induced shutoff of host transcription remains obscure. ... host RNA polymerase-associated protein. ... 2A shows the 35S-labeled proteins present in a DNA-free.
Vol. 60, No. 3
JOURNAL OF VIROLOGY, Dec. 1986, p. 1145-1147
0022-538X/86/121145-03$02.00/0 Copyright © 1986, American Society for Microbiology
The Bacteriophage T4 Regulatory Protein gpunflalc Binds to DNA in the Absence of RNA Polymerase D. PETER SNUSTAD,* NANCY HAAS, AND DAVID G. OPPENHEIMER
Department of Genetics and Cell Biology, College of Biological Sciences, University of Minnesota, St. Paul, Minnesota 55108-1095 Received 23 June 1986/Accepted 15 August 1986
DNA-cellulose chromatography and two-dimensional gel electrophoresis have been used to demonstrate the DNA-binding capacity of bacteriophage T4 gpunf7alc. The unflalc protein does not bind to DNA via an association with RNA polymerase; gpunflalc was shown to bind to DNA after separation from RNA polymerase and other large proteins by Sephadex chromatography. Infection of Escherichia coli with bacteriophage T4 has long been known to result in the rapid arrest of host transcription. Yet the mechanism responsible for this virusinduced shutoff of host transcription remains obscure. Sirotkin et al. (12) concluded that the product of the T4 unflalc gene, the only T4 gene implicated in the shutoff of host transcription by the results of in vivo experiments, is a host RNA polymerase-associated protein. After failing to detect any interaction between gpunflalc and RNA polymerase by affinity chromatography, we began to investigate the possibility that gpunflalc might exert its effect on transcription via a direct interaction with the DNA template. The results reported here show that gpunflalc binds to doublestranded DNA, and does so in the absence of RNA polymerase. These results suggest that the primary site of action of gpunftalc is the DNA template, not RNA polymerase. The product of the phage T4 unflalc gene has four welldocumented phenotypic effects in infected cells: (i) unfolding of the host nucleoid (12, 15, 18), (ii) inhibition of transcription of deoxycytosine-containing phage T4 and phage A DNAs (8, 11, 16), (iii) rapid shutoff of host transcription (12, 14, 18), and (iv) the occurrence of late DNA replication in restrictive hosts (E. coli strains harboring plasmid pR386) (7). These effects may all result from one primary gpunftalc activity (14). Studies on the role of gpunflalc have focused almost exclusively on its putative interaction with RNA polymerase (12, 14). Sirotkin et al. (12) concluded that gpunftalc is a 15,000-dalton polypeptide known to bind to E. coli RNA polymerase (17). However, this conclusion has not been confirmed (14). Instead, subsequent studies have shown gpunflalc to have a molecular size of slightly over 18,000 daltons (6, 9). We used RNA polymerase affinity chromatography to investigate the interaction between T4 proteins and RNA polymerase. Although several T4 proteins which are synthesized during the first 12 min after infection were shown to interact with RNA polymerase in these experiments, gpunftalc was not one of them (N. Haas and D. P. Snustad, unpublished data). These results prompted us to investigate the possibility that gpunflalc might regulate transcription through a direct interaction with DNA. The DNA-binding property of gpunflalc has been demonstrated by DNA-cellulose chromatography as described by Alberts and co-workers (1, 2). DNA-free lysates (2) were prepared in 50 mM NaCl from T4D+-infected cells labeled *
with 3S from 4 to 10 min postinfection (13) and were subjected to calf thymus DNA-cellulose chromatography (1, 2). The 35S-labeled T4 proteins retained by DNA-cellulose in 50 mM NaCl were subsequently eluted with 2 M NaCl, separated by two-dimensional isoelectric focusing and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (10) and recorded by scintillation autoradiography (3). A typical DNA-cellulose elution profile is shown in Fig. 1. The results shown in Fig. 2A to C demonstrate that gpunflalc was quantitatively retained by DNA-cellulose when present in 50 mM NaCl and was subsequently eluted by 2 M NaCl. Figure 2A shows the 35S-labeled proteins present in a DNA-free lysate labeled from 4 to 10 min after infection with T4D+; the location of gpunflalc on two-dimensional gels has been previously established by studies of spontaneous revertants of the mutant unf39x5 (6). Figure 2B shows the labeled proteins in the lysate shown in Fig. 2A that are not bound to DNA-cellulose in 50 mM NaCl (fractions 2 to 7 of Fig. 1). Figure 2C shows the bound proteins that are released in 2 M NaCl (fractions 18 and 19 of Fig. 1). These results demonstrate that gpunflalc is quantitatively bound to DNAcellulose when present in 50 mM NaCl. The results shown in Fig. 2D demonstrate that the protein labeled gpunflalc (Fig. 2A and C) is indeed gpunflalc. This 10 2M
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Corresponding author.
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molecular-weight proteins. A 75-ml Sephadex G-75 column (2 by 45 cm) was prepared and calibrated with Blue Dextran, Phenol Red, bovine serum albumin, and soybean trypsin inhibitor as described by Cooper (4), with a flow rate of 33 ml/h. 35S-labeled RNA polymerase was purified (Fig. 3A) from uninfected E. coli B/5 cells by the procedure of Gross et al. (5) and shown to elute in the excluded volume of the Sephadex column (Fig. 3B). The elution profile of the 35S-labeled proteins present in a DNA-free lysate of E. coli B/5 cells labeled from 4 to 10 min after infection with T4D+ is shown in Fig. 3C. The fractions containing the lowmolecular-weight proteins (fractions 26 to 40) were pooled and subjected to DNA-cellulose chromatography as described above. Figure 2F shows an autoradiograph of a two-dimensional isoelectric-focusing sodium dodecyl sulfate-polyacrylamide gel of the labeled DNA-binding proteins present in the pooled low-molecular-weight fractions from the Sephadex column. The results clearly demonstrate that gpunflalc binds to DNA in the absence of RNA polymerase and other large proteins. We have no information on the binding specificity or association constants for gpunflalc-DNA interactions. However, Alberts and Herrick (2) have calculated that to remain bound to DNA in 50 mM NaCl under the conditions used, even proteins with one binding site per 10 nucleotide pairs must have dissociation constants of less than 10-5 M.
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autoradiograph shows the DNA-binding proteins present in a lysate processed exactly like those yielding the results shown in Fig. 2C, except that the infecting phage was the mutant unf39x5 (15). The results shown in Fig. 2E demonstrate that gpunflalc binds to DNA, not to cellulose. One-half of a T4D+-infected cell lysate was chromatographed on DNA-cellulose (Fig. 2A to C); the other half was subjected to DNA-free cellulose chromatography. Figure 2E shows the labeled proteins in the lysate that bound to DNA-free cellulose in 50 mM NaCl and subsequently eluted in NaCl. As mentioned above, gpunflalc was thought to interact with RNA polymerase (12). It was critical, therefore, to demonstrate that the retention of gpunflalc by DNAcellulose was not mediated by an association with RNA polymerase. This was accomplished by separating gpunflalc (z18,000 daltons) from RNA polymerase (=480,000 daltons) and other large proteins by Sephadex G-75 chromatography and by demonstrating the DNA-binding capacity of gpunflalc present in pooled fractions containing only low-
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FIG. 3. Documentation of Sephadex G-75 chromatographic separation of gpunf7alc from E. coli RNA polymerase. (A) Autoradiograph of an 8.75% linear sodium dodecyl sulfate-polyacrylamide gel of the E. coli RNA polymerase preparation used to demonstrate its presence in the excluded volume of the Sephadex G-75 column. (B) Sephadex G-75 elution profiles of Phenol Red, Blue Dextran, and RNA polymerase. OD540, optical density at 540 nm. (C) Sephadex G-75 elution profiles of bovine serum albumin (BSA), soybean trypsin inhibitor (STI), and the 35S-labeled proteins present in the DNA-free T4D+-infected cell lysate. Fractions 26 to 40 of the cell lysate were pooled and used to demonstrate the DNA-binding capacity of gpunflalc in the absence of RNA polymerase by DNAcellulose chromatography (see Fig. 2F). OD280, optical density at 280 nm.
VOL. 60, 1986
Proteins that bind selectively at specific sites on DNA would require even lower dissociation constants to be retained by DNA-cellulose under these conditions. Thus, the affinity of gpunflalc for DNA must be substantial. Since phage T4 DNA normally contains glucosylated 5-hydroxymethylcytosine whereas E. coli DNA contains unmodified cytosine, we believe that the effects of gpunflalc on transcription may well be explained by the differential affinity of gpunflalc for glucosylated 5-hydroxymethylcytosine- and deoxycytosine-containing DNAs. We are no longer pursuing this project, but we hope that someone will investigate this possibility. This research was supported initially by Public Health Service grant GM25417 from the National Institutes of Health and subsequently by a grant from the Graduate School of the University of Minnesota. LITERATURE CITED 1. Alberts, B. M., F. J. Amodio, M. Jenkins, E. D. Gutmann, and F. L. Ferris. 1968. Studies with DNA-cellulose chromatography. I. DNA-binding proteins from Escherichia coli. Cold Spring Harbor Symp. Quant. Biol. 33:289-305. 2. Alberts, B. M., and G. Herrick. 1971. DNA-cellulose chromatography. Methods Enzymol. 21:198-217. 3. Bohner, W. M., and R. A. Laskey. 1974. A film detection methlod for tritium-labeled proteins and nucleic acids in polyacrylamide gels. Eur. J. Biochem. 46:83-88. 4. Cooper, T. G. 1977. The tools of biochemistry. John Wiley & Sons, Inc., New York. 5. Gross, C., F. Engbaek, T. Flammang, and R. Burgess. 1976. Rapid micromethod for the purification of Escherichia coli ribonucleic acid polymerase and the preparation of bacterial extracts active in ribonucleic acid synthesis. J. Bacteriol. 128:382-389. 6. Herman, R. E., N. Haas, and D. P. Snustad. 1984. Identification of the bacteriophage T4 unf (=alc) gene product, a protein involved in the shutoff of host transcription. Genetics 108:305-317. 7. Herman, R. E., and D. P. Snustad. 1985. Bacteriophage T4 unf (=alc) gene function is required for late replication in the presence of plasmid pR386. J. Virol. 53:430-439.
NOTES
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8. Kutter, E. M., D. Bradley, R. Schenck, B. S. Guttman, and R. Laiken. 1981. Bacteriophage T4 Alc gene product: general inhibitor of transcription from cytosine-containing DNA. J. Virol. 40:822-829. 9. Kutter, E., R. Drivdahl, and K. Rand. 1984. Identification and characterization of the alc gene product of bacteriophage T4. Genetics 108:291-304. 10. O'Farrell, P. H. 1975. High resolution two-dimensional electrophoresis of proteins. J. Biol. Chem. 250:4007-4021. 11. Pearson, R. E., and L. Snyder. 1980. Shutoff of lambda gene expression by bacteriophage T4: role of the T4 alc gene. J. Virol. 35:194-202. 12. Sirotkin, K., J. Wei, and L. Snyder. 1977. T4 bacteriophagecoded RNA polymerase subunit blocks host transcription and unfolds the host chromosome. Nature (London) 265:28-32. 13. Snustad, D. P., C. J. H. Bursch, K. A. Parson, and S. H. Hefeneider. 1976. Mutants of bacteriophage T4 deficient in the ability to induce nuclear disruption: shutoff of host DNA and protein synthesis, gene dosage experiments, identification of a restrictive host, and possible biological significance. J. Virol. 18:268-288. 14. Snustad, D. P., L. Snyder, and E. Kutter. 1983. Effects on host genome structure and expression, p. 40-55. In C. K. Mathews, E. M. Kutter, G. Mosig, and P. B. Berget (ed.), Bacteriophage T4. American Society for Microbiology, Washington, D.C. 15. Snustad, D. P., M. A. Tigges, K. A. Parson, C. J. H. Bursch, F. M. Caron, J. F. Koerner, and D. J. Tutas. 1976. Identification and preliminary characterization of a mutant defective in the bacteriophage T4-induced unfolding of the Escherichia coli nucleoid. J. Virol. 17:622-641. 16. Snyder, L., L. Gold, and E. Kutter. 1976. A gene of bacteriophage T4 whose product prevents true late transcription on cytosine-containing T4 DNA. Proc. Natl. Acad. Sci. USA 73:3098-3102. 17. Stevens, A. 1972. New small polypeptides associated with DNA-dependent RNA polymerases of Escherichia coli after infection with bacteriophage T4. Proc. Natl. Acad. Sci. USA 69:603-607. 18. Tigges, M. A., C. J. H. Bursch, and D. P. Snustad. 1977. Slow switchover from host RNA synthesis to bacteriophage RNA synthesis after infection of Escherichia coli with a T4 mutant defective in the bacteriophage T4-induced unfolding of the host nucleoid. J. Virol. 24:775-785.
