From the
Adenovirus (Ad) genome DNA is complexed with viral core proteins
in the virus particle and in host cells during the early stages of
infection. This DNA protein complex, called Ad core, is thought to be
the template for transcription and DNA replication in infected cells.
The Ad core functioned as template for DNA replication in the cell-free
system consisting of viral replication proteins, uninfected HeLa
nuclear extracts, and a novel factor, template activating factor-I
(TAF-I) that we have isolated from uninfected HeLa cytoplasmic
fractions. The Ad core did not function as an efficient template in the
cell-free transcription system with nuclear extracts of uninfected HeLa
cells. The addition of TAF-I resulted in the stimulation of
transcription from E1A and ML promoters on the Ad core. TAF-I was
required, at least, for the formation of preinitiation complexes. These
observations suggest that, in addition to factors essential for
transcription on naked DNA template, the factor such as TAF-I needed
for replication of the Ad core is also required for transcription from
the Ad genome in a chromatin-like structure.
In general, the transcription level of an individual gene is
regulated by the quantity and the combination of a variety of
transcription factors. However, the kinetical alteration of
transcription pattern, for instance, in the early development or in the
cell cycle progression, is thought to be dependent on changes of not
only transcription factors but also the template DNA structure. Namely,
the amount of active transcription factors in nucleus and the
accessibility of the transcription factors to the regulatory regions on
DNA play important roles in gene regulation. It has not been well known
how the genes that are transcriptionally repressed presumably by
forming tight chromatin structure are activated. Recently, the
existence of mechanisms of the direct access of the transcription
factor to the repressed chromatin by nucleosome displacement is
suggested (reviewed by Adams and Workman
(1) ). Accordingly, the
recent suggestion that acetylation of core histones exerts an influence
on the accessibility of chromatin to the transcription factor
(2) is of interest.
Adenovirus (Ad)
We have been studying the correlation between the regulations of Ad
DNA replication and transcription in static ways
(9, 10, 11) . Here we developed a cell-free
transcription system using the Ad core as template and examined the
effect of TAF-I on transcription from promoters in the Ad core. TAF-I,
which is essential for DNA replication of the Ad core, also stimulated
transcription from Ad E1A and ML promoters on the Ad core in the
cell-free transcription system. Furthermore, transcription from the ML
promoter in the Ad core was further stimulated when DNA replication of
the Ad core proceeded prior to the transcription reaction. Ad
transcription units can be divided into early and late genes, based on
their timing of expression after infection. Thomas and Mathews
(12) have revealed that the Ad late gene products appear in the
infected cells only after Ad DNA replication. Therefore, this system is
promising for the analysis of the molecular mechanism for activation of
transcription of the genes in the Ad core, as one of models for the
activation of repressed genes.
It has been suggested that the Ad genome DNA complexed with
basic proteins (Ad core) is a bona fide template for early
stage transcription and DNA replication
(5) . Therefore we have
first established the cell-free replication system with the Ad core as
a template
(8) and purified a novel host factor, TAF-I, that
stimulates DNA replication of Ad core (Fig. 1). The
denaturation-renaturation experiment indicates that a major portion of
TAF-I complementation activity corresponds to the 39-kDa peptide, while
the 41-kDa peptide shows less activity than the 39-kDa peptide. TAF-I
would be an acidic protein, judging from its elution profiles on ion
exchange chromatographies during purification
(8) . It is
possible that TAF-I activates the Ad core for DNA replication by
interacting with basic core proteins V and/or VII and by inducing
structural change(s) of the Ad core. The same could be true for
activation of transcription from the Ad core.
Using Ad core as the
template (Fig. 4), a quite low level of transcription from E1A
promoter was detected. We could not observe the transcription activity
from ML promoter on the Ad core. The addition of TAF-I stimulated the
transcription activities from both E1A and ML promoters. The level of
stimulation by TAF-I was greater for E1A promoter than for ML promoter.
The reason for this difference is presently unclear. In the replication
reaction, DNA synthesis is found to proceed from the terminus to the
inside depending on the increasing amounts of TAF-I
(8) . It is,
therefore, presumed that TAF-I could interact with the Ad core from the
terminus where the E1A promoter resides. Analysis using Sarkosyl
(Fig. 5) indicated that TAF-I is required at least for the step
of the formation of initiation complexes. ATP was not needed for TAF-I
action. Since TAF-I stimulates two different mechanisms on DNA,
i.e. replication and transcription, TAF-I could interact
directly with core proteins rather than the mechanisms involved in each
reaction. Accordingly, it has been reported that the addition of core
protein VII repressed the cell-free DNA replication of Ad DNA-prot
(22) and the cell-free transcription of the Ad naked DNA
(23) . It is possible that treatment by TAF-I induces a
structural change of the Ad core or dissociates the core proteins from
DNA and thereby makes the means for replication and transcription
accessible to its association sites.
Transcription units of many DNA
viruses, including papovaviruses
(24) , herpesviruses
(25) , and adenoviruses
(12) can be divided into early
and late genes depending on the onset of their expression before or
after DNA replication of the genome, respectively. It has been shown
that the activation of transcription from Ad ML promoter is dependent
on the Ad DNA replication in infected cells. Thomas and Mathews
(12) showed by superinfection experiments that late gene
products from secondarily infected viruses are not detected before
their own DNA replication even when the host cells are in ``the
late stage'' by the first infection. This observation suggests
that late stage-specific change(s) of environment, i.e. change(s) in activity of transcription factor(s), is not enough
for the onset of transcription of Ad ML promoter. We developed the
coupled cell-free system that reproduced the DNA replication-dependent
stimulation of transcription from ML promoter using the Ad core as
template (Fig. 6). This DNA replicational activation of the late
gene transcription was not detected using the naked Ad DNA-prot as
template (data not shown). The exact mechanism of the DNA replicational
activation of Ad ML promoter transcription remains to be resolved.
Replication of the Ad genome and transcription of E1A gene, whose
cis-acting elements are present in the terminal region of Ad DNA, are
readily activated by the addition of TAF-I. It is hypothesized that
transcription factors and/or transcription machineries become
accessible to the ML promoter region only when DNA polymerase proceeds.
Workman et al. (26) have shown that ML transcription
factor present during nucleosome assembly enhances the ability of
transcription factor IID for binding to the TATA-box in competition
with nucleosomes. Toth et al. (27) have shown that in
Ad-infected cells transcription factors ML transcription factor and
transcription factor IID bind to Ad ML promoter only in the late stage
of infection. Although it is unclear whether the core proteins are
associated with Ad DNA after the Ad core DNA replication, the DNA
replication should provide the opportunity that the structure of the Ad
core is perturbed, as previously suggested for disruption of nucleosome
structure by DNA replication
(28, 29) .
Recently
static and dynamic correlations between DNA-mediated mechanisms have
been revealed in, for instance, transcription and recombination
(30) and transcription and repair (reviewed by Buratowski
(31) ). Mechanisms for activation of genes transcriptionally
repressed are not yet well known. Access of transcription factors to
such the repressed genes directly
(32) or by modifying the
chromosomal proteins
(2) is suggested. Here, we have proven a
novel mechanism of transcription regulation in that a host factor, such
as TAF-I, could play a role in activation of transcription of repressed
genes, and DNA replication would disrupt the repressed chromatin
structure and exert the influence of the accessibility of the chromatin
to transcription apparatus. Although much remains to be done, the study
using this cell-free system must provide a useful cue for the
understanding of transcription regulation not only by changes of
transcription factors in quantity and quality but also by the
structural change of template DNA.
We thank Dr. A. Ishihama for his support at the
initial stage of this work and Drs. A. Kikuchi and Y. Ishimi and our
colleagues in RIKEN for useful discussions.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(
)
is a double-stranded DNA virus of which DNA replication and
transcription have been studied extensively as a good model of the
eukaryotic system. The Ad genome DNA in the virion associates closely
with viral basic proteins VII and V in nucleosome-like structure
(3, 4) . It is reported that the Ad genome DNA is
associated with protein VII in the nuclei of infected cells
(5) , suggesting that the Ad core is the bona fide template for replication and transcription in infected cells.
Russell and co-workers revealed that the Ad core can act as template
for the initiation of Ad DNA replication in a cell-free system,
although the elongation of DNA replication is quite limited to only to
0.1 the length of the genome
(6, 7) . In the course of
experiments to test the possibility that the Ad core is the functional
template for early transcription and DNA replication in cell-free
systems, we found that a novel host factor, template activating factor
I (TAF-I), greatly stimulates the elongation of DNA replication using
the Ad core as template in the cell-free system consisting of viral
replication proteins and uninfected HeLa nuclear extracts
(8) .
Templates
The Ad core was prepared by heating Ad
type 5 virions in the presence of 0.5% sodium deoxycholate and purified
as described elsewhere
(8) . Ad DNA-prot was prepared from Ad
type 2 virions as previously described
(13) . Plasmid pLA1 (6572
base pairs) contains 3290 base pairs of Ad type 5 DNA left terminus
(14, 15) . pSmaF is a plasmid-containing SmaIF
fragment of Ad type 2
(16) . Plasmid DNAs were prepared from
transformants by the alkali-SDS method and purified by CsCl
centrifugation
(17) .
Denaturation-Renaturation Protocol of TAF-I
TAF-I
fraction eluted from MonoQ column chromatography
(8) was
electrophoresed in 12.5% SDS-polyacrylamide gel. The gel was excised,
and the proteins were eluted and renatured under conditions as
described elsewhere
(18) . To remove guanidine HCl, the proteins
were dialyzed against 10 m
M Tris-HCl, pH 7.8, 0.1 m
M
EDTA, 10% glycerol, and 0.1 m
M dithiothreitol. Renatured
proteins were assayed for TAF-I activity using the cell-free Ad core
DNA replication system
(8) . The Ad core (60 ng of DNA) was
incubated with Ad pol/pTP, Ad DBP, uninfected HeLa nuclear extracts,
and the renatured fractions in 12.5 µl of reaction mixture
containing 25 m
M Hepes, pH 7.5, 4 m
M dithiothreitol,
5 m
M MgCl, 5 m
M creatine phosphate, 20
µg/ml phosphocreatinekinase, 200 µg/ml bovine serum albumin, 20
m
M NaCl, 40 µ
M each dATP, dGTP, and dTTP, 4
µ
M dCTP, 5 µCi of
[
-
P]dCTP, and 3 m
M ATP at 30
°C for 2 h. The products were digested with KpnI and
electrophoresed on 0.8% agarose gel. The gel was dried and subjected to
autoradiography.
Cell-free Transcription
Nuclear extracts were
prepared from uninfected HeLa cells according to the method described
by Dignam et al. (19) . Cell-free transcription using
HeLa nuclear extracts was performed essentially as previously described
(9) . After the transcription mixture was incubated at 30 °C
for 1 h, the transcripts were analyzed by primer extension. Primers
PE1A (5`-GGCAGATAATATGTCTC-3`, nucleotide sequence at positions
577-561 of Ad type 2) for E1A transcript, and PML
(5`-AATATCAAATCCTCCTCGT-3`, 6158-6140) or PML2
(5`-GCATCACCGCGGGCCAGGTGAATATCAAAT-3`, 6178-6149) for transcript
from ML promoter were end-labeled. The purified RNAs were incubated at
65 °C for 10 min with the primers in 10 µl of reaction mixture
containing 50 m
M Tris-HCl, pH 8.3, 75 m
M KCl, 3
m
M MgCl, 10 m
M dithiothreitol, and 1
m
M each 4 dNTPs, followed by gradual cooling to room
temperature. The primer annealed with transcript was extended by 80
units of Moloney murine leukemia virus RNase H free reverse
transcriptase (Life Technologies Inc.) at 42 °C for 1 h. The
products were precipitated with ethanol and electrophoresed on 8%
polyacrylamide gel in the presence of urea. The gels were dried and
subjected to autoradiography.
Coupled Cell-free System
For replication prior to
the transcription reaction, the Ad core (480 ng of DNA) was incubated
with Ad pol/pTP, Ad DBP, TAF-I, and HeLa nuclear extracts (7 µg of
protein) in 40 µl of reaction mixture as above at 30 °C for 2
h, except that [-
P]dCTP was omitted and the
concentration of dCTP was raised to 40 µ
M. For
transcription, the Ad core which had been subjected to the replication
reaction was mixed with HeLa nuclear extracts (50 µg of protein) in
80 µl of reaction mixture under the conditions suitable for
transcription by the addition of MgCl
(final concentration,
7.5 m
M), creatine phosphate (final concentration, 7.5
m
M), RNase inhibitor, GTP, CTP, and UTP. The final
concentration of ATP was lowered to 1.5 m
M, since higher
concentration of ATP inhibits transcription
(10) . After the
mixtures were incubated at 30 °C for 1 h, the transcripts were
analyzed by primer extension as the section above.
Stimulation of the Ad Core DNA Replication by TAF-I in
the Cell-free System
Recently we found that the elongation of
DNA replication in a cell-free system using the Ad core as template was
greatly stimulated by the addition of cytoplasmic fractions prepared
from uninfected HeLa cells. We purified the stimulatory factor of the
Ad core DNA replication from HeLa cytoplasmic fractions
(8) .
The SDS-polyacrylamide gel of the fraction of the final purification
step revealed proteins with molecular mass of 41, 39, and 32 kDa (Fig.
1 A). In order to identify the protein(s) that carries the
stimulatory activity of the Ad core DNA replication, the proteins that
were eluted from the gel and renatured were assayed for the
complementing activity for the cell-free Ad core DNA replication system
(Fig. 1 B). The stimulatory activity was detected in the
fractions 1, 2, and 3 and the maximal activity existed in the fraction
2. The SDS-polyacrylamide gel of the eluted proteins revealed that the
39-kDa protein is commonly present in the fractions 1, 2, and 3
(Fig. 1 C). Therefore, we concluded that the activity is
mainly associated to the 39-kDa protein and designated this activity as
TAF-I, although it is possible that the 39-kDa protein is a product of
proteolysis from the 41-kDa protein. Fig. 1 E shows the
elution profile of the purified TAF-I through a Superose 2PC column.
Proteins with molecular mass of 41 and 39 kDa formed a peak
corresponding to an apparent molecular mass of about 240 kDa. This
observation suggests that TAF-I exists as either hetero- or homohexamer
of 41- and 39-kDa polypeptides.
Figure 1:
The stimulatory activity (TAF-I) for
the Ad core DNA replication purified from HeLa cytoplasmic fractions.
A, TAF-I fraction eluted from Mono Q column chromatography (8)
was electrophoresed in 12.5% SDS-polyacrylamide gel. The molecular mass
of the detected proteins were 41, 39, and 32 kDa from left to
right. The gel was excised into five fragments shown by
brackets ( 1-5) and the proteins were eluted and
renatured. B, the Ad core DNA replication assay was performed
in the absence ( lane -) or presence ( lanes
1-5) of the eluted and renatured proteins. The products were
digested with KpnI and separated in a 0.8% agarose gel. The
fragments of KpnI digestion of Ad type 5 are indicated on the
left of the gel. The G and H fragments are
located at the ends of the genome DNA in which replication starts (see
D). C, the SDS-polyacrylamide gel pattern of the
TAF-I fraction ( lane T) and the eluted proteins ( lanes
1-5). Lane M indicates molecular mass markers
corresponding to proteins with molecular masses of 43 and 31 kDa. The
gel was stained with silver. The common band between 41 and 39 kDa seen
in lanes 1-5 is probably derived from the bovine serum
albumin fraction added to the all fractions in the renaturation step.
D, KpnI fragments of Ad type 5 DNA. E,
elution profile of the TAF-I protein through gel filtration. One
microgram of the Mono-Q fraction of the TAF-I protein was loaded onto
Pharmacia Superose 2PC column equilibrated with a buffer containing 30
m
M Hepes-NaOH, pH 7.8, 0.5 m
M EDTA, 10% glycerol, and
50 m
M NaCl. Aliquots of the indicated fractions
( 7-25) were analyzed by electrophoresis on 10%
polyacrylamide gel in the presence of SDS. The gel was stained with a
silver stain kit (Bio-Rad). Lane TAF-I indicates the sample
before gel filtration. For calibration of the molecular mass,
thyrogloblin (670 kDa), -globin (158 kDa), ovalbumin (44 kDa), and
myoglobin (17 kDa) were analyzed under the same conditions, and their
positions are indicated by
arrowheads.
Cell-free Transcription on Cloned DNA in
Plasmid
Next, we examined transcription activities from Ad E1A
and ML promoters as representatives of early and late promoters in the
cell-free transcription system with uninfected HeLa nuclear extracts.
Transcription activities on truncated plasmid DNA were compared with
those on closed circular plasmid DNA in the cell-free system to examine
whether the trans-acting factors which bind to the cis-acting elements
in these promoters are active in our system (Fig. 2). The
products from the cell-free transcription were analyzed by primer
extension.
Figure 2:
Transcription activity of the truncated
templates. The cell-free transcription using uninfected HeLa nuclear
extracts was performed without DNA ( no DNA), with 500 ng of
DNA in the form of supercoiled ( ccc) or truncated DNA as shown
at the top of the figure as template. Transcripts were
analyzed by primer extension using end-labeled primer PE1A ( Panel
A) or PML ( Panel B). The products were electrophoresed in
8% polyacrylamide gel in the presence of urea. Lane M indicates HpaII-digested pUC18 DNA as markers. The
positions of expected products of accurate transcription from E1A or ML
promoter are indicated.
For the Ad E1A promoter, plasmid pLA1 which contains 3290
base pairs of Ad type 5 DNA left terminus was used as the template DNA
(Fig. 2 A). When the plasmid DNA was digested with
EcoRI which cut at the left end of Ad DNA or with
BalI which cut at -231 relative to the transcription
initiation site (+1), the transcription activity on these
truncated DNAs was similar to that on supercoiled template DNA
(Fig. 2 A, lanes 1-3). In contrast, only a
slight activity was detected with pLA1 digested with PvuII
which cuts at -47 and +124 of E1A promoter ( lane
4). In this case, a TATA box is the only known cis-regulatory
element in the upstream of the promoter. These results suggest that the
region between the BalI and PvuII sites is essential
for the effective transcription from Ad type 5 E1A promoter
(20) . Plasmid pSmaF was used for testing the Ad ML promoter
activity in the cell-free transcription system (Fig. 2 B). In
this case, negatively supercoiled template DNA was the most active
among the template structures tested (Fig. 2 B). Linearization
by SmaI resulted in decrease of the transcription activity to
about a half level of that on supercoiled template DNA
(Fig. 2 B, lanes 2 and 3). pSmaF
digested with PmaCI showed a faint signal of the transcription
from the ML promoter ( lane 4), while ScaI digestion
of the plasmid leaded to no signal in primer extension ( lane
5). PmaCI digests at the site recognized by ML
transcription factor which is the most important factor involved in
transcription of the ML promoter, and ScaI-truncated template
does not contain the primer site. These data indicate that nuclear
extracts from uninfected HeLa cells contain all the essential
transcription factors which support efficient transcription from both
E1A and ML promoters, although it has been reported that the Ad
infection induces transcription factor(s) which increase(s) the
efficiency of the transcription from the ML promoter
(21) .
Since the primer extension experiment using the primer PML in
Fig. 2
resulted in a product even without template DNA ( lane
1), we used another primer PML2 in the latter experiments with
which a 140-nucleotide long product is synthesized.
Transcription Activity on Ad DNA Genome
Ad genome
is a linear DNA covalently linked to TP at the 5` ends, which is called
Ad DNA-prot. It is possible that TPs at the end of genome DNA play
roles in transcription from E1A and ML promoters. Thus we next examined
the transcription activities from E1A and ML promoters on Ad DNA-prot.
As shown in Fig. 3, the transcription on Ad DNA-prot appeared to
be as much as that on plasmid DNA, since the apparent level of
transcripts in Fig. 3would reflect the molar ratio between the
plasmids and Ad DNA-prot used. These results suggest that TP had no
effect on transcription activity from Ad DNA.
Figure 3:
Transcription activity of Ad DNA-prot. The
cell-free transcription was performed with supercoiled plasmid DNAs
(300 ng) or Ad type 2 DNA-prot (300 ng DNA) as template. Transcripts
were analyzed by primer extension using end-labeled primer PE1A
( Panel A) or PML2 ( Panel B). The positions of
expected products of accurate transcription from E1A or ML promoter are
indicated.
In contrast to naked
DNAs, Ad core functioned only poorly as the template of transcription
(Fig. 4 B). Transcription from E1A promoter was observed
to be inefficient, whereas ML promoter was virtually inactive on Ad
core. It is possible that TAF-I would have an effect on transcription
from Ad core as it does stimulate the replication reaction from the Ad
core. This was indeed the case as shown in Fig. 4 B.
TAF-I stimulated transcription from E1A promoter effectively, and it
also activated transcription from the ML promoter on the Ad core less
efficiently than that from E1A promoter. TAF-I had no effect on
transcription from naked DNAs, both of plasmids, and Ad genome DNA
(Fig. 4 A). The data presented so far suggest that the
factor required for DNA replication of Ad core facilitates
transcription from transcription promoters on Ad core.
Figure 4:
Stimulation of transcription from Ad core
DNA by template activating factor-I. The cell-free transcription was
performed with supercoiled plasmid DNAs (150 ng of pLA1 and 150 ng of
pSmaF, lanes 1-3 in Panel A and lane 1 in Panel B), 300 ng Ad type 2 DNA-prot ( lanes
4-6 in Panel A and lanes 2 and 3 in Panel B) or Ad core (300 ng for lane 4 in
Panel B and 600 ng for lanes 5-7 in Panel
B) in the absence ( lanes 1 and 4 in Panel A and lanes 1, 2, 4, and 5 in
Panel B) or presence (0.04 unit, lanes 2 and 5 in Panel A and lane 6 in Panel B; 0.12
unit, lanes 3 and 6 in Panel A and lanes
3 and 7 in Panel B) of TAF-I. Transcripts were
analyzed by primer extension as shown in Fig.
3.
To clarify
the step affected by TAF-I, the level of the formation of transcription
initiation complexes on the Ad core was measured in the presence or
absence of TAF-I (Fig. 5). The Ad core was first incubated with
the components indicated, and then the remaining components and
Sarkosyl, which inhibits the formation of additional initiation
complexes but not the process of RNA synthesis from preformed
initiation complexes, were added. TAF-I was required during the
formation of initiation complexes ( lanes 2 and 3).
This step did not need ATP. Therefore, it is suggested that TAF-I is
necessary at least for the formation of initiation complexes, although
it is unclear whether or not TAF-I is required for the elongation
process.
Figure 5:
Effect of
TAF-I on the formation of initiation complex. The Ad core (300 ng) was
first incubated at 30 °C for 30 min in nuclear extracts with 0.04
unit of TAF-I ( lanes 2 and 3) in the presence
( lanes 3 and 5) or absence ( lanes 1,
2, and 4) of ATP. At the zero time ( 0 min)
Sarkosyl at the final concentration of 0.025% and the remaining
components indicated in the figure were added and further incubated at
30 °C for 60 min. Transcripts were analyzed by primer extension as
described above. Lane M indicates size markers. The positions
of transcripts from E1A and ML promoters are
indicated.
Stimulation of Transcription from Ad ML Promoter by Ad
DNA Replication in the Coupled Cell-free System
Ad transcription
units can be divided into early and late genes, based on their timing
of expression after infection. Thomas and Mathews
(12) have
revealed that the Ad late gene products appear in the infected cells
only after Ad DNA replication. Since transcription from ML promoter on
the Ad core template by TAF-I was less than that from E1A promoter, we
next tried to reproduce the DNA replication-dependent stimulation of
transcription from Ad ML promoter in a cell-free system. Because the
reaction conditions of Ad DNA replication disagree with those of the
transcription in the cell-free systems (data not shown), we used the
two-step reaction in which the DNA replication was performed, followed
by the transcription. The Ad core was incubated with the DNA
replication enzymes in the presence of TAF-I, dNTPs, and ATP for DNA
replication reaction. Then, uninfected HeLa nuclear extracts, GTP, CTP,
and UTP, were added to the reaction mixture, and the second incubation
was carried out for transcription. The transcripts from E1A and ML
promoters were analyzed by primer extension (Fig. 6). Without DNA
replication E1A promoter was more active than ML promoter in agreement
with results shown in Figs. 4 B and 5 ( lanes 5 versus
6). When the transcription reaction was performed after DNA
replication, the transcription from ML promoter was activated
( lanes 1-4). The level of the activation was increased
as a function of the amount of TAF-I added in the replication reaction.
In the standard replication reaction using 60 ng of Ad core, the amount
of TAF-I needed for the saturation level of replication was
0.01-0.02 unit
(8) . In the coupled system using 480 ng of
Ad core, 0.13-0.2 unit ( lanes 3 and 4) of TAF-I
was needed for the maximal transcription from ML promoter. These
results suggest that the stimulation of DNA replication by TAF-I acts
as the primary mediator of the activation of transcription from ML
promoter, and TAF-I does not function independently as an activator.
The transcription from ML promoter was repressed to the similar level
with reaction without replication, when aphidicholin, which has been
shown to be an inhibitor of elongation of the cell-free Ad DNA
replication
(8) , was added to the replication reaction (data
not shown). Thus this cell-free system reproduced the DNA
replication-dependent stimulation of late transcription.
Figure 6:
The DNA replication-dependent activation
of late transcription in the cell-free system. For replication prior to
the transcription reaction ( lanes 1-4), the Ad core (480
ng) was incubated with Ad pol/pTP, Ad DBP, and HeLa nuclear extracts in
the absence ( lane 1) or presence of 0.07 ( lane 2),
0.13 ( lane 3), and 0.2 ( lane 4) unit of TAF-I at 30
°C for 2 h. For transcription, 480 ng of DNA of the Ad core which
had been subjected to the replication reaction ( lanes
1-4), or 480 ng of DNA of the unreplicated Ad core
( lanes 5 and 6) were mixed with HeLa nuclear extracts
in the absence ( lane 5) or presence ( lane 6) of 0.13
unit of TAF-I under the conditions suitable for transcription. After
the mixtures were incubated at 30 °C for 1 h, the transcripts were
analyzed by primer extension with end-labeled PE1A and PML2. The
products were electrophoresed on 8% polyacrylamide gel in the presence
of urea. The gel was dried and subjected to
autoradiography.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.