observed on incubation with the enzyme and no reduction in the size of RNA made in vitro with E. coli RNA polymerase upon further incubation with the enzyme. The main contaminant, revealed by polyacrylamide gels, was actin. One unit of enzyme corresponds to the incorporation of 1 nmol of UMP incorporated in 10 min at 37°C with single-stranded calf thymus DNA as template. The protein concentration of the purified enzyme was not measured. (b) Electron
microscopy
RNA polymerase binding to polyoma DNA was monitored by the electron microscopy technique of Dubochet et al. (1971) as previously described (by Lescure et al., 1976). (c) Conditions
for binding
of RNA polymerase synthesis
to polyoma
DNA
and for RNA
Identical ionic conditions were used for RNA polymerase binding and for RNA synthesis. The incubation mixture for E. coli RNA polymerase contained : 20 miw-Tris . HCl (pH 7.9), 10 mM-MgCl,, 150 m&r-Kcl, 0.2 mM-dithiothreitol. The following concentrations of ribotriphosphates: 0.2 mM-GTP, -CTP, -ATP and 0.1 miw-[3H]UTP (100 &X/pmol) were added when transcription was studied. The incubation mixture for calf thymus RNA polymerase II contained: 20 mM-Tris*HCl (pH 7*9), 2.5 mivr-M&l,, 20 m&r-(NH,)&),, 0.5 mnrdithiothreitol, 0.4 mM-GTP, -CTP, -ATP and 0.2 mM-[3H]UTP (100 &ip/mol) were added for transcription. For binding studies RNA polymerase was incubated with DNA for 5 min at 37°C in the ionic conditions described above. Transcription was initiated after 5 min by addition of the ribonucleoside triphosphates. The reaction was stopped with 5% trichloroacetic acid, samples were filtered on nitrocellulose membranes, dried and counted by liquid scintillation. (d) Complementary
RNA
pur$cation
For preparation of complementary RNA, the reaction w&s stopped by addition of EDTA to a flnal concentration of 20 mM and extracted with an equal volume of water-saturated
POLYOMA
VIRUS
DNA
TRANSCRIPTION
IA’
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73
II
twice with an equal volume of chloroform. The aqueous phase was re-extracted The final aqueous phase was precipitated with 2 vol. ethanol at, - 20°C. After centrifugation, the precipitate was dissolved in 10 mM-Tris.HCl (pH 7.9), 5 mM-MgClz and incubated for 1 h at 37°C with 20 pg DNase I/ml (RNaae-free). The incubation mixture was reextracted with phenol and the RNA precipitated with ethanol. The final precipitate was dissolved in 10 mM-Tris.HCl (pH 7.9), 1 mM-EDTA.
phenol.
3. (a) Transcription
of superhelical
Results and linear
polyoma
DNA
virus
in vitro
Superhelical polyoma DNA was transcribed in vitro with calf thymus RNA polymerase 11. As shown in Table 1, the incorporation observed was about lOoA, of that obtained when the optimal amount of E. coli RNA polymerase holoenzyme transcribed the same template (Lescure et al., 1976). A further increase in the amount of enzyme I1 added did not result in higher incorporation of triphosphates. That incorporation is greatly affected by the superhelicity of the DNA is shown by the fact that linear double-stranded molecules of polyoma DNA produced by EcoRI treatment were seven to eightfold less effective than superhelical DNA template, at the same ratio of enzyme to DNA (Table 1). In an attempt to analyse the multiple steps involved in the transcription process and to elucidate the differences between procaryotic and eucaryotic RNA polymerases, we studied the formation and the stabilit’) of initiation complexes as well as the RNA elongation step. TABLE 1 Transcription
of polyom,a
DNA
FI
or FIII in vitro
by calf thymus
RNA
polym,era.se
II
UMP incorporated in cRN.4 with 0.2 unit of RNA polymerase II (pmol) 4 min A min 15 min
Template
Polyoma
DNA
FI (1.5 pg)
Polyoma
DNA
FIII
(1.5 pg)
53
9x
114
7
13
16
Thr conditions used for transcription are described in Material and Methods. At various times of synthesis, the radioactivity incorporated into cRNA was monitored by liquid scintillation counting.
(b) Formation
of enzyme-DNA
com,plexes
Binary complexes were formed between RNA polymerase 11 and FIT or linear polyoma DNA at a ratio of enzyme to DNA of 0.2 unit/pg. Complexes were spread for electron microscopy (Fig. 1) and the number of enzyme molecules bound on each kind of template was scored. An average of 3.6 enzyme molecules were bound to superhelical DNA (Figs l(a) and Z(a)). When linear polyoma DNA, produced by EcoRI treatment of circular DNA was used, many of the RNA polymerase 11 molecules were bound to the sticky ends of the DNA molecules (Fig. l(b)). The average number of enzyme molecules bound per DNA molecule was l-28 if all bound t Abbreviations used: FI DNA, superhelical polyoma DN8; FIlI DXA. cRNA, complamcnt,ary RNA, i.p. RNA synthesized in w&o from polyoma.
linear
polpoma
DKA
:
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FIG. 1. Vizualisation by electron microscopy of calf thymus RNA polymerase II molecules bound to polyoma DNA. The complexes were formed as described in Materials and Methods, at a weight ratio of enzyme to DNA of 0.2 unit of enzyme/pg of (a) polyoma DNA FI or (b) polyoma DNA FIII. After 5 min of binding at 37X’, glutaraldehyde was added to a final concn of 10-2 M, incubation continued for 15 min at 37°C and the samples were spread for electron microscopy as previously described (Lescure et al., 1976). The bar represents 100 nm.
molecules were counted and 0.78 if enzyme molecules on the ends were excluded from the calculation (Fig. 2(b)). Thus it appears that RNA polymerase II displays a three to fourfold lower tendency to bind to untwisted DNA relative to negatively superhelical DNA. (c) EJfect of heparin on
the
transcription
of polyom
DNA
The polyanion heparin is a potent inhibitor of E. coli RNA polymerase and of nuclear eucaryotic RNA polymerases I, II and III (Walter et al., 1967; Cox, 1973; Ferencz & Seifart, 1975; Long & Crippa, 1976). As expected, when heparin was added either to E. coli or to RNA polymerase II prior to the DNA template RNA synthesis was completely inhibited with both enzymes (Fig. 3(a) and (b)). When heparin was added before initiation to preformed enzyme-DNA complexes, the results differed for the two enzymes. In agreement with the previously observed resistance of binary complexes to the polyanion, E. coli RNA polymerase was only slightly affected (Zillig et al., 1971; Schgfer et al., 1973). In contrast, under the same conditions, RNA polymerase II dependent synthesis was totally inhibited (Fig. 3). In another experiment heparin was added 15 seconds after the initiation of RNA synthesis. Only a slight inhibition was observed with E. coli RNA polymerase during the four minutes of synthesis (Fig. 3(a), curve 4). With the calf thymus RNA polymerase B, an inhibition of about 50% is observed in these conditions, indicating that initiated
POLYOMA
VIRUS
DNA
TRANSCRIPTION
IN
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i;
;I-&I )246802468
No. of RNA polymerose n-decules/ DNA molecule
FIG.
2. Binding
of calf
thymus
(cl
(b)
(a) RNA
polymerwe
II
to polyoma
DNA
FI
or FIII.
The number of polymerase molecules bound to superhelical or linear template at, a ratio o enzyme to DNA of 0.2 unit/pg was monitored by electron microscopy. (a) RNA polymerase molecules on superhelical template; (b) RNA polymerase molecules on linear template including molecules bound to the stickv ends of the EcoRI linear molecules: fcl RNA nolvmerase molecules * ” on linear template excluding molecules bound to the end of linear DNA molecules. \
I
Time (min)
Time (mm1
(a)
(b)
Fro. 3. Effect of heparin on in vitro transcription of polyoma IjN.4 PI by (a) E’. coli KS:2 polymerase or (b) calf thymus RNA polymerase. r3H]RNA was synthesized as described in Materials and Methods, at a weight ratio of enzyme’ to DNA of 3 rg/l rg for E. coli RNA polymerase or 0.2 unit/l pg for calf thymus RNA polymerasc II. At various intervals [3H]oRNA synthesis was estimated. Curve 1, initial RNA synthesis in the absence of heparin. Curve 2, heparin at a final concn of 10 pg/ml was added for 2 min at 37°C to reformed complexes between RNA polymerase and polyoma DNA FI prior to the addition of ribotriphosphates. Curve 3, heparin was added for 2 min at 37% to free RNA polymerase prior to the addition of polyoma DNA FI. Curve 4, heparin was added 16 s after initiation of synthesis. Curve 5, synthesis was pre-initiated by adding GpU (0.4 mM) and CTP (0.2 mix) to reformed complexes. After 6 min of incubation at 37”C, heparin was added at a final concn of 10 pg/ml. incubation continued 6 min at 37°C prior to initiation of RNA elongation by the addition of the other 3 ribotriphosphates.
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No. of RNA polymerme molecules/DNA (0)
M. YANIV
ib)
molecule (ci
FIN. 4. Studies by electron microscopy of the effect of heparin on binary and ternary complexes formed between calf thymus RNA polymerase II and polyoma DNA FI, or calf thymus RNA polymerme II polyoma DNA FI and GpUpC. (a) Number of calf thymus RNA polymerase molecules bound to polyoma DNA FI at a ratio of enzyme to DNA of 0.1 unit/l pg. (b) Number of calf thymus RNA polymerase molecules bound to polyoma DNA FI at the same ratio of enzyme to DNA after incubation during 2 min at 37°C of preformed complexes with heparin at a final concn of 10 pg/ml. (c) Enzyme molecules bound to polyoma DNA FI after pre-incubation with GpU and CTP, followed by heparin treatment (10 pg/ml) for 2 min at 37’C.
complexes are either partially or totally resistant to the action of heparin (Fig. 3(b), curve 4). Two hypotheses can be postulated to explain these results : (i) elongation is limited with RNA polymerase II ; after two minutes of synthesis RNA chains were terminated, the enzyme DNA complexes dissociated and the free RNA polymerase inhibited by heparin. (ii) RNA polymerase II engaged in ternary complexes (enzymeDNA-RNA) is sensitive to heparin ; however the inhibition is less effective than that obtained with binary complexes (Fig. 3(b), curve 2). The second hypothesis can be excluded by the following experiment: we have shown that for the two enzymes, some of the in vitro initiation sites on polyoma DNA start with the sequence GpUpC (Lescure et al., accompanying paper). Using RNA polymerase II, we initiated the transcription by a preincubation in the presence of GpU and CTP (the number of C residues incorporated that could include one to several residues was not determined). Heparin was then added to the ternary complexes of enzyme-DNA-GpUpC for five minutes at 37°C before the addition of the other three ribotriphosphates. A short synthesis of RNA occurred during roughly two minutes (Fig. 3(b), curve 5). The mechanism of action of heparin is confirmed by the following electron microscopy observations. An average number of about two molecules of RNA polymerase II were bound to polyoma DNA using a ratio of enzyme to DNA of O-1 unit/pg (Fig. 4(a)). After incubation with heparin for two minutes at 37”C, almost all the enzyme molecules were removed from the template (Fig. 4(b)). However if transcription was initiated using GpU and CTP, prior to the addition of heparin, a large fraction of RNA polymerase molecules (33%) remained associated with the template (Fig. 4(c)) confirming that the ternary complexes became partially resistant to the action of the polyanion. (d) Rate of dissociation of RNA polymerwe-DNA binary complexes different methods, previously applied with E. coli RNA polymerase, were used to measure the stability of enzymes-DNA complexes in the present study. In Two
POLYOMA
VIRUS
DNA
TRANSCRIPTION
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I I Time (min) (a)
I
I,!I
59
II
I
I
2 3” IO 20 30 Time (mm) (b)
Fro. 5. Stability of binary complexes formed between RNA polymerase and polyoma DNA Fl. -O-O--, calf thymus RNA polymerase Il. -O-e-, E. r&i RNA polymerase; (a) Nitrocellulose filter assay (Hinkle & Chamberlin, 1971). The reaction was performed in a final vol. of 200 pl. E. coli RNA polymerase or calf thymus RNA polymerase II w&s incubated at 37°C with polyoma [3H]DNA FI in the standard conditions (Materials and Methods) at a ratio of enzyme to DNA of 4 pg/l rg for the E. coli enzyme or 0.2 unit/l rg for the calf thymus enzyme. After 5 min, 100 pg of denatured calf thymus were added, samples of 40 ~1 were removed, diluted to 1 ml with the standard medium for each enzyme and filtered on nitrocellulose filters. Polyoma [3H]DNA retained on the filter was estimated at various times. (b) Residual activity in the presence of heparin. The method used has been described previously (Lescure et al., 1976). The ratio of enzyme to DNA was the same as that used for the nitrocellulose filter assay.
the nitrocellulose filter binding assay (Hinkle & Chamberlin, 1971), the rate of dissociation of previously formed complexes between E. coli RNA polymerase or calf thyms RNA polymerase II and [3H]DNA polyoma FI was measured by adding a hundredfold weight excess of denatured calf thymus DNA. Samples were removed at intervals and filtered on nitrocellulose membranes to determine the amount of radioactive DNA retained (Fig. 5(a)). Almost no dissociation was detected after an eight-minute incubation with the complexes formed between E. coli RNA polymerase and polyoma DNA FI. In contrast, 50% of complexes formed with RNA polymerase II had dissociated after two minutes of incubation. The second method measures the residual activity, in the presence of heparin (Richardson, 1975), that inactivates free enzyme molecules which dissociate from the template. Recent studies however have shown that heparin can attack polymerase in binary complexes directly without prior dissociation from the template (Pfeffer et al., 1977 ; Giacomoni et al., 1977). In this case, both techniques may in fact measure the accessibility of binary complexes to attack by polyanions. Heparin was added to previously formed complexes, samples werca removed at intervals and residual activity measured (Fig. 5(b)). Using this technique. the stability of complexes formed with E. coli RNA polymerase was again very long (half-life time of 30 h). Although resistant complexes can be detected with RNA polymerase II, 50% of these complexes were inactivated after 30 seconds of incubation at 37°C in the presence of heparin. When linear double-stranded polyoma DNA was used, re-initiation could be blocked either by heparin or by addition of 0.75 M-KU alone. The rate of dissociation of the RNA polymerase II was too fast to be measured. Under similar conditions (using poly(rI) s,s polyanion), E. c& RNA polymerasr dissociated with a half-life time of five hours (Lescure et ~2.. 1976).
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4s
16 S
235
M. YANIV
,
I
20
30
I
Fmction no.
FIG. 6. Sucrose gradient analysis of cRNA synthesized. [3H]cRNA was synthesized and purified as described in Materials and Methods. Before sedimentation analyses cRNA was treated for 1 min at 100°C and quiokly cooled to 0°C. Samples were layered onto S-ml linear 6% to 20% sucrose gradients in 0.1 M-NaCl, 0.02 M-Tris.HCl (pH 7.4) and 0.6 mix-EDTA and centrifuged for 180 min at 48,000 revs/min at 4°C inithe Spinco SWKO.l rotor. Radioactivity from gradient fractions was estimated by scintillation counting. -O-O--, Transcription of polyoma DNA FI by E. co.5 enzyme; -@-a--, transcription of polyoma DNA FI by calf thymue RNA polymerase II.
(e) Analysis
of complementary
RNA
The experiments described in Figure 3 suggest that RNA polymerase II is limited in elongation, we therefore tried to characterize the RNA made in vitro. Under the transcription conditions used with polyoma DNA FI the RNA was largely symmetric ; 60 to 70% of the cRNA was resistant to treatment by RNaseI in 2 x SSC (SSC is 0.15 iw-NaCl, 0*015 M-sodium citrate) after self-annealing. Hence, as with E. coli RNA polymerase (Lescure et al., 1976), at least two initiation sites for RNA synthesis in vitro are located on opposite strands of polyoma DNA. The size of the cRNA was analysed by sedimentation on a sucrose gradient. The peak of radioactivity was heterogeneous with a maximum at about 9 S (Fig. 6). This size is far below a complete transcript of the genome and smaller than that obtained when E. coli RNA polymerase transcribes the same template (Fig. 6). The smaller size of the RNA synthesized by RNA polymerase II was not due to the presence of RNase in the enzyme preparation since the size of the RNA synthesized by E. coli RNA polymerase was not altered by post-incubation with RNA polymer&se II. To confirm these observations on the limited elongation, we have estimated the RNA chain length using [Y-~~P]ATP, [Y-~~P]GTP and [3H]UTP as radioactive precursors. The average chain length of the RNA synthesized by E. coli RNA polymerase and by RNA polymerase II was calculated from the ratio of 3H/32P incorporated, to give values of 2500 and 660 nucleotides, respectively (Table 2). The continued synthesis of RNA with RNA polymerase II in the absence of heparin (Table 1) is certainly due to chain reinitiation. (f) Formation
of UNA-RNA
hybrids during elongation
in vitro
Nascent RNA molecules can be completely released in vitro from linear DNA templates, by treatment with agents such as detergents that denature proteins (Bremer $ Konrad, 1964; Richardson, 1966). However, when the template is a negative superhelical DNA, denaturation of the RNA polymerase does not release all of the RNA (Hayashi, 1965 ; Champoux & McConaughy, 1975 ; Lescure et al., 1976). The positive free energy of the superhelical DNA is responsible for the formation of
II
1.40
0.39
0.27
plus [y-=P]GTP in cRNA (pmol) 8 min
0.85
[y-=P]ATP incorporated 4 min
40
580
4 min
13H]UTP
incorporated
synthesized in vitro
2
Polyoma DNA FI was transcribed by E. coli RN-4 polymerase or by calf thymus RNA polymerase labelled by [3H]UTP (100 mCi/mmol) [y-32P]ATP (2300 mCi/mmol) and [y-32P]GTP (2800 mCi/mmol). ditions, 32P and 3H radioactivity incorporated in cRNA were estimated by liquid scint,illation counting.
polymerase
RNA
Calf
thymus
polymerase
E. coli RNA
Enzyme
Chain length of RNA
TABLE
(pmol) Mean
664
2500
chain length (nucleotides) after 8 min of synthesis
II as described in Materials and Methods. cRNA After a synthesis for 4 or 8 min in the standard
65
875
8 min
in cRNA
w&s con-
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stable hybrids between the RNA and the template (Vinograd et al., 1968; Wang, 1974). However, it was shown recently that native E. coli RNA polymerase can prevent the formation of such hybrids if the intact structure of t,he enzyme is maintained (Richardson, 1975). Polyoma DNA FI was transcribed for one minute at, 37°C by h’. coli RNA polymerase or by RNA polymerase II and the incubation was terminated by addition of EDTA alone (non denaturing conditions) or by addition of EDTA and sodium dodecyl sulfate as described by Richardson (1975). The resistance to RNase I digestion was then measured in 2 x SSC or in 0.1 x SSC to estimate the percentage of RNA-DNA hybrids (Table 3). When E. coli RNA polymerase was used, 32% of the newly synthesized RNA was resistant to RNase I digestion in 2 x SSC, if the transcription was terminated with EDTA and sodium dodecyl sulfate. On the contrary, only 2.1% of the RNA was resistant when the incubation was terminated with EDTA alone. When RNA polymerase II was used, 28% of the newly synthesized RNA was resistant to RNase I when the transcription was terminated with EDTA and sodium dodecyl sulfate and 21% when EDTA alone was added. In contrast to the observations with the E. coli RNA polymerase, the native form of calf thymus RNA polymerase II is less efficient in preventing the formation of RNA-DNA hybrids during the transcription of a superhelical template.
TABLE 3 Effect of detergent on nascent RNA
Digestion E. coli RNA
polymerase
Calf thymus RNA polymerase
II
in
hybridized
to the DNA
template
Percentage of product resistance to RNase A digestion after stopping the reaction with EDTA EDTA + SDS
0.1 x ssc 2 x ssc
0.9 2.1
0.7 32
0.1 x ssc 2 x ssc
0.7 21
1.0 28
The method applied was essentially that described by Richardson (1974). Two pg of polyoma DNA FI were transcribed for 1 min at 37°C with 4 pg of E. coli RNA polymerase or with 0.4 unit of calf thymus RNA polymerase II in a final volume of 200 ~‘1. RNA synthesis was terminated by the addition of 16 ~1 of 0.1 M-EDTA, or 16 ~1 of 0.1 M-EDTA + 8 ~1 of 10% sodium dodecyl sulfate (SDS). The incubation was continued for 2 min at 37°C. After chilling in an ice bath, the SDS precipitate was removed by centrifugation. The supernatant solution was carefully removed and samples were treated for 30 min at 37°C with RNase A (20 pg/ m 1) and RNase T, (1 unit/ml) after dilution in 2 ml of 2 x SSC or 0.1 x SSC.
4. Discussion The mechanism of initiation by E. coli RNA polymerase was divided by Chamberlin (1974) into three steps: (1) initial recognition and formation of a closed promoter complex; (2) activation by formation of an open promoter complex (rapid start complex) ; (3) initiation and formation of a ternary complex. The rate of dissociation of the open complex is much slower than that of the closed complex. The stability of the open complexes is temperature-dependent, they are favoured at 37°C as compared
POLYOiWA
VIRUS
DNA
TRANSCRIPTIOS
IN
VlTHO
II
RR
to 0°C. In addition, the formation of open complexes is favoured in negatively supercoiled DNA molecules. The present studies analyse the steps of initiation and elongation with a eucaryotic DNA in its closed superhelical and linear forms in an attempt to compare the properties of procaryotic and eucaryotic RNA pal-ymerases. Double-stranded superhelical polyoma DNA is a moderate template for the in vitro transcription by calf thymus RNA polymerase .II. under optimal conditions for transcription, the level of activity with this enzyme is only lO?i to 15% of that obtained with E. coEi RNA polymerase (Lescure et aZ., 1976). Since the extent of the polymerization of triphosphates depends on the rates of initiation and of elongat,ion we analysed these two steps separately. Two approaches were used to measure t,hr formation of stable rapid start enzyme-DNA binary complexes: residual activity in the presence of a polyanion and nitrocellulose membrane filtration. The electron microscopy studies reported here show that inactivation with the polyanion heparin and the removal of the enzyme from the DNA template occur concomitantly. Nonet,lirless. recent results (Pfeffer et al., 1977; Giacomoni et al., 1977) indicate that heparin can also attack E. coli RNA poiymerase in binary complexes, causing dissociation of the complex and the inactivation of the enzyme. We cannot exclude the possibility that the same is true for RNA polymerase 1 I. In such a case, direct attack by the polyanion will inactivate the enzyme and remove it from the DNA. The dissociation rate measured will be the rate of attack of the binary complex by the polpanion. Both approaches show that the complexes formed between RNA polyInerase II and FI polyoma DNA have a very short half-life (30 to 60 s) at 37°C as compared to a half-life of 30 hours for the E. coli enzyme with the same ternplater. Similar results were obtained by Huet et al. (1976) for complexes formed between yeast RNA polymerase II and SV40 DNA FI. Hossenlop et al. (1974) measured thta stabi1it.v of calf thymus RNA polymerase II-SV40 DNA Fl complexes by nitrocellulose membrane filtration in the presence of excess of competitor double-stranded DNA. The half-life time they found in the presence of 40 mM-ammonium sulfate was shorter than I.5 minutes at 37°C. Removal of the ammonium sulfate increased thtb stability of these complexes. Eventually, some factor(s) participating in the formation of stable initiation complexes is lost during eucaryotic RNA polgmerase I I isolation. :Utlernatively, stable initiation complexes can only be formed wit,h chromatin in the presence of specific non-histone proteins. Thus, among t,he rucaryotic enzymes oni\ RNA polymerase II1 from Xelzopus Zuevis forms heparin-resistant binary complexes (Long & Crippa, 1976). whereas RNA polymerase II and RNA polymerase I (Ferencz 8~Seifarb. 1975) are unable to form such resistant complexes. It is ou1.v upon initiat,ion of t’ranscription that the ternary complexes obtained with polymerase II becomcb of a single or very few phosphoresistant t,o the action of polyanions. The formation die&r bonds is sufficient, for the stabilization of the complex. In agreement with t,tltb observations of Dausse et nl. (1975) with E. coli RNA polymerase, these ternar! complexes are &able at high ionic strength and low tcmperaturc (Lescurc et rrl.. accompan.ving paper). Transcript,ion complexes, isolated from viral-infect’etl cells in t ht. prescncr of Sarkosyl. resemble the tertiary complexrls \\‘(t form i/r vitm in thr,ir rclsist,anct+ to thr a&ion of the polyanion (Gariglio. 19iA: Grcc>n & Brooks. 1976). ()ur t+ctron microscopy studies demonstrate that, after t,hc action of heparin no tanzynie molecules remain bound to the DNA template. In contrast. aRrr a preinitiation wit’h GpU and CTP, a considerable fract,ion of t,he c>nzyrne molc~cul~ rc~mainerl associat,ctl wit,h t,tre t,emplatc in t,hr prcsenctz of hepa~rin.
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Previous studies have shown that superhelical DNA is a more active template than relaxed DNA when transcribed with E. coli RNA polymerase (Westphal, 1971; Botchan et al., 1973; Richardson, 1974; Lescure et al., 1976). In agreement with the report of Mandel & Chambon (1974), we show a similar effect of superhelicity on RNA synthesis with calf thymus RNA polymerase II. The electron microscopy data reported in this paper show that the lower activity of the linear DNA results from a smaller number of binary complexes formed with polymerase II. Similar conclusions were obtained with the E. coli enzyme and phage PM2 DNA (Richardson, 1975) or polyoma DNA (Lescure et aE., 1976) as templates. The introduction of negatively superhelical turns into DNA (Vinograd et al., 1968) increases its affinity for “DNA-unwinding proteins”. The increase observed in the affinity of the enzyme is compatible with the formation of open complexes by both RNA polymerases. Saucier & Wang (1972) showed that the E. coli enzyme can in fact unwind the DNA duplex. Similar experiments are not feasible with RNA polymerase II due to the much shorter half-life time of the binary complexes. In addition to the low affinity of polymerase II for the DNA, the lower incorporation of triphosphates is caused in part by the synthesis of shorter products when compared to that obtained with E. coli RNA polymerase. Similar observations were reported by Mandel & Chambon (1974) using the same enzyme and SV40 DNA as template. Interestingly, many of the in vitro transcription stimulatory factors for eucaryote RNA polymerases act at the elongation rather than the initiation step (Seifart et al., 1973; Link & Richter, 1977). Stable RNA-DNA hybrids are obtained between nascent RNA and superhelical DNA templates when incubation is terminated with denaturing agents (Richardson, 1975). The stabilization of the cRNA-DNA hybrids by negative superhelical turns is expe&ed from consideration of the free energy of superhelix formation (Vinograd et al., 1968). As shown by Richardson (1975) and by our present results these hybrids are formed only after the denaturation of E. coli RNA polymerase in the transcription complex, suggesting that this enzyme is able to prevent the formation of such hybrids as long as it is in the native conformation. On the contrary, calf thymus RNA polymerase II is unable to prevent the formation of RNA-DNA hybrids during the transcription of polyoma superhelical DNA in vitro. The formation of RNA-DNA hybrids during elongation may cause premature termination with RNA polymerase IT. Presumably the factor(s) lacking in the initiation and elongation steps will prevent the formation of RNA-DNA hybrids during transcription in vivo by RNA polymerase 11. This work was supported by grants from the Centre National de la Recherche Scientifique (L.A. no. 269-ATP no. 2876), the Institut National pour la Sante et la Recherche MBdicale (C.C.P. no. 021) and the Fondation pour la Recherche MBdicale Franpaise. We are indebted to Odile Croissant for providing us with electron microscope facilities and for help in these studies. We thank C. Dauget and D. Cany for technical assistance, C. Maczuka for help in the preparation of the manuscript and S. Busby for valuable criticisms of the manuscript.
REFERENCES Acheson, N. (1976). Cell, 8, 1-12. Botchan, P., Wang, J. C. & Echols, H. (1973). Proc. Nat. Acad. hi., U.S.A. 70, 3077 3081. Bremer, H. & Konrad, M. W. (1964). Proc. Nat. Acad. Sci., U.S.A. 51, 801-808.
POLYOMA
VIRUS
DNA
TRANSCRIPTION
IN
I’ITRO
II
8.5
(%amberlin, M. (1974). Annu. Rev. Biochem. 43, 721--788. Champoux, ,J. J. $ McConaughy, B. L. (1975). Biochemielry, 14, 307 318. Contreras, R.. Rogiers, R., Van de Voorde, A. & Fiers, W. (1977). Cell. 12, 524 -538. Cox, .K. F. (1973). Eur. J. Biochem. 39, 49-62. Dausne, ,J. P., Sentenac, A. & Fromageot, P. (1975). Eur. ,J. Biochem. 57, 569-577. Dhar. K., Subramanian, K. N., Pan, J. & Weissman, S. M. (1977). I’roc. I%‘&. Acarl. Sci., I’.S.A. 74, 827-831. Dltbochet, J ., Ducommun, M., Zollinger, M. & Kellenberper. E. ( 1971). ./. r%rast~uct. Hes. 35, 147.. 167. l~unn. J. & Studier, F. W. (1973). E’TOC. Nat. Acad. Sci., C.S.A. 70, 155!1- 1564. Ferencz, A. & Seifart, K. H. (1975). Eur. J. Biochem. 53, 605. 612. (:ariplio, P. (1976). U$erenliation, 5, 179-183. Giacomoni, P., Delain, E. & Le Pecq, J. B. (1977). Eur. ,J. Biochem. 78, 215-220. (:me~r. M. & Brooks, ,J. (1976). C’irology, 72, llO- 120. Hayashi, M. (1965). Proc. Nat. Acad.Sci., U.S.A. 54, 1736-~1743. Hildebrandt,, A. & Sugden, B. (1972). J. Viral. 10, 1086-1089 Hinklc. D. & Chamberlin, M. J. (1971). Cold Spring Harbor Symp. f&ant. Biol. 35, 65.--76. Hossenlop, P.. Oudet, P. & Chambon, P. (1974). Eur. .J. Biochem. 41, 397-411. Huet. ,J., Dezelee, S., Borva, I., Buhler, J. M., Sentenac. A. & Fromageot,, P. (1976). Biochimie, 58, 7 l--80. Humphries. P.. McConnel, D. J. & Gordon, It. L. (1973). Biochem. .J. 133, 201 203. Kedinger, C. & Chambon, P. (1972). Eur. J. Biochem. 28, 283%298. Lescure, B., Oudet, P., Chambon, P. & Yaniv, M. (1976). .I. Mol. Biol. 108, 83- 95. Long. E. & Crippa. M. (1976). PEBS Letters, 72, 67-70. Link, G. &. Richter, G. (1977). Ew. J. Biochem. 76, 119-128. Mandel, J. I,. & Chambon, P. (1974). Eur. J. Biochem. 41, 367 378. Pfeffer, K. S., Stahl, S. J. 8: Chamberlin, M. J. (1977). J. Hiol. Chem. 252, 5403 -5407. l’ribnow, D. (1975). Proc. Nat. Acud. Sci., C.S.A. 72, 784 788. I’rice. IZ. & Penman, S. (1972). J. V’irol. 9, 621-626. Richardson, J. I’. (1966). J. Mol. Biol. 21, 83-91. Richttrdeon, J. P. (1974). Biochemistry, 13, 3164.-3169. R’ichardson, J. P. (1975). J. Mol. Biol. 98, 565.-579. Saucier. *J. M. & Wang, J. C. (1972). Nature New Biol. 239, 167~~170. Schiifer, H., Kriimer, R., Zilling, W. & Cudny, H. (1973). Eur. J. Biochem. 40, 367.--376. Seifart, K. H., *Juhasz, P. P. & Benecke, B. J. ( 1973). Eur. J. Biochem. 33, 181-l 90. Sekiya. T., van Ormont, H. & Khorana, H. (1975). .I. Biol. Chem. 250, 1087-1098. Vinograd. J.. Lebowitz, J. & Watson, R. (1968). J. a%lol. Riol. 33, 172 197. Walter, G., Zilling, W.. Palm, P. & Fuchs, E. (1967). Eur. .J. Biochem. 3, 194-203. Wang, J. C. (1974). J. Mol. Biol. 87, 797-816. Wc,stphal. H. (I 97 1). In The Biology of Oncogenic I’irwses, Lepetit Colloq. (Silvestri, I,. G., c,d.), pp. 183 187. North-Holland, Amsterdam. Zilling, W., Zechel, D. Rabussay, M., Schachner, U.S., Sethi. I’. Palm, A., Neil, A. & Seifrrt. IT. (1971). Cold Spring Harbor Symp. f&ant. Biol. 35, 47Yi8.
Transcription II.? Transcription
of Polyoma Virus DNA in Vitro of Superhelical and Linear Polyoma DNA by RNA Polymerase II
BERNARD LESCURE, ANNICK CHESTIER AND MOSHE YANIV Department of Molecular Bioloyy Institut Pasteur 25 rue du Dr Roux 75015 Paris, Prance (Received 3 January
1978)
Several steps in the transcription of double-stranded polyoma DNA by calf thymus RNA polymerase II(B) were analysed. Electron microscopy observations show that, as found for the Eecherichia coli enzyme, the eucaryotic RNA polymerase exhibits a higher affinity for superhelical DNA as compared to linear DNA. The half-life time of complexes formed at 37°C between the enzyme and superhelical DNA is relatively short (30 to 60 s), compared with 30 h for the E. coli enzyme under identical conditions. Binary complexes formed with linear DNA have a faster dissociation rate. The same results were obtained by using the nitrocellulose binding assay or by measuring the decay of heparinresistant complexes. Only ternary complexes, enzyme-DNA-RNA, obtained after the formation of the first phosphodiester bond, are resistant to polyanions. These results suggest that, as for the procaryotic enzyme, RNA polymerase II has to melt locally the double-stranded DNA during initiation, but that it lacks a sigma type factor necessary to stabilise the open initiation complexes. RNA polymerase II synthesizes shorter RNA chains than E. coli RNA polymerase. The formation of RNase I resistant RNA-DNA hybrids during transcription may explain the reduced rate of chain elongation.
1. Introduction The circular double-stranded DNA of the papovaviruses (simian virus 40, polyoma, etc.) is transcribed in vivo by RNA polymerase II (B) as suggested by the sensitivity of viral RNA synthesis to a-amanitin (Price & Penman, 1972; Hildebrandt & Sugden, 1972). The small size of these DNAs, the abundant information about physical and functional maps of SV40 and polyoma (Acheson, 1976), and sequence information (Dhar et aE., 1977 ; Contreras et al., 1977 ; Griffin, personal communication), make t,his template a useful tool for the study of enzyme-DNA interactions. Several steps have been defined in the process of recognition of double-stranded DNA by procaryotic RNA polymerase (Hinkle & Chamberlin, 1971: Dunn & Studier, 1973) : entry, formation of closed complexes, formation of rapid start open complexes and initiation. The signals for the recognition of the DNA by this enzyme were characterized on a t Paper I in this series is Lescure et al. (1976). 73 0022-2836/78/250073-13 $02.00/O
0 1978 Academic Press Inc. (London) Ltd.
74
B. LESCURE,
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large number of bacterial promoters (Pribnow, 1975; Sekiya et al., 1975). The fact that the formation of stable initiation complexes requires the opening of the doublestranded DNA (Saucier & Wang, 1972) explains the increased affinity of Escherichia coZi RNA polymerase for superhelical DNA (Richardson, 1975 ; Lescure et al., 1976). In the present studies, we set out to characterize better the interactions between the eucaryotic RNA polymerase II and the double-stranded viral DNA. The ability to form stable initiation complexes and to transcribe polyoma virus DNA was studied. We show that RNA polymerase II exhibits a higher affinity for superhelical DNA than for relaxed DNA, and that stable initiation complexes can be obtained only after initiation with a dinucleotide and a single ribonucleoside triphosphate. The elongation with E. coli RNA polymerase, with RNA polymerase II is less than that obtained possibly because of RNA-DNA hybrids formed during transcription with the former enzyme.
2. Materials and Methods (a) DNA
and enzyme preparations
Superhelical and linear polyoma virus DNA were purified as previously described (Lescure et al., 1976). E. coli RNA polymerase was purified from E. coli MRE 600 cells as described by Humphries et al. (1973). The enzyme had a spec. act. of 400 units/mg (1 unit equals the incorporation of 1 nmol UTP in 10 min at 37°C). Calf thymus RNA polymerase B (II) was either a gift from Dr P. Chambon or purified according to the method described by Kedinger C Chambon (1972) applying the following steps: DEAE-cellulose and phosphocellulose columns followed by glycerol gradient centrifugation. The enzyme obtained was free of DNase and RNase contamination: no conversion of superhelical to relaxed polyoma DNA was observed on incubation with the enzyme and no reduction in the size of RNA made in vitro with E. coli RNA polymerase upon further incubation with the enzyme. The main contaminant, revealed by polyacrylamide gels, was actin. One unit of enzyme corresponds to the incorporation of 1 nmol of UMP incorporated in 10 min at 37°C with single-stranded calf thymus DNA as template. The protein concentration of the purified enzyme was not measured. (b) Electron
microscopy
RNA polymerase binding to polyoma DNA was monitored by the electron microscopy technique of Dubochet et al. (1971) as previously described (by Lescure et al., 1976). (c) Conditions
for binding
of RNA polymerase synthesis
to polyoma
DNA
and for RNA
Identical ionic conditions were used for RNA polymerase binding and for RNA synthesis. The incubation mixture for E. coli RNA polymerase contained : 20 miw-Tris . HCl (pH 7.9), 10 mM-MgCl,, 150 m&r-Kcl, 0.2 mM-dithiothreitol. The following concentrations of ribotriphosphates: 0.2 mM-GTP, -CTP, -ATP and 0.1 miw-[3H]UTP (100 &X/pmol) were added when transcription was studied. The incubation mixture for calf thymus RNA polymerase II contained: 20 mM-Tris*HCl (pH 7*9), 2.5 mivr-M&l,, 20 m&r-(NH,)&),, 0.5 mnrdithiothreitol, 0.4 mM-GTP, -CTP, -ATP and 0.2 mM-[3H]UTP (100 &ip/mol) were added for transcription. For binding studies RNA polymerase was incubated with DNA for 5 min at 37°C in the ionic conditions described above. Transcription was initiated after 5 min by addition of the ribonucleoside triphosphates. The reaction was stopped with 5% trichloroacetic acid, samples were filtered on nitrocellulose membranes, dried and counted by liquid scintillation. (d) Complementary
RNA
pur$cation
For preparation of complementary RNA, the reaction w&s stopped by addition of EDTA to a flnal concentration of 20 mM and extracted with an equal volume of water-saturated
POLYOMA
VIRUS
DNA
TRANSCRIPTION
IA’
VITRO
73
II
twice with an equal volume of chloroform. The aqueous phase was re-extracted The final aqueous phase was precipitated with 2 vol. ethanol at, - 20°C. After centrifugation, the precipitate was dissolved in 10 mM-Tris.HCl (pH 7.9), 5 mM-MgClz and incubated for 1 h at 37°C with 20 pg DNase I/ml (RNaae-free). The incubation mixture was reextracted with phenol and the RNA precipitated with ethanol. The final precipitate was dissolved in 10 mM-Tris.HCl (pH 7.9), 1 mM-EDTA.
phenol.
3. (a) Transcription
of superhelical
Results and linear
polyoma
DNA
virus
in vitro
Superhelical polyoma DNA was transcribed in vitro with calf thymus RNA polymerase 11. As shown in Table 1, the incorporation observed was about lOoA, of that obtained when the optimal amount of E. coli RNA polymerase holoenzyme transcribed the same template (Lescure et al., 1976). A further increase in the amount of enzyme I1 added did not result in higher incorporation of triphosphates. That incorporation is greatly affected by the superhelicity of the DNA is shown by the fact that linear double-stranded molecules of polyoma DNA produced by EcoRI treatment were seven to eightfold less effective than superhelical DNA template, at the same ratio of enzyme to DNA (Table 1). In an attempt to analyse the multiple steps involved in the transcription process and to elucidate the differences between procaryotic and eucaryotic RNA polymerases, we studied the formation and the stabilit’) of initiation complexes as well as the RNA elongation step. TABLE 1 Transcription
of polyom,a
DNA
FI
or FIII in vitro
by calf thymus
RNA
polym,era.se
II
UMP incorporated in cRN.4 with 0.2 unit of RNA polymerase II (pmol) 4 min A min 15 min
Template
Polyoma
DNA
FI (1.5 pg)
Polyoma
DNA
FIII
(1.5 pg)
53
9x
114
7
13
16
Thr conditions used for transcription are described in Material and Methods. At various times of synthesis, the radioactivity incorporated into cRNA was monitored by liquid scintillation counting.
(b) Formation
of enzyme-DNA
com,plexes
Binary complexes were formed between RNA polymerase 11 and FIT or linear polyoma DNA at a ratio of enzyme to DNA of 0.2 unit/pg. Complexes were spread for electron microscopy (Fig. 1) and the number of enzyme molecules bound on each kind of template was scored. An average of 3.6 enzyme molecules were bound to superhelical DNA (Figs l(a) and Z(a)). When linear polyoma DNA, produced by EcoRI treatment of circular DNA was used, many of the RNA polymerase 11 molecules were bound to the sticky ends of the DNA molecules (Fig. l(b)). The average number of enzyme molecules bound per DNA molecule was l-28 if all bound t Abbreviations used: FI DNA, superhelical polyoma DN8; FIlI DXA. cRNA, complamcnt,ary RNA, i.p. RNA synthesized in w&o from polyoma.
linear
polpoma
DKA
:
76
B. LESCURE,
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AND
M. YANIV
FIG. 1. Vizualisation by electron microscopy of calf thymus RNA polymerase II molecules bound to polyoma DNA. The complexes were formed as described in Materials and Methods, at a weight ratio of enzyme to DNA of 0.2 unit of enzyme/pg of (a) polyoma DNA FI or (b) polyoma DNA FIII. After 5 min of binding at 37X’, glutaraldehyde was added to a final concn of 10-2 M, incubation continued for 15 min at 37°C and the samples were spread for electron microscopy as previously described (Lescure et al., 1976). The bar represents 100 nm.
molecules were counted and 0.78 if enzyme molecules on the ends were excluded from the calculation (Fig. 2(b)). Thus it appears that RNA polymerase II displays a three to fourfold lower tendency to bind to untwisted DNA relative to negatively superhelical DNA. (c) EJfect of heparin on
the
transcription
of polyom
DNA
The polyanion heparin is a potent inhibitor of E. coli RNA polymerase and of nuclear eucaryotic RNA polymerases I, II and III (Walter et al., 1967; Cox, 1973; Ferencz & Seifart, 1975; Long & Crippa, 1976). As expected, when heparin was added either to E. coli or to RNA polymerase II prior to the DNA template RNA synthesis was completely inhibited with both enzymes (Fig. 3(a) and (b)). When heparin was added before initiation to preformed enzyme-DNA complexes, the results differed for the two enzymes. In agreement with the previously observed resistance of binary complexes to the polyanion, E. coli RNA polymerase was only slightly affected (Zillig et al., 1971; Schgfer et al., 1973). In contrast, under the same conditions, RNA polymerase II dependent synthesis was totally inhibited (Fig. 3). In another experiment heparin was added 15 seconds after the initiation of RNA synthesis. Only a slight inhibition was observed with E. coli RNA polymerase during the four minutes of synthesis (Fig. 3(a), curve 4). With the calf thymus RNA polymerase B, an inhibition of about 50% is observed in these conditions, indicating that initiated
POLYOMA
VIRUS
DNA
TRANSCRIPTION
IN
C’ITRO
II
i;
;I-&I )246802468
No. of RNA polymerose n-decules/ DNA molecule
FIG.
2. Binding
of calf
thymus
(cl
(b)
(a) RNA
polymerwe
II
to polyoma
DNA
FI
or FIII.
The number of polymerase molecules bound to superhelical or linear template at, a ratio o enzyme to DNA of 0.2 unit/pg was monitored by electron microscopy. (a) RNA polymerase molecules on superhelical template; (b) RNA polymerase molecules on linear template including molecules bound to the stickv ends of the EcoRI linear molecules: fcl RNA nolvmerase molecules * ” on linear template excluding molecules bound to the end of linear DNA molecules. \
I
Time (min)
Time (mm1
(a)
(b)
Fro. 3. Effect of heparin on in vitro transcription of polyoma IjN.4 PI by (a) E’. coli KS:2 polymerase or (b) calf thymus RNA polymerase. r3H]RNA was synthesized as described in Materials and Methods, at a weight ratio of enzyme’ to DNA of 3 rg/l rg for E. coli RNA polymerase or 0.2 unit/l pg for calf thymus RNA polymerasc II. At various intervals [3H]oRNA synthesis was estimated. Curve 1, initial RNA synthesis in the absence of heparin. Curve 2, heparin at a final concn of 10 pg/ml was added for 2 min at 37°C to reformed complexes between RNA polymerase and polyoma DNA FI prior to the addition of ribotriphosphates. Curve 3, heparin was added for 2 min at 37% to free RNA polymerase prior to the addition of polyoma DNA FI. Curve 4, heparin was added 16 s after initiation of synthesis. Curve 5, synthesis was pre-initiated by adding GpU (0.4 mM) and CTP (0.2 mix) to reformed complexes. After 6 min of incubation at 37”C, heparin was added at a final concn of 10 pg/ml. incubation continued 6 min at 37°C prior to initiation of RNA elongation by the addition of the other 3 ribotriphosphates.
78
B. LESCURE,
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EO1-
I
AND
I
No. of RNA polymerme molecules/DNA (0)
M. YANIV
ib)
molecule (ci
FIN. 4. Studies by electron microscopy of the effect of heparin on binary and ternary complexes formed between calf thymus RNA polymerase II and polyoma DNA FI, or calf thymus RNA polymerme II polyoma DNA FI and GpUpC. (a) Number of calf thymus RNA polymerase molecules bound to polyoma DNA FI at a ratio of enzyme to DNA of 0.1 unit/l pg. (b) Number of calf thymus RNA polymerase molecules bound to polyoma DNA FI at the same ratio of enzyme to DNA after incubation during 2 min at 37°C of preformed complexes with heparin at a final concn of 10 pg/ml. (c) Enzyme molecules bound to polyoma DNA FI after pre-incubation with GpU and CTP, followed by heparin treatment (10 pg/ml) for 2 min at 37’C.
complexes are either partially or totally resistant to the action of heparin (Fig. 3(b), curve 4). Two hypotheses can be postulated to explain these results : (i) elongation is limited with RNA polymerase II ; after two minutes of synthesis RNA chains were terminated, the enzyme DNA complexes dissociated and the free RNA polymerase inhibited by heparin. (ii) RNA polymerase II engaged in ternary complexes (enzymeDNA-RNA) is sensitive to heparin ; however the inhibition is less effective than that obtained with binary complexes (Fig. 3(b), curve 2). The second hypothesis can be excluded by the following experiment: we have shown that for the two enzymes, some of the in vitro initiation sites on polyoma DNA start with the sequence GpUpC (Lescure et al., accompanying paper). Using RNA polymerase II, we initiated the transcription by a preincubation in the presence of GpU and CTP (the number of C residues incorporated that could include one to several residues was not determined). Heparin was then added to the ternary complexes of enzyme-DNA-GpUpC for five minutes at 37°C before the addition of the other three ribotriphosphates. A short synthesis of RNA occurred during roughly two minutes (Fig. 3(b), curve 5). The mechanism of action of heparin is confirmed by the following electron microscopy observations. An average number of about two molecules of RNA polymerase II were bound to polyoma DNA using a ratio of enzyme to DNA of O-1 unit/pg (Fig. 4(a)). After incubation with heparin for two minutes at 37”C, almost all the enzyme molecules were removed from the template (Fig. 4(b)). However if transcription was initiated using GpU and CTP, prior to the addition of heparin, a large fraction of RNA polymerase molecules (33%) remained associated with the template (Fig. 4(c)) confirming that the ternary complexes became partially resistant to the action of the polyanion. (d) Rate of dissociation of RNA polymerwe-DNA binary complexes different methods, previously applied with E. coli RNA polymerase, were used to measure the stability of enzymes-DNA complexes in the present study. In Two
POLYOMA
VIRUS
DNA
TRANSCRIPTION
11v VITRO
I I Time (min) (a)
I
I,!I
59
II
I
I
2 3” IO 20 30 Time (mm) (b)
Fro. 5. Stability of binary complexes formed between RNA polymerase and polyoma DNA Fl. -O-O--, calf thymus RNA polymerase Il. -O-e-, E. r&i RNA polymerase; (a) Nitrocellulose filter assay (Hinkle & Chamberlin, 1971). The reaction was performed in a final vol. of 200 pl. E. coli RNA polymerase or calf thymus RNA polymerase II w&s incubated at 37°C with polyoma [3H]DNA FI in the standard conditions (Materials and Methods) at a ratio of enzyme to DNA of 4 pg/l rg for the E. coli enzyme or 0.2 unit/l rg for the calf thymus enzyme. After 5 min, 100 pg of denatured calf thymus were added, samples of 40 ~1 were removed, diluted to 1 ml with the standard medium for each enzyme and filtered on nitrocellulose filters. Polyoma [3H]DNA retained on the filter was estimated at various times. (b) Residual activity in the presence of heparin. The method used has been described previously (Lescure et al., 1976). The ratio of enzyme to DNA was the same as that used for the nitrocellulose filter assay.
the nitrocellulose filter binding assay (Hinkle & Chamberlin, 1971), the rate of dissociation of previously formed complexes between E. coli RNA polymerase or calf thyms RNA polymerase II and [3H]DNA polyoma FI was measured by adding a hundredfold weight excess of denatured calf thymus DNA. Samples were removed at intervals and filtered on nitrocellulose membranes to determine the amount of radioactive DNA retained (Fig. 5(a)). Almost no dissociation was detected after an eight-minute incubation with the complexes formed between E. coli RNA polymerase and polyoma DNA FI. In contrast, 50% of complexes formed with RNA polymerase II had dissociated after two minutes of incubation. The second method measures the residual activity, in the presence of heparin (Richardson, 1975), that inactivates free enzyme molecules which dissociate from the template. Recent studies however have shown that heparin can attack polymerase in binary complexes directly without prior dissociation from the template (Pfeffer et al., 1977 ; Giacomoni et al., 1977). In this case, both techniques may in fact measure the accessibility of binary complexes to attack by polyanions. Heparin was added to previously formed complexes, samples werca removed at intervals and residual activity measured (Fig. 5(b)). Using this technique. the stability of complexes formed with E. coli RNA polymerase was again very long (half-life time of 30 h). Although resistant complexes can be detected with RNA polymerase II, 50% of these complexes were inactivated after 30 seconds of incubation at 37°C in the presence of heparin. When linear double-stranded polyoma DNA was used, re-initiation could be blocked either by heparin or by addition of 0.75 M-KU alone. The rate of dissociation of the RNA polymerase II was too fast to be measured. Under similar conditions (using poly(rI) s,s polyanion), E. c& RNA polymerasr dissociated with a half-life time of five hours (Lescure et ~2.. 1976).
80
B. LESCURE.
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AND
,--/
IO
4s
16 S
235
M. YANIV
,
I
20
30
I
Fmction no.
FIG. 6. Sucrose gradient analysis of cRNA synthesized. [3H]cRNA was synthesized and purified as described in Materials and Methods. Before sedimentation analyses cRNA was treated for 1 min at 100°C and quiokly cooled to 0°C. Samples were layered onto S-ml linear 6% to 20% sucrose gradients in 0.1 M-NaCl, 0.02 M-Tris.HCl (pH 7.4) and 0.6 mix-EDTA and centrifuged for 180 min at 48,000 revs/min at 4°C inithe Spinco SWKO.l rotor. Radioactivity from gradient fractions was estimated by scintillation counting. -O-O--, Transcription of polyoma DNA FI by E. co.5 enzyme; -@-a--, transcription of polyoma DNA FI by calf thymue RNA polymerase II.
(e) Analysis
of complementary
RNA
The experiments described in Figure 3 suggest that RNA polymerase II is limited in elongation, we therefore tried to characterize the RNA made in vitro. Under the transcription conditions used with polyoma DNA FI the RNA was largely symmetric ; 60 to 70% of the cRNA was resistant to treatment by RNaseI in 2 x SSC (SSC is 0.15 iw-NaCl, 0*015 M-sodium citrate) after self-annealing. Hence, as with E. coli RNA polymerase (Lescure et al., 1976), at least two initiation sites for RNA synthesis in vitro are located on opposite strands of polyoma DNA. The size of the cRNA was analysed by sedimentation on a sucrose gradient. The peak of radioactivity was heterogeneous with a maximum at about 9 S (Fig. 6). This size is far below a complete transcript of the genome and smaller than that obtained when E. coli RNA polymerase transcribes the same template (Fig. 6). The smaller size of the RNA synthesized by RNA polymerase II was not due to the presence of RNase in the enzyme preparation since the size of the RNA synthesized by E. coli RNA polymerase was not altered by post-incubation with RNA polymer&se II. To confirm these observations on the limited elongation, we have estimated the RNA chain length using [Y-~~P]ATP, [Y-~~P]GTP and [3H]UTP as radioactive precursors. The average chain length of the RNA synthesized by E. coli RNA polymerase and by RNA polymerase II was calculated from the ratio of 3H/32P incorporated, to give values of 2500 and 660 nucleotides, respectively (Table 2). The continued synthesis of RNA with RNA polymerase II in the absence of heparin (Table 1) is certainly due to chain reinitiation. (f) Formation
of UNA-RNA
hybrids during elongation
in vitro
Nascent RNA molecules can be completely released in vitro from linear DNA templates, by treatment with agents such as detergents that denature proteins (Bremer $ Konrad, 1964; Richardson, 1966). However, when the template is a negative superhelical DNA, denaturation of the RNA polymerase does not release all of the RNA (Hayashi, 1965 ; Champoux & McConaughy, 1975 ; Lescure et al., 1976). The positive free energy of the superhelical DNA is responsible for the formation of
II
1.40
0.39
0.27
plus [y-=P]GTP in cRNA (pmol) 8 min
0.85
[y-=P]ATP incorporated 4 min
40
580
4 min
13H]UTP
incorporated
synthesized in vitro
2
Polyoma DNA FI was transcribed by E. coli RN-4 polymerase or by calf thymus RNA polymerase labelled by [3H]UTP (100 mCi/mmol) [y-32P]ATP (2300 mCi/mmol) and [y-32P]GTP (2800 mCi/mmol). ditions, 32P and 3H radioactivity incorporated in cRNA were estimated by liquid scint,illation counting.
polymerase
RNA
Calf
thymus
polymerase
E. coli RNA
Enzyme
Chain length of RNA
TABLE
(pmol) Mean
664
2500
chain length (nucleotides) after 8 min of synthesis
II as described in Materials and Methods. cRNA After a synthesis for 4 or 8 min in the standard
65
875
8 min
in cRNA
w&s con-
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AND
M. YANIP
stable hybrids between the RNA and the template (Vinograd et al., 1968; Wang, 1974). However, it was shown recently that native E. coli RNA polymerase can prevent the formation of such hybrids if the intact structure of t,he enzyme is maintained (Richardson, 1975). Polyoma DNA FI was transcribed for one minute at, 37°C by h’. coli RNA polymerase or by RNA polymerase II and the incubation was terminated by addition of EDTA alone (non denaturing conditions) or by addition of EDTA and sodium dodecyl sulfate as described by Richardson (1975). The resistance to RNase I digestion was then measured in 2 x SSC or in 0.1 x SSC to estimate the percentage of RNA-DNA hybrids (Table 3). When E. coli RNA polymerase was used, 32% of the newly synthesized RNA was resistant to RNase I digestion in 2 x SSC, if the transcription was terminated with EDTA and sodium dodecyl sulfate. On the contrary, only 2.1% of the RNA was resistant when the incubation was terminated with EDTA alone. When RNA polymerase II was used, 28% of the newly synthesized RNA was resistant to RNase I when the transcription was terminated with EDTA and sodium dodecyl sulfate and 21% when EDTA alone was added. In contrast to the observations with the E. coli RNA polymerase, the native form of calf thymus RNA polymerase II is less efficient in preventing the formation of RNA-DNA hybrids during the transcription of a superhelical template.
TABLE 3 Effect of detergent on nascent RNA
Digestion E. coli RNA
polymerase
Calf thymus RNA polymerase
II
in
hybridized
to the DNA
template
Percentage of product resistance to RNase A digestion after stopping the reaction with EDTA EDTA + SDS
0.1 x ssc 2 x ssc
0.9 2.1
0.7 32
0.1 x ssc 2 x ssc
0.7 21
1.0 28
The method applied was essentially that described by Richardson (1974). Two pg of polyoma DNA FI were transcribed for 1 min at 37°C with 4 pg of E. coli RNA polymerase or with 0.4 unit of calf thymus RNA polymerase II in a final volume of 200 ~‘1. RNA synthesis was terminated by the addition of 16 ~1 of 0.1 M-EDTA, or 16 ~1 of 0.1 M-EDTA + 8 ~1 of 10% sodium dodecyl sulfate (SDS). The incubation was continued for 2 min at 37°C. After chilling in an ice bath, the SDS precipitate was removed by centrifugation. The supernatant solution was carefully removed and samples were treated for 30 min at 37°C with RNase A (20 pg/ m 1) and RNase T, (1 unit/ml) after dilution in 2 ml of 2 x SSC or 0.1 x SSC.
4. Discussion The mechanism of initiation by E. coli RNA polymerase was divided by Chamberlin (1974) into three steps: (1) initial recognition and formation of a closed promoter complex; (2) activation by formation of an open promoter complex (rapid start complex) ; (3) initiation and formation of a ternary complex. The rate of dissociation of the open complex is much slower than that of the closed complex. The stability of the open complexes is temperature-dependent, they are favoured at 37°C as compared
POLYOiWA
VIRUS
DNA
TRANSCRIPTIOS
IN
VlTHO
II
RR
to 0°C. In addition, the formation of open complexes is favoured in negatively supercoiled DNA molecules. The present studies analyse the steps of initiation and elongation with a eucaryotic DNA in its closed superhelical and linear forms in an attempt to compare the properties of procaryotic and eucaryotic RNA pal-ymerases. Double-stranded superhelical polyoma DNA is a moderate template for the in vitro transcription by calf thymus RNA polymerase .II. under optimal conditions for transcription, the level of activity with this enzyme is only lO?i to 15% of that obtained with E. coEi RNA polymerase (Lescure et aZ., 1976). Since the extent of the polymerization of triphosphates depends on the rates of initiation and of elongat,ion we analysed these two steps separately. Two approaches were used to measure t,hr formation of stable rapid start enzyme-DNA binary complexes: residual activity in the presence of a polyanion and nitrocellulose membrane filtration. The electron microscopy studies reported here show that inactivation with the polyanion heparin and the removal of the enzyme from the DNA template occur concomitantly. Nonet,lirless. recent results (Pfeffer et al., 1977; Giacomoni et al., 1977) indicate that heparin can also attack E. coli RNA poiymerase in binary complexes, causing dissociation of the complex and the inactivation of the enzyme. We cannot exclude the possibility that the same is true for RNA polymerase 1 I. In such a case, direct attack by the polyanion will inactivate the enzyme and remove it from the DNA. The dissociation rate measured will be the rate of attack of the binary complex by the polpanion. Both approaches show that the complexes formed between RNA polyInerase II and FI polyoma DNA have a very short half-life (30 to 60 s) at 37°C as compared to a half-life of 30 hours for the E. coli enzyme with the same ternplater. Similar results were obtained by Huet et al. (1976) for complexes formed between yeast RNA polymerase II and SV40 DNA FI. Hossenlop et al. (1974) measured thta stabi1it.v of calf thymus RNA polymerase II-SV40 DNA Fl complexes by nitrocellulose membrane filtration in the presence of excess of competitor double-stranded DNA. The half-life time they found in the presence of 40 mM-ammonium sulfate was shorter than I.5 minutes at 37°C. Removal of the ammonium sulfate increased thtb stability of these complexes. Eventually, some factor(s) participating in the formation of stable initiation complexes is lost during eucaryotic RNA polgmerase I I isolation. :Utlernatively, stable initiation complexes can only be formed wit,h chromatin in the presence of specific non-histone proteins. Thus, among t,he rucaryotic enzymes oni\ RNA polymerase II1 from Xelzopus Zuevis forms heparin-resistant binary complexes (Long & Crippa, 1976). whereas RNA polymerase II and RNA polymerase I (Ferencz 8~Seifarb. 1975) are unable to form such resistant complexes. It is ou1.v upon initiat,ion of t’ranscription that the ternary complexes obtained with polymerase II becomcb of a single or very few phosphoresistant t,o the action of polyanions. The formation die&r bonds is sufficient, for the stabilization of the complex. In agreement with t,tltb observations of Dausse et nl. (1975) with E. coli RNA polymerase, these ternar! complexes are &able at high ionic strength and low tcmperaturc (Lescurc et rrl.. accompan.ving paper). Transcript,ion complexes, isolated from viral-infect’etl cells in t ht. prescncr of Sarkosyl. resemble the tertiary complexrls \\‘(t form i/r vitm in thr,ir rclsist,anct+ to thr a&ion of the polyanion (Gariglio. 19iA: Grcc>n & Brooks. 1976). ()ur t+ctron microscopy studies demonstrate that, after t,hc action of heparin no tanzynie molecules remain bound to the DNA template. In contrast. aRrr a preinitiation wit’h GpU and CTP, a considerable fract,ion of t,he c>nzyrne molc~cul~ rc~mainerl associat,ctl wit,h t,tre t,emplatc in t,hr prcsenctz of hepa~rin.
84
B. LESCURE,
A CHESTIER
AND
M. YANIV
Previous studies have shown that superhelical DNA is a more active template than relaxed DNA when transcribed with E. coli RNA polymerase (Westphal, 1971; Botchan et al., 1973; Richardson, 1974; Lescure et al., 1976). In agreement with the report of Mandel & Chambon (1974), we show a similar effect of superhelicity on RNA synthesis with calf thymus RNA polymerase II. The electron microscopy data reported in this paper show that the lower activity of the linear DNA results from a smaller number of binary complexes formed with polymerase II. Similar conclusions were obtained with the E. coli enzyme and phage PM2 DNA (Richardson, 1975) or polyoma DNA (Lescure et aE., 1976) as templates. The introduction of negatively superhelical turns into DNA (Vinograd et al., 1968) increases its affinity for “DNA-unwinding proteins”. The increase observed in the affinity of the enzyme is compatible with the formation of open complexes by both RNA polymerases. Saucier & Wang (1972) showed that the E. coli enzyme can in fact unwind the DNA duplex. Similar experiments are not feasible with RNA polymerase II due to the much shorter half-life time of the binary complexes. In addition to the low affinity of polymerase II for the DNA, the lower incorporation of triphosphates is caused in part by the synthesis of shorter products when compared to that obtained with E. coli RNA polymerase. Similar observations were reported by Mandel & Chambon (1974) using the same enzyme and SV40 DNA as template. Interestingly, many of the in vitro transcription stimulatory factors for eucaryote RNA polymerases act at the elongation rather than the initiation step (Seifart et al., 1973; Link & Richter, 1977). Stable RNA-DNA hybrids are obtained between nascent RNA and superhelical DNA templates when incubation is terminated with denaturing agents (Richardson, 1975). The stabilization of the cRNA-DNA hybrids by negative superhelical turns is expe&ed from consideration of the free energy of superhelix formation (Vinograd et al., 1968). As shown by Richardson (1975) and by our present results these hybrids are formed only after the denaturation of E. coli RNA polymerase in the transcription complex, suggesting that this enzyme is able to prevent the formation of such hybrids as long as it is in the native conformation. On the contrary, calf thymus RNA polymerase II is unable to prevent the formation of RNA-DNA hybrids during the transcription of polyoma superhelical DNA in vitro. The formation of RNA-DNA hybrids during elongation may cause premature termination with RNA polymerase IT. Presumably the factor(s) lacking in the initiation and elongation steps will prevent the formation of RNA-DNA hybrids during transcription in vivo by RNA polymerase 11. This work was supported by grants from the Centre National de la Recherche Scientifique (L.A. no. 269-ATP no. 2876), the Institut National pour la Sante et la Recherche MBdicale (C.C.P. no. 021) and the Fondation pour la Recherche MBdicale Franpaise. We are indebted to Odile Croissant for providing us with electron microscope facilities and for help in these studies. We thank C. Dauget and D. Cany for technical assistance, C. Maczuka for help in the preparation of the manuscript and S. Busby for valuable criticisms of the manuscript.
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POLYOMA
VIRUS
DNA
TRANSCRIPTION
IN
I’ITRO
II
8.5
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