(Received for publication, September 27, 1994; and in revised form, December 21, 1994)
From the
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.
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). ()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.
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.
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.
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).
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.
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.
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.
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.
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). ()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 (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
1 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.