|
|
|
|||||||
|
|||||||||
(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 ( 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). (
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
(
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). ( 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 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.
Volume 270,
Number 9,
Issue of March 3, 1995 pp. 4405-4411
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Initiator
of the
Baculovirus Polyhedrin Promoter Also Binds Specifically to the Coding
Strand (*)
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
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.
)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.
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.
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
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
, 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
cpm/ml) for 16 h, washed, and
subjected to autoradiography (Burma et al., 1994).
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.
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.
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.
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.
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.
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) 
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).
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.
)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. (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.
))
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.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
This article has been cited by other articles:
|
|
 |
|
  P. Viswanathan, B. Venkaiah, M. S. Kumar, S. Rasheedi, S. Vrati, M. D. Bashyam, and S. E. Hasnain The Homologous Region Sequence (hr1) of Autographa californica Multinucleocapsid Polyhedrosis Virus Can Enhance Transcription from Non-baculoviral Promoters in Mammalian Cells J. Biol. Chem., December 26, 2003; 278(52): 52564 - 52571. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Acharya and K. P. Gopinathan Identification of an enhancer-like element in the polyhedrin gene upstream region of Bombyx mori nucleopolyhedrovirus J. Gen. Virol., November 1, 2001; 82(11): 2811 - 2819. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Ghosh, A. Jain, B. Mukherjee, S. Habib, and S. E. Hasnain The Host Factor Polyhedrin Promoter Binding Protein (PPBP) Is Involved in Transcription from the Baculovirus Polyhedrin Gene Promoter J. Virol., September 1, 1998; 72(9): 7484 - 7493. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Habib and S. E. Hasnain A 38-kDa Host Factor Interacts with Functionally Important Motifs within the Autographa californica Multinucleocapsid Nuclear Polyhedrosis Virus Homologous Region (hr1) DNA Sequence J. Biol. Chem., November 8, 1996; 271(45): 28250 - 28258. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Ramachandran, A. Jain, P. Arora, M. D. Bashyam, U. Chatterjee, S. Ghosh, V. K. Parnaik, and S. E. Hasnain Novel Sp Family-like Transcription Factors Are Present in Adult Insect Cells and Are Involved in Transcription from the Polyhedrin Gene Initiator Promoter J. Biol. Chem., June 22, 2001; 276(26): 23440 - 23449. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| All ASBMB Journals | Molecular and Cellular Proteomics |
| Journal of Lipid Research | ASBMB Today |