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The bovine papillomavirus type 1 transcriptional activator E2 protein binds to its DNA recognition sequence as a dimer
VIROLOGY
169,236-238
(1989)
The Bovine Papillomavirus Type 1 Transcriptional Activator E2 Protein Binds to Its DNA Recognition Sequence as a Dimer CHRISTOPHER A. MOSKALUK AND DEEPAK BASTIA’ Department
of Microbiology
and Immunology,
Received September
Duke University Medical Center, Durham, North Carolina 27710 12, 1988; accepted November
16, 1988
The transcriptional trans-activator E2 protein from bovine papillomavirus type 1 has been shown to bind to the DNA consensus sequence ACCNBGGT. We have produced ‘the DNA-binding domain of the E2 protein as a recombinant protein in Escherichia co/i. The E2 DNA-binding domain was purified as two different molecular weight forms. Using these purified proteins, we show that two molecules of the E2 protein bind to a single DNA consensus binding site. 0 1999 Academic
Press, Inc.
tion was separated on a cation exchange column (Pharmacia, mono S) into two peaks (Fig. 1). The first eluting peak comprised two forms of E2Kpn380, one minor and one major form as detected on a SDS-polyacrylamide gel stained with Coomassie blue (Fig. 1, insert). The major form migrated as a 2 1-kDa protein. The proteins in this peak were termed the L forms of E2Kpn380. The product of the second eluting peak was homogeneous, and migrated as a 16-kDa protein. This protein was termed the S form of E2Kpn380. The S form appears to be the limit digest of collagenase, because the L forms could be converted to the S form by incubating further with collagenase (data not shown). A previous study using a collagen-@-galactosidase fusion protein has shown that limit digestion with collagenase removes all but three amino acids of the collagen linker (18). The limit collagenase digestion product migrates at a slower rate than expected for the predicted mass of 14 kDa, but anomalous migration of recombinant E2 protein has been reported by other researchers (16). The two forms of the E2Kpn380 protein were used to determine DNA-binding stoichiometry in a gel retardation assay. An oligonucleotide pair (5’-GATCACCGCTAGCGGT-3’,5’-TCGAACCGCTAGCGGT-3’ was synthesized, hybridized, and cloned into BamH l/Sail cut pUC19 plasmid (19). The resulting plasmid was cut with HindIll and fcoR1, resulting in a 59-bp fragment wiith a single E2 DNA-binding consensus sequence. This fragment was purified and radioactively labeled with [a-32P]deoxyadenosine triphosphate and the Klenow fragment of DNA polymerase I. This DNA probe was incubated with an equal number of units of the S form and the L form, and an equal unit mixture of the S and L forms of the E2Kpn380 protein. The S and L protein solutions had been titrated in gel retardation
The E2 open reading frame (ORF) of the bovine papillomavirus type 1 (BPV-1) has been shown to encode a frans-acting factor that activates a BPV-1 transcriptional enhancer (1). Human papillomaviruses (HPV) contain E2 ORFs with strong sequence homology to the BPV ORF, and which code for interchangeable transcriptional activating factors (2-5). The c&acting element in the E2-dependent enhancer consists of multiple copies of the nucleotide sequence ACCNGGGT, where N is any nucleotide (6-8). The protein product of the E2 ORF has been shown to bind specifically to this DNA consensus sequence (7- 14). The E2 ORF codes for different sized E2 proteins in papillomavirus-infected cells (15, 16). The DNA-binding domain of the E2 protein is contained in the carboxy-terminal portion of the protein (12, 13). The truncated carboxy E2 fragment (E2C) has been implicated in down-regulation of E2 transcriptional activation (3, 11, 17). E2C is presumed to compete for DNA-binding sites with the activator form of E2. In this paper we show that the E2 DNA-binding domain binds to a single ACCNGGGT DNA consensus sequence with a stoichiometry of two. The 3’terminus of the BPV-1 E2 ORF was cloned into the bacterial expression vector pDSlO0 as described previously (13). This produced a fusion gene consisting of 380 nucleotides of the E2 ORF, a portion of a collagen gene, and the lac Z gene. The fusion protein was expressed in fscherichia co/i and purified in two chromatographic steps as described by Moskaluk and Bastia (14). The fusion protein was subjected to limited digestion with collagenase. This separated the E2 domain (termed E2Kpn380) from the P-galactosidase protein. Portions of the collagen linker remained attached to the E2Kpn380 protein. This protein popula’ To whom requests for reprints should be addressed. 0042.6822/89
$3.00
Copynght 0 1999 by Academic Press. Inc. All rights of reproduction I” any form resewed.
236
SHORT COMMUNICATIONS
mono S cation exchange 0.10
.0.75
8 2 0.50 0.05
= 0 2
0.25
0 ,
0
d0
retention
!
40
volume (ml)
FIG. 1. Purification of the S and L forms of the E2 DNA-binding domain. The mono S column elution profile is shown. There are two protein peaks that elute with NaCl off of the cation exchange column. The Coomassie blue-stained SDS-polyacrylamide electrophoresis gel of the two peaks (L and S) is shown in the insert. Molecularweight markers are shown with their mass given in kilodaltons (kDa).
assays previously, with one unit of DNA-binding activity arbitrarily defined as the amount of protein solution needed to shift 50% of a labeled DNA fragment containing an E2-binding site into a complexed form, with one unit equaling approximately 6 ng of each protein. Gel electrophoresis of the reaction products resolved the free DNA probe from that complexed with proteins. In previous studies using the S protein preparation, a combination gel retardation/DNase footprinting analysis on three different ACCN,GGT sites revealed that every gel retarded complex observed corresponded to a sequence-specific DNA-protein interaction (14). As expected, complexes with the larger L protein migrated more slowly than the complexes with the smaller S protein (Fig. 2). It should be noted that the gel shift contribution of the minor Lform of the E2Kpn380 protein cannot be discerned. Mixing the S and L forms together resulted in the appearance of a third DNA-protein complex, with a gel migration that was intermediate that of the S and L complexes (Fig. 2). This observation indicates that two molecules of E2 proteins can bind to a single ACCNG-GGT DNA recognition site. Denaturing conditions were not required to form the intermediate complex, so the S and L forms of the E2 DNA-binding
237
domain must not form stable associations in solution in the absence of DNA. The complexes do not form in equimolar amounts although equimolar amounts of DNA-binding activity were added. Specifically, the L complex is reduced in comparison to the SL and S complexes. This suggests that the collagen peptide attached to the L form of the protein partially interferes with the formation of protein dimers on the DNA recognition site, thus selecting for dimers of two S forms or an SL dimer. In an analogous experiment Hope and Struhl, using two different sized forms of the yeast GCN4 protein, showed that such an intermediate DNA-protein complex was indeed composed of a heterodimer of the two protein forms (20). The partial inverted repeat structure of the ACCNGGGT DNA consensus sequence was predictive of interactions with an even number of protein subunits. In addition, symmetric DNA contacts with the E2 protein were found on both halves of the consensus binding site (14). Taken together, these results indicate that two E2 protein molecules contact the binding site on roughly the same side of the DNA helix (74). It is known that E2 protein is found in various sizes in BPV-l-infected cells (16) and that the smaller E2C form is antagonistic to the larger E2 activator form (3, 11, 17). This observation, that heterodimers of different sized E2 proteins can bind to a single site in vitro, suggests that a similar phenomenon may occur in viva. The formation of E2-E2C heterodimers may cause down-regulation of the rrans-activator function as well as the E2C di-
c
s
L
SL
free *DNA
FIG. 2. E2Kpn380 protein-DNA complexes. Of a ‘*P end-labeled DNA probe that contains one E2-binding site (lane C) 10,000 Cerenkov cpm (approximately 0.01 pmol) was incubated with one unit of the S form (lane S), one unit of the L form (lane L). and a mixture of one unit each of the S and Lforms (lane SL) of the E2Kpn380 protein in a 50 ~1 volume containing 25 mM 2-(N-morpholino)ethanesulfonic acid (MES). pH 6.0, 7.5 mM MgCIZ, 50 mM KCI, 100 mM NaCI, 0.05 mM EDTA, 4 mM dithiothreitol, and 0.5 pg of sheared calf thymus DNA (Sigma). The reactions were incubated at ambient room temperature for 20 min. Prior to electrophoresis, glycerol was added to 10% (v/v) and an additional 10 pg of calf thymus DNA was added. Gel electrophoresis of the DNA-protein reactions as described by Moskaluk and Bastia (13) separated the different sized DNA-protein complexes.
SHORT COMMUNICATIONS
238
mers. This may allow the E2C to be a more effective “genetic switch,” allowing modulation of the E2 activator at a lower E2C concentration than if the E2C dimer concentration alone were the critical determining factor. ACKNOWLEDGMENTS This work was supported by grants from the National Institutes of Health and the National Cancer Institute. CM. is supported by the National Institutes of Health Medical Scientist Training Program. D.B. is an Established Investigator of the American Heart Association.
REFERENCES 1. SPALHOLZ, B. A., YANG, Y. C., and HOWLEY, P. M., Cell 42, 183191 (1985). 2. HIROCHIKA.H., BROKER,T. R., and CHOW, L.T.,/. L&o/. 61,25992606 (1987). 3. GRIPE, T. P., HAUGEN, T. H., TURK, J. P., TABATABAI, F., SCHMID, P. G., DURST, M., GISSMANN, L.. ROMAN, A., and TUREK, L. P., EMBO/. 6,3745-3753 (1987). 4. THIERRY,F., andYANIV, M., EMf3OJ. 6, 3391-3397 (1987). 5. PHELPS, W. C., and HOWLEY, P. M., f. I/ire/. 61, 1630-l 638 (1987).
6. SPALHOLZ, B. A., LAMBERT, P. F., YEE, C. L., and HOWLEY, P. M., 1. Viral. 61, 2128-2137 (1987). 7. HAWLEY-NELSON,P., ANDROPHY, E. J., Lowv, D. R., and SCHILLER, J.T., EA&IO/. 7, 525-531 (1988). 8. HIROCHIKA, H., HIROCHIKA, R., BROKER, T. R., and CHOW, L. T., Genes Oev. 2,54-67 (1988). 9. ANDROPHY, E. J., Lowv, D. R., and SCHILLER,J. T., Nature (Londonj 325, 70-73 (1987). 10. GIRI, I., and YANIV, M.,/. Viral. 62, 1573-l 581 (1988). 11. CHIN, M. T., HIROCHIKA, H., BROKER,T. R., and CHOW, L. T., 1. Viral. 62, 2994-3002 (1988). 72. MCBRIDE, A. A., SCHLEGEL, R., and HOWLEY, P. M., En/l50 J. 7, 533-539 (1988). 13. MOSKALUK, C. A., and BASIIA, D., Proc. Nat/. Acad. Sci. USA 85, 1826-1830(1988). 14. MOSKALUK. C. A., and BASTIA, D.,I. Vifol. 62, 1925-1931 (1988). 15. LI, C. H., GILDEN, R. V., SHOWALTER,S. D., and SHAH, K. V., J. Virol. 62, 606-609 (1988). 16. HUBEERT,N. L., SCHILLER,J. T., Lowv, D. R., and ANDROPHY, E. J., Proc. Nat/. Acad. Sci. USA 85, 5864-5868 (I 988). 17. LAMBERT, P. F., SPALHOLZ, B.A., and HOWLEY. P. M., Ce//50,6978 (1987). 18. BASTIA, D., GERMINO, 1.. MUKHERJEE,S., and VANAMAN, T., In “Genetic Engineering” (J. Setlow and A. Hollaender, Eds.), Vol. 8, pp. 333-351. Plenum, New York, 1986. 19. YANISCH-PERRON,C., VIEIRA, J., and MESSING, J.. Gene 33, 1031 19 (1985). 20. HOPE, I. A., and STRUHL. K., EMBO/. 6,2781-2784 (1987).
169,236-238
(1989)
The Bovine Papillomavirus Type 1 Transcriptional Activator E2 Protein Binds to Its DNA Recognition Sequence as a Dimer CHRISTOPHER A. MOSKALUK AND DEEPAK BASTIA’ Department
of Microbiology
and Immunology,
Received September
Duke University Medical Center, Durham, North Carolina 27710 12, 1988; accepted November
16, 1988
The transcriptional trans-activator E2 protein from bovine papillomavirus type 1 has been shown to bind to the DNA consensus sequence ACCNBGGT. We have produced ‘the DNA-binding domain of the E2 protein as a recombinant protein in Escherichia co/i. The E2 DNA-binding domain was purified as two different molecular weight forms. Using these purified proteins, we show that two molecules of the E2 protein bind to a single DNA consensus binding site. 0 1999 Academic
Press, Inc.
tion was separated on a cation exchange column (Pharmacia, mono S) into two peaks (Fig. 1). The first eluting peak comprised two forms of E2Kpn380, one minor and one major form as detected on a SDS-polyacrylamide gel stained with Coomassie blue (Fig. 1, insert). The major form migrated as a 2 1-kDa protein. The proteins in this peak were termed the L forms of E2Kpn380. The product of the second eluting peak was homogeneous, and migrated as a 16-kDa protein. This protein was termed the S form of E2Kpn380. The S form appears to be the limit digest of collagenase, because the L forms could be converted to the S form by incubating further with collagenase (data not shown). A previous study using a collagen-@-galactosidase fusion protein has shown that limit digestion with collagenase removes all but three amino acids of the collagen linker (18). The limit collagenase digestion product migrates at a slower rate than expected for the predicted mass of 14 kDa, but anomalous migration of recombinant E2 protein has been reported by other researchers (16). The two forms of the E2Kpn380 protein were used to determine DNA-binding stoichiometry in a gel retardation assay. An oligonucleotide pair (5’-GATCACCGCTAGCGGT-3’,5’-TCGAACCGCTAGCGGT-3’ was synthesized, hybridized, and cloned into BamH l/Sail cut pUC19 plasmid (19). The resulting plasmid was cut with HindIll and fcoR1, resulting in a 59-bp fragment wiith a single E2 DNA-binding consensus sequence. This fragment was purified and radioactively labeled with [a-32P]deoxyadenosine triphosphate and the Klenow fragment of DNA polymerase I. This DNA probe was incubated with an equal number of units of the S form and the L form, and an equal unit mixture of the S and L forms of the E2Kpn380 protein. The S and L protein solutions had been titrated in gel retardation
The E2 open reading frame (ORF) of the bovine papillomavirus type 1 (BPV-1) has been shown to encode a frans-acting factor that activates a BPV-1 transcriptional enhancer (1). Human papillomaviruses (HPV) contain E2 ORFs with strong sequence homology to the BPV ORF, and which code for interchangeable transcriptional activating factors (2-5). The c&acting element in the E2-dependent enhancer consists of multiple copies of the nucleotide sequence ACCNGGGT, where N is any nucleotide (6-8). The protein product of the E2 ORF has been shown to bind specifically to this DNA consensus sequence (7- 14). The E2 ORF codes for different sized E2 proteins in papillomavirus-infected cells (15, 16). The DNA-binding domain of the E2 protein is contained in the carboxy-terminal portion of the protein (12, 13). The truncated carboxy E2 fragment (E2C) has been implicated in down-regulation of E2 transcriptional activation (3, 11, 17). E2C is presumed to compete for DNA-binding sites with the activator form of E2. In this paper we show that the E2 DNA-binding domain binds to a single ACCNGGGT DNA consensus sequence with a stoichiometry of two. The 3’terminus of the BPV-1 E2 ORF was cloned into the bacterial expression vector pDSlO0 as described previously (13). This produced a fusion gene consisting of 380 nucleotides of the E2 ORF, a portion of a collagen gene, and the lac Z gene. The fusion protein was expressed in fscherichia co/i and purified in two chromatographic steps as described by Moskaluk and Bastia (14). The fusion protein was subjected to limited digestion with collagenase. This separated the E2 domain (termed E2Kpn380) from the P-galactosidase protein. Portions of the collagen linker remained attached to the E2Kpn380 protein. This protein popula’ To whom requests for reprints should be addressed. 0042.6822/89
$3.00
Copynght 0 1999 by Academic Press. Inc. All rights of reproduction I” any form resewed.
236
SHORT COMMUNICATIONS
mono S cation exchange 0.10
.0.75
8 2 0.50 0.05
= 0 2
0.25
0 ,
0
d0
retention
!
40
volume (ml)
FIG. 1. Purification of the S and L forms of the E2 DNA-binding domain. The mono S column elution profile is shown. There are two protein peaks that elute with NaCl off of the cation exchange column. The Coomassie blue-stained SDS-polyacrylamide electrophoresis gel of the two peaks (L and S) is shown in the insert. Molecularweight markers are shown with their mass given in kilodaltons (kDa).
assays previously, with one unit of DNA-binding activity arbitrarily defined as the amount of protein solution needed to shift 50% of a labeled DNA fragment containing an E2-binding site into a complexed form, with one unit equaling approximately 6 ng of each protein. Gel electrophoresis of the reaction products resolved the free DNA probe from that complexed with proteins. In previous studies using the S protein preparation, a combination gel retardation/DNase footprinting analysis on three different ACCN,GGT sites revealed that every gel retarded complex observed corresponded to a sequence-specific DNA-protein interaction (14). As expected, complexes with the larger L protein migrated more slowly than the complexes with the smaller S protein (Fig. 2). It should be noted that the gel shift contribution of the minor Lform of the E2Kpn380 protein cannot be discerned. Mixing the S and L forms together resulted in the appearance of a third DNA-protein complex, with a gel migration that was intermediate that of the S and L complexes (Fig. 2). This observation indicates that two molecules of E2 proteins can bind to a single ACCNG-GGT DNA recognition site. Denaturing conditions were not required to form the intermediate complex, so the S and L forms of the E2 DNA-binding
237
domain must not form stable associations in solution in the absence of DNA. The complexes do not form in equimolar amounts although equimolar amounts of DNA-binding activity were added. Specifically, the L complex is reduced in comparison to the SL and S complexes. This suggests that the collagen peptide attached to the L form of the protein partially interferes with the formation of protein dimers on the DNA recognition site, thus selecting for dimers of two S forms or an SL dimer. In an analogous experiment Hope and Struhl, using two different sized forms of the yeast GCN4 protein, showed that such an intermediate DNA-protein complex was indeed composed of a heterodimer of the two protein forms (20). The partial inverted repeat structure of the ACCNGGGT DNA consensus sequence was predictive of interactions with an even number of protein subunits. In addition, symmetric DNA contacts with the E2 protein were found on both halves of the consensus binding site (14). Taken together, these results indicate that two E2 protein molecules contact the binding site on roughly the same side of the DNA helix (74). It is known that E2 protein is found in various sizes in BPV-l-infected cells (16) and that the smaller E2C form is antagonistic to the larger E2 activator form (3, 11, 17). This observation, that heterodimers of different sized E2 proteins can bind to a single site in vitro, suggests that a similar phenomenon may occur in viva. The formation of E2-E2C heterodimers may cause down-regulation of the rrans-activator function as well as the E2C di-
c
s
L
SL
free *DNA
FIG. 2. E2Kpn380 protein-DNA complexes. Of a ‘*P end-labeled DNA probe that contains one E2-binding site (lane C) 10,000 Cerenkov cpm (approximately 0.01 pmol) was incubated with one unit of the S form (lane S), one unit of the L form (lane L). and a mixture of one unit each of the S and Lforms (lane SL) of the E2Kpn380 protein in a 50 ~1 volume containing 25 mM 2-(N-morpholino)ethanesulfonic acid (MES). pH 6.0, 7.5 mM MgCIZ, 50 mM KCI, 100 mM NaCI, 0.05 mM EDTA, 4 mM dithiothreitol, and 0.5 pg of sheared calf thymus DNA (Sigma). The reactions were incubated at ambient room temperature for 20 min. Prior to electrophoresis, glycerol was added to 10% (v/v) and an additional 10 pg of calf thymus DNA was added. Gel electrophoresis of the DNA-protein reactions as described by Moskaluk and Bastia (13) separated the different sized DNA-protein complexes.
SHORT COMMUNICATIONS
238
mers. This may allow the E2C to be a more effective “genetic switch,” allowing modulation of the E2 activator at a lower E2C concentration than if the E2C dimer concentration alone were the critical determining factor. ACKNOWLEDGMENTS This work was supported by grants from the National Institutes of Health and the National Cancer Institute. CM. is supported by the National Institutes of Health Medical Scientist Training Program. D.B. is an Established Investigator of the American Heart Association.
REFERENCES 1. SPALHOLZ, B. A., YANG, Y. C., and HOWLEY, P. M., Cell 42, 183191 (1985). 2. HIROCHIKA.H., BROKER,T. R., and CHOW, L.T.,/. L&o/. 61,25992606 (1987). 3. GRIPE, T. P., HAUGEN, T. H., TURK, J. P., TABATABAI, F., SCHMID, P. G., DURST, M., GISSMANN, L.. ROMAN, A., and TUREK, L. P., EMBO/. 6,3745-3753 (1987). 4. THIERRY,F., andYANIV, M., EMf3OJ. 6, 3391-3397 (1987). 5. PHELPS, W. C., and HOWLEY, P. M., f. I/ire/. 61, 1630-l 638 (1987).
6. SPALHOLZ, B. A., LAMBERT, P. F., YEE, C. L., and HOWLEY, P. M., 1. Viral. 61, 2128-2137 (1987). 7. HAWLEY-NELSON,P., ANDROPHY, E. J., Lowv, D. R., and SCHILLER, J.T., EA&IO/. 7, 525-531 (1988). 8. HIROCHIKA, H., HIROCHIKA, R., BROKER, T. R., and CHOW, L. T., Genes Oev. 2,54-67 (1988). 9. ANDROPHY, E. J., Lowv, D. R., and SCHILLER,J. T., Nature (Londonj 325, 70-73 (1987). 10. GIRI, I., and YANIV, M.,/. Viral. 62, 1573-l 581 (1988). 11. CHIN, M. T., HIROCHIKA, H., BROKER,T. R., and CHOW, L. T., 1. Viral. 62, 2994-3002 (1988). 72. MCBRIDE, A. A., SCHLEGEL, R., and HOWLEY, P. M., En/l50 J. 7, 533-539 (1988). 13. MOSKALUK, C. A., and BASIIA, D., Proc. Nat/. Acad. Sci. USA 85, 1826-1830(1988). 14. MOSKALUK. C. A., and BASTIA, D.,I. Vifol. 62, 1925-1931 (1988). 15. LI, C. H., GILDEN, R. V., SHOWALTER,S. D., and SHAH, K. V., J. Virol. 62, 606-609 (1988). 16. HUBEERT,N. L., SCHILLER,J. T., Lowv, D. R., and ANDROPHY, E. J., Proc. Nat/. Acad. Sci. USA 85, 5864-5868 (I 988). 17. LAMBERT, P. F., SPALHOLZ, B.A., and HOWLEY. P. M., Ce//50,6978 (1987). 18. BASTIA, D., GERMINO, 1.. MUKHERJEE,S., and VANAMAN, T., In “Genetic Engineering” (J. Setlow and A. Hollaender, Eds.), Vol. 8, pp. 333-351. Plenum, New York, 1986. 19. YANISCH-PERRON,C., VIEIRA, J., and MESSING, J.. Gene 33, 1031 19 (1985). 20. HOPE, I. A., and STRUHL. K., EMBO/. 6,2781-2784 (1987).