JOURNAL OF VIROLOGY, May 1975, p. 1286-1288 Copyright 0 1975 American Society for Microbiology
Vol. 15, No. 5 Printed in U.S.A.
DNA Strand Specificity of Transcripts Produced In Vivo and In Vitro by RNA Polymerase from SP82-Infected Bacillus subtilis JONATHON M. LAWRIE, GEORGE B. SPIEGELMAN, AND H. R. WHITELEY* University of Washington, School of Medicine, Department of Microbiology, Seattle, Washington 98195
Received for publication 16 December 1974
Phage-specific RNA synthesized early in the infection of Bacillus subtilis with SP82 hybridizes to both heavy (H) and light (L) strands of SP82 DNA nearly equally. Phage RNA synthesized during the middle of the infection hybridizes preferentially to the H strand. The ratio of H/L strand binding of RNAs synthesized in vitro by RNA polymerases isolated from uninfected and infected cells resembles the ratios of early and middle phage RNA classes, respectively. This supports the conclusion that a modified RNA polymerase is required for the transcription of middle RNA classes. The synthesis of six temporal classes of phage-specific RNA during the infection of Bacillus subtilis by phage SP82 (8) and by the related phage SPOl (4, 5) can be distinguished by competition-hybridization. Evidence has been obtained that a modified RNA polymerase (9) catalyzes the synthesis of classes of RNA produced in the middle of the phage infection. The polymerase isolated from SP82-infected cells consisted of the core subunits (0', a2), little or no a subunit, and several (3 to 5) small polypeptides, whereas the enzyme from uninfected cells had the expected subunit composition (,B',B, a, a2). A similar modified subunit structure has also been found for polymerase isolated from SPOl-infected cells (2, 4). Competition-hybridization experiments showed that the RNA produced in vitro by the SP82-modified enzyme contained temporal classes characteristic of the middle of the phage infection; RNA synthesized in vitro by the host enzyme had only early RNAs (8). In the present communication, isolated heavy (H) and light (L) strands of SP82 DNA have been used to analyze the strand specificity of transcription during the normal lytic cycle and during in vitro synthesis by host and modified RNA polymerases. Centrifugation of denatured SP82 DNA in CsCl yields two bands with buoyant densities of 1.751 g/cm3 (L strand) and 1.761 g/cm 3 (H strand); DNA of native buoyant density, 1.742 g/cm3, is produced when material from these two bands is hybridized (7). The difference in densities of the two strands can be increased to 45 mg/cm3 by reaction with polyguanylic acid [poly(G); 10], thereby providing better strand separation. To isolate H and L strands for use in hybridization experiments, 3 mg of purified
SP82 DNA were denatured and complexed with poly(G) (Sigma Chemical Co., St. Louis, Mo.) according to Champoux and Hogness (1). CsCl was added to a final concentration of 60% (wt/vol) and the preparation was centrifuged at 96,600 x g for 60 h at 10 to 15 C in an angle head 50 rotor using a Beckman model L centrifuge. The gradients were fractionated and H- and L-strand fractions were pooled, concentrated, and dialyzed. Prior to use in hybridization, each preparation was self-annealed in 0.3 M NaCl, 0.03 M sodium citrate at 65 C for 8 h to complex any DNA of the opposite strand. RNA-DNA hybridizations were performed according to Nygaard and Hall (6) with the following modifications: 3 ug of H- or L-strand DNA and 20 ,g of 3H-labeled RNA were incubated at 65 C for 18 h in a total volume of 0.25 ml of 0.3 M NaCl and 0.03 M sodium citrate, and then treated for 1 h at 37 C with RNase (10 U of Ti per ml and 10 jig of RNase A per ml, both from Sigma Chemical Co., St. Louis, Mo.). RNase-resistant material was collected by filtration on B-6 membrane filters (Schleicher and Schuell, Inc., Keene, N.H.) and washed with 75 ml of 0.5 M KCl-0.01 M Tris buffer, pH 7.5. The filters were dried and the radioactivity was determined in a scintillation counter using a toluenebased scintillant. Each experiment was performed at least three times. Self-annealing of 3H-labeled RNAs was determined by hybridization in the absence of DNA. The methods used for pulse labeling and extracting RNA, for isolation of RNA synthesized in vitro, for purification and assay of RNA polymerases, and for the preparation of SP82 DNA have been described (8, 9). Table 1 compares the hybridization of H and
1286
1287
NOTES
VOL. 15, 1975
TABLE 1. Hybridization of RNAs synthesized in vivo and in vitro to isolated H and L strands of SP82 DNA
'H-labeled RNA synthesized: In vivo - 2 to 3 minc In vivo 0 to 4 minc In vivo 0 to 5 minc In vivo 0 to 7 minc In vivo 5 to 7 minc In vivo 9 to 13 minc In vivo 12 to 15 minc In vivo 17 to 20 minc In vivo 21 to 25 minc In vivo CM 2 to 8 mind In vitro host enzyme In vitro SP82-modified enzyme
H strand % Counts/mm 47 1,669 73 3,110 80 4,242 94 23,198 95 19,569 95 24,439 97 10,178 97 20,133 98 26,468 57 3,062
2,226 5,767
73 95
L strand Counts/mm
1,862 1,174 1,071 1,607
Self-annealing'
2,267
53 27 20 6 5 5 3 3 2 43
1.2 0.7 0.5 0.9 2.3 1.7 3.5 4.8 1.7 4.0
825 295
27 5
4.0 4.3
936
1,188 320 522 498
Percentage of total bound radioactivity. Expressed as percentage of input radioactivity which is RNase resistant after hybridization in the absence of DNA. ['H Juridine (1 AsCi/ml) (36.4 Ci/mmol, New England Nuclear) was added at different intervals, expressed as minutes after infection with SP82. d Chloramphenicol (CM) was added 2 min before SP82, ['H ]uridine was added 2 min after addition of SP82, and the culture was harvested 6 min later. a
I
L strands of SP82 DNA with pulse-labeled, 'H-labeled RNAs extracted at various times during the infection of B. subtilis with SP82. RNA synthesized during the first few minutes of the infection hybridized to both strands of SP82 DNA. Thereafter, there was a gradual shift to transcription from the H strand and preferential transcription from the H strand continued throughout the rest of the infection. A similar pattern of synthesis has been found for B.
RNA synthesized in vitro by the polymerase isolated from SP82-infected cells (i.e., SP82modified enzyme) hybridized predominantly to the H strand. This indicates that the modified enzyme has the strand specificity characteristic of in vivo RNA synthesis during the middle and latter part of the lytic cycle and supports our earlier conclusion that the modified enzyme has an altered transcriptional specificity. Preliminary experiments (Spiegelman, Lawrie, and subtilis infected with SPOl (4). Competition- Whiteley, unpublished observations) indicate hybridization experiments employing a series of that the ratio of H/L strand transcription varies RNA preparations isolated at different times in depending on the complement of small polypepthe infection show that the small amount of tides copurified with the modified enzyme. radioactivity binding to the L strand late in the Competition-hybridization experiments using infection represents synthesis of two temporal separated H and L strands are in progress to RNA classes (J. M. Lawrie et al., manuscript in determine the pattern of transcription of the six preparation). Table 1 also shows that when temporal RNA classes detected during phage chloramphenicol was added prior to phage in- infection and to analyze the product RNAs fection, the switch to H strand transcription did synthesized in vitro by different polymerase not occur. Thus, as in SPOl infections (4), preparations. transcription beyond early genes requires proThis research was supported by grant VC-46B from the tein synthesis. American Cancer Society and by Public Health Service grant The RNA produced in vitro by the host GM-20784 from the National Institute of General Medical holoenzyme hybridized to both strands yielding Sciences. HRW is a recipient of Research Career Award a ratio of H/L transcription which was similar to K6-GM-442 from the National Institute of General Medical that obtained with RNA produced in vivo Sciences. during the first 5 min of infection and similar to LITERATURE CITED RNA produced in the presence of chloramphen- 1. Champoux, J. J., and D. S. Hogness. 1972. The topograicol. These results, together with earlier obserphy of lambda DNA:polyriboguanylic acid binding vations on the classes of RNA detected by sites and base composition. J. Mol. Biol. 71:383-405. J. J., and E. P. Geiduschek. 1973. Transcription competition-hybridization experiments (8), are 2. Duffy, specificity of an RNA polymerase fraction from bacteconsistent with the interpretation that host riophage SPOl-infected B. subtilis. FEBS Lett. polymerase transcribed only the early sequences 34:172-174. in vitro. 3. Fox, T. D., and J. Pero. 1974. New phage-SPOl-induced
1288
4. 5.
6. 7.
NOTES
polypeptides associated with Bacillus subtilis RNA polymerase. Proc. Natl. Acad. Sci. U.S.A. 71:27612765. Gage, L. P., and E. P. Geiduschek. 1971. RNA synthesis during bacteriophage SPOl development: six classes of SPOl RNA. J. Mol. Biol. 57:279-300. Gage, L. P., and E. P. Geiduschek. 1971. RNA synthesis during bacteriophage SPOl development. II. Some modulations and prerequisites of the transcription program. Virology 44:200-210. Nygaard, A. P., and B. D. Hall. 1963. A method for the detection of RNA-DNA complexes. Biochem. Biophys. Res. Commun. 12:98-104. Sheldrick, P., and W. Szybalski. 1967. Distribution of
J. VIROL.
pyrimidine "clusters" between the complementary DNA strands of certain Bacillus bacteriophages. J. Mol. Biol. 29:217-228. 8. Spiegelman, G. B., and H. R. Whiteley. 1974. In vivo and in vitro transcription by ribonucleic acid polymerase from SP82-infected Bacillus subtilis. J. Biol. Chem. 249:1483-1489. 9. Spiegelman, G. B., and H. R. Whiteley. 1974. Purification of ribonucleic acid polymerase from SP82-infected Bacillus subtilis. J. Biol. Chem. 249:1476-1482. 10. Truffaut, N., B. Revet, and M.-O. Soulie. 1970. Etude comparative des DNA de phages 2C, SP8*, SP82, 0e, SPOl et SP50. Eur. J. Biochem. 15:391-400.
Vol. 15, No. 5 Printed in U.S.A.
DNA Strand Specificity of Transcripts Produced In Vivo and In Vitro by RNA Polymerase from SP82-Infected Bacillus subtilis JONATHON M. LAWRIE, GEORGE B. SPIEGELMAN, AND H. R. WHITELEY* University of Washington, School of Medicine, Department of Microbiology, Seattle, Washington 98195
Received for publication 16 December 1974
Phage-specific RNA synthesized early in the infection of Bacillus subtilis with SP82 hybridizes to both heavy (H) and light (L) strands of SP82 DNA nearly equally. Phage RNA synthesized during the middle of the infection hybridizes preferentially to the H strand. The ratio of H/L strand binding of RNAs synthesized in vitro by RNA polymerases isolated from uninfected and infected cells resembles the ratios of early and middle phage RNA classes, respectively. This supports the conclusion that a modified RNA polymerase is required for the transcription of middle RNA classes. The synthesis of six temporal classes of phage-specific RNA during the infection of Bacillus subtilis by phage SP82 (8) and by the related phage SPOl (4, 5) can be distinguished by competition-hybridization. Evidence has been obtained that a modified RNA polymerase (9) catalyzes the synthesis of classes of RNA produced in the middle of the phage infection. The polymerase isolated from SP82-infected cells consisted of the core subunits (0', a2), little or no a subunit, and several (3 to 5) small polypeptides, whereas the enzyme from uninfected cells had the expected subunit composition (,B',B, a, a2). A similar modified subunit structure has also been found for polymerase isolated from SPOl-infected cells (2, 4). Competition-hybridization experiments showed that the RNA produced in vitro by the SP82-modified enzyme contained temporal classes characteristic of the middle of the phage infection; RNA synthesized in vitro by the host enzyme had only early RNAs (8). In the present communication, isolated heavy (H) and light (L) strands of SP82 DNA have been used to analyze the strand specificity of transcription during the normal lytic cycle and during in vitro synthesis by host and modified RNA polymerases. Centrifugation of denatured SP82 DNA in CsCl yields two bands with buoyant densities of 1.751 g/cm3 (L strand) and 1.761 g/cm 3 (H strand); DNA of native buoyant density, 1.742 g/cm3, is produced when material from these two bands is hybridized (7). The difference in densities of the two strands can be increased to 45 mg/cm3 by reaction with polyguanylic acid [poly(G); 10], thereby providing better strand separation. To isolate H and L strands for use in hybridization experiments, 3 mg of purified
SP82 DNA were denatured and complexed with poly(G) (Sigma Chemical Co., St. Louis, Mo.) according to Champoux and Hogness (1). CsCl was added to a final concentration of 60% (wt/vol) and the preparation was centrifuged at 96,600 x g for 60 h at 10 to 15 C in an angle head 50 rotor using a Beckman model L centrifuge. The gradients were fractionated and H- and L-strand fractions were pooled, concentrated, and dialyzed. Prior to use in hybridization, each preparation was self-annealed in 0.3 M NaCl, 0.03 M sodium citrate at 65 C for 8 h to complex any DNA of the opposite strand. RNA-DNA hybridizations were performed according to Nygaard and Hall (6) with the following modifications: 3 ug of H- or L-strand DNA and 20 ,g of 3H-labeled RNA were incubated at 65 C for 18 h in a total volume of 0.25 ml of 0.3 M NaCl and 0.03 M sodium citrate, and then treated for 1 h at 37 C with RNase (10 U of Ti per ml and 10 jig of RNase A per ml, both from Sigma Chemical Co., St. Louis, Mo.). RNase-resistant material was collected by filtration on B-6 membrane filters (Schleicher and Schuell, Inc., Keene, N.H.) and washed with 75 ml of 0.5 M KCl-0.01 M Tris buffer, pH 7.5. The filters were dried and the radioactivity was determined in a scintillation counter using a toluenebased scintillant. Each experiment was performed at least three times. Self-annealing of 3H-labeled RNAs was determined by hybridization in the absence of DNA. The methods used for pulse labeling and extracting RNA, for isolation of RNA synthesized in vitro, for purification and assay of RNA polymerases, and for the preparation of SP82 DNA have been described (8, 9). Table 1 compares the hybridization of H and
1286
1287
NOTES
VOL. 15, 1975
TABLE 1. Hybridization of RNAs synthesized in vivo and in vitro to isolated H and L strands of SP82 DNA
'H-labeled RNA synthesized: In vivo - 2 to 3 minc In vivo 0 to 4 minc In vivo 0 to 5 minc In vivo 0 to 7 minc In vivo 5 to 7 minc In vivo 9 to 13 minc In vivo 12 to 15 minc In vivo 17 to 20 minc In vivo 21 to 25 minc In vivo CM 2 to 8 mind In vitro host enzyme In vitro SP82-modified enzyme
H strand % Counts/mm 47 1,669 73 3,110 80 4,242 94 23,198 95 19,569 95 24,439 97 10,178 97 20,133 98 26,468 57 3,062
2,226 5,767
73 95
L strand Counts/mm
1,862 1,174 1,071 1,607
Self-annealing'
2,267
53 27 20 6 5 5 3 3 2 43
1.2 0.7 0.5 0.9 2.3 1.7 3.5 4.8 1.7 4.0
825 295
27 5
4.0 4.3
936
1,188 320 522 498
Percentage of total bound radioactivity. Expressed as percentage of input radioactivity which is RNase resistant after hybridization in the absence of DNA. ['H Juridine (1 AsCi/ml) (36.4 Ci/mmol, New England Nuclear) was added at different intervals, expressed as minutes after infection with SP82. d Chloramphenicol (CM) was added 2 min before SP82, ['H ]uridine was added 2 min after addition of SP82, and the culture was harvested 6 min later. a
I
L strands of SP82 DNA with pulse-labeled, 'H-labeled RNAs extracted at various times during the infection of B. subtilis with SP82. RNA synthesized during the first few minutes of the infection hybridized to both strands of SP82 DNA. Thereafter, there was a gradual shift to transcription from the H strand and preferential transcription from the H strand continued throughout the rest of the infection. A similar pattern of synthesis has been found for B.
RNA synthesized in vitro by the polymerase isolated from SP82-infected cells (i.e., SP82modified enzyme) hybridized predominantly to the H strand. This indicates that the modified enzyme has the strand specificity characteristic of in vivo RNA synthesis during the middle and latter part of the lytic cycle and supports our earlier conclusion that the modified enzyme has an altered transcriptional specificity. Preliminary experiments (Spiegelman, Lawrie, and subtilis infected with SPOl (4). Competition- Whiteley, unpublished observations) indicate hybridization experiments employing a series of that the ratio of H/L strand transcription varies RNA preparations isolated at different times in depending on the complement of small polypepthe infection show that the small amount of tides copurified with the modified enzyme. radioactivity binding to the L strand late in the Competition-hybridization experiments using infection represents synthesis of two temporal separated H and L strands are in progress to RNA classes (J. M. Lawrie et al., manuscript in determine the pattern of transcription of the six preparation). Table 1 also shows that when temporal RNA classes detected during phage chloramphenicol was added prior to phage in- infection and to analyze the product RNAs fection, the switch to H strand transcription did synthesized in vitro by different polymerase not occur. Thus, as in SPOl infections (4), preparations. transcription beyond early genes requires proThis research was supported by grant VC-46B from the tein synthesis. American Cancer Society and by Public Health Service grant The RNA produced in vitro by the host GM-20784 from the National Institute of General Medical holoenzyme hybridized to both strands yielding Sciences. HRW is a recipient of Research Career Award a ratio of H/L transcription which was similar to K6-GM-442 from the National Institute of General Medical that obtained with RNA produced in vivo Sciences. during the first 5 min of infection and similar to LITERATURE CITED RNA produced in the presence of chloramphen- 1. Champoux, J. J., and D. S. Hogness. 1972. The topograicol. These results, together with earlier obserphy of lambda DNA:polyriboguanylic acid binding vations on the classes of RNA detected by sites and base composition. J. Mol. Biol. 71:383-405. J. J., and E. P. Geiduschek. 1973. Transcription competition-hybridization experiments (8), are 2. Duffy, specificity of an RNA polymerase fraction from bacteconsistent with the interpretation that host riophage SPOl-infected B. subtilis. FEBS Lett. polymerase transcribed only the early sequences 34:172-174. in vitro. 3. Fox, T. D., and J. Pero. 1974. New phage-SPOl-induced
1288
4. 5.
6. 7.
NOTES
polypeptides associated with Bacillus subtilis RNA polymerase. Proc. Natl. Acad. Sci. U.S.A. 71:27612765. Gage, L. P., and E. P. Geiduschek. 1971. RNA synthesis during bacteriophage SPOl development: six classes of SPOl RNA. J. Mol. Biol. 57:279-300. Gage, L. P., and E. P. Geiduschek. 1971. RNA synthesis during bacteriophage SPOl development. II. Some modulations and prerequisites of the transcription program. Virology 44:200-210. Nygaard, A. P., and B. D. Hall. 1963. A method for the detection of RNA-DNA complexes. Biochem. Biophys. Res. Commun. 12:98-104. Sheldrick, P., and W. Szybalski. 1967. Distribution of
J. VIROL.
pyrimidine "clusters" between the complementary DNA strands of certain Bacillus bacteriophages. J. Mol. Biol. 29:217-228. 8. Spiegelman, G. B., and H. R. Whiteley. 1974. In vivo and in vitro transcription by ribonucleic acid polymerase from SP82-infected Bacillus subtilis. J. Biol. Chem. 249:1483-1489. 9. Spiegelman, G. B., and H. R. Whiteley. 1974. Purification of ribonucleic acid polymerase from SP82-infected Bacillus subtilis. J. Biol. Chem. 249:1476-1482. 10. Truffaut, N., B. Revet, and M.-O. Soulie. 1970. Etude comparative des DNA de phages 2C, SP8*, SP82, 0e, SPOl et SP50. Eur. J. Biochem. 15:391-400.
