selective dinucleotide-primed in vitro transcription of a cloned fragment of cauliflower mosaic...

Eur. J. Biochem. 157,83 - 89 (1986) 0 FEBS 1986

Selective dinucleotide-primed in vitro transcription of a cloned fragment of cauliflower mosaic virus DNA is dependent on a limited region of the viral genome Richard COOKE and Paul PENON Laboratoire de Physiologie Vkgktale, Universitk de Perpignan

(Received December 6, 1985/February 3, 1986) - EJB 85 1350

We have previously shown that plant RNA polymerase I1 preferentially forms ternary transcription complexes on a cloned fragment of the cauliflower mosaic virus genome in the presence of a particular dinucleotide/purine NTP combination (ApG + ATP). This preferential interaction is observed when the viral sequences are present on a discrete circular molecule. Deletion of a 205-bases-pair region abolishes this selectivity. The deleted region contains a considerable number of symmetrical or repeating elements. The use of nuclease S1 as a probe shows that this region contains a homopurine-homopyrimidine sequence which is extremely sensitive to this enzyme, indicating its capacity to adopt a non-B DNA conformation. A possible alternative structure of these sequences, which may explain the preferential interaction with the RNA polymerase, is presented.

An understanding of the mechanisms involved in the transcriptional process is essential in order to understand the control of gene expression. While the role played by the procaryotic RNA polymerase in transcription has been rela- tively well defined, our knowledge of the reactions catalysed by the eucaryotic enzymes remains extremely limited. The use of animal cell-free systems [l, 21 has allowed the detection of template sequences and certain protein cofactors which are essential for accurate transcription initiation ([3], and references therein), although the role played by the RNA polymerase remains unclear.

We are interested in defining the steps in the transcription process which can be catalysed by purfied plant RNA polymerase I1 and those which require the presence of additional cofactors. Results obtained by other workers [4-91 and by ourselves [lo], showing that purified RNA polymerase I1 of animal and plant origin is capable of showing a highly selective interaction with particular sequences on cloned homologous templates, suggest that the conclusion that transcription by purified enzymes of native templates is non-specific [I 11 was perhaps somewhat hastily drawn. The particular conditions used in these recent studies (low enzyme : DNA ratio, low nucleoside triphosphate concentrations, defined supercoiled templates, dinucleotide primers etc.) probably explain the apparent contradiction with previous results.

We have shown that higher plant RNA polymerase I1 forms stable binary complexes at a limited number of sites on cloned cauliflower mosaic virus (CaMV) DNA [12] and is capable, like its yeast counterpart [4], of selective abortive initiation on a supercoiled recombinant plasmid, pCa8, containing a fragment of the CaMV genome, in the presence

Correspondence to R. Cooke, Laboratoire de Physiologie Vkgktale, Universitk de Perpignan, Avenue de Villeneuve, F-66025 Perpignan Cedex, France

Abbreviations. C a m , cauliflower mosaic virus; bp, base pairs.

of a particular dinucleoside monophosphate/purine NTP combination (ApG + ATP, [lo]). Coarse mapping of the se- lectively transcribed region of pCa8 by hybridisation of labelled transcription products to restriction fragments of the template showed that this preferential abortive initiation occurs within the gene coding for the viral reverse transcriptase [13, 141, several hundred base pairs upstream from the known in vivo transcription control signals.

We were interested in determining which features of sequence or structure are involved in this preferential interaction of RNA polymerase I1 with a limited region of the CaMV genome present on a supercoiled plasmid. The extraction from infected cells of C a m DNA in a supercoiled form [I51 suggestes that the torsional stress induced in such molecules may intervene in the expression of the viral genome, as has been proposed for eucaryotic systems by Smith [16]. It has been shown that structural changes induced by superhelical tension in recombinant plasmids can lead to the formation of altered DNA structures, which are similar to those found in chromatin [17].

We show here that ternary transcription complex formation is maintained even when bacterial and viral sequences are present on separate supercoiled molecules. The selectivity can be totally abolished by the deletion of a 205-base-pair (bp) region which contains a remarkable concentration of symmetry and other strucutral elements. Using nuclease S1 as a probe we show that the region contains sequences capable of adopting an alternative structure under torsional stress and which may be involved in interaction with the RNA polymerase.

MATERIALS AND METHODS Restriction endonucleases were obtained from Boehringer

Mannheim or Amersham (France) and used according to the suppliers. Nuclease S1 was from Boehringer Mannheim,

84

nuclease Ba131 and DNA polymerase I (Klenow fragment) from BRL and bacterial alkaline phosphatase, T4 polynucleotide kinase, DNA ligase and radioactive products from Amersham (France). The experiments described in this article were carried out using wheat germ RNA polymerase I1 pro- vided by D. and C. Job, M. Teissere and J.Dietrich (CBM, CNRS Marseille) and purified as described [18]. The enzyme has a specific activity of 300-400 units/mg. The characteristics of ternary transcription complex formation by this enzyme were identical to those previously described for the soybean enzyme [lo]. The formation and analysis of ternary transcription complexes were as previously described [lo] at (NH&S04 concentrations indicated in figure legends. All analyses were carried out in duplicate.

Circularisation of DNA fragments

The preparation of supercoiled fragments was carried out essentially as described by Schon et al. [19]. Preparations which were to be labelled were first treated with bacterial alkaline phosphatase (20 units/pmol 5' termini) and labelled with T4 polynucleotide kinase as described by the suppliers using (Y-~~PIATP (6000 Ci/mmol). Ligation was carried out at 1-2 pg/ml in 25 mM Tris/HC1, pH 7.5, 10 mM MgCl,, 10 mM dithiothreitol, 0.2 mM spermidine, 0.5 mM ATP, 1 pg/ml ethidium bromide and 25 pg/ml bovine serum albumin using 5 Weiss units/ml T4 DNA ligase. After overnight incubation at 4°C the DNA was deproteinised by phenol extraction and concentrated by three extractions with butanol before ethanol precipitation. ATP was removed by passage through Sephadex (3-50 followed by ethanol precipitation.

Deletion with Ba131 nuclease and selection of deleted clones

10 pg XbaI-digested pCa8 was treated with 4 units Ba131 nuclease for 1 min at 30°C in 100 p140 mM Tris/HCl, pH 8.0; 24mM CaC1,; 24mM MgC12, 400mM NaCl and 2mM EDTA and the reaction stopped by the addition of 10 pl 500 mM EDTA. After phenol extraction the DNA was pre- cipitated with two volumes ethanol and redissolved in 50 p1 50 mM Tris/HCl, pH 7.5; 10 mM MgS04; 0.1 mM dithiothreitol; 50 pg/ml bovine serum albumin; 50 pM dATP, dGTP, dCTP, TTP. After addition of 1 unit Klenow fragment of DNA polymerase I the reaction was incubated for 30 min at room temperature and stopped by the addition of 1 pl 500mM EDTA. After phenol extraction and ethanol precipitation, DNA was redissolved in 50 p1 66 mM Tris/HCl, pH 7.6; 1 mM ATP, 1 mM spermidine; 10 mM MgCl,; 15 mM dithiothreitol; 200 pg/ml bovine serum albumin; synthetic XbaI linker (Amersham, molar ratio 1O:l) and 2.5 units T4 DNA ligase were added. Incubation was for 16 h at 20°C and the reaction was stopped by phenol extraction followed by ethanol precipitation. The DNA was redissolved in 10 mM Tris/HCl, pH 7.5; 1 mM EDTA and aliquots used to transformed competent Escherichia coli HB 101 cells. Ampicillin-resistant clones were tested for the presence of the HindII and HindIII restriction sites flanking the XbaI site and for the insertion of the XbaI linker. One clone, pCa 4.2, was selected and the exact limits of the deletion determined by sequencing away from the new XbaI termini. This plasmid has 205 bp from 491 5 to 51 19 bp deleted, compared with pCa8, and has the 8-bp XbaI linker between these two extremities.

/

Fig. 1. Physical map of plasmid pCa8. The inserted CaMV sequences (fragment Bg[IIB) are shown by a thick line. Figures inside the circle indicate thousands of base pairs, zero being the unique EcoRI site of the vector plasmid pKC7. Digestion sites for restriction enzymes HindII (w), Hind111 (+) XbaI ( V ) and BgDI ( 7 ) are shown, with their coordinates on C a w DNA. The localisation of the two fragments, HindIID and HindIIIB, to which dinucleotide-primed RNAs hybridise, is shown. The figure also shows the extent of the deletion (0) in plasmid pCa4.2 and the localisation of the Sau3AI fragment used in this work (see text). The arrow (+) shows the transcription initiation site for gene VI (see text)

SI nicking of supercoiled circles S1 digestion was carried out in 40 pl final volume

containing 1 pg total DNA, this concentration being obtained by addition of unlabelled plasmid pCa8. Digestion was for 1 min in 50 mM sodium acetate, pH 4.5; 0.2 M NaCl; 1 mM ZnS04 at 37"C, using 3000 units/ml nuclease S1. Samples were incubated in parallel in the absence of nuclease S1 as 'minus-S1' controls to detect bands due to non-specific nicking contaminants in subsequent restriction enzyme digestion. After phenol extraction and ethanol precipitation, the DNA was redigested with Sau3AI or Sau3AI plus Hind11 and the reaction products analysed on polyacrylamide/urea gels [20].

RESULTS Plasmid pCa8 contains the 1861-bp BglII B fragment of

the cabbage B-JI isolate of CaMV DNA, which covers the 3' extremity of gene V, located at 5672 bp, and the beginning of gene VI (coding for the viral inclusion body protein), as well as the intergenic region containing the promoter of the latter gene, whose transcription is initiated at 5764 bp. Fig. 1 shows a physical map of this plasmid and resumes the results of our previous work, in which we demonstrated by ternary transcription complex analysis that, in the presence of a particular dinucleotide/purine NTP combination, a plant RNA polymerase I1 is capable of highly selective abortive initiation within a limited region of the CaMV sequences on the supercoiled form of pCa8, primed transcription products hybridising to restriction fragments HindIII B and HindII D [lo]. This selectivity is most striking for the combination (ApG + ATP), other combinations priming transcription to a greater extent or even exclusively on vector sequences. We were interested in determining the features of sequence or structure which lead to this selective interaction between a particular region of the CaMV genome and RNA polymer-

85

Fig. 2. Transcription complex formation on religated fragments of pCa8. (a) 0.5 pg native pCa8 were incubated with 0.1 unit RNA polymerase for 5 min at 30°C in the presence of 1 pM ATP and 0.3 mM ApG in a buffer containing 50 mM Tris/HCl, pH 7.9; 5 mM dithiothreitol; 1 mM MnClz and 25 mM (NH4)$04. Poly(inosinic acid) was added to 25 pg/ml and incubation conhnued for a further 5 min. GTP and CTP ( 5 pM) and UTP (2 pM, 4 pCi/test) were added and incubation continued for 2 min. Elongation was blocked by the addition of cordycepin triphosphate to 2 pM and a-amanitin to 10 pg/ ml. 0.1 volume 10 x concentrated BgnI buffer was added with 8 units BgnI and reactions incubated 1 h at 37°C. EDTA and bromophenol blue were added to 5 mM and 0.1% respectively and fragments separated on a 1 % agarose gel (16 h, 30 mA) in 36 mM Tris/HCl, pH 7.5, 30mM NaH2P04, 1 mM EDTA. Gels were dried and autoradiographed. Lane 1 , ethidium-bromide-stained gel of BgZII- digested pCa8. Lane 2 autoradiography of lane 1 . V = vector, I = insertion. (b) Plasmid pCa8 was digested with BgAI and religated in the presence of ethidium bromide as described. Transcription complexes were formed on 0.5 pg religated material under the conditions described above for native pCa8, except that, after blocking elongation with cordycepin triphosphate and a-amanitin, 0.1 O h bromophenol blue was added and the reaction mixture loaded on an agarose gel and electrophoresis carried out as described. Lane 3, ethidium-bromide-stained gel. Lane 4, autoradiography of lane 3. Is = supercoiled insertion, IR = relaxed insertion, Vs = supercoiled vector, VR = relaxed vector, pCa 8s = supercoiled pCa 8 reformed during ligation

aseII, and in defining more precisely the DNA sequences involved in this interaction.

Selectivity is maintained when vector and insertion sequences are present on sepurate molecules

We first asked whether the preferential interaction with the viral insertion sequences is maintained when the two fragments are present as discrete, supercoiled molecules. Plasmid pCa8 was digested with BglII to separate procaryotic and eucaryotic sequences and religated at a low DNA concentration in the presence of ethidium bromide. After removal of ethidium bromide the major products of ligation are supercoiled insert and vector DNAs with low amounts of the relaxed open-circular forms (Fig. 2, lane 3). Formation of ternary transcription complexes on this mixed population in the presence of the dinucleotide/purine NTP combination (ApG + ATP) allows us to test directly the affinity of the RNA polymerase for initiation sites on the vector and insertion molecules. Fig. 2 shows that the level of complex formation on insertion sequences is similar, whether complexes are

formed on pCa8, followed by BgnI digestion (lane 2), or directly on the discrete molecules in the ligation mix (lane 4). The same is also true for vector sequences. Taking into account the relative sizes of the vector and insertion molecules (5868bp and 1861 pb respectively) we can conclude that transcription complexes are preferentially formed on the viral sequences. We consistently observe complexes on the supercoiled insertion sequences in the autoradiograph as a fairly broad band migrating noticeably slower than the insertion detected by fluorescence (cf. lanes 3 and 4). In some experiments this band is seen to be a doublet with a fainter, faster-migrating band corresponding to the fluorescent material. This displacement probably corresponds to the fmation on one molecule of DNA of one or more RNA polymerase molecules. The lack of retardation of the vector and reformed pCa8 molecules may be due to a lower level of polymerase binding to these molecules or simply to the fact that a slower migration rate would be less noticeable in the higher- molecular-mass region on the gel.

Deletion of a short region eliminates preferential complex formation

As shown in Fig. 1, coarse mapping of (ApG + ATP)- primed transcription by hybridisation of primed RNA to restriction fragments localised the selective initiation sites within a limited region of the viral insertion of pCa8, probably between the HindII site at 4838 bp and the HindIII site at 5151 bp [lo]. We therefore decided to delete portions of this region in order to delimit more clearly the sequences involved in selective abortive initiation. Plasmid pCa8 contains a unique XbaI site (situated at 4979 bp on CaMV DNA), almost equidistant from the HindII and HindIII sites. Deletions were constructed as described, using nuclease Ba131, and the re- sulting plasmids screened for the conservation of these two Hind sites. We selected one deleted plasmid, pCa 4.2, in which viral sequences from 491 5 to 51 19 (205 bp) had been removed (see Fig. 1). The ability of RNA polymerase I1 to form (ApG + ATP)-primed ternary transcription complexes on this plasmid was tested.

Fig. 3 (lanes 1 and 6) shows that, even under low stringency conditions [lo mM (NH&S04], the level of formation of complexes on the viral sequences of pCa4.2 is consider- ably lower than that on the insertion of pCa8, while the vector sequences are transcribed to the same extent. Densitometric scanning of the autoradiogram indicates a ratio of 7: 1 between the level of complexes on vector and insertion fragments of pCa4.2, while the ratio is 1 : 1 for pCa8. Thus, the level of transcription complex formation on the insertion of pCa4.2 is even below that expected from random initiation on the plasmid, whereas we once again observe a selective transcription of the insertion sequences of pCa8, even under low salt, low stringency conditions. Increasing the stringency conditions by increasing the salt concentration [up to 50 mM (NH&S04] drastically decreases the level of complex formation on vector sequences and on the pCa4.2 insert (lanes 8 - lo), while, after an initial decrease, the level on the insertion of pCa8 remains remarkably stable at increasing ionic strengths (lanes 3 - 5) . This effect is not simply due to the removal by deletion of potential, random initiation sites, as the sequences deleted in pCa4.2 contain only 15 of the 101 ApGpA sequences on the pCa8 insert and two others are incorporated with the insertion of the XbaI linker. In any case the results presented in Fig. 3 clearly demonstrate the existence of two

86

Characteristics of the deleted sequences

The above results indicate that sequences within a 205-bp region of the CaMV DNA in pCa8 are implicated in the selective interaction of RNA polymerase I1 and the formation of transcription complexes on the viral sequences of this plasmid. The preferential formation of complexes on the supercoiled form of the plasmid [lo] suggests that torsional stress due to supercoiling may be necessary to provoke conformational changes in particular sequences on the DNA, which are thus available for interaction with the RNA polymerase. It has been shown that torsional stress is capable of generating a number of alternative DNA structures at the level of particular sequences (cruciforms within palindromic regions [22], Z-DNA in alternating purine-pyrimidine sequences [23], denaturation of AtT-rich regions [24] or ‘open’ structures in homopurine-homopyrimidine sequences [25]). Fig. 4 shows that the 205-bp region deleted in pCa4.2 contains a surprising number of symmetry elements (inverted repeats or potential cruciform structures) and direct repeats, particularly concentrated between 4925 bp and 5005 bp. This latter region is equally characterised by alternating runs of homopurine and homopyrimidine residues.

Alternative, non-B structures of DNA can often be detected using as a probe nuclease S1. Using the method of Schon et al. [19], we attempted to detect nuclease-S1-sensitive structures within the region of interest. We chose a SadAI fragment of 375 bp (4895 - 5270), whose extremities coincide as closely as possible with the Hind11 and Hind111 sites which delimit the region to which (ApG + ATP)-primed RNAs hybridise (Fig. 5). The fragment was 5’-end-labelled and converted to a supercoiled circle as described in Materials and Methods.

The kinetics of digestion by nuclease S1 (Fig. 5 ) show that the supercoiled, circular fragment is rapidly converted to the relaxed form by nicking of one strand, followed by a much slower conversion to the linear form through digestion of the complementary strand opposite the nicks. This indicates that the fragment contains certain sites which are extremely sensitive to S1 nuclease digestion on supercoiled molecules. We chose to use a digestion time of 1 min, which gives a large majority of relaxed circular molecules. Redigestion of the

Fig. 3. Transcription complex formation onpCa8 andpCa4.2 at varying ionic strength. Transcription complexes were formed on 0.5 pg pCa8 (lanes 1 - 5) or pCa4.2 (lanes 6- 10) and BglII digestion and analysis carried out as described in the legend to Fig. 2, except that (NH4)2S04 concentrations were varied as indicated below. (NH&S04 concentrations were 10 mM (lanes 1 and 6), 30 mM (2,7), 40 mM (3,8), 45 mM (4,9) and 50 mM ($10). V = vector, I = insertion. Autoradiography was for 16 h

classes of complex. Those formed on pCa4.2 and the vector of pCa8 are only observed at low ionic strength and may correspond to initiations within regions which are easily denaturable under these conditions and which have been shown to be sites for preferential fixation of RNA polymer- aseII [21]. In contrast, a considerable proportion of the complexes formed on the insert of pCa8 are stable at increasing ionic strengths and thus form a second class, whose selective formation is dependent on the presence of sequences within the 205-bp region, which is deleted in pCa4.2.

4931) 4951) -L - 2

3 3

497? 499:

5 t - 5

4 L - - 1 1 3 3,

2 2, - Sol? 5030 505y 5070

I

~ ~ ~ ~ T ~ ~ ~ ~ ~ ~ ~ ~ C C C ~ ~ ~ ~ ~ CACATICTTA . 2 - 1 ,-

& 5 - - Fig. 4. Structural features of the region deleted in pCa4.2. The figures shows palindromic sequences (above the sequence), direct (+) and inverted (-+ c) repeats (below) and homopurine or homopyrimidine stretches (underlined). Coordinates on viral DNA are indicated

87

H2 S(4895) H3 S(5270) . - 1

4806-- 5006 5206

Fig. 5. Nuclease SI nicking of” the circular 375-bp Sau3AZ fragment. The 375-bp fragment (4895 - 5270 bp) was isolated, labelled and circularised as described in Materials and Methods. The upper part of the figure shows the location of this fragment with respect to the Hind11 and Hind111 sites used in the selection of deleted plasmids. The lower part shows the kinetics of conversion of the supercoiled (S) fragment (lane 1 ) to relaxed (R) and linear (L) molecules after 1, 2, 4, 8, 15 and 30 min digestion with nuclease S1 (lanes 2-7 respectively). Nuclease digestion was carried out as described in Materials and Methods and the reaction products were analysed on a non-denaturing 3.5% polyacrylamide gel. After electrophoresis the gel was fixed with 10% acetic acid, dried and autoradiographed for 30 min

nicked molecules by Suu3AI followed by analysis in denaturing conditions allows us to detect the sites of preferential S1 nicking (Fig. 6 a, lanes 1 and 2). It is essential to process a minus-S1 control, as restriction enzymes or contaminating nucleases often introduce nicks at specific sequences, which are detected as bands present in both plus and minus lanes.

We detect only one region of S1 nicking within the fragment, which is visible as a nest of bands of varying intensity, each differing from its neighbours by one nucleotide in length. Secondary digestion of the Sau3AI fragment by Hind111 allows us to separate the two labelled extremities. Analysis of the Suu3AI-Hind111 fragment (4895 - 51 51 bp) shows the same nicking profile as the intact fragment (Fig. 6a, lanes 3 and 4), indicating that the nicking sites lie between 50 and 70 nucleotides from the Suu3AI site at 4895 bp on the upper, non-transcribed strand of the viral DNA. We have never detected nicking of the other strand within this region (results not shown), in agreement with the observations of Schon et al. [19] that nicking is often confined to one strand, nor have we detected other ‘nicking boxes’ within this fragment.

A comparison of the nicking box with the purine sequence ladder of the same labelled fragment allows us to position exactly the sites of nicking (Fig. 6 b, lanes 5 and 6). The length of the sensitive region is at least 17 nucleotides and covers the sequence CTCTTCAAGAAGAAGAT. Densitometric scanning of the autoradiograph allows us to measure the relative intensity of nicking within the sensitive sequences (Fig. 6c). The most sensitive region is at the junction between the homopyrimidine and homopurine sequences, no other obvious preference being detected.

DISCUSSION

The results presented here confirm and extend our previous observation that, in the presence of a particular dinucleotide/purine NTP combination, plant RNA poly-

merase I1 is capable of a high degree of selectivity of transcription initiation sites within a limited region of the CaMV sequences on plasmid pCa8, in the gene coding for the viral reverse transcriptase, some 800 bp upstream from known transcription control sequences. Two explanations are possible for our demonstration that selectivity is maintained even when the vector and insertion sequences are present on separate molecules. The viral insertion may contain sequences for which free RNA polymerase has considerable affinity, leading to preferential binding of the enzyme to the insertion molecules. On the other hand, bound polymerase may show a higher abortive initiation efficiency at certain sites wich differ from other potential initiation sites in their sequence or structure, leading to a higher level of initiation on the insertion. Our results do not allow us to distinguish with certitude between these two possibilities. Nevertheless, the presence of the dinucleotide/ATP combination apparently allows abortive initiation to occur at sites at which initiation is otherwise not possible, as transcription complex formation in the presence of only ATP or GTP on pCa8 [lo] or the ligation mix (our unpublished results) shows no preferential transcription of the viral sequences.

The use of plasmid pCa4.2 allows us to localise the sequences involved in the preferential formation of transcription complexes to a 205-bp region lying within the gene coding for the viral reverse transcriptase [13,14]. The study of ternary transcription complex formation on vector and insertion fragments of pCa8 and pCa4.2 at increasing ionic strengths shows that this short region contains sequences at which RNA polymerase I1 is capable of forming particularly stable complexes, transcription complex formation being practically abolished in selective conditions in the absence of these sequences. The ratio of the number of ApGpA sequences within the 205-bp fragment to that within the viral insertion as a whole cannot possibly explain these results. On the other hand, we were intrigued by the surprising number of sequences which are present in this short region and which are capable

88

Fig. 6. S1 nickingputtern of the 375-bp fragment. The circularised 375-bp fragment was digested with S1 nuclease for 1 min as described (lanes 2 and 4). ‘Minus-S1’ controls (lanes 1 and 3) were incubated under the same conditions in the absence of nuclease S1. After phenol extraction and ethanol precipitation the DNA was digested with Suu3AI (lanes 1 and 2) or SuufAI and Hind111 (lanes 3 and 4). In the latter case the Suu3AI-Hind111 fragment (4895 - 51 51) was recovered from an agarose gel before further analysis. (a) Digestion products were analysed on a denaturing polyacrylamide/urea gel, which was fixed, dried and autoradiographed. Figures a t the left show the sizes of molecular mass markers (in bases). (b) Lanes 5 and 6 show respectively in an enlargement of a longer exposure of lane 2 and the A + G sequencc laddcr of the same fragment. (c) The nicking box is shown, with the length of the arrows corresponding to the intensity of nicking at different sites. This was determined by densitometric scanning of the autoradiograms

of adopting alternative structures under certain conditions. In the light of our observation that ternary transcription complexes are formed almost exclusively on the supercoiled form of pCa8 [lo], we considered the possibility that supercoiling could induce structural changes within this region, which could be involved in the interaction with the RNA polymerase.

Our results show that the most stable alternative structure which is formed on a supercoiled fragment containing the 205-bp region is a 17-bp sequence in which a short run of pyrimidine residues is followed by 10 purine residues. This does not exclude the possibility that other, less stable structures may be formed at other sites within the fragment, as the method used detects only the most frequently represented alternative structure in the population [19], S1 nicking re- moving the torsional constraints which induce such structures. While computer analysis predicts several stable cruciform structures within the 205 bp and the entire region may be folded into a surprisingly stable secondary structure, the localisation and unilateral pattern of nicking sites are incom- patible with these possibilities. In contrast, the nicking pattern is strikingly similar to that which has been discribed by several authors [15,19,26 - 291 within homopurine-homopyrimidine sequences. Similar structures have been found in the 5’-flanking regions of several genes as well as in other transcriptional control sequences such as the simian virus 40

enhancer [29], and it is possible that they may be involved in gene regulation.

Several possible structures have been proposed to explain the formation of S1-hypersensitive structures, including left- handed helices [25], whose formation may be induced by pro- tonation of C residues [29], or formation of single-stranded loops by slippage in repeated sequences [27]. However, these models do not satisfactorily explain the frequent observation that nicking is limited to one strand. Lee et al. [30] have recently proposed the formation within homopurine- homopyrimidine regions of triple helices, in which one such sequence would be folded back onto an inverted repeat of the same sequence, the polypyrimidine strand being incorporated into a triple helix and the purine strand remaining unpaired. It should be emphasized that, as a triple helix incorporating two polypurine strands is not formed (301, the polypyrimidine strand is never susceptible to S1 digestion, in agreement with the unilateral nicking pattern. It is interesting to note that an almost perfect inverted repeat of the S1-hypersensitive structures, which we describe here, is present immediately upstream (see Fig. 4) and that formation of a triple helix at this site would leave unpaired a region which corresponds closely to the hypersensitive sequences (Fig. 7). Lee et al. [30] propose that such structures may serve as sites of interaction with RNA polymerase, in agreement with our observation of preferential abortive initiation.

89

t t t t t t t Fig. 7. Possible triple-helical structure of the nuclease S1-hypersensitive region detected an the insertion of pCa8. Coordinates are from 4932 to 4969 on the viral genome. Arrows show the sites of nuclease S1 nicking. (Based on Lee et al. [30])

Experiments similar to those described above, using other Suu3AI fragments derived from the insertion of pCa8, failed to detect comparable hypersensitive sites (results not shown), which is compatible with the absence of homologous sequences within the rest of the insertion. While we have no evidence for a direct interaction between RNA polymerase I1 and the hypersensitive region, it is interesting to note that, of the 15 ApGpA sequences within the 205-bp region, four lie within the nicking box, nine within a 55-bp region surrounding the box and five of the six others in a region in which 11 out of 14 residues are identical to the hypersensitive region. Further, Zhu et al. [28] have demonstrated a close correlation between sites which are hypersensitive to several nucleases, including nuclease S1, both in supercoiled plasmids and in chromatin, and minor transcription initiation sites of several globin genes which are located upstream from the major cap site. This observation indicates that RNA polymerase I1 is capable of interaction and initiation of transcription in vivo at the level of sequences showing the same structural particularities as those which we have detected on the CaMV genome. Despite considerable efforts, information on the transcription pattern of CaMV DNA in vivo remains extremely fragmentary [31] and it is thus impossible to relate our observations directly to the expression of the viral genome. Only two promoters have so far been demonstrated to be functional on the viral genome and only one of these gives rise to a translatable messenger, the other being involved in viral replication [31]. The region within which we have shown preferential transcription complex formation lies some 800 bp upstream from the former promoter, but we have no evidence at present allowing us to link this preferential interaction to the expression of the viral genome.

This work was supported by grants from the Centre National de la Recherche Scientifique. The authors gratefully acknowledge expert technical assistance from Georges Villelongue, Alain Got and Marie- ThCrtse Lacoste.

REFERENCES 1 . Manley, J . L., Fire, A., Cano, A., Sharp, P. A. & Gefter, M. L.

2. Matsui, T., Segall, J., Weil, A. & Roeder, R. G. (1980) J. Biol.

3. Jones, K . A., Yamamoto, K. R. &Tjian. R. (1985) Cell42, 559-

4. Lescure, B., Williamson, V. & Senetenac, A. (1981) Nucleic Acids

5. Lescure, B., Bennetzen, J. & Sentenac, A. (1981) J . Biol. Chem.

6. Lescure, B. (1983) J . Biol. Chem. 258, 946-952. 7. Ballario, P., Buongiorno-Nardelli, M., Carnevali, F., di Mauro,

E. & Pedone, F. (1981) Nucleic Acids Res. 9, 3959-3978. 8. Carnevali, F., Caserta, M. & di Mauro, E. (1983) J . Mol. Biol.

9 . Tsuda,M.&Suzuki,Y.(1982) J . Biol. Chem.257,12367-12372. 10. Cooke, R., Penon, P., Got, C. & Miassod, R. (1983) Eur. J.

Biochem. 137, 365- 371. 1 1 . Lewis, M. K . & Burgess, R. R. (1982) in The enzymes (Boyer, P.

D., ed.) 3rd edn, vol. 15B, pp. 109- 153, Academic Press, New York.

12. Grellet, F., Cooke, R., Teissere, M., Delseny, M., Xech, .I. & Penon, P. (1981) Nucleic Acids Res. 9, 3927-3939.

13. Toh, H. , Hayashida, H. & Miyata, T. (1983) Nature (Lond.) 305,

14. Volovitch, M., Modjtahedi, N., Yot, P. & Brun, G. (1984) EMBO

15. Olszewski, N., Hagen, G. & Guilfoyle, T. J. (1982) Cell 29,395 -

16. Smith, G. R. (1981) Cell24, 599-600. 17. Larsen, A. & Weintraub, H. (1982) Cell29,609-622. 18. Job, D., Durand, R., Job, C., Teissere, M . (1984) Nucleic Acids

19. Schon, E., Evans, J., Welsh, J. & Efstratiadis, A. (1983) Cell35,

20. Maxam, A. M. &Gilbert, W. (1980) Methods Enzymol. 65,499-

21. Saragosti, S., Croissant, 0. & Yaniv, M. (1980) Eur. J . Biochem.

22. Lilley, D. M. J . (1980) Proc. Natl Acad. Sci. USA 77.6468 -6472. 23. Singleton, C. K . &Wells, R. D. (1982) J. Biol. Chem. 257,6292-

24. Lilley, D. M . J. (1981) Nucleic Acids Res. 9, 1271 -1289. 25. Cantor, C. R., Efstratiadis, A. (1984) Nucleic Acids Res. 12,

26. Shen, C.-K. J . (1983) Nucleic Acids Res. 11, 7899-7910. 27. Mace, H . A. F., Pelham, H. R. B. & Travers, A. A. (1983) Nature

28. Zhu, J., Allan, M. 81 Paul, J. (1984) Nuckic Acids Res. 12.9191 -

29. Evans, T., Schon, E., Gora-Maslak, G., Patterson, J. &

30. Lee, J . S., Woodworth, M. L., Latimer, L. J. P. & Morgan, A. R.

31. Covey, S. N. (1985) CRC Crit. Rev., in the press.

(1980) Proc. Natl Acad. Sci USA 77, 3855-3859.

Chem. 255, 11 992 - 1 1 996.

512.

Res. 9, 31 -45.

256, 1 1 018 - 1 1 024.

165. 59-77.

a21 - 829.

J. 3, 309-314.

402.

Res. 12, 6155-6170.

837 - 848.

560.

106,25-31.

6295.

8059- 8072.

(Lond.) 304,555 - 557.

9204.

Efstratiadis, A. (1984) Nucleic Acid.y Rex 12, 8043 -8058.

(1984) Nucleic Acids Res. 12, 6603-6614.

selective dinucleotide-primed in vitro transcription of a cloned fragment of cauliflower mosaic...

Documents