J. Mol. Biol. (1976) 108, 83-97

Transcription of Polyoma Virus D N A in vitro L o c a l i z a t i o n o f Escherichia coli R N A P o l y m e r a s e I n i t i a t i o n Sites BERNARD LESCURE 1, PIERRE 0UD:ET 2, PIERRE ~HAMBON 2 AND MOSHE "YANIV 1

1Department of Molecular Biology, Institut Pasteur 25, rue du Docteur Roux, 75015 Paris, France and 2Institut de Chimie biologique, Facultd de Mgdecine 67085 Strasbourg, France (Received 2 February 1976, and in revised form 7 July 1976) Localization of Escherichia cell RNA polymerase binding sites and initiation sites on superhelical or linear polyoma virus DNA was achieved by electron microscopy and by hybridization of short synthesis complementary RNA to polyoma DNA HpaII fragments. These sites are located in positions 0.06, 0.25, 0"57, 0.85 and 0"98 on the physical map of polyoma DNA (Griffin et al., 1974). With an equal ratio of enzyme to DNA, threefold less enzyme is bound to linear DNA than to superhelical DNA ; however, the distribution of bound enzyme molecules in the different sites was identical on both templates. In addition, the complex formed with linear DNA is less stable than the complex formed with superhelical DNA. These findings are correlated with the decreased template activity of the linear DNA relative to superhelical DNA. Electron mierographs of complexes, obtained in the presence of a high ratio of enzyme to DNA, demonstrate the unwhlding properties of the RNA polymerase. 1. I n t r o d u c t i o n Much recent progress in the s t u d y of the genome of D N A tumour viruses comes from the identification and mapping of cytoplasmic messenger R N A on the viral physical map. The early transcription products of simian virus 40 (SV40) or polyoma DIgA is found in the cytoplasm as an R N A species sedimenting at 19 S. I t comprises the sequences from about 50% of the D N A early strand of the viral genome. The same m R N A species is present in transformed cells (Lindstrom & Dulbeceo, 1972; Sambrook et al., 1972,1973; K h o u r y et al., 1973; Dhar et al., 1974a,b; K a m e n et al., 1974). Late in infection, two novel partially overlapping species of viral RIGA, 19 S and 16 S, transcribed from the DNA late strand have been detected (Weinberg & IGewbold, 1974; May et al., 1975; K a m e n & Shure, 1976). These observations suggest a possible control of transcription during the infection cycle and the presence of at least two initiation sites for transcription on opposite DIGA strands. A certain ambiguity remains, however, as to the exact nature of the p r i m a r y transcription products in the nucleus (Aloni, 1974; Beard et al., 1976). 83

84

B. L E S C U R E E T A L .

The DNA of papovaviruses is associated in vivo with histones in a relaxed nucleoprotein complex (Seebeck & Weft, 1974; Griffith, 1975; Germond et al., 1975; Cr4misi et al., 1976). Removal of these histones results in the formation of superhelical DNA. Any study of viral D N A transcription has to take into account the variation in template activity due to the generation of negative superlielieal tm'ns. Studies on the transcription of the viral D N A in vitro should help to identify the initiation sites for transcription in vivo and provide information about the possible control mechanisms of gene expression. The circular double helical D N A of papovaviruses is an efficient template for both Escherichia coli and eukaryotic DNA-dependent R N A polymerases (Westphal, 1970; Chambon et al., 1973; Mandel & Chambon, 1974a,b; Hosseulopp et al., 1974; K a m e n et al., 1974). A strong initiation site for the E. coli R N A polymerase has been located on SV40 D N A at position 0.17 (Westphal et al., 1973; Dhar et al., 1974a,b; Allet, 1975), with a sequence which resembles t h a t of an E. coli or bacteriophage promoter (Dhar et al., 1974a,b; Pribnow, 1975). Its location on the SV40 genome m a y be fortuitous or it m a y be related to the initiation of early transcription. With the aim of identifying the possible initiation sites for transcription on polyoma virus DNA, we a t t e m p t e d to localize by electron microscopy all possible binding sites for E. coli R N A polymerase and for eukaryotic R N A polymerase. I n this paper we present results obtained with E. coli RNA polymerase. We have studied the effects of superhelicity on the activity of polyoma D N A and on the location of the polymerase binding sites. Our results demonstrate the presence of five specific E. coli RNA polymerase binding sites on polyoma DNA. The same binding sites, although ~ i t h a lower stability of the bound enzyme are observed on a linear template. Most of these sites are located in ( A + T ) - r i c h regions of the D N A (Lescure & Yaniv, 1975). I n the presence of a large excess of enzyme, polyoma D N A can bind up to 50 R N A polymerase molecules and appears as a relaxed circle when examined b y electron microscopy.

2. Materials and Mvthods (a) Preparation of polyoma virus D N A Superhelical polyoma DNA was extracted by the method of I-Iirt (1967) from primary mouse kidney cell cultures infected with a multiplicity of 40 plaque-forming units/cell with LP polyoma virus derived from recent plaque isolates. The superhelical DNA was purified by isopycnic centrifugation in CsC1/ethidinm bromide gradients. The digestion pattern of this DNA by H p a I I restriction enzy~ne is identical to that described by Fried et al. (1974) for the A-3 type of polyoma virus. Linear polyoma DNA was prepared by treatment of FI DNA~ with an excess of EcoRl endonuclease (Yaniv et al., 1974). After extraction with phenol and precipitation with ethanol, the EcoR1 linear molecules were purified by sedimentation in a 5% to 20% sucrose gradient. The peak fractions corresponded to lmit length linear molecules as confirmed by electron microscopic measurements. Polyoma DNA H p a I I fragments were prepared by treating I)NA form I with H p a I I restriction enzyme (Griffin et al., 1974). After extraction with phenol and precipitation with ethanol, polyoma H p a I I fragments were separated by electrophoresis on a 2.5% to 10% polyacrylamide gradient slab gel (Jeppeson, 1974). Each band was eluted electrophoretically from the gel and purified by chromatography on hydroxyapatite. Abbreviations used: FI DNA, superhelieal polyoma DNA; F I I I DNA, linear polyoma DNA; complementary RNA, RNA synthesized in vitro from polyoma DNA; eRNA, complementary RNA.

T R A N S C R I P T I O N OF P O L Y O M A DNA I N

VITRO

85

(b ) Enzyme pu~ification E. cell R N A polymerase was purified from E. cell MRE600 as described by Humphries et al. (1973). Frozen ceils were disrupted b y grinding with a h u n i n a a n d the extract was fractionated sequentiaUy b y precipitation with polyethylene glycol, chromatography on DNA-ceUulose a n d separation on a glycerol gradient at high ionic strength. The enzyme is over 97~/o pure a n d contains 0"85 to 0"90 equivalent of sigma, as determined b y polyacrylamide gel electrophoresis in the presence of sodium dodeeyl sulphate. EcoR1 restriction endonuelease was purified from E. cell RY13 according to the procedure of Yoshimori (1971), up to a point where no c o n t a m i n a n t endonuclease activity could be detected (as checked b y conversion into form I I of bacteriophage PM2 component I DNA, which lacks the EeoR1 site (Mertz & Davis, 1972)). The mixture of H i n d I I a n d H i n d I I I endonucleases was purified from Haemophilus influenzae R d (exo- m u t a n t ) (Smith & Wilcox, 1970) b y gel filtration on Biogel A -- 0"5 M followed b y chromatography on a phosphoceilulose column. H p a I I endonuelease was purified from Haemophilus parainfluenzae by gel filtration a n d phosphocellulose column chromatography (Sharp et al., 1973). (e) E. cell R N A polymerase binding to polyoma D N A

E. eoli R N A polymerase was incubated for 5 rain at 37~ with polyoma DNA F I or F I I I iu binding buffer containing 30 mM-Tris.HC1 (pH 7-9), 10 rn~-MgCl2, 150 mra-KC1, 0.2 rn~-dithiothreitol in a volume of 200 t*l. After 5 rain incubation, glutaraldehyde was added to a final concn of 10 na~ a n d incubation was continued for 15 m i n at 37~ The D N A - R N A polymerase complexes were dialysed overnight against 10 m_~-Tris.ttC1 (pH 7.9}, 1 m ~ - E D T A a n d then treated with EcoR1 restriction endonuclease (Yaniv et al., 1974). The cleaved complexes were then diluted to a final DNA conch of 0.1 tzg/ml with l0 mM-Tris.HCl (7"9), 1 mM-EDTA a n d spread for electron microscopy. (d) Electron microscopy R N A polymerase binding to polyoma DNA was monitored by the electron microscopy technique of Duboehet ct al (1971) using carbon-coated grids charged in amylamine vapour. After adsorption of the D N A - R N A polymerase complexes, the grids were stained with 2% (w/v) u r a n y l acetate, rotary shadowed with P t / P d at a n angle of 8 ~ a n d examined with a Siemens Elmiskop 101 microscope. Photographs were taken at a magnification of 20,000 • D N A - R N A polymerase complexes were measured on photographic enlargements using a laboratory made co-ordinatometer connected to a PDP-8 digital computer.

(e) R N A synthesis and analysis The standard transcription assay contained: 20 rn~-Tris (pH 7-9), 10 mM-MgC12, 150 m~-KCI, 0.2 mM-dithiothreitol, 0-2 n ~ - G T P , A T P and CTP, 0.1 mM-[3tt]UTP (1 Ci/mmol) and various amounts of R N A polymerase a n d polyoma virus DNA. The order of additions were: (1) D N A was first added to the buffer, (2) R N A polymerase was then added to the mixture a n d incubated at 37~ for 5 rain, (3} the reaction was started by addition of ribonueleotide triphosphates or MgC12 previously omitted. The reaction was stopped with 5~/o (w/v) triehloroacetie acid. After at least 10 min at 0~ precipitated material was collected and washed on nitrocelhdose filters. The radioactivity on dried filters was monitored by liquid scintillation counting. For preparation of eRNA for further studies, the reaction was stopped b y addition of E D T A a n d sodium dodecyl sulphate at final conchs of 20 mM a n d 0"5~/o (w/v), respectively. The resulting solution was extracted with a n equal volume of water-saturated phenol. The aqueous phase was re-extracted twice with a n equal volume of chloroform. The fmal aqueous phase was precipitated with 2 vol. absolute ethanol at --20~ After centrifugation, the precipitate was dissolved in 10 m~-Tris (7.9), 1 m ~ - E D T A . Short synthesis (30 s) [3H]cRNA was released from its [14C]DNA template b y heat d e n a t u r a t i o n for 1 m i n at 100~ followed b y quick-cooling to 0~ a n d separated from the D N A b y zone sedimentation in a 5~/o to 20~/o sucrose gradient. The size of cRNA was analysed on 2"3~o (w/v) acrylamide, 0-2~/o (w]v) N, N ' methylene bis-acrylamide gel electrophoresis using total E. cell R N A as a molecular weight marker.

B. L E S C U R E E T A L .

86

3. R e s u l t s (a) Transcription of superhelical and linear polyoma virus D N A in vitro Polyoma virus superhelical D N A was transcribed in vitro b y E. coli DNA-dependent R N A polymerase holoenzyme. The size of the cRNA after 30 minutes of synthesis was found to be heterogeneous with a peak at 14 S (data not shown), as determined b y gel electrophoresis in non-aqueous solution (Staynov et al., 1972). Under conditions where SV40 D N A in vitro transcription was asymmetric (Westphal, 1970), R N A synthesized on polyoma virus D N A F I displayed a high degree of self-annealing (Table 1) (Kamen et al., 1974). EcoR1 linear molecules of polyoma DI~A were twoto threefold less effective t h a n F I polyoma D N A template at the same ratio of TABLE 1

Transcription of polyoma D N A F I or F I I I by E. coli R N A polymerase in v i t r o

Template

Polyoma DNA FI (1.5 ~g) Polyoma DNA F I I I (1.5 ~g)

UMP incorporated in cRNA with Self-annealing 4 ~g of RNA polymerase (nmol) of cRNA 5 min 10 rain 30 rain (%) 0-55 0.25

0-85 0-40

1.60 0.65

47 40

The method used for transcription in vitro is described in Materials and Methods. At various times of synthesis radioactivity incorporated into cRI~A was monitored by liquid scintillation counting. Self-annealing of cRNA obtained after 30 rain of synthesis was carried out in 4 • SSC at 65~ for 8 h. RNA-RNA hybrids were measured by precipitation with trichloroaeetic acid after pancreatic RNAase digestion (20 ~g/ml) for 30 min at 37~ in 2 • SSC (see section (b), below). enzyme to DNA (Table 1). The degree of self-complementarity of cRNA synthesized on F I or F I I I polyoma D N A was similar (Table 1). These results suggested t h a t no further selection of initiation sites, for transcription in vitro, occurs when the lower effective linear template was used. This was corroborated b y comparative localization of binding sites for E. coli R N A polymerase on both templates (this paper). Therefore, in non-restrictive conditions for transcription in vitro (Kamen et al., 1974), at least two promotors for R N A synthesis in vitro are located on opposite strands of both templates. Moreover, during a 30-minute period of transcription in vitro at 37~ serf-annealing occurred in the test-tube between complementary strands of the cRNA produced, and up to 40% of the long synthesis product was RNAase resistant without further serf-annealing (data not shown). (b) The formation of D N A - R N A hybrids in vitro Superhelical polyoma D N A was transcribed for 30 seconds at 25~ after a binding period of five minutes at 37~ Under these conditions, cRNA of an average length of approximately 240 nucleotides was produced as shown b y acrylamide gel electrophoresis (Fig. l(a), peak 2). A large amount of cRNA remained associated with the template after sodium dodeeyl sulphate t r e a t m e n t and extraction with phenol (Fig. l(a), peak 1). This DNA-associated R N A (1) banded at the D N A position in a Cs2S0 4 isopycnic gradient (not shown), (2) it was 40~/o resistant to RNAase digestion in 2 • SSC (SSC is 0-15 M-NaC1, 0.015 M-:Na3 citrate, p H 7.0), whereas the entire cRNA from peak 2 (Fig. l(a)) was solubilized b y the same treatment, (3) it was

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d i s s o c i a t e d from t h e t e m p l a t e b y h e a t i n g a t 100~ for one m i n u t e followed b y quickcooling a t 0~ (Fig. l(b)). F r o m these d a t a , we c o n c l u d e d t h a t , following t r a n s c r i p t i o n of p o l y o m a virus F I D N A , h i g h l y s t a b l e D N A - R N A h y b r i d s were f o r m e d a f t e r s o d i u m d o d e c y l s u l p h a t e t r e a t m e n t , as a l r e a d y r e p o r t e d w i t h v a r i o u s n e g a t i v e superhelical t e m p l a t e s ( H a y a s h i , 1965; W a n g , 1974; L e b o w i t z & Bloodgood, 1975; R i c h a r d s o n , 1975b). T o t a l [ 3 H ] c R N A (peaks 1 a n d 2, Fig. l(a)) from s h o r t s y n t h e s i s e x p e r i m e n t s could be s e p a r a t e d from t h e [*4C]DNA t e m p l a t e b y t h e r m a l d e n a t u r a t i o n followed b y s e d i m e n t a t i o n on a 5~/o to 20~o sucrose g r a d i e n t . (c) Stability of R N A polymerase bound to polyoma D N A T h e kinetics of dissociation of t h e R N A p o l y m e r a s e h o l o e n z y m e - p o l y o m a D N A complexes were studied. E. cell R N A p o l y m e r a s e was b o u n d for five m i n u t e s a t 37~ ~dtli p o l y o m a D N A F I or F I I I , p o l y ( r I ) was t h e n a d d e d to i n a c t i v a t e free e n z y m e a n d t h e f r a c t i o n of a c t i v e e n z y m e remailfing was d e t e r m i n a t e d a t v a r i o u s t i m e s (Fig. 2). W h e n linear p o l y o m a v i l ~ s D N A was used as t e m p l a t e (Fig. 2(a)), t h e half-life of t h e s t a b l e e n z y m e - D N A c o m p l e x e s was equal to five hours a t 37~ a n d to t h r e e m i n u t e s a t 0~ These results are in good a g r e e m e n t w i t h t h a t f o u n d u n d e r t h e s a m e ionic s t r e n g t h conditions for E. cell p o l y m e r a s e - T 7 D N A c o m p l e x e s b y H i n l d e & C h a m b e r l i n (1972) using t h e nitrocellulose filter assay. W h e n superhelical p o l y o m a D N A was used as t e m p l a t e (Fig. 2(b)), t h e half-life o f t h e s t a b l e e n z y m e - D N A complexes was a t least 30 hours a t 37~ a n d 50 m i n u t e s a t 0~ Thus, t h e s t a b i l i t y of E. cell R N A p o l y m e r a s e - D N A complexes is lower w i t h a linear t h a n with a supercoiled t e m p l a t e . Moreover, t h e difference in t h e s t a b i l i t y was i n c r e a s e d a t lower t e m p e r a t u r e s .

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FIe. 2. RNA polymerase-DNA complex dissociation rates. The method applied was essentially that described by Richardson (1974), except that poly(rl) replaced heparin as a competitor. RNA polymeraso was bound to DNA form I I I or form I in the standard buffer (20 mM-Tris.HCl (pH 7.9) l0 mM-MgC12 100 mM-KC1, 0"2 mM-dithiothreitol) for 5 min at 37~ Poly(rI) was then added (10 /zg/ml) and incubation continued at 37~ ( - - Q - - C ) - - ) or 0~ ( - - Q - - O - - ) . At various intervals, the fraction of bound active enzyme was determinated by addition of the 4 nucleotide triphosphates. RNA synthesis was terminated after 5 min at 37~ and acid-insoluble radioactivity was determined. The initial RNA polymerase activity on DNA F I or F I I I was measm'ed by simultaneous addition of the 4 triphosphates and poly(rI) after 5 min binding at 37~ The rapid initial dissociation of about 20% of the enzyme may be due to a small contamination of the RNA polymerase preparation with core enzyme. (a) DNA form I I I ; (b) DNA form I.

T R A N S C R I P T I O N OF POLYOMA DNA I N V I T R O

89

(d) Electron microscopy studies of E. coli R N A polymerase binding to polyoma

DNA Previous studies on the interaction of R N A polymerase with polyoma DNA F I by sucrose gradient analysis (Petitjolm & Kamiya, 1967) showed that at high weight ratios of enzyme to DNA (30:1 to 100 : 1), large amounts of R N A polymerase, only limited by available space on the DNA (according to these authors), were able to attach to superhelieal DNA. At lower ratios of enzyme to DNA (2.5 : 1 to 5 : 1) or at higher ionic strengths, the maximum amount of enzyme that can bind to DNA was much smaller, indicating that the enzyme had specificity for only few sites under these conditions. We have made use of novel electron microscopy techniques that visualize nucleic acid-protein complexes (Dubochet et al., 1971 ; Bordier & Dubochet, 1974) and permit a more detailed study of the interaction of R N A polymerase with nucleic acids. Binding at various ratios of enzyme to DNA was carried out for five minutes at 37~ in the standard binding buffer, the e n z y m e - D N A complexes were fixed with glutaraldehyde and then spread for electron microscopy as described in Materials and Methods. At low ratios of enzyme to DNA (0.5 : 1 to 8 : 1), only a few enzyme molecules were bound to the DNA (Figs 3(a) and 4(a)). These enzyme molecules are located at specific sites on the genome as demonstrated in section (e) below. At a weight ratio of enzyme to DNA of 4 : 1, with an equimolar concentration of polyoma DNA F I and F I I I , the average number of enzyme molecules observed on each kind of template was different (Fig. 5). E. coli R N A polymerase holoenzyme exhibits higher affinity for superhelical than for linear templates. At very high weight ratios of enzyme to DNA (30:1) a maximum number of 50 enzyme molecules was bound on both superhelical and linear DNA molecules (Fig. 3(b)). These complexes appear as relaxed structures when compared to supercoiled DNA or to complexes containing three to nine bound enzyme molecules spread under identical ionic conditions (Figs 3(a) and 4(a)) t. Analysis of the polyoma DNA by alkaline sucrose gradients proved that no nicks were introduced during incubation with excess enzyme. These images demonstrate the unwinding properties of E. coli R N A polymerase holoenzyme previously described by Saucier & Wang (1972). Another process of unwinding was recently demonstrated b y electron microscopy. The formation of about 20 nucleosomes on superhelical viral D N A with the concomitant decrease in the superhelicity of the complex gives rise to relaxed structures when observed by electron microscopy (Germond et al., 1975; Cr~misi et al., 1976).

(e) Location of specific E. cell R N A polymerase binding sites on superhelical and linear polyoma virus D N A When E. cell R N A polymerase holoenzyme was bound to superhelieal polyoma DNA at a weight ratio of 4 : 1, an average number of four enzymes were observed per D N A molecule (Figs 4(a) and 5). To permit the location on the genome of bound t Although electron microscopy images cannot be used to measure the exact number of superhelical turns in a circular DNA, the average number of cross-overs of the DNA chain can be used as a qualitative estimate of the degree of superhelicity. FI DNA can be easily distinguished from relaxed or nicked circular polyoma D:NA by this criterion. No appreciable artefacts are produced (luring the preparation of the samples for electron microscopy. Analysis of viral DNA samples by alkaline gradients, by agarose gel electrophoresis or by electron microscopy gave identical results with respect to their content in superhelical, relaxed and linear DNA.

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