©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The 30-kDa Protein Binding to the Initiator of the Baculovirus Polyhedrin Promoter Also Binds Specifically to the Coding Strand (*)

(Received for publication, September 27, 1994; and in revised form, December 21, 1994)

Bipasha Mukherjee (§) Sandeep Burma (§) Seyed E. Hasnain (¶)

From the Eukaryotic Gene Expression Laboratory, National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi 110067, India

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

We previously reported the purification and characterization of the polyhedrin promoter-binding protein (PPBP), an unusual DNA-binding protein that interacts with transcriptionally important motifs of the baculovirus polyhedrin gene promoter (S. Burma, B. Mukherjee, A. Jain, S. Habib, and S.E. Hasnain, J. Biol. Chem.(1994) 269, 2750-2757. PPBP also exhibits a sequence-specific single-stranded DNA-binding activity. Gel retardations and competition analyses with double- and single-stranded oligonucleotides indicated that PPBP binds the coding strand and not the noncoding strand of the promoter. This was further confirmed by UV cross-linking and Southwestern blotting experiments. Gel retardations with mutated oligonucleotides indicated that both dsDNA and ssDNA binding involve common AATAAATAAGTATT motifs. However, ssDNA binding is dependent upon ionic interactions unlike dsDNA binding, which is mainly through nonionic interactions. The affinity of PPBP for the coding strand appears to be higher than that for duplex promoter DNA. Interestingly, the PPBP-coding strand complex has a longer half-life (60 min) than the PPBP-duplex promoter complex (15 min). PPBP represents a unique example of an ``initiator'' promoter-binding protein with dual dsDNA and ssDNA binding activities, and this reconciles very well with the unusual binding characteristics displayed by it. The formation of the PPBP-coding strand complex in vivo may be a crucial step for the exceptionally high and repeated rounds of transcriptional activity of the baculovirus polyhedrin gene promoter.


INTRODUCTION

In the baculovirus expression vector system, the very late polyhedrin gene promoter of the Autographa californica nuclear polyhedrosis virus is used to direct the expression of foreign genes (Luckow, 1991; Jarvis and Summers, 1992; O' Reilly et al., 1992). Although the baculovirus expression vector system is widely used for heterologous gene expression, little is known about the regulation of the polyhedrin promoter and the mechanism responsible for hypertranscription from this promoter.

The polyhedrin promoter of the Autographa californica nuclear polyhedrosis virus consists of a 69-base pair region (-1 to -69) just upstream of the ATG start codon (Matsuura et al., 1987; Possee and Howard, 1987; Rankin et al., 1988). The transcription start point (at -50) lies within a highly conserved TAAGTATT motif that is absolutely essential for transcription initiation (Ooi et al., 1989). Morris and Miller(1994) have recently shown that an 18-base pair region surrounding the transcription start point (-42 to -59) is sufficient for ``minimal'' promoter activity, whereas the sequences encoding the untranslated mRNA leader are required for the very late burst of expression. The structure of the polyhedrin promoter is, therefore, similar to that of certain eukaryotic TATA-less promoters where the initiator (the sequence around the transcription start site) acts as a minimal promoter capable of directing basal levels of transcription (Smale and Baltimore, 1989).

We previously reported (Burma et al., 1994) the identification and characterization of a 30-kDa host factor, the polyhedrin promoter-binding protein (PPBP). (^1)The transcriptionally essential TAAGTATT sequence is important for PPBP binding in association with an AATAAA motif present just upstream from it (Fig. 1), which is known to be important for promoter activity (linker scan mutations affecting the AATAAA motif decreased promoter activity by about 65% (Ooi et al., 1989)). The minimal promoter described by Morris and Miller (1994) consists essentially of these two hexa- and octanucleotide motifs. PPBP was purified from Sf21 (Spodoptera frugiperda) insect cell line and appeared to be an unusual DNA-binding protein with respect to its stability, high binding affinity, and high specificity. The correlation of functional promoter sequences and the factor-binding site argues that the interaction of PPBP with the polyhedrin promoter may be an important event in the initiation of transcription from this promoter. We proposed that PPBP might function in a manner analogous to the TATA-binding protein in recruiting the virus-specific RNA polymerase and/or trans-acting factor(s) to the polyhedrin promoter.


Figure 1: Nucleotide sequence of the coding strand of the polyhedrin gene promoter (Possee et al., 1991). The translation start site is designated as +1. The transcription start point, marked with a bent arrow, is at -50. The PPBP-binding region (Burma et al., 1994), consisting of the octanucleotide (outlined) and hexanucleotide motifs, has been enlarged. The minimal promoter (Morris and Miller, 1994), (-42 to -59) has been highlighted by a hatched bar. Boundaries of the oligonucleotide used in gel retardations (-32 to -63) are indicated by a double arrow.



The unusual structure of the polyhedrin promoter (with the initiator acting as the minimal promoter) and the binding characteristics of PPBP involving the transcription start point poses the problem of whether binding would be maintained even after the melting of DNA at this point during transcription initiation. We, therefore, carried out experiments to investigate whether PPBP exhibits any single-stranded DNA-binding activity. Such an activity would allow PPBP to maintain its position when the DNA helix melts during the initiation of transcription. We show here that in addition to binding duplex promoter DNA, PPBP binds the coding strand but not the noncoding strand of the promoter in a sequence-specific manner involving common cognate motifs. Unlike duplex-promoter binding, ssDNA binding appears to involve ionic interactions. Furthermore, the affinity of PPBP for the coding strand of the promoter is higher than that for duplex promoter DNA. Interestingly, the PPBP-coding strand complex has a longer half-life (60 min) compared with the PPBP-duplex promoter complex (15 min). These features of PPBP may have important implications in the regulation of hypertranscription from the polyhedrin promoter.


MATERIALS AND METHODS

Gel Retardation Assays

Sf21 cells were maintained in TNM-FH medium supplemented with 10% fetal calf serum (O'Reilly et al., 1992). Nuclear protein extracts were prepared using a modification of the method of Dignam et al.(1983) as described earlier (Burma et al., 1994). Synthetic oligonucleotides were labeled by T4 polynucleotide kinase using [-P]ATP. 2 µg of crude nuclear extract was incubated at 25 °C for 15 min with 1 ng of dsDNA or 0.5 ng of ssDNA (10^4 cpm) in the presence of 10 mM Hepes-NaOH (pH 7.5), 200 mM NaCl, 0.5 mM dithiothreitol, and 1 µg of poly[d(I-C)] in a final volume of 20 µl (Chodosh, 1988a). The DNA-protein complex was resolved by electrophoresis at 4 °C in a 5% (29:1 acrylamide/bisacrylamide) polyacrylamide gel in TAE buffer (7 mM Tris-HCl (pH 7.5), 3 mM sodium acetate, 1 mM EDTA). After electrophoresis, the gel was dried and autoradiographed. For competition experiments, an appropriate amount of unlabeled ssDNA or dsDNA was added to the reaction mixture.

UV Cross-linking of DNA-Protein Complex

The binding reaction was carried out as described above. After 15 min the incubation mixture was placed on ice and UV-irradiated (254 nm) at a distance of 1 cm for 30 min (Chodosh, 1988b). Following irradiation, the mixture was electrophoresed in a 15% SDS-polyacrylamide gel. After electrophoresis, the gel was dried and autoradiographed.

Southwestern Blotting

All operations were carried out at 4 °C. 50 µg of nuclear extract was fractionated on a 15% SDS-polyacrylamide gel and electrophoretically transferred to a nitrocellulose membrane in a buffer containing 25 mM Tris and 190 mM glycine for 16 h at 30 mA. The filter was first incubated with blocking buffer (2% nonfat dry milk, 1% bovine serum albumin, 10 mM Hepes-NaOH (pH 7.5), 200 mM NaCl, 50 mM MgCl(2), 0.1 mM EDTA, 16 µg/ml sonicated salmon sperm DNA) for 2 h and then incubated with binding buffer (blocking buffer with 0.2% nonfat dry milk) containing labeled ssDNA or dsDNA (10^6 cpm/ml) for 16 h, washed, and subjected to autoradiography (Burma et al., 1994).


RESULTS

PPBP Exhibits Dual dsDNA and ssDNA Binding Activities

Oligonucleotides corresponding to a 32-base pair region of the polyhedrin promoter (-63 to -32) (Fig. 1) encompassing the 18-base pair minimal promoter (Morris and Miller, 1994) and bearing the PPBP-cognate motifs (Burma et al., 1994) were chemically synthesized, annealed, radioactively labeled, and used in gel retardation assays. The PPBP-dsDNA complex obtained (Fig. 2A, lane 2) could be competed out with a 30-fold excess of cold double-stranded promoter DNA (Fig. 2A, lane 3) and also with a 30-fold molar excess of the coding strand (Fig. 2A, lane 5) but not with the noncoding strand (Fig. 2A, lane 6) of the promoter. Nonspecific dspUC18 DNA or sspUC18 DNA (generated by heating and quick cooling dspUC18) could not compete for complex formation (Fig. 2A, lanes 4 and 7, respectively). These results indicated that PPBP also binds the coding strand but not the noncoding strand of the promoter.


Figure 2: A, the coding strand of the polyhedrin promoter can compete for the PPBP-duplex promoter complex. Labeled duplex promoter was incubated alone (lane 1) or with 2 µg of nuclear extract from Sf21 cells (lanes 2-7). Competition was performed with a 30-fold molar excess of duplex promoter (lane 3), dspUC18 (lane 4), coding strand (lane 5), noncoding strand (lane 6), or sspUC18 (lane 7). B, binding of PPBP to the coding strand is sequence-specific, whereas the noncoding strand cannot bind. Labeled coding strand was incubated alone (lane 1) or with 2 µg of nuclear extract (lanes 2-7). Competition was performed with a 30-fold molar excess of coding strand (lane 3), noncoding strand (lane 4), sspUC18 (lane 5), duplex promoter (lane 6), or dspUC18 (lane 7). Labeled noncoding strand was incubated alone (lane 8) or with 2 µg of nuclear extract (lane 9). C, dsDNA binding is not caused by denaturation of the duplex promoter DNA. The binding reaction was carried out with 2 µg of nuclear extract and duplex promoter with both strands labeled (lane 1), only coding strand labeled (lane 2), or only noncoding strand labeled (lane 3).



Gel retardations and cold competition assays with the labeled coding or noncoding strand of the promoter (Fig. 2B) revealed that the former elicited a complex similar to that obtained with duplex promoter DNA (Fig. 2B, lane 2). The complex obtained could be competed out with a 30-fold excess of unlabeled coding strand DNA (Fig. 2B, lane 3) but not with a similar excess of the noncoding strand or sspUC18 DNA (Fig. 2B, lanes 4 and 5, respectively). The complex could also be competed out with a 30-fold molar excess of unlabeled duplex promoter DNA (Fig. 2B, lane 6) but not by a similar excess of dspUC18 DNA (Fig. 2B, lane 7). The ability of the coding strand to compete for the complex obtained with duplex promoter DNA (Fig. 2A, lane 5) and vice versa (Fig. 2B, lane 6) confirmed that the same factor, PPBP, can bind both dsDNA and ssDNA. The labeled noncoding strand did not show any evidence of complex formation in a gel retardation assay (Fig. 2B, lane 9), even after prolonged exposure of the film. The observation that PPBP had a distinct preference for the coding strand rather than the complementary noncoding strand validated that the binding of PPBP to the coding strand is sequence-specific.

Before proceeding further, it was important to exclude the possibility that the observed dsDNA binding reflected binding to a small population of denatured DNA molecules. Double-stranded probes prepared individually by labeling either the coding strand (Fig. 2C, lane 2) or the noncoding strand (Fig. 2C, lane 3) formed identical complexes regardless of which strand was labeled. Since the noncoding strand did not bind, no complex formation should have been observed in lane 3, where the coding strand was not labeled, if the observed dsDNA binding was caused by denaturation of the double-stranded probe. Thus, double-stranded DNA binding activity truly represented binding to duplex DNA structure rather than to contaminating single strands.

The Same 30-kDa Factor Binds both dsDNA and ssDNA

To characterize the relationship between the ssDNA and dsDNA binding activities of PPBP, UV cross-linking and Southwestern assays were performed with double-stranded and single-stranded probes. The DNA-protein complex was UV-irradiated (254 nm) for 30 min in the presence or absence of competitor DNA. After separation on a 15% SDS-polyacrylamide gel followed by autoradiography (Fig. 3A) it was apparent that the molecular mass of both of the binding activities was about 30 kDa. A cross-linked complex with an expected mass of about 30 kDa (Burma et al., 1994) was obtained with labeled double-stranded promoter DNA (Fig. 3A, lane 1). The cross-linked complex was not obtained with labeled noncoding strand DNA (Fig. 3A, lane 2) or when the labeled coding strand was irradiated alone in the absence of nuclear extract (Fig. 3A, lane 3). A 30-kDa cross-linked band was obtained upon irradiation of the PPBP-coding strand complex (Fig. 3A, lane 4). Complex formation was greatly reduced in the presence of an excess of cold coding strand or duplex promoter DNA (Fig. 3A, lanes 5 and 8) but was unaffected in the presence of an excess of cold noncoding strand DNA or sspUC18 DNA (Fig. 3A, lanes 6 and 7, respectively), thereby demonstrating the specificity of the cross-linked complex.


Figure 3: A, the same 30-kDa factor binds both dsDNA and ssDNA. 2 µg of nuclear extract was incubated with labeled duplex promoter (lane 1), noncoding strand (lane 2), or coding strand (lanes 4-8) and UV-irradiated. In lane 3 the labeled coding strand was irradiated in the absence of nuclear extract. Competition of the PPBP-coding strand complex (lane 4) was performed with a 30-fold molar excess of coding strand (lane 5), noncoding strand (lane 6), sspUC18 (lane 7), or duplex promoter (lane 8). The position of a 30-kDa protein molecular size marker is shown by an arrowhead. (The free probe has been cut out from the bottom of the gel.) B, Southwestern blotting confirms the molecular mass of the dsDNA/ssDNA binding activity. Nuclear extract was probed with labeled duplex promoter (panel a), coding strand (panel b), or noncoding strand (panel c). The position of a 30-kDa protein molecular size marker has been shown by an arrowhead.



Southwestern analyses of the nuclear extract, using radiolabeled duplex promoter, coding strand, or noncoding strand as probe, was carried out to confirm the molecular mass of the dsDNA/ssDNA binding activity. Nuclear extracts were fractionated on a 15% SDS-polyacrylamide gel, blotted onto nitrocellulose, and probed (Fig. 3B). A common band was obtained in the 30-kDa region when the blots were probed with labeled duplex promoter DNA or ssDNA corresponding to the coding strand (Fig. 3B, panels a and b, respectively). The band was specific since it did not appear when a similar blot was probed with noncoding strand DNA (Fig. 3B, panel c). These results reinforced the idea that the same factor, PPBP, displayed both of these binding activities.

Both dsDNA and ssDNA Binding Involve Common Cognate Motifs

An octanucleotide motif (TAAGTATT) present at the transcription start point and a hexanucleotide motif (AATAAA) present immediately upstream of the octanucleotide (Fig. 1) are essential for PPBP binding (Burma et al., 1994). Mutated versions of the coding strand, mutOct-c (TAAGTATT substituted with GCCTGCGG) and mutHex-c (AATAAA substituted with CCGCCC), were used in gel retardation assays (Fig. 4). No binding was observed with labeled mutOct-c or mutHex-c (Fig. 4, lanes 2 and 3, respectively), indicating that both of these motifs are essential for ssDNA binding (Fig. 4, lane 1). A complementary experiment, where the complex formed by the labeled wild type coding strand (Fig. 4, lane 4) could be competed out with an excess of cold wild type coding strand DNA (Fig. 4, lane 5) but not with the mutated versions of the same (Fig. 4, lanes 6 and 7), confirmed the involvement of the octa/hexa motifs (essential for ds DNA binding) in ssDNA binding also.


Figure 4: PPBP binds to the hexa- and octanucleotide motifs of the coding strand. The binding reaction was carried out with 2 µg of nuclear extract and labeled coding strand (lane 1), mutOct-c (lane 2), or mutHex-c (lane 3). The complex obtained with labeled coding strand (lane 4) was competed with a 30-fold molar excess of coding strand (lane 5), mutOct-c (lane 6), or mutHex-c (lane 6).



PPBP-DNA Complex Formation Does Not Involve Any RNA Component

There have been reports of sequence-specific binding of single-stranded nucleic acids by heterogenous nuclear ribonucleoproteins (Wilusz and Shenk, 1990; Kumar et al., 1986). Nuclear extracts (2 µg) were pre-treated with 2 µg of proteinase K or 2 µg of pancreatic RNase at 37 °C for 60 min and assayed for dsDNA and ssDNA binding activities by gel retardation. Proteinase K abolished both dsDNA and ssDNA binding activities (Fig. 5, lanes 2 and 5), whereas RNase treatment did not affect DNA binding in any way (Fig. 5, lanes 3 and 6). Thus, dsDNA/ssDNA binding by PPBP does not involve any RNA component.


Figure 5: PPBP does not have any RNA component. Nuclear extracts (2 µg) were mock-treated (lanes 1 and 4) or pretreated with proteinase K (lanes 2 and 5) or RNase (lanes 3 and 6) and assayed for dsDNA/ssDNA binding activities by gel retardation using labeled duplex promoter (lanes 1-3) or coding strand (lanes 4-6) as probe.



ssDNA Binding, Unlike dsDNA Binding, Possibly Involves Ionic Interactions

PPBP can bind duplex promoter DNA over a very wide salt range, i.e. 200-2000 mM NaCl (Burma et al., 1994). Since complex formation was not affected by such a wide fluctuation in NaCl concentration, it appeared that the dsDNA-PPBP association was predominantly through nonionic interactions. Surprisingly, we found that the interaction of PPBP with the coding strand showed lower levels of salt tolerance (Fig. 6A). PPBP could bind to the coding strand only at salt concentrations ranging from 200 to 500 mM NaCl (Fig. 6A, lanes 1 and 2). Complex formation was greatly reduced at 1 M NaCl (Fig. 6A, lane 3) and was abolished at 2 M NaCl (Fig. 6A, lane 4). Therefore, it appears that ssDNA binding, unlike dsDNA binding, is dependent on ionic interactions.


Figure 6: A, ssDNA binding is dependent on ionic interactions. The binding reaction was carried out with 2 µg of nuclear extract and labeled coding strand at NaCl concentrations of 0.2 M (lane 1), 0.5 M (lane 2), 1 M (lane 3), or 2 M (lane 4). B, divalent cations are not required for ssDNA binding. The binding reaction was carried out with 2 µg of nuclear extract and labeled coding strand in the absence (lane 1) or presence (lane 2) of 100 mM EDTA.



Divalent cations are not required for dsDNA binding (Burma et al., 1994). Divalent cations are also not required for ssDNA binding as evident from the observation (Fig. 6B, lane 2) that the addition of 100 mM EDTA did not affect complex formation in any way.

PPBP Displays Relatively Stronger Binding to the Coding Strand

A competition analysis was performed to directly assess the relative affinities of dsDNA and ssDNA binding by PPBP. Binding reactions (with labeled coding strand DNA) were performed in the presence of graded amounts of cold coding strand or duplex promoter DNA (0.25-4-fold excess). Bound probe was analyzed by the gel retardation assay (Fig. 7, inset). Relative amounts of bound material were quantitated by phosphor image analysis (Bio-Rad GS-250 molecular imager) and plotted (Fig. 7) as % maximal binding ((amount bound in presence of competitor/amount bound in absence of competitor) times 100). The coding strand-PPBP complex was competed to half-maximal binding by 2-fold lower molar quantities of coding strand DNA, compared with duplex promoter DNA (evident from three independent experiments).


Figure 7: PPBP displays relatively stronger binding to the coding strand. Labeled coding strand was incubated with 2 µg of nuclear extract in the presence of graded amounts of cold coding strand or duplex promoter (0.25-4-fold excess) and analyzed by gel retardation (inset: - indicates no competitor, whereas arrows indicate increasing concentrations of cold coding strand or duplex promoter DNA). Relative amounts of bound probe were plotted as % maximal bindingversus -fold excess of competitor added.



The PPBP-coding Strand Complex has a Longer Half-life Than the PPBP-Duplex Promoter Complex

The half-life of a DNA-protein complex is usually determined by challenging a preformed complex of protein and labeled probe with an excess of unlabeled probe (Choo and Klug, 1993). In this method the dissociated protein is trapped in a new nonradioactive complex and thus prevented from rebinding to the probe. The decay of radioactivity in the original complex as a function of time would then reflect the half-life of the complex. Preformed dsDNA-PPBP (Fig. 8, panel a) or ssDNA-PPBP complexes (Fig. 8, panel b) were challenged with an excess of cold duplex promoter or coding strand DNA, respectively, and reactions were loaded onto a running gel over a time period ranging from 0 to 60 min. The decay of radioactivity in the original complexes was quantitated by phosphor-image analysis, and % maximal binding was plotted against time (Fig. 8). The half-life of the dsDNA-PPBP complex was estimated to be only 15 min, whereas that of the ssDNA-PPBP complex was 60 min.


Figure 8: The PPBP-coding strand complex has a longer half-life than the PPBP-duplex promoter complex. Preformed dsDNA-PPBP (panel a) or ssDNA-PPBP (panel b) complexes were challenged with an excess of cold duplex promoter or coding strand, respectively. Reactions were loaded onto a running gel at various time points (in min) indicated above each lane (inset). The dissociation of the original complex was plotted as % maximal bindingversustime.




DISCUSSION

A knowledge of the factors interacting with the polyhedrin gene promoter and the mechanism of such interactions is a prerequisite for understanding polyhedrin promoter activation. Seven Autographa californica nuclear polyhedrosis virus genes (lef-1 to lef-7) are now known to be involved in expression from the very late polyhedrin promoter (Li et al., 1993; Passarelli and Miller, 1993a, b, c; Morris et al., 1994). (^2)However, it is not known whether the lef gene products act at the level of replication, transcription, or translation. To date, PPBP is the only factor known to directly interact with the polyhedrin promoter. Given the unusual structure of this promoter, its temporal pattern of activation, and the exceptionally high levels of polyhedrin gene expression it was pertinent to investigate the interaction of PPBP with the promoter. The structure of the polyhedrin promoter is similar to that of certain eukaryotic promoters where the initiator is capable of acting as the minimal promoter. The unique structure of such promoters is believed to be necessary for directing transcription of a subset of genes that require strict activation and inactivation during cellular differentiation and development (Smale and Baltimore, 1989). The structure of the polyhedrin promoter might ensure the very late pattern of polyhedrin gene expression in a similar manner. Although several initiator-binding proteins have been identified (Weis and Reinberg, 1992), the pathways for the assembly of transcription complexes at such promoters remain to be worked out, and it is not known whether these alternate pathways require the standard set of basal factors for initiation (Buratowski, 1994). Because of its position, the initiator element is susceptible to DNA melting very early in the transcription initiation process (Goodrich and Tjian, 1994). Therefore, it is tempting to speculate that key initiator-binding factors must possess dual dsDNA/ssDNA binding activities that would enable them to maintain their positions at the initiator irrespective of the double- or single-stranded state of the DNA.

Crick(1971) published a model for chromosomes of higher organisms in which ``the recognition sites needed for control purposes are mainly unpaired single-stranded stretches of double-stranded DNA'' and hypothesized that sequence-specific ssDNA binding proteins would be found and would play an important role in gene regulation. Reports have indicated that regulatory regions of chromatin may be sensitive to nuclease S(1) (Larsen and Weintraub, 1982; Johnson et al., 1988), indicating the presence of ssDNA. Surprisingly, there have been few well documented reports of ssDNA binding transcription-regulatory proteins. Examples of transcription factors with dual dsDNA and ssDNA binding activities are even more limited: (i) the estrogen receptor selectively binds the coding strand of an estrogen receptor element (Lannigan and Notides, 1989); (ii) muscle factor 3 and MyoD interact with double- and single-stranded forms of muscle gene elements (Santoro et al., 1991); (iii) sterol regulatory element-binding factor recognizes both double-stranded and single-stranded forms of a sterol regulatory element, SRE-1 (Stark et al., 1992); (iv) the yeast alpha1 and MCM1 proteins bind either single-stranded or duplex DNA representing their cognate upstream activation sequence (Grayhack, 1992); (v) complex formation of nuclear proteins with repeated elements in the external transcribed spacer of Cucumis sativus ribosomal DNA does not depend upon the single- or double-stranded state of the DNA (Zentgraf and Hemleben, 1992); and (vi) oviduct nuclear proteins binding to the steroid-dependent regulatory elements of the chicken ovalbumin gene prefer ssDNA in a sequence-specific manner (Nordstrom, 1993). To our knowledge, PPBP is the first example of a ``core'' promoter-binding protein with dual dsDNA and ssDNA binding activities.

There are three possible hypotheses to explain how a protein might bind both duplex and single-stranded DNA: (i) the protein might exclusively contact bases on one strand of the DNA. Such a protein might either impart on the ssDNA a conformation similar to that of the same strand in the duplex state or confer a distinct conformation to both dsDNA and ssDNA; (ii) the protein might recognize a common secondary structure formed by both dsDNA and ssDNA; or (iii) the protein might possess two domains, one recognizing dsDNA and the other recognizing ssDNA. Binding to ssDNA could then be caused by either a sequence-specific ssDNA binding domain or the combined action of a nonspecific ssDNA binding domain and the sequence-specific dsDNA binding domain, which retains some base-specific contacts in the cognate motif. It is important to reiterate here the observed differences in the kinetics of ssDNA and dsDNA binding.

It is possible that, in vivo, PPBP initially binds to the duplex promoter. Following transcription initiation and ``open complex'' formation, PPBP would bind more tightly and stably to the coding strand of the promoter. Thus, PPBP can maintain its position at the transcription start point in spite of DNA melting. Experiments described by Mollegaard et al.,(1994) suggest that DNA melting might enhance promoter recognition by RNA polymerase. Therefore, PPBP could keep the promoter region single-stranded after the polymerase has passed in the elongation process and, thereby, enhance the next transcription round. Thus, the formation of the PPBP-coding strand complex may be a crucial step for repeated rounds of transcription to take place and could, perhaps, explain the exceptionally high levels of polyhedrin gene expression observed.

Regulatory proteins are likely to interact with the transcription apparatus by looping, bending, twisting, and unwinding of DNA to bring the two sets of proteins into alignment (Ptashne, 1988). Thus, proteins like PPBP that tolerate structural changes in DNA may be able to regulate transcription more efficiently (the polyhedrin promoter is exceptionally hyperactive very late in the viral infection cycle). Indeed, ssDNA represents more structural pliability, as well as a greater number of contact points for specific DNA-protein interactions. Achieving the high sequence specificity necessary to accurately control gene expression in eukaryotes may thus be made easier by the use of such interactions. Therefore, it may be tempting to cite PPBP as another example where eukaryotic transcriptional activation is controlled not only by the more canonical transcription factors that bind dsDNA motifs but through another level of control, which involves regulatory proteins with dual double- and single-stranded DNA binding activities.


FOOTNOTES

*
This work was supported by Research Grant BT/MS/01/001/89/silk/baculo (to S. E. H.) from the Department of Biotechnology, Government of India. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
B. M. and S. B. acknowledge the award of research fellowships from the Council for Scientific and Industrial Research, Government of India.

To whom correspondence should be addressed. Tel.: 9111-686-3004 (ext. 301); Fax: 9111-686-2125; ehtesham{at}nii.ernet.in.

(^1)
The abbreviations used are: PPBP, polyhedrin promoter-binding protein; ssDNA, single-stranded DNA; dsDNA, double-stranded DNA.

(^2)
A. L. Passarelli and L. K. Miller, submitted for publication.


ACKNOWLEDGEMENTS

We are grateful to Drs. V. Kumar and S. Mukherjee from the International Centre for Genetic Engineering and Biotechnology, New Delhi, and S. Rath from the National Institute of Immunology for critically reviewing this manuscript. We thank Manoj Kumar and Owes Ahmad for technical assistance.


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  23. O'Reilly, D. R., Miller, L. K., and Luckow, V. A. (1992) Baculovirus Expression Vectors: A Laboratory Manual , W. H. Freeman and Co., New York
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  27. Possee, R. D., and Howard, S. C. (1987) Nucleic Acids Res. 15, 10233-10248 [Abstract]
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  30. Rankin, C., Ooi, B. G., and Miller, L. K. (1988) Gene (Amst.) 70, 39-49 [CrossRef][Medline] [Order article via Infotrieve]
  31. Santoro, I. M., Yi, T.-M., and Walsh, K. A. (1991) Mol. Cell. Biol. 11, 1944-1953 [Medline] [Order article via Infotrieve]
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  36. Zentgraf, U., and Hemleben, V. (1992) Nucleic Acids Res. 20, 3685-3691 [Abstract]

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