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Terminal strand-switching of E. coli RNA polymerase transcribing a truncated DNA fragment
446
Biochimica et Biophysica Acta, 655 (1981) 446-448
Elsevier/North-Holland Biomedical Press
BBA Report BBA 91525 TERMINAL STRAND-SWITCHING OF E. COLI R N A POLYMERASE TRANSCRIBING A T R U N C A T E D DNA FRAGMENT BEN A. OOSTRA, ANNIKA C. ARNBERG, GEERT AB and MAX GRUBER Biochemisch Laboratorium, R i/ksuniversiteit Groningen, Nijenborgh 16, 9747 A G Groningen (The Netherlands}
(Received June llth, 1981)
Key words: RNA polymerase; Strand-switching; Transcription in vitro; rRNA; Promoter," {E. coliJ
When transcribing a restriction fragment containing the promoters and the first part of the rrnE operon of Escherichia coil, RNA polymerase holoenzyme starts exclusively on the promoters. Besides run-off transcripts, molecules longer than template-size are formed by terminal strand switch.
In our earlier studies on the regulation of rRNA synthesis in Escherichia coli we have used a hybridization-competition assay for specific rRNA transcripts formed in vitro [1]. In another approach, focussing on the initiation step of rRNA transcription, we have now used the so-called truncated template system. DNA restriction fragments carrying the first part of a gene including its promoter are transcribed in vitro and the synthesis of run-off transcripts of discrete size is followed to assess specific initiation. We used an 1.4 kilobase EcoRI fragment of phage )~rnetA20 DNA carrying the tandem promoters, P1
EcoRl
EcoRI
1 I
1 P,, P' 437
r//~l 6"sfr "DNA ~/'/A
109 174
670
100 bp Fig. 1. Map of the 1.4 kilobase EcoRI fragment from XmetA20. The distances, given in basepairs, are taken from Ref. 2. Isolation of the fragment was as described in Ref. 2. Open area, E. coli chromosomal DNA; hatched area, E. coli ribosomal DNA. bp, basepair.
and P2, of the rrnE operon (Fig. 1). Transcription was performed in a system with RNA polymerase and the DNA fragment as the only macromolecules. The size of the transcripts was determined by gel electrophoresis and autoradiography. Prior to analysis the RNAs were denatured; renaturation was prevented by glyoxylation [3]. With RNA polymerase core enzyme one band of 1400 nucleotides was observed, most probably representing end-to-end transcripts (Fig. 2, lane 1). With the RNA polymerase holoenzyme, saturated with the sigma subunit, the RNA transcripts are quite different (Fig. 2, lane 2). A broad band of about 900 nucleotides is seen which is in some instances resolved in two components of equal intensity. Most likely they are the run-off transcripts started on the initiation sites P1 and P2, which appear to be used in about equal frequency under these conditions (see also Ref. 4). The transcripts of the rRNA tandem promoters would be 950 and 850 nucleotides long. No transcripts of 1400 nucleotides are found; apparently, the holoenzyme does not start at an end. Surprisingly, also transcripts of 2200-2300 nucleotides are found. These longer transcripts constitute about 5-10% of the molecules synthesized. The occurrence of longer transcripts has also been noticed with other templates but their nature has not
0005-2787/81/0000-0000/$02.50 © 1981 Elsevier/North-Holland Biomedical Press
447
lS
S
_
16
S
_
__
2500b
_
~ b
O
effectively maintained when RNA polymerase is passing by. To decide between these two possibilities, RNA was analysed under non-denaturing conditions. In the terminal strand switch model only one kind of 2200nucleotide RNA chain is expected, which will form a hairpin structure. Such molecules were demonstrated by electron microscopy of the material eluted from the agarose gel, banding between 18 S and 16 S. Fig. 3A shows a molecule having a double-stranded stretch of about 800 basepairs connected to a 500-nueleotide single-stranded tail. Upon denaturation these molecules yield single-stranded structures with a total length of 2200 nucleotides (Fig. 3C). Renaturation of
9 S _
RN~A
DNA
Fig. 2. Agarose gel electrophoresis of RNA synthesized in vitro. RNA was synthesized in 50 /~1 reaction mixture [1] with 0.1 rrtM [a-32PIUTP (1.4 Ci/mmol). After phenol extraction and ethanol precipitation in the presence of wheat germ tRNA Glu (100 /~g/ml) the RNAs were glyoxylated and separated on 1.25% agarose gel [3]. Lane 1: RNA synthesized with RNA polymerase core enzyme; lanes 2 - 4 : RNA synthesized by RNA polymerase holoenzyme. Lane3: ppGpp present (0.5 mM); lane 4: Sl nuelease-treated DNA template used. b, bases.
been analysed further [5,6]. To explain formation of RNA chains longer than template.size two possibilities can be envisaged. First, RNA polymerase having synthesized a 850- or 950-nucleotide chain, and reaching the end of the template, switches to the complementary strand and extends the RNA chain by another 1400 nucleotides. A similar phenomenon has been described for reverse transcriptase [7]. Secondly, RNA polymerase could read through on the same molecule circularized, or on another molecule in a dimer. Dimer formation or circularization which may occur by base.pairing of the 'sticky ends', will not yield long-lived structures in view of the small number of basepairs involved. Moreover, it is difficult to conceive how base-pairing at the DNA termini is
Fig. 3. Electron microg~aphs of the 2200-nucleotide transcript. Unlabeled transcripts were separated under non-denaturing conditions on a 1.25% agarose gel [3]. RNA was electroeluted from the gel according to Goodman and McDonald [8] and ethanol-precipitated. RNA was dissolved in Tris-HC1 (pH 7.5)/0.1 raM EDTA and spread from 40% formamide/1.6 M urea/90 mM Tris-HCl (pH 8,5)/2 mM EDTA/0.004% cytochrome c on a hypophase of triple-distilled water. To unfold the RNA, the solution was incubated at 100°C for 1 rain, quenched on ice and spread as described above. The denatured RNA was renatured in 70% formamide/ 0.3 M NaC1/10 mM Tris-HC1 (pH 8_5)/1 mM EDTA for 15 rain at 50°C. The RNA was spread from 52% formamide/1 M urea/94 mM Tris-HC1 (pH 8_5)/2.4 mM EDTA/0.12 M NaC1/ 0.004% cytochrome c. A. Native molecule. B. Schematic drawing of A. C. Unfolded RNA molecule. The bar represents 0.1 ~m.
448
the melted RNA restores the original, partially double-stranded structure. If the 2200.nucleotide chains had been formed by the read-through mechanism, two kinds of molecule would be found: hairpin structures indistinguishable from those observed synthesized on taft-to-tail dimers, but also extended chains synthesized on the equally probably head-to-tail dimers. Molecules transcribed from circles would be only of the second, non-hairpin type. Since in non-denaturing gels only one component is observed (data not shown), we feel that the read-through mechanism can be excluded. The terminal strand switch of RNA polymerase appears to require protruding ends of a template, since DNA fragments treated with $1 nuclease do not yield the 2200-nucieotide product when transcribed (Fig. 2, lane 4). Even after prolonged exposure, no such band becomes visible in lane 4 (data not shown). Whatever is the mechanism of their formation, it is clear that the long transcripts are initiated on the promoters of the rRNA operon. Experiments in vitro have implicated ppGpp as a direct effector of rRNA synthesis [9,10]. This nucleotide tetraphosphate acts on the initiation step of the rRNA synthesis [9,10]. In our system, transcription is also inhibited by ppGpp (Fig. 2, lane 3). There is a decrease in transcripts of 900 as well as of 2300 nucleotides. This supports our conclusion that transcription is initiated solely at P1 and P2Thus, RNA synthesis on a DNA fragment by holo-
enzyme starts only on the promoters. This system will allow kinetic and mechanism studies of effectors, e.g., ppGpp. The strand switching artifact does not interfere with the analysis of specific promoter sites. We thank Adri van Vliet for his excellent technical assistance and Mr. K. Gilissen for printing the photographs. This investigation was carried out under the auspices of the Netherlands Foundation for Chemical Research (S.O.N.) with the financial aid from the Netherlands Organization for the Advancement of Pure Research (Z.W.O.). References
10ostra, B.A., AB, G. and Gruber, M. (1980) Mol. Gem Genet. 177,291-295 2 Hamming, J., Gruber, M. and AB, G. (1979) Nucleic Acids Res. 7, 1019-1034 • 3 McMaster, G.K. and Carmichael, C.G. (1977) Proc. Natl. Acad Sci. USA 74, 4835-4838 4 Hamming, J., Arnberg, A.C., AB, G. and Gruber, M. (1981 ) Nucleic Acids Res. 9, 1339-1350 5 Edens, L., Konings, R.N.H. and Schoenmakers, J.G.G. (1978) Virology 86,354-368 6 Sugimoto, K., Sugisaki, H., Okamoto, T. and Takanami, M. (1977) J. Mol. Biol. 111,487-507 7 Salser, W.A. (1974) Annu. Rev. Biochem. 43,923-965 8 Goodman, H.M. and McDonald, R.J. (1979) Methods Enzymol. 68, 75-90 90ostra, B.A., Van Ooyen, A.J.J. and Gruber, M. (1977) Mol. Gen. Genet. 152, 1-6 10 Van Ooyen, A.J.J., Gmber, M. and J~brgenscn, P. (1976) Cell 8,123-128
Biochimica et Biophysica Acta, 655 (1981) 446-448
Elsevier/North-Holland Biomedical Press
BBA Report BBA 91525 TERMINAL STRAND-SWITCHING OF E. COLI R N A POLYMERASE TRANSCRIBING A T R U N C A T E D DNA FRAGMENT BEN A. OOSTRA, ANNIKA C. ARNBERG, GEERT AB and MAX GRUBER Biochemisch Laboratorium, R i/ksuniversiteit Groningen, Nijenborgh 16, 9747 A G Groningen (The Netherlands}
(Received June llth, 1981)
Key words: RNA polymerase; Strand-switching; Transcription in vitro; rRNA; Promoter," {E. coliJ
When transcribing a restriction fragment containing the promoters and the first part of the rrnE operon of Escherichia coil, RNA polymerase holoenzyme starts exclusively on the promoters. Besides run-off transcripts, molecules longer than template-size are formed by terminal strand switch.
In our earlier studies on the regulation of rRNA synthesis in Escherichia coli we have used a hybridization-competition assay for specific rRNA transcripts formed in vitro [1]. In another approach, focussing on the initiation step of rRNA transcription, we have now used the so-called truncated template system. DNA restriction fragments carrying the first part of a gene including its promoter are transcribed in vitro and the synthesis of run-off transcripts of discrete size is followed to assess specific initiation. We used an 1.4 kilobase EcoRI fragment of phage )~rnetA20 DNA carrying the tandem promoters, P1
EcoRl
EcoRI
1 I
1 P,, P' 437
r//~l 6"sfr "DNA ~/'/A
109 174
670
100 bp Fig. 1. Map of the 1.4 kilobase EcoRI fragment from XmetA20. The distances, given in basepairs, are taken from Ref. 2. Isolation of the fragment was as described in Ref. 2. Open area, E. coli chromosomal DNA; hatched area, E. coli ribosomal DNA. bp, basepair.
and P2, of the rrnE operon (Fig. 1). Transcription was performed in a system with RNA polymerase and the DNA fragment as the only macromolecules. The size of the transcripts was determined by gel electrophoresis and autoradiography. Prior to analysis the RNAs were denatured; renaturation was prevented by glyoxylation [3]. With RNA polymerase core enzyme one band of 1400 nucleotides was observed, most probably representing end-to-end transcripts (Fig. 2, lane 1). With the RNA polymerase holoenzyme, saturated with the sigma subunit, the RNA transcripts are quite different (Fig. 2, lane 2). A broad band of about 900 nucleotides is seen which is in some instances resolved in two components of equal intensity. Most likely they are the run-off transcripts started on the initiation sites P1 and P2, which appear to be used in about equal frequency under these conditions (see also Ref. 4). The transcripts of the rRNA tandem promoters would be 950 and 850 nucleotides long. No transcripts of 1400 nucleotides are found; apparently, the holoenzyme does not start at an end. Surprisingly, also transcripts of 2200-2300 nucleotides are found. These longer transcripts constitute about 5-10% of the molecules synthesized. The occurrence of longer transcripts has also been noticed with other templates but their nature has not
0005-2787/81/0000-0000/$02.50 © 1981 Elsevier/North-Holland Biomedical Press
447
lS
S
_
16
S
_
__
2500b
_
~ b
O
effectively maintained when RNA polymerase is passing by. To decide between these two possibilities, RNA was analysed under non-denaturing conditions. In the terminal strand switch model only one kind of 2200nucleotide RNA chain is expected, which will form a hairpin structure. Such molecules were demonstrated by electron microscopy of the material eluted from the agarose gel, banding between 18 S and 16 S. Fig. 3A shows a molecule having a double-stranded stretch of about 800 basepairs connected to a 500-nueleotide single-stranded tail. Upon denaturation these molecules yield single-stranded structures with a total length of 2200 nucleotides (Fig. 3C). Renaturation of
9 S _
RN~A
DNA
Fig. 2. Agarose gel electrophoresis of RNA synthesized in vitro. RNA was synthesized in 50 /~1 reaction mixture [1] with 0.1 rrtM [a-32PIUTP (1.4 Ci/mmol). After phenol extraction and ethanol precipitation in the presence of wheat germ tRNA Glu (100 /~g/ml) the RNAs were glyoxylated and separated on 1.25% agarose gel [3]. Lane 1: RNA synthesized with RNA polymerase core enzyme; lanes 2 - 4 : RNA synthesized by RNA polymerase holoenzyme. Lane3: ppGpp present (0.5 mM); lane 4: Sl nuelease-treated DNA template used. b, bases.
been analysed further [5,6]. To explain formation of RNA chains longer than template.size two possibilities can be envisaged. First, RNA polymerase having synthesized a 850- or 950-nucleotide chain, and reaching the end of the template, switches to the complementary strand and extends the RNA chain by another 1400 nucleotides. A similar phenomenon has been described for reverse transcriptase [7]. Secondly, RNA polymerase could read through on the same molecule circularized, or on another molecule in a dimer. Dimer formation or circularization which may occur by base.pairing of the 'sticky ends', will not yield long-lived structures in view of the small number of basepairs involved. Moreover, it is difficult to conceive how base-pairing at the DNA termini is
Fig. 3. Electron microg~aphs of the 2200-nucleotide transcript. Unlabeled transcripts were separated under non-denaturing conditions on a 1.25% agarose gel [3]. RNA was electroeluted from the gel according to Goodman and McDonald [8] and ethanol-precipitated. RNA was dissolved in Tris-HC1 (pH 7.5)/0.1 raM EDTA and spread from 40% formamide/1.6 M urea/90 mM Tris-HCl (pH 8,5)/2 mM EDTA/0.004% cytochrome c on a hypophase of triple-distilled water. To unfold the RNA, the solution was incubated at 100°C for 1 rain, quenched on ice and spread as described above. The denatured RNA was renatured in 70% formamide/ 0.3 M NaC1/10 mM Tris-HC1 (pH 8_5)/1 mM EDTA for 15 rain at 50°C. The RNA was spread from 52% formamide/1 M urea/94 mM Tris-HC1 (pH 8_5)/2.4 mM EDTA/0.12 M NaC1/ 0.004% cytochrome c. A. Native molecule. B. Schematic drawing of A. C. Unfolded RNA molecule. The bar represents 0.1 ~m.
448
the melted RNA restores the original, partially double-stranded structure. If the 2200.nucleotide chains had been formed by the read-through mechanism, two kinds of molecule would be found: hairpin structures indistinguishable from those observed synthesized on taft-to-tail dimers, but also extended chains synthesized on the equally probably head-to-tail dimers. Molecules transcribed from circles would be only of the second, non-hairpin type. Since in non-denaturing gels only one component is observed (data not shown), we feel that the read-through mechanism can be excluded. The terminal strand switch of RNA polymerase appears to require protruding ends of a template, since DNA fragments treated with $1 nuclease do not yield the 2200-nucieotide product when transcribed (Fig. 2, lane 4). Even after prolonged exposure, no such band becomes visible in lane 4 (data not shown). Whatever is the mechanism of their formation, it is clear that the long transcripts are initiated on the promoters of the rRNA operon. Experiments in vitro have implicated ppGpp as a direct effector of rRNA synthesis [9,10]. This nucleotide tetraphosphate acts on the initiation step of the rRNA synthesis [9,10]. In our system, transcription is also inhibited by ppGpp (Fig. 2, lane 3). There is a decrease in transcripts of 900 as well as of 2300 nucleotides. This supports our conclusion that transcription is initiated solely at P1 and P2Thus, RNA synthesis on a DNA fragment by holo-
enzyme starts only on the promoters. This system will allow kinetic and mechanism studies of effectors, e.g., ppGpp. The strand switching artifact does not interfere with the analysis of specific promoter sites. We thank Adri van Vliet for his excellent technical assistance and Mr. K. Gilissen for printing the photographs. This investigation was carried out under the auspices of the Netherlands Foundation for Chemical Research (S.O.N.) with the financial aid from the Netherlands Organization for the Advancement of Pure Research (Z.W.O.). References
10ostra, B.A., AB, G. and Gruber, M. (1980) Mol. Gem Genet. 177,291-295 2 Hamming, J., Gruber, M. and AB, G. (1979) Nucleic Acids Res. 7, 1019-1034 • 3 McMaster, G.K. and Carmichael, C.G. (1977) Proc. Natl. Acad Sci. USA 74, 4835-4838 4 Hamming, J., Arnberg, A.C., AB, G. and Gruber, M. (1981 ) Nucleic Acids Res. 9, 1339-1350 5 Edens, L., Konings, R.N.H. and Schoenmakers, J.G.G. (1978) Virology 86,354-368 6 Sugimoto, K., Sugisaki, H., Okamoto, T. and Takanami, M. (1977) J. Mol. Biol. 111,487-507 7 Salser, W.A. (1974) Annu. Rev. Biochem. 43,923-965 8 Goodman, H.M. and McDonald, R.J. (1979) Methods Enzymol. 68, 75-90 90ostra, B.A., Van Ooyen, A.J.J. and Gruber, M. (1977) Mol. Gen. Genet. 152, 1-6 10 Van Ooyen, A.J.J., Gmber, M. and J~brgenscn, P. (1976) Cell 8,123-128