Volume 7 Number 6 1979
Nucleic Acids Research
Plasmid replication functions. IV. Promoters in the replication region of plasmid R6-5
Patrick M.Slocombe, Susan Ely and Kenneth N.Timmis Max-Planck-Institut fUr Molekulare Genetik, Ihnestr. 63-73, 1000 Berlin-West 33, GFR
Received 24 August 1979 ABSTRACT Eight RNA polymerase binding sites have been shown to map within the EcoRI fragment E-2 (replication region RepA EcoRI fragment) of plasmid R6-5 and all but one have been shown to contain active promoters of transcription. Three of the identified promoters are located within a 2.7 kb region essential for controlled, autonomous plasmid replication and may be involved in the functional expression of the three R6-5 replication determinants that have thus far been identified, namely the origin of vegetative replication, oriV, the replication control gene, cop, and the determinant of an essential, positive-acting element, designated RepA. INTRODUCTION
The replication of cellular genetic elements, chromosomes and extrachromosomes, is strictly regulated and coordinated with cell growth. This means that for every cell doubling there is a parallel doubling in the number of each type of cellular replicon, such that in growing cell cultures the average number of each replicon per cell remains constant. This fundamental property of the regulation of replicon duplication is poorly understood despite the considerable effort invested in past years. Recently, attention has focussed on bacterial plasmids, in particular low copy number plasmids such as R6-5, Rl and F, as convenient models for studies of replicon control. The control of plasmid replication is mediated by a plasmidspecified element, the copy control (cop) gene product. This product, in the case of R6-5 and other plasmids of the incFII group, is probably a repressor protein that regulates negatively the initiation of replication at the plasmid origin of replication (oriV) and that is responsible for plasmid incompatibility (inc)
C) Information Retrieval Limited 1 Falconberg Court London Wl V 5FG England
1469
Nucleic Acids Research (1-4). This repressor is assumed to regulate the initiation of plasmid replication by inhibition of (a) plasmid attachment to a replication structure, or assembly of a replication structure on the plasmid, (b) the synthesis of an obligatory RNA primer, (c) the synthesis of a positive-acting plasmid-specified and plasmidspecific protein that participates in the initiation of DNA synthesis (i.e. acts at the RNA-DNA switch point), (d) the activity of such a protein, or (e) events, such as transcription, which "activate" the origin structure. An understanding of the control of initiation of DNA replication requires the characterization of origin of vegetative replication sequences and the proteins and controlling elements that interact with one another and with these sequences, and the analysis of the patterns of synthesis of these replication elements. This type of study requires at the outset the identification of promoters of the replication determinants and their corresponding transcription products. In this communication we report the identification of RNA polymerase binding sites in the RepA replication region of the incFII antibiotic resistance plasmid R6-5, and show by transcription studies that these sites represent active promoters. MATERIALS AND METHODS
Bacterial strains and plasmids have been described previously, as have experimental conditions for the purification of DNA samples and their cleavage with restriction endonucleases, and for the analytical and preparative electrophoresis of DNA fragments in agarose gels and their subsequent extraction from the gels (1,5). Restriction endonucleases were obtained from New England Biolabs (AvaI, glI, BglII, HaeII, PvuII, SalI), Boehringer-Mannheim (EcoRI, HincII, SmaI), and Dr. H.Mayer (PstI). Unlabelled triphosphates were obtained from Boehringer-Mannheim whereas radioactive triphosphates were purchased from AmershamBuchler. RNA polymerase binding assays: Approximately 5 pg of restriction endonuclease-generated DNA fragments were incubated with 1 ig of RNA polymerase holoenzyme prepared by Dr. K. Yoshinaga according to the method of Burgess and Jendrisak (6), in
1470
Nucleic Acids Research 200 pl of RNA polymerase binding buffer (RPBB): 20 mM Tris, pH 7.9, 50mM KCl, 10 mM MgCl2, 1 mM CaCl2, 0.1 mM EDTA, 0.1 mM dithiothreitol, and 5% glycerol (7). The reaction mixture was incubated for 10 min at 37 0 C, after which heparin was added to a final concentration of 0.5 mg/ml (8) and incubation continued for an additional 10 min. The mixture was then diluted with RPBB to 1 ml and filtered slowly through a cellulose nitrate filter (pore size 0.45 tm, Sartorius). The filter was washed three times with 2.5 ml 50mM NaCl, 20 mM Tris, pH 7.9, 8mM MgCl2, and three times with 2.5 ml of 20 mM Tris, pH 7.9, 8mM MgCl2. In some experiments 25pM rATP and 25pM rGTP were included in the binding reaction; in this case, filters were first washed three times with 2.5 ml 1 M NaCl, 20 mM Tris, pH 7.9, 8mM MgCl2 before proceeding as indicated above. RNA polymerase-bound DNA fragments were eluted by 2 washes with 0.5 ml 0.5% SDS, 10 mM Tris, pH 7.9, and collected by ethanol precipitation. Bound fragments were identified by co-electrophoresis in agarose gels with unreacted DNA fragments from the original restriction endonuclease digest. In vitro transcription reactions: 1 ig of purified linear (plasmid fragments) or supercoiled (complete plasmids) DNA template was incubated with one unit of RNA polymerase holoenzyme in 100 pl of RPBB, containing 10 pM rATP, 10 pM rGTP, 2.5 pM UTP, and 30 -50 pCi [a- 32 P]rCTP (specific activity 590 Ci/mmole). The reaction was allowed to proceed at 37 0C for the time indicated, and was terminated by phenol extraction. The extracted RNA was mixed with carrier tRNA and 1/9 vol of 3M Na-acetate, pH 5.5, and precipitated by addition of 2 vol ethanol. The precipitate was collected by centrifugation, air dried, and resuspended in 50 pl of 1 mM Tris, pH 8.0, 0.1 mM EDTA. The transfer of electrophoretically-separated DNA fragments from agarose gels to nitrocellulose sheets (Schleicher and Schull) and subsequent hybridization with radioactive RNA were performed essentially as described by Southern (9). RESULTS RNA Polymerase Binding Sites in the R6-5 RepA Replication Region. The generation of miniplasmids in vitro from restriction endonuclease fragments of the R6-5 plasmid has demonstrated that
1471
Nucleic Acids Research all functions essential for the regulated autonomous replication of this replicon are clustered on EcoRI fragment 2 - RepA EcoRI and, within this fragment, on two adjacent PstI fragments, P-4 and P-6 (5,10,11,1). RNA polymerase binding reactions were carried out initially with PstI-digested DNA from either the miniplasmid pSC102 (5) or the purified RepA EcoRI fragment (Fig.1). Seven of the eleven PstI fragments generated from pSC102 were found to bind RNA polymerase (Fig.2); strong binding of the enzyme to fragments P-1 (Km resistance fraament; ref.1), P-2, P-3, P-4 (oriV fragment; ref.12) and P-6 (cop/inc fragment; refs.1,11) Km <
Nucleic Acids Research
Plasmid replication functions. IV. Promoters in the replication region of plasmid R6-5
Patrick M.Slocombe, Susan Ely and Kenneth N.Timmis Max-Planck-Institut fUr Molekulare Genetik, Ihnestr. 63-73, 1000 Berlin-West 33, GFR
Received 24 August 1979 ABSTRACT Eight RNA polymerase binding sites have been shown to map within the EcoRI fragment E-2 (replication region RepA EcoRI fragment) of plasmid R6-5 and all but one have been shown to contain active promoters of transcription. Three of the identified promoters are located within a 2.7 kb region essential for controlled, autonomous plasmid replication and may be involved in the functional expression of the three R6-5 replication determinants that have thus far been identified, namely the origin of vegetative replication, oriV, the replication control gene, cop, and the determinant of an essential, positive-acting element, designated RepA. INTRODUCTION
The replication of cellular genetic elements, chromosomes and extrachromosomes, is strictly regulated and coordinated with cell growth. This means that for every cell doubling there is a parallel doubling in the number of each type of cellular replicon, such that in growing cell cultures the average number of each replicon per cell remains constant. This fundamental property of the regulation of replicon duplication is poorly understood despite the considerable effort invested in past years. Recently, attention has focussed on bacterial plasmids, in particular low copy number plasmids such as R6-5, Rl and F, as convenient models for studies of replicon control. The control of plasmid replication is mediated by a plasmidspecified element, the copy control (cop) gene product. This product, in the case of R6-5 and other plasmids of the incFII group, is probably a repressor protein that regulates negatively the initiation of replication at the plasmid origin of replication (oriV) and that is responsible for plasmid incompatibility (inc)
C) Information Retrieval Limited 1 Falconberg Court London Wl V 5FG England
1469
Nucleic Acids Research (1-4). This repressor is assumed to regulate the initiation of plasmid replication by inhibition of (a) plasmid attachment to a replication structure, or assembly of a replication structure on the plasmid, (b) the synthesis of an obligatory RNA primer, (c) the synthesis of a positive-acting plasmid-specified and plasmidspecific protein that participates in the initiation of DNA synthesis (i.e. acts at the RNA-DNA switch point), (d) the activity of such a protein, or (e) events, such as transcription, which "activate" the origin structure. An understanding of the control of initiation of DNA replication requires the characterization of origin of vegetative replication sequences and the proteins and controlling elements that interact with one another and with these sequences, and the analysis of the patterns of synthesis of these replication elements. This type of study requires at the outset the identification of promoters of the replication determinants and their corresponding transcription products. In this communication we report the identification of RNA polymerase binding sites in the RepA replication region of the incFII antibiotic resistance plasmid R6-5, and show by transcription studies that these sites represent active promoters. MATERIALS AND METHODS
Bacterial strains and plasmids have been described previously, as have experimental conditions for the purification of DNA samples and their cleavage with restriction endonucleases, and for the analytical and preparative electrophoresis of DNA fragments in agarose gels and their subsequent extraction from the gels (1,5). Restriction endonucleases were obtained from New England Biolabs (AvaI, glI, BglII, HaeII, PvuII, SalI), Boehringer-Mannheim (EcoRI, HincII, SmaI), and Dr. H.Mayer (PstI). Unlabelled triphosphates were obtained from Boehringer-Mannheim whereas radioactive triphosphates were purchased from AmershamBuchler. RNA polymerase binding assays: Approximately 5 pg of restriction endonuclease-generated DNA fragments were incubated with 1 ig of RNA polymerase holoenzyme prepared by Dr. K. Yoshinaga according to the method of Burgess and Jendrisak (6), in
1470
Nucleic Acids Research 200 pl of RNA polymerase binding buffer (RPBB): 20 mM Tris, pH 7.9, 50mM KCl, 10 mM MgCl2, 1 mM CaCl2, 0.1 mM EDTA, 0.1 mM dithiothreitol, and 5% glycerol (7). The reaction mixture was incubated for 10 min at 37 0 C, after which heparin was added to a final concentration of 0.5 mg/ml (8) and incubation continued for an additional 10 min. The mixture was then diluted with RPBB to 1 ml and filtered slowly through a cellulose nitrate filter (pore size 0.45 tm, Sartorius). The filter was washed three times with 2.5 ml 50mM NaCl, 20 mM Tris, pH 7.9, 8mM MgCl2, and three times with 2.5 ml of 20 mM Tris, pH 7.9, 8mM MgCl2. In some experiments 25pM rATP and 25pM rGTP were included in the binding reaction; in this case, filters were first washed three times with 2.5 ml 1 M NaCl, 20 mM Tris, pH 7.9, 8mM MgCl2 before proceeding as indicated above. RNA polymerase-bound DNA fragments were eluted by 2 washes with 0.5 ml 0.5% SDS, 10 mM Tris, pH 7.9, and collected by ethanol precipitation. Bound fragments were identified by co-electrophoresis in agarose gels with unreacted DNA fragments from the original restriction endonuclease digest. In vitro transcription reactions: 1 ig of purified linear (plasmid fragments) or supercoiled (complete plasmids) DNA template was incubated with one unit of RNA polymerase holoenzyme in 100 pl of RPBB, containing 10 pM rATP, 10 pM rGTP, 2.5 pM UTP, and 30 -50 pCi [a- 32 P]rCTP (specific activity 590 Ci/mmole). The reaction was allowed to proceed at 37 0C for the time indicated, and was terminated by phenol extraction. The extracted RNA was mixed with carrier tRNA and 1/9 vol of 3M Na-acetate, pH 5.5, and precipitated by addition of 2 vol ethanol. The precipitate was collected by centrifugation, air dried, and resuspended in 50 pl of 1 mM Tris, pH 8.0, 0.1 mM EDTA. The transfer of electrophoretically-separated DNA fragments from agarose gels to nitrocellulose sheets (Schleicher and Schull) and subsequent hybridization with radioactive RNA were performed essentially as described by Southern (9). RESULTS RNA Polymerase Binding Sites in the R6-5 RepA Replication Region. The generation of miniplasmids in vitro from restriction endonuclease fragments of the R6-5 plasmid has demonstrated that
1471
Nucleic Acids Research all functions essential for the regulated autonomous replication of this replicon are clustered on EcoRI fragment 2 - RepA EcoRI and, within this fragment, on two adjacent PstI fragments, P-4 and P-6 (5,10,11,1). RNA polymerase binding reactions were carried out initially with PstI-digested DNA from either the miniplasmid pSC102 (5) or the purified RepA EcoRI fragment (Fig.1). Seven of the eleven PstI fragments generated from pSC102 were found to bind RNA polymerase (Fig.2); strong binding of the enzyme to fragments P-1 (Km resistance fraament; ref.1), P-2, P-3, P-4 (oriV fragment; ref.12) and P-6 (cop/inc fragment; refs.1,11) Km <
