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Transcription by Escherichia coli RNA polymerase of a single-stranded fragment by bacteriophage φX174 DNA 48 residues in length
J. Mol. Biol. (1975) 93, 367-374
Transcription by Escherichia coli RNA Polymerase of a Single-stranded Fragment by Bacteriophage 4X174 DNA 48 Residues in Length ELIZABETH H. BLACKBURN Me&cd Research Council Laboratory of Molecular Biology Hills Road, Cambridge CB2 2&H, England (Received 26 March 1974, and in revisrd jorm IS November 1974) A single-stranded fragment of bacteriophage -$X174 DNA, 48 residues in length and of known sequence, has been transcribed by Escherichia coli DNA-dependent RNA polymerase. After transcription for eight hours at 37°C the molar yield of RNA synthesized was 20 to 40 times the molar amount of template DNA present. The products of transcription were very heterogeneous in size, indicating multiple points of initiation and termination along the template. Sequence analysis of the heterogeneous transcripts included a two-dimensional fingerprinting procedure designed to seperate oligonucleotides containing initiation and t,ermination points from internal, complete oligonucleotides produced by digestion with T, or pancreatic ribonuclease. Overlapping sequences were obtained by partial digestion with T1 ribonuclease and it w&s shown that the sequence of all but the first five 5’-terminal residues of the template could be determined by this method.
1. Introduction Single-stranded DNA can be used as a template for transcription by Escherichia coli DNA-dependent RNA polymerase in vitro (Hurwitz et al., 1962 ; Chamberlin & Berg, 1962). Under these conditions the enzyme shows considerably less specificity for sites of initiation and termination of transcription than on a double-stranded DNA template (Berg et Cal.,1965; Wood & Berg, 1964; Bremer et al., 1966; see also review by Richardson, 1969). Therefore, regions of DNA, which may not be transcribed from the double-stranded template, can be copied in vitro from single-stranded or denatured DNA into labelled RNA of high specific activity, suitable for sequence analysis by established techniques (Sanger et al., 1965 ; Brownlee & Sanger, 1967,1969 ; Brownlee et al., 1968). This method has been used to determine the sequence of the lac operator region (Gilbert & Maxam, 1973; Gilbert et al., 1973). A detailed characterization of the products of transcription of a synthetic oligodeoxyribonucleotide, 29 residues in length, has been carried out (Terao et al., 1972), in which the effect of priming with an oligoribonucleotide was also examined. In the work described here the products of transcription from a larger single-stranded fragment of DNA were characterized, to determine the extent and fidelity of copying by E. coli RNA polymerase, in order to test the usefulness of unprimed transcription as a method for sequencing singlestranded DNA. The template chosen was a pure fragment of +X174 single-stranded DNA, 43 residues in length, whose sequence had been determined by direct methods (Ziff et al., 1973; Galibert et al., 1974). 367
368
E. H. BLACKBURN
2. Materials and Methods (a) DNA Single-stranded viral DNA (a gift from Dr H. L. Weith) was prepared from 4x174 am3cs70 (lysis defective). 32P-labelled $X174 arn3cs70 DNA (a gift from Dr J. W. Sedat) was prepared as described by Galibert et al. (1974). For use as template DNA, a specific fragment of single-stranded #X DNA, 48 residues in length, was prepared by limited endonuclease IV digestion of the viral-strand DNA, to which 32P-labelled $X DNA was added as tracer, as described by Galibert et al. (1974). (b) RNA polynaerme DNA-dependent RNA polymerase holoenzyme from E. coli was a gift from Dr A. A. Trevers (Burgess & Travers, 1971). ATPase-free core polymerase prepared from E. coli was a gift from Dr C. Goff (Burgess, 1969; Goff, 1974). (c) Transcription incubatimw Reaction mixtures (20 to 50 ~1) contained 20 to 50 pmol template DNA, 100 pmol RNA polymer&se (holoenzyme or core poiymerase), 26 maa-Tris*HCl (pH 7-S), 8 m&r-ECl, 10 mM-MgCl,, 6 m&r-dithiothreitol, 0.5 mm-EDTA, 08 maa-potassium phosphate, three m&belled ribonuoleoside triphosphates (Sigma) at a concentration of O-5 mM and one [u-32P]ribonuoleoside triphosphate (New Enghmd Nuclear) at a concentration of 10 to 50 pM. Incubations were carried out at 37°C for times up to 8 h. Reaction was stopped by the addition of 5 ~1 of 0.1 M-EDTA. RNA and DNA were precipitated at - 70°C for 1 h by addition of 0- 1 vol. 3 M-sodium acetate, 50 pg of tRNA as carrier and 2.5 vol. ethanol. Unincorporated triphosphates were removed either by a second precipitation in ethanol or by acid precipitation in 0.4 M-HCl for 15 min at 0% (Ling, unpublished data). The precipitate was washed twice with ice-cold ethanol, dissolved in 20 ~1 of 30% triethylamine bicarbonate solution, and lyophilized. The residue was then twice dissolved in water and lyophilized. (d) Digestion of RNA and fractionation of oligonudeotides (i) S%ngmprinting procedure Transcription products were digested with either RNAase A (Worthington Biochemical Corporation, N.J., U.S.A.) or RNAase T1 (Srmkyo, Tokyo, Japan) for 46 min at 37”C, at an enzyme-to-carrier tRNA weight ratio of 1: 20. The products of digestion were separated by 8 2dimensional fingerprinting procedure. The first dimension was eleotrophoresis in 7 M-urea (pH 3.5) on cellulose acetate as described by Sanger et al. (1965). The oligonucleotides were transferred to PEl-cellulose thin-layer plates (Sohleicher & Schuell, Dassel, W. Gernnmy) and separated in the second dimension by development in I.5 Mpyridinium formate (pH 3.5), in 7 M-urea. After autoradiography the oligonucleotides were eluted as described for DEAE-cellulose thin-layer plates by Barrel1 (1971). (ii) Partial digestion with ribonuclease Tl The transcription product, labelled by incorporation of [c+~~P]GTP, RNAase T, at an enzyme-to-carrier tRNA weight ratio of 1:2000 for the presence of 10 mM-MgCl,. The resulting partial digestion products by the standard 2-dimensional thin-layer system (Brownlee & Sanger,
was digested with 15 min at O”C, in were fractionated 1969).
(e) Sequence analysis Sequence analysis of the oligonucleotides produced by complete digestion with RNAase T, was carried out by digestion with pancreatic RNAase or with RNAase Us (Sankyo, Tokyo, Japan) as described by Barrel1 (1971). The products of pancrerttic RNAase digestion were hydrolysed in alkali to yield nearest-neighbour information. RNAase U, digestion products were further enalysed by digestion with pancreatic RNAsse (to preserve runs of adenylic acid residues).
TRANSCRIPTION
OF
SINGLE-STRANDED
DNA
369
The oligonucleotides from pancreatic RNAase fingerprints were analysed by digestion with RNAase Ti followed by alkaline hydrolysis of the products. Oligonucleotides from partial digestion of the transcript with RNAase T1 were divided in two and each fraction analyaed by complete T1 or pancreatic RNAase digestion. The products were fractionated on DEAE-paper by electrophoresis in 7% formic acid, eluted and digested with either pancreatic or Ti RNAase. The resulting oligonucleotides were
separated on DEAE-paper by electrophoresis at pH 3.5.
3. Results (a)
Kinetics of RNA synthesis
With the fragment of single-stranded +X174 DNA, 48 residues in length, as template, transcription continued for up to eight hours at 37°C (Fig. 1). Each DNA molecule was calculated to be used, on average, 20 to 40 times as template in the course of a typical reaction lasting several hours. After 30 minutes, one, two, four and eight hours incubation, T, fingerprints of the RNA synthesized showed similar relative amounts of complete and incomplete T, products. The distribution of sizes among the transcription products also did not change with time from 30 minutes up to four hours incubation (Plate I), although after four hours products larger than the
Time(h)
Fm. 1. Kinetics of RNA synthesis from single-stranded DNA tempIate 48 residues in length. Transcription incubations were as described in the text, with [cz-~~P]CTP inoorporsted to label the transcript. RNA synthesis was measured by diluting samples of the reaction mixture into 40 ~1 of bovine serum albumin (20 mg/ml) followed by addition of 2 ml of oold (0“C) 5% triohloroaoetio acid containing 6 g tetrssodium pyrophosphate/l. After standing at 0°C for 16 min, the precipitates were collected on OF/C filters (Wbetman), which were then washed with the same triohloroacetio acid solution, dried and oounted with 2 ml of scintillation fluid (0.4% BBOT in toluene) in a Beckman 260 liquid sointillation aounter.
template began to be evident. Plate I shows that the synthesized RNA was heterogeneous in length, and gave a number of rather diffuse bands on polyacrylamide gel electrophoresis. However, analysis of these bands by digestion with T, RNAase showed that they were not pure species; indeed in each band all of the T, RNAase digestion products described in Table 1 were present, strongly suggesting that each band was a mixture of transcripts resulting from copying different regions of the template. These results indicate that there are several initiation and termination points on the template and that transcription was asynchronous in that reinitiation occurred many times during the course of the reaction.
370
E. H.
BLACKBURN
TABLES
Oligonucleotides produced by complete ribonuclease T, cligestion Sequance
No. Tl T2 T3 T4 TS
A-C-C-A-A-U-C-U-G(A) U-A-A-U-A-A-G(A) A-C-C-A-G(C) C-C-A-A-G(A) C-A-A-G(G)
No.
Sequence
Tf3 T7 T8 T9 TlO
A-A-G(C) A-U-G(G) A-C-G(A) G(G) G(A)
(b) Sequence analysis of transcription products Two-dimensional fingerprints of T, RNAase digests of transcription products, labelled separately with each of the four ribonucleoside triphosphates, are shown in Plate II. Table 1 shows the sequences of the complete T, products, and in Table 2 a detailed sequence ana.lysis of one of the T, oligonucleotides (Tl) is given. TABLE 2
Sample sequenceanalysis of oligonucleotide no. Tl Pancreatic
Label
C U
A G
A-A-U(C) A-A-U(not A-A-U(not
U(G)
Uz products
products
+ A-C(C) U) + C(U) A) + C(A) + G(A)
A(C) + C-C-A-A + U-C-U-G U-C-U-G + C-C-A-A(U) U-C-U-G(A) + C-C-A-A
Deduced sequenoe
A-C-C-A-A-U-C-U-G(A)
U-C-U-G
Similarly, the pancreatic RNAase digestion products of the labelled transcripts were fingerprinted (Plate III) and analysed (Table 3). In contrast to the fingerprints obtained from the products of digestion with T, RNAase, the fingerprints shown in Plate III contained products, some in high yield, that could only be accounted for as the result of unspecific nuclease activity or overdigestion. All of these “extra” spots were eluted and analysed, and consisted of sequences forming parts of the complete pancreatic RNAase digestion products. In different experiments the products shown in Table 3 appeared consistently; the “extra” spots were much more varied in intensity, depending on the conditions of digestion. The original 5’-triphosphate termini of the transcripts were found, after fingerprinting and analysis, as 5’-monophosphate groups. This was confirmed by alkaline hydrolysis and electrophoresis on DEAE-paper at pH 3.5. The products pAp and pGp were identified in the hydrolysates from their mobilities and characteristic streaking properties on electrophoresis. On fingerprinting, these 5’-phosphate termini remained at the origin of the second dimension and migrated faster in the first dimension than “internal” completed digestion products carrying 5’-hydroxyl and 3’-phosphate groups. These latter fell on graticules comparable to those occurring in tigerprints in which the second dimension is electrophoresis on DEAE-paper in 7% formic acid
I’LATES
I-III
L'LATE 1. I’olyaorylamide gel electrophoresis of RNA transcribed from single-stranded DNA template 48 residues in length. Transcription incubat,ions (as described in the text,) were for 0.5. 1, 4 and 8 h at 37”C, with [a-32P]CTP incorporated to label the transcript. Electrophoresis on a 19?; polyacrylamide 8 M-urea slab gel was carried out as described by Galibert et nl. (1974). ‘rho position of the t’emplate DNA (T) and the bromphenol blur marker dye (B) are indicated for rc~fhmce.
PLATE 1 I. Aut,oradiographs of two-dimensional fractionations of ohgonucleotides produced hy complete RNAase T, digestion of transcripts. RNA was labelled by incorporation of [Q-s*P] ATP (A), [u-~~P]CTP (C), [w3sP]GTP (G) and [u-~~P]U’I’P (U). Oligonuoleotide sequences are tlewribed in Tables 1 and 2. Electrophoresis was from right to left on cellulose acetate in 7 M-urea, pH 3.5. Thin-layer chromatography was on PEl-cellulose plates, developed from the bott)om upwards in 1.5 M-pyridinium formate (pH 3.5) in 7 v-urea.
I'LATE III. Autoradiographs of two-dimensional fract,ionations of oligonucleotides produced by nxnplete pancreatic RNAase digestion of transcript,s. RNA was labelled by incorporation of [ a-=P]ATP (A), [G(-~~P]CTP (C), [c+P]GTP (G) and 1CZ-~“P]UTP (U). Oligonuoleotide sequences arc described in Table 3. Electrophoresis was from right to left on cellulose acetate in 7 M-urea, pH 3.5. Thin-layer chromatography was on PEl-cellulose plates, developed from t,he bot,tom upwar
Time Cl11
Origin-
0.5
1
YLAI’E
I.
R
T-
.-4
0
I:”
pH 3.5 electrophoresis
4
T3+T4
P3
pH 3-5 electrophoresis 1’1.\TK I I I.
4
TRANSCRIPTION
OF
SINGLE-STRANDED
DNA
371
TABLE 3
No.
Sequence
Pl P2 P3 P4 P6
A-A-G-G-A-A-G-C(C) A-A-G-A-U(G) A-A-G-A-C(G) A-A-U(C) A-A-U(A)
No.
Sequenoe
P6 P7 PS P9 I’10 Pll
C(A) cm G-A-C(C) A-G-C(A) U(G) G-G-U(A)
(Brownlee & Sanger, 1967; author’s unpublished results). 3’-hydroxyl termini were also separated from completed oligonucleotide digestion products as they have a decreased mobility in the first dimension (Brownlee & Sanger, 1967) and were found to migrate closer to the solvent front in the second dimension. Overlapping sequences were obtained from the products of partial digestion of the transcript with T, RNAase, and are shown in Table 4. Quantitation of the complete digestion products shown in Tables 1 and 2 was only possible after analysis of individual, fractionated products of partial T, RNAase digestion, since the complete digestion products obtained from total RNA synthesized were found in yields reflecting the frequency of copying the complementary region of the template rather than the frequency of their occurrence in the sequence. Thus the sequence shown in Figure 2 TABLET Oligonmleotidea produced by partial digestion with ribonu&a.se T, T, RNAase (see Table 1)
Pancreatic RNA-0 (see Table 3)
1
T3 Tb T6 T4
P9 Pl A-A-G
2
T8 Tl T3
A-C(G) PlO A-G
3
T6 T4 T7 T9
A-A-G(C) P2 G-G
4
Tl, T2, T3, T4 TB, T6, T7, T8
P3, P9 PIO, Pl, P2
6
T8 TL
A-C(G) PlO
E. H.
372
BLACKBUR’N DNA
J’~~TT-T-T-A-C-C-A-~.T-A.T.T-C-T.G-C-T-G-G-T-T-A-G-A-C-T-G-G-T-C-G-T-T-C-C-T-T-C-G-G-T-T-C-T-A-C-C-CP~’
. . .
“-G-G-“-A-A-U-A-A-G-A-C-G-A-C-C-A-A-”-C-”-G-A-C-C-A-G-C-A-A-G-G-A-A-G-C-C-A-A-G-A-~!-G-G-G
I
I
2 II
I II
I
5
I
I
8 I 3
I
I
I II
8
5
III
4
710
I
I
5106
II
8
4
2
II
3 I
6
I
9
T, RNAose products
4--?-+ I
I
5
Pancreatic products
I
2
--T---f
I
I
’
!
RNAose
Overlapping fragments obtained from limited T, digest
FIU. 2. Sequence analysis of transcripts of a single-stranded DNA fragment. The sequence the template is shown in the top line (Ziff et al., 1973; Galibert et al., 1974). The products complete digestion with T1 and pancreatic ribonucleases are numbered as in Tables 1 and respectively. Overlapping sequences obtained by limited digestion with T1 ribonuclease (Table are indicated.
of of 3, 4)
was deduced. It is apparent that the information obtained by standard fingerprinting and sequence analysis procedures carried out on the RNA synthesized was sufficient to determine the sequence of all but the five 5’-terminal residues of the template.
4. Discussion By characterizing the products of transcription of a small single-stranded DNA template of known sequence, it has been shown that virtually the whole of the template was transcribed. The RNA synthesized was heterogeneous with respect to size, suggesting several sites for initiation and termination-in agreement with the results of Terao et al. (1972). It is clear that with a template of 48 residues in length (compared with the one 29 residues in length studied by these authors) there were many more positions at which initiation and termination occurred, as judged by the complexity of the transcription products. The finding that the transcripts fell into roughly defined size classes suggests that the phenomena observed by Terao et al. (1972) were also occurring in the experiments described here. However, these authors did not find any increase in the number of moles of synthesized RNA over the number of template molecules, as also described by Gilbert & Maxam (1973). This may be accounted for by the fact that the highest temperature used by Terao et al. was 25°C compared with 37°C in the present work, and 35°C in the case of the experiments of Gilbert & Maxam, suggesting that at the higher temperatures previously formed transcripts are more readily displaced by RNA polymerase in the process of synthesizing new transcripts. Terao et al. (1972) have also demonstrated that a small proportion of the transcript RNA is longer than the template. This has been observed in some experiments in the present work (Plate I) and it is possible that it is the result of formation of selfcomplementary RNA, a phenomenon previously observed when single-stranded bacteriophage templates were transcribed using an excess of E. coli RNA polymerase (Tabak $ Borst, 1971; Robertson, 1971). In the present work, in two experiments in which transcription incubations were carried out at a high molar ratio of polymerase to template (50: l), incorporating [cr-32P]uridine triphosphate to label the product, extra oligonucleotides appeared in good yield in the T, fingerprint. The sequences of
TRANSCRIPTION
OF SINGLE-STRANDED
DNA
373
these oligonucleotides were determined as C-U-U-C-C-U-U-G, C-U-G, A-U-U-G and G(U) ; that is, complementary to sequences that appear normally in the RNA transcript. At low molar ratios of polymerase to template (less than 5: l), no self-complementary sequences could be identified. The presence of transcription products larger than the template, which persist in polyacrylamide gels containing 8-3 M-urea, or in columns run under denaturing conditions (Terao et al., 1972), suggests that formation of self-complementary RNA may not occur by re-initiation of RNA synthesis in the manner suggested by Bishop (1969) but rather by the formation of a hairpin loop of double-stra,nded RNA. Clarification of this point, however, will require demonstration of the presence or absence of self-complementary sequences in transcription products larger than the template. One purpose of the work described here has been to evaluate the method of unprimed transcription to sequence single-stranded DNA. Analysis of the transcripts after one-dimensional polyacrylamide gel electrophoresis suggested that a two-dimensional fractionation would be necessary to purify the different transcription products. However, it has been shown that complete sequence information for a large part of the template can be obtained by analysis of the total RNA synthesized. Therefore, only the completed products of T, and pancreatic RNAase digestion were analysed. This obviates the need for a more complex analysis which would take into account the many 5’-phosphate and 3’-hydroxyl termini of the transcripts that were separated from the completed digestion products by the fingerprinting procedure used. To derive the sequence shown in Figure 2 from the data given, it was necessary to know the size of the template, which could be obtained (for example) by gel electrophoresis (Maniatis & Ptashne, 1973). This was required in this case because the breakdown products shown in the fingerprints of Plate III created difficulties in interpreting these results, although the results from complete and partial T, RNAase digestion were quite clear. In addition, the results obtained using high molar ratios of RNA polymerase to template suggest that, with a small single-stranded DNA template, the conditions of transcription should be carefully defined in order to minimize the formation of products larger than the template and, possibly, selfcomplementary RNA sequences.
I thank my supervisor, Dr F. Sanger, for his encouragement and helpful discussions, and Drs J. W. Sedat and E. B. Ziff for valuable comments and suggestions. Support by a Gowrie Travelling Research Scholarship is gratefully acknowledged.
REFERENCES Barre& B. G. (1971). In Procedures in Nucleic Acid Research (Cantoni, G. L. & Davies, D. R., eds), vol. 2, pp. 751-779, Harper & Row, New York. Berg, P., Kornberg, R. D., Fancher, H. $ Dieckmann, M. (1965). Rio&em. Biophys. I&s. Commun. 18, 932-942. Bishop, J. 0. (1969). B&him. Biophys. A&z, 174, 636-652. Bremer, H., Konrad, M. & Bruner, R. (1966). J. Mol. Biol. 16, 104-117. Brownlee, G. G. & Sanger, F. (1967). J. Mol. Biol. 23, 337-353. Brownlee, G. G. & Sanger, F. (1969). Eur. J. Biochem. 11, 395-399. Brownlee, G. G., Sanger, F. & Barrel& B. G. (1968). J. Mol. Biol. 34, 379-412. Burgess, R. R. (1969). J. BioZ. Chem. 244, 6160-6167.
374
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Burgess, R. R. & Travers, A. A. (1971). In Procedures in Nucleic Acid Research (Cantoni, G. L. & Davies, D. R., eds), vol. 2, pp. 861-863, Harper & Row, New York. Chemberlin, M. & Berg, P. (1962). Proc. Nat. Aoad. Soi., U.S.A. 48, 81-94. Caliber%, F., Sedat, J. W. & Ziff, E. B. (1974). J. MoZ. Biol. 87, 377-407. Gilbert, W. & Maxam, A. (1973). Proc. Nat. Acad. Sci., U.S.A. 70, 3681-3684. Gilbert, W., Maizels, N. & Max-, A. (1973). Cold Spring Harbor Syrnp. Quati. Biol. 38, 846-866. Goff, C. (1974). J. Biol. Chem. 249, 6181-6190. Hurwitz, J., Furth, J. J., Anders, M. & Evans, A. (1962). J. Biol. Chena. 237, 3762-3769. Maniatis, T. & Ptsshne, M. (1973). Proc. Nut. Acad. Sci., U.S.A. 70, 1631-1636. Richardson, J. P. (1969). In Progreee Nucleic Acids Research and Molecular Biology (Davidson, J. N. & Cohn, W. E., ads), vol. 9, pp. 76-l 16, Academic Press, New York. Robertson, H. D. (1971). Na&re New Biol. 229, 169-172. Sanger, F., Brownlee, G. G. & Barrell, B. G. (1966). J. Mol. Biol. 13, 373-398. Tabak, H. F. & Borst, P. (1971). Biochim. Biophya. Acta, 246, 460-467. Terao, T., Dahlberg, J. E. & Khorana, H. G. (1972). J. Biol. Chem. 247. 6167-6166. Wood, W. B. & Berg, P. (1964). J. Mol. Biol. 9, 462-471. Ziff, E. B., Sedat, J. W. BEGalibert, F. (1973). Nature New Biol. 241, 34-37.
Transcription by Escherichia coli RNA Polymerase of a Single-stranded Fragment by Bacteriophage 4X174 DNA 48 Residues in Length ELIZABETH H. BLACKBURN Me&cd Research Council Laboratory of Molecular Biology Hills Road, Cambridge CB2 2&H, England (Received 26 March 1974, and in revisrd jorm IS November 1974) A single-stranded fragment of bacteriophage -$X174 DNA, 48 residues in length and of known sequence, has been transcribed by Escherichia coli DNA-dependent RNA polymerase. After transcription for eight hours at 37°C the molar yield of RNA synthesized was 20 to 40 times the molar amount of template DNA present. The products of transcription were very heterogeneous in size, indicating multiple points of initiation and termination along the template. Sequence analysis of the heterogeneous transcripts included a two-dimensional fingerprinting procedure designed to seperate oligonucleotides containing initiation and t,ermination points from internal, complete oligonucleotides produced by digestion with T, or pancreatic ribonuclease. Overlapping sequences were obtained by partial digestion with T1 ribonuclease and it w&s shown that the sequence of all but the first five 5’-terminal residues of the template could be determined by this method.
1. Introduction Single-stranded DNA can be used as a template for transcription by Escherichia coli DNA-dependent RNA polymerase in vitro (Hurwitz et al., 1962 ; Chamberlin & Berg, 1962). Under these conditions the enzyme shows considerably less specificity for sites of initiation and termination of transcription than on a double-stranded DNA template (Berg et Cal.,1965; Wood & Berg, 1964; Bremer et al., 1966; see also review by Richardson, 1969). Therefore, regions of DNA, which may not be transcribed from the double-stranded template, can be copied in vitro from single-stranded or denatured DNA into labelled RNA of high specific activity, suitable for sequence analysis by established techniques (Sanger et al., 1965 ; Brownlee & Sanger, 1967,1969 ; Brownlee et al., 1968). This method has been used to determine the sequence of the lac operator region (Gilbert & Maxam, 1973; Gilbert et al., 1973). A detailed characterization of the products of transcription of a synthetic oligodeoxyribonucleotide, 29 residues in length, has been carried out (Terao et al., 1972), in which the effect of priming with an oligoribonucleotide was also examined. In the work described here the products of transcription from a larger single-stranded fragment of DNA were characterized, to determine the extent and fidelity of copying by E. coli RNA polymerase, in order to test the usefulness of unprimed transcription as a method for sequencing singlestranded DNA. The template chosen was a pure fragment of +X174 single-stranded DNA, 43 residues in length, whose sequence had been determined by direct methods (Ziff et al., 1973; Galibert et al., 1974). 367
368
E. H. BLACKBURN
2. Materials and Methods (a) DNA Single-stranded viral DNA (a gift from Dr H. L. Weith) was prepared from 4x174 am3cs70 (lysis defective). 32P-labelled $X174 arn3cs70 DNA (a gift from Dr J. W. Sedat) was prepared as described by Galibert et al. (1974). For use as template DNA, a specific fragment of single-stranded #X DNA, 48 residues in length, was prepared by limited endonuclease IV digestion of the viral-strand DNA, to which 32P-labelled $X DNA was added as tracer, as described by Galibert et al. (1974). (b) RNA polynaerme DNA-dependent RNA polymerase holoenzyme from E. coli was a gift from Dr A. A. Trevers (Burgess & Travers, 1971). ATPase-free core polymerase prepared from E. coli was a gift from Dr C. Goff (Burgess, 1969; Goff, 1974). (c) Transcription incubatimw Reaction mixtures (20 to 50 ~1) contained 20 to 50 pmol template DNA, 100 pmol RNA polymer&se (holoenzyme or core poiymerase), 26 maa-Tris*HCl (pH 7-S), 8 m&r-ECl, 10 mM-MgCl,, 6 m&r-dithiothreitol, 0.5 mm-EDTA, 08 maa-potassium phosphate, three m&belled ribonuoleoside triphosphates (Sigma) at a concentration of O-5 mM and one [u-32P]ribonuoleoside triphosphate (New Enghmd Nuclear) at a concentration of 10 to 50 pM. Incubations were carried out at 37°C for times up to 8 h. Reaction was stopped by the addition of 5 ~1 of 0.1 M-EDTA. RNA and DNA were precipitated at - 70°C for 1 h by addition of 0- 1 vol. 3 M-sodium acetate, 50 pg of tRNA as carrier and 2.5 vol. ethanol. Unincorporated triphosphates were removed either by a second precipitation in ethanol or by acid precipitation in 0.4 M-HCl for 15 min at 0% (Ling, unpublished data). The precipitate was washed twice with ice-cold ethanol, dissolved in 20 ~1 of 30% triethylamine bicarbonate solution, and lyophilized. The residue was then twice dissolved in water and lyophilized. (d) Digestion of RNA and fractionation of oligonudeotides (i) S%ngmprinting procedure Transcription products were digested with either RNAase A (Worthington Biochemical Corporation, N.J., U.S.A.) or RNAase T1 (Srmkyo, Tokyo, Japan) for 46 min at 37”C, at an enzyme-to-carrier tRNA weight ratio of 1: 20. The products of digestion were separated by 8 2dimensional fingerprinting procedure. The first dimension was eleotrophoresis in 7 M-urea (pH 3.5) on cellulose acetate as described by Sanger et al. (1965). The oligonucleotides were transferred to PEl-cellulose thin-layer plates (Sohleicher & Schuell, Dassel, W. Gernnmy) and separated in the second dimension by development in I.5 Mpyridinium formate (pH 3.5), in 7 M-urea. After autoradiography the oligonucleotides were eluted as described for DEAE-cellulose thin-layer plates by Barrel1 (1971). (ii) Partial digestion with ribonuclease Tl The transcription product, labelled by incorporation of [c+~~P]GTP, RNAase T, at an enzyme-to-carrier tRNA weight ratio of 1:2000 for the presence of 10 mM-MgCl,. The resulting partial digestion products by the standard 2-dimensional thin-layer system (Brownlee & Sanger,
was digested with 15 min at O”C, in were fractionated 1969).
(e) Sequence analysis Sequence analysis of the oligonucleotides produced by complete digestion with RNAase T, was carried out by digestion with pancreatic RNAase or with RNAase Us (Sankyo, Tokyo, Japan) as described by Barrel1 (1971). The products of pancrerttic RNAase digestion were hydrolysed in alkali to yield nearest-neighbour information. RNAase U, digestion products were further enalysed by digestion with pancreatic RNAsse (to preserve runs of adenylic acid residues).
TRANSCRIPTION
OF
SINGLE-STRANDED
DNA
369
The oligonucleotides from pancreatic RNAase fingerprints were analysed by digestion with RNAase Ti followed by alkaline hydrolysis of the products. Oligonucleotides from partial digestion of the transcript with RNAase T1 were divided in two and each fraction analyaed by complete T1 or pancreatic RNAase digestion. The products were fractionated on DEAE-paper by electrophoresis in 7% formic acid, eluted and digested with either pancreatic or Ti RNAase. The resulting oligonucleotides were
separated on DEAE-paper by electrophoresis at pH 3.5.
3. Results (a)
Kinetics of RNA synthesis
With the fragment of single-stranded +X174 DNA, 48 residues in length, as template, transcription continued for up to eight hours at 37°C (Fig. 1). Each DNA molecule was calculated to be used, on average, 20 to 40 times as template in the course of a typical reaction lasting several hours. After 30 minutes, one, two, four and eight hours incubation, T, fingerprints of the RNA synthesized showed similar relative amounts of complete and incomplete T, products. The distribution of sizes among the transcription products also did not change with time from 30 minutes up to four hours incubation (Plate I), although after four hours products larger than the
Time(h)
Fm. 1. Kinetics of RNA synthesis from single-stranded DNA tempIate 48 residues in length. Transcription incubations were as described in the text, with [cz-~~P]CTP inoorporsted to label the transcript. RNA synthesis was measured by diluting samples of the reaction mixture into 40 ~1 of bovine serum albumin (20 mg/ml) followed by addition of 2 ml of oold (0“C) 5% triohloroaoetio acid containing 6 g tetrssodium pyrophosphate/l. After standing at 0°C for 16 min, the precipitates were collected on OF/C filters (Wbetman), which were then washed with the same triohloroacetio acid solution, dried and oounted with 2 ml of scintillation fluid (0.4% BBOT in toluene) in a Beckman 260 liquid sointillation aounter.
template began to be evident. Plate I shows that the synthesized RNA was heterogeneous in length, and gave a number of rather diffuse bands on polyacrylamide gel electrophoresis. However, analysis of these bands by digestion with T, RNAase showed that they were not pure species; indeed in each band all of the T, RNAase digestion products described in Table 1 were present, strongly suggesting that each band was a mixture of transcripts resulting from copying different regions of the template. These results indicate that there are several initiation and termination points on the template and that transcription was asynchronous in that reinitiation occurred many times during the course of the reaction.
370
E. H.
BLACKBURN
TABLES
Oligonucleotides produced by complete ribonuclease T, cligestion Sequance
No. Tl T2 T3 T4 TS
A-C-C-A-A-U-C-U-G(A) U-A-A-U-A-A-G(A) A-C-C-A-G(C) C-C-A-A-G(A) C-A-A-G(G)
No.
Sequence
Tf3 T7 T8 T9 TlO
A-A-G(C) A-U-G(G) A-C-G(A) G(G) G(A)
(b) Sequence analysis of transcription products Two-dimensional fingerprints of T, RNAase digests of transcription products, labelled separately with each of the four ribonucleoside triphosphates, are shown in Plate II. Table 1 shows the sequences of the complete T, products, and in Table 2 a detailed sequence ana.lysis of one of the T, oligonucleotides (Tl) is given. TABLE 2
Sample sequenceanalysis of oligonucleotide no. Tl Pancreatic
Label
C U
A G
A-A-U(C) A-A-U(not A-A-U(not
U(G)
Uz products
products
+ A-C(C) U) + C(U) A) + C(A) + G(A)
A(C) + C-C-A-A + U-C-U-G U-C-U-G + C-C-A-A(U) U-C-U-G(A) + C-C-A-A
Deduced sequenoe
A-C-C-A-A-U-C-U-G(A)
U-C-U-G
Similarly, the pancreatic RNAase digestion products of the labelled transcripts were fingerprinted (Plate III) and analysed (Table 3). In contrast to the fingerprints obtained from the products of digestion with T, RNAase, the fingerprints shown in Plate III contained products, some in high yield, that could only be accounted for as the result of unspecific nuclease activity or overdigestion. All of these “extra” spots were eluted and analysed, and consisted of sequences forming parts of the complete pancreatic RNAase digestion products. In different experiments the products shown in Table 3 appeared consistently; the “extra” spots were much more varied in intensity, depending on the conditions of digestion. The original 5’-triphosphate termini of the transcripts were found, after fingerprinting and analysis, as 5’-monophosphate groups. This was confirmed by alkaline hydrolysis and electrophoresis on DEAE-paper at pH 3.5. The products pAp and pGp were identified in the hydrolysates from their mobilities and characteristic streaking properties on electrophoresis. On fingerprinting, these 5’-phosphate termini remained at the origin of the second dimension and migrated faster in the first dimension than “internal” completed digestion products carrying 5’-hydroxyl and 3’-phosphate groups. These latter fell on graticules comparable to those occurring in tigerprints in which the second dimension is electrophoresis on DEAE-paper in 7% formic acid
I’LATES
I-III
L'LATE 1. I’olyaorylamide gel electrophoresis of RNA transcribed from single-stranded DNA template 48 residues in length. Transcription incubat,ions (as described in the text,) were for 0.5. 1, 4 and 8 h at 37”C, with [a-32P]CTP incorporated to label the transcript. Electrophoresis on a 19?; polyacrylamide 8 M-urea slab gel was carried out as described by Galibert et nl. (1974). ‘rho position of the t’emplate DNA (T) and the bromphenol blur marker dye (B) are indicated for rc~fhmce.
PLATE 1 I. Aut,oradiographs of two-dimensional fractionations of ohgonucleotides produced hy complete RNAase T, digestion of transcripts. RNA was labelled by incorporation of [Q-s*P] ATP (A), [u-~~P]CTP (C), [w3sP]GTP (G) and [u-~~P]U’I’P (U). Oligonuoleotide sequences are tlewribed in Tables 1 and 2. Electrophoresis was from right to left on cellulose acetate in 7 M-urea, pH 3.5. Thin-layer chromatography was on PEl-cellulose plates, developed from the bott)om upwards in 1.5 M-pyridinium formate (pH 3.5) in 7 v-urea.
I'LATE III. Autoradiographs of two-dimensional fract,ionations of oligonucleotides produced by nxnplete pancreatic RNAase digestion of transcript,s. RNA was labelled by incorporation of [ a-=P]ATP (A), [G(-~~P]CTP (C), [c+P]GTP (G) and 1CZ-~“P]UTP (U). Oligonuoleotide sequences arc described in Table 3. Electrophoresis was from right to left on cellulose acetate in 7 M-urea, pH 3.5. Thin-layer chromatography was on PEl-cellulose plates, developed from t,he bot,tom upwar
Time Cl11
Origin-
0.5
1
YLAI’E
I.
R
T-
.-4
0
I:”
pH 3.5 electrophoresis
4
T3+T4
P3
pH 3-5 electrophoresis 1’1.\TK I I I.
4
TRANSCRIPTION
OF
SINGLE-STRANDED
DNA
371
TABLE 3
No.
Sequence
Pl P2 P3 P4 P6
A-A-G-G-A-A-G-C(C) A-A-G-A-U(G) A-A-G-A-C(G) A-A-U(C) A-A-U(A)
No.
Sequenoe
P6 P7 PS P9 I’10 Pll
C(A) cm G-A-C(C) A-G-C(A) U(G) G-G-U(A)
(Brownlee & Sanger, 1967; author’s unpublished results). 3’-hydroxyl termini were also separated from completed oligonucleotide digestion products as they have a decreased mobility in the first dimension (Brownlee & Sanger, 1967) and were found to migrate closer to the solvent front in the second dimension. Overlapping sequences were obtained from the products of partial digestion of the transcript with T, RNAase, and are shown in Table 4. Quantitation of the complete digestion products shown in Tables 1 and 2 was only possible after analysis of individual, fractionated products of partial T, RNAase digestion, since the complete digestion products obtained from total RNA synthesized were found in yields reflecting the frequency of copying the complementary region of the template rather than the frequency of their occurrence in the sequence. Thus the sequence shown in Figure 2 TABLET Oligonmleotidea produced by partial digestion with ribonu&a.se T, T, RNAase (see Table 1)
Pancreatic RNA-0 (see Table 3)
1
T3 Tb T6 T4
P9 Pl A-A-G
2
T8 Tl T3
A-C(G) PlO A-G
3
T6 T4 T7 T9
A-A-G(C) P2 G-G
4
Tl, T2, T3, T4 TB, T6, T7, T8
P3, P9 PIO, Pl, P2
6
T8 TL
A-C(G) PlO
E. H.
372
BLACKBUR’N DNA
J’~~TT-T-T-A-C-C-A-~.T-A.T.T-C-T.G-C-T-G-G-T-T-A-G-A-C-T-G-G-T-C-G-T-T-C-C-T-T-C-G-G-T-T-C-T-A-C-C-CP~’
. . .
“-G-G-“-A-A-U-A-A-G-A-C-G-A-C-C-A-A-”-C-”-G-A-C-C-A-G-C-A-A-G-G-A-A-G-C-C-A-A-G-A-~!-G-G-G
I
I
2 II
I II
I
5
I
I
8 I 3
I
I
I II
8
5
III
4
710
I
I
5106
II
8
4
2
II
3 I
6
I
9
T, RNAose products
4--?-+ I
I
5
Pancreatic products
I
2
--T---f
I
I
’
!
RNAose
Overlapping fragments obtained from limited T, digest
FIU. 2. Sequence analysis of transcripts of a single-stranded DNA fragment. The sequence the template is shown in the top line (Ziff et al., 1973; Galibert et al., 1974). The products complete digestion with T1 and pancreatic ribonucleases are numbered as in Tables 1 and respectively. Overlapping sequences obtained by limited digestion with T1 ribonuclease (Table are indicated.
of of 3, 4)
was deduced. It is apparent that the information obtained by standard fingerprinting and sequence analysis procedures carried out on the RNA synthesized was sufficient to determine the sequence of all but the five 5’-terminal residues of the template.
4. Discussion By characterizing the products of transcription of a small single-stranded DNA template of known sequence, it has been shown that virtually the whole of the template was transcribed. The RNA synthesized was heterogeneous with respect to size, suggesting several sites for initiation and termination-in agreement with the results of Terao et al. (1972). It is clear that with a template of 48 residues in length (compared with the one 29 residues in length studied by these authors) there were many more positions at which initiation and termination occurred, as judged by the complexity of the transcription products. The finding that the transcripts fell into roughly defined size classes suggests that the phenomena observed by Terao et al. (1972) were also occurring in the experiments described here. However, these authors did not find any increase in the number of moles of synthesized RNA over the number of template molecules, as also described by Gilbert & Maxam (1973). This may be accounted for by the fact that the highest temperature used by Terao et al. was 25°C compared with 37°C in the present work, and 35°C in the case of the experiments of Gilbert & Maxam, suggesting that at the higher temperatures previously formed transcripts are more readily displaced by RNA polymerase in the process of synthesizing new transcripts. Terao et al. (1972) have also demonstrated that a small proportion of the transcript RNA is longer than the template. This has been observed in some experiments in the present work (Plate I) and it is possible that it is the result of formation of selfcomplementary RNA, a phenomenon previously observed when single-stranded bacteriophage templates were transcribed using an excess of E. coli RNA polymerase (Tabak $ Borst, 1971; Robertson, 1971). In the present work, in two experiments in which transcription incubations were carried out at a high molar ratio of polymerase to template (50: l), incorporating [cr-32P]uridine triphosphate to label the product, extra oligonucleotides appeared in good yield in the T, fingerprint. The sequences of
TRANSCRIPTION
OF SINGLE-STRANDED
DNA
373
these oligonucleotides were determined as C-U-U-C-C-U-U-G, C-U-G, A-U-U-G and G(U) ; that is, complementary to sequences that appear normally in the RNA transcript. At low molar ratios of polymerase to template (less than 5: l), no self-complementary sequences could be identified. The presence of transcription products larger than the template, which persist in polyacrylamide gels containing 8-3 M-urea, or in columns run under denaturing conditions (Terao et al., 1972), suggests that formation of self-complementary RNA may not occur by re-initiation of RNA synthesis in the manner suggested by Bishop (1969) but rather by the formation of a hairpin loop of double-stra,nded RNA. Clarification of this point, however, will require demonstration of the presence or absence of self-complementary sequences in transcription products larger than the template. One purpose of the work described here has been to evaluate the method of unprimed transcription to sequence single-stranded DNA. Analysis of the transcripts after one-dimensional polyacrylamide gel electrophoresis suggested that a two-dimensional fractionation would be necessary to purify the different transcription products. However, it has been shown that complete sequence information for a large part of the template can be obtained by analysis of the total RNA synthesized. Therefore, only the completed products of T, and pancreatic RNAase digestion were analysed. This obviates the need for a more complex analysis which would take into account the many 5’-phosphate and 3’-hydroxyl termini of the transcripts that were separated from the completed digestion products by the fingerprinting procedure used. To derive the sequence shown in Figure 2 from the data given, it was necessary to know the size of the template, which could be obtained (for example) by gel electrophoresis (Maniatis & Ptashne, 1973). This was required in this case because the breakdown products shown in the fingerprints of Plate III created difficulties in interpreting these results, although the results from complete and partial T, RNAase digestion were quite clear. In addition, the results obtained using high molar ratios of RNA polymerase to template suggest that, with a small single-stranded DNA template, the conditions of transcription should be carefully defined in order to minimize the formation of products larger than the template and, possibly, selfcomplementary RNA sequences.
I thank my supervisor, Dr F. Sanger, for his encouragement and helpful discussions, and Drs J. W. Sedat and E. B. Ziff for valuable comments and suggestions. Support by a Gowrie Travelling Research Scholarship is gratefully acknowledged.
REFERENCES Barre& B. G. (1971). In Procedures in Nucleic Acid Research (Cantoni, G. L. & Davies, D. R., eds), vol. 2, pp. 751-779, Harper & Row, New York. Berg, P., Kornberg, R. D., Fancher, H. $ Dieckmann, M. (1965). Rio&em. Biophys. I&s. Commun. 18, 932-942. Bishop, J. 0. (1969). B&him. Biophys. A&z, 174, 636-652. Bremer, H., Konrad, M. & Bruner, R. (1966). J. Mol. Biol. 16, 104-117. Brownlee, G. G. & Sanger, F. (1967). J. Mol. Biol. 23, 337-353. Brownlee, G. G. & Sanger, F. (1969). Eur. J. Biochem. 11, 395-399. Brownlee, G. G., Sanger, F. & Barrel& B. G. (1968). J. Mol. Biol. 34, 379-412. Burgess, R. R. (1969). J. BioZ. Chem. 244, 6160-6167.
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Burgess, R. R. & Travers, A. A. (1971). In Procedures in Nucleic Acid Research (Cantoni, G. L. & Davies, D. R., eds), vol. 2, pp. 861-863, Harper & Row, New York. Chemberlin, M. & Berg, P. (1962). Proc. Nat. Aoad. Soi., U.S.A. 48, 81-94. Caliber%, F., Sedat, J. W. & Ziff, E. B. (1974). J. MoZ. Biol. 87, 377-407. Gilbert, W. & Maxam, A. (1973). Proc. Nat. Acad. Sci., U.S.A. 70, 3681-3684. Gilbert, W., Maizels, N. & Max-, A. (1973). Cold Spring Harbor Syrnp. Quati. Biol. 38, 846-866. Goff, C. (1974). J. Biol. Chem. 249, 6181-6190. Hurwitz, J., Furth, J. J., Anders, M. & Evans, A. (1962). J. Biol. Chena. 237, 3762-3769. Maniatis, T. & Ptsshne, M. (1973). Proc. Nut. Acad. Sci., U.S.A. 70, 1631-1636. Richardson, J. P. (1969). In Progreee Nucleic Acids Research and Molecular Biology (Davidson, J. N. & Cohn, W. E., ads), vol. 9, pp. 76-l 16, Academic Press, New York. Robertson, H. D. (1971). Na&re New Biol. 229, 169-172. Sanger, F., Brownlee, G. G. & Barrell, B. G. (1966). J. Mol. Biol. 13, 373-398. Tabak, H. F. & Borst, P. (1971). Biochim. Biophya. Acta, 246, 460-467. Terao, T., Dahlberg, J. E. & Khorana, H. G. (1972). J. Biol. Chem. 247. 6167-6166. Wood, W. B. & Berg, P. (1964). J. Mol. Biol. 9, 462-471. Ziff, E. B., Sedat, J. W. BEGalibert, F. (1973). Nature New Biol. 241, 34-37.