From the Department of Biochemistry, University of Illinois, Urbana, Illinois 61801
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ABSTRACT |
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A human RNA polymerase II (pol II) complex was isolated from a HeLa-derived cell line that conditionally expresses an epitope-tagged RPB9 subunit of human pol II. The isolated FLAG-tagged pol II complex (f:pol II) contains a subset of general transcription factors but is devoid of TFIID and TFIIA. In conjunction with TATA-binding protein (TBP) or TFIID, f:pol II is able to mediate both basal and activated transcription by Gal4-VP16 when a transcriptional coactivator PC4 is also provided. Interestingly, PC4, in the absence of a transcriptional activator, actually functions as a repressor to inhibit basal transcription. Remarkably, TBP is able to mediate activator function in this transcription system. The presence of TBP-associated factors, however, helps overcome PC4 repression and further enhance the level of activation mediated by TBP. Alleviation of PC4 repression can also be achieved by preincubation of the transcriptional components with the DNA template. Sarkosyl disruption of preinitiation complex formation further illustrates that PC4 can only inhibit transcription prior to the assembly of a functional preinitiation complex. These results suggest that PC4 represses basal transcription by preventing the assembly of a functional preinitiation complex, but it has no effect on the later steps of the transcriptional process.
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INTRODUCTION |
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In eukaryotes, transcription of protein-encoding genes requires general transcription factors (GTFs)1 and RNA polymerase II (pol II), which are assembled on the promoter region to form a preinitiation complex (PIC) capable of producing RNAs when both ribonucleoside triphosphates and energy sources are provided (1, 2). In vivo, the PIC assembly is a rate-limiting step for the initiation of transcription and is often facilitated by gene-specific transcriptional activators (3, 4). Currently, there are two proposed pathways for the PIC assembly on TATA-containing promoters. In the sequential pathway, TFIID binding to the TATA box is followed, in a stepwise fashion, by the joining of TFIIB, pol II/TFIIF, TFIIE, and TFIIH, whereas in the two-component pathway, binding of TFIID is accompanied by a preassembled pol II complex that contains pol II, a subset of GTFs, SRBs (suppressors of RNA polymerase B mutations (5, 6)), and other proteins involved in chromatin remodeling, DNA repair, or mRNA processing (5-13). Transcriptional activators, in most cases, are able to increase the level of initiation by enhancing the recruitment of TFIID and/or other components of the basal transcription machinery to the promoter region (3, 4). This activation process often requires transcriptional coactivators. Thus far, two major classes of general coactivators required for activator function have been identified in mammalian cell-free transcription systems. One is TBP-associated factors (TAFs) in TFIID (14-16), and the other is protein cofactors derived from the upstream stimulatory activity (USA) found in the phosphocellulose P11 0.85 M KCl fraction of HeLa nuclear extracts (17, 18).
Positive cofactor 4 (PC4) was isolated from a crude USA fraction and was able to substitute for USA to mediate activator-dependent transcription in vitro (19-21). PC4 is a nonspecific DNA-binding protein, which shows a higher affinity toward single-stranded (ss) DNA molecule (20-22). The ssDNA binding activity of PC4 can replace human ssDNA-binding protein (HSSB, also called replication protein A (RPA)) in supporting the T antigen-catalyzed unwinding of SV40 origin-containing duplex DNA (23). Nevertheless, PC4 cannot substitute for HSSB in other aspects of replication activities mediated by HSSB (23). Likewise, the transcriptional activity of PC4 cannot be replaced by other ssDNA-binding proteins (19). The coactivator function of PC4 seems to correlate with its double-stranded DNA binding activity (21) and its interactions with transcriptional activators and with components of the general transcription machinery such as TFIIA (19). Surprisingly, gene inactivation of a PC4 homologue in yeast does not lead to cell death, indicating that PC4 is nonessential in yeast (24, 25). Since yeast PC4 also exhibits distinct properties from that of human PC4 (24, 25), it is likely that PC4 may function differentially in various organisms. This remains to be further investigated.
Using an in vitro transcription system reconstituted with either TBP or TFIID and a preassembled pol II complex, we found that PC4 could function as a repressor to suppress basal transcription in the absence of an activator. Interestingly, TBP was able to mediate Gal4-VP16 activation in the absence of TAFs. This finding suggests that human TBP can indeed mediate activator function, as observed in the yeast system (26-28). To understand the molecular mechanism of PC4 repression, we carried out template challenge and Sarkosyl disruption experiments using our two-component transcription system. The results indicate that PC4 represses transcription by preventing the assembly of a functional preinitiation complex when an activator is not present.
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EXPERIMENTAL PROCEDURES |
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Plasmid Constructions--
A tetracycline-regulated human
RPB9-expressing plasmid, pTetCMV-Fo:hRPB9, was first
constructed by cloning the RPB9 cDNA (29), isolated from
pBn-Fo:14.5 (provided by H. Ge) between NdeI and
XbaI sites, to pTetCMV-Fo(AS) (30) at the same
enzyme-cutting sites. The expression plasmid, pF:TFIIA(55)-11d, was
made by transferring the p55 insert from pET11aN1-376 (31) into
pF:TBP-11d (32) after removing the TBP insert between NdeI
and EcoRI sites. Similarly, plasmids pF:TFIIA(1-274)-11d, pF:TFIIA(275-376), and pF:TFIIA(hp12) were constructed by swapping individual TFIIA inserts from pJD1-274, pJDGEX2t(L)275-376, and pGEX2t(L)hp12 (33) with the TBP insert from pF:TBP-11d between NdeI and BamHI sites.
Establishment of a Tetracycline-regulated Clonal Human Cell Line-- Ten µg of PvuI-linearized pTetCMV-Fo:hRPB9 DNA was cotransfected with 50 µg of sheared calf thymus DNA and with 0.5 µg of SacI-linearized pREP4 (Invitrogen) into HtTA-1 (34), a HeLa-derived cell line that constitutively expresses a tetracycline-controlled transactivator. Detailed procedures for establishing the tetracycline-regulated clonal cell lines had been described (30). A cell line (hRPB9-3) that showed induced expression of FLAG-tagged hRPB9 in the absence of tetracycline was isolated after screening a dozen hygromycin-resistant cellular clones.
Western Blotting-- To detect the presence of individual GTFs as shown in Fig. 1A, 50 µl of HeLa nuclear extracts and 200 µl of f:pol II were mixed separately with an equal volume of the 2× protein sample buffer. The protein mixture was then separated by 10% SDS-polyacrylamide gel electrophoresis using a preparative mini-gel apparatus (Bio-Rad). After transfer to the nitrocellulose filters, samples were divided into multiple lanes using a Mini-Protean II multiscreen apparatus (Bio-Rad). Each sample lane was then incubated with a primary antibody, usually diluted 1000-fold unless otherwise specified, in a final volume of 600 µl. The rest of the procedures for Western blotting were performed as described (30).
Protein Purification-- The f:pol II complex was purified from hRPB9-3 as follows. The hRPB9-3 cell line was maintained in suspension culture with Joklik medium containing 5% calf serum in the presence of tetracycline (1 µg/ml) and selected with G418 (0.6 mg/ml, total weight) for 4 days before expansion for the preparation of nuclear extracts and S100. To induce protein expression, cells were pelleted and washed at least 3 times with 1× phosphate-buffered saline to remove tetracycline. Cells were then resuspended in fresh Joklik medium plus 5% calf serum. Nuclear extracts and S100 were prepared from hRPB9-3 cells, 4 days after removing tetracycline, as described (35). To purify f:pol II, 10 ml of the hRPB9-3 nuclear extract or S100 was incubated with 250-500 µl of anti-FLAG M2-agarose beads (Kodak) at 4 °C for 6-12 h. Bound proteins were then washed and eluted with the synthetic FLAG peptide with 100 mM KCl-containing buffer as described previously for TFIID purification (36).
Recombinant PC4 was purified from bacteria harboring pET11a/PC4, obtained from H. Ge, as described (37). Purification of recombinant FLAG-tagged basal transcription factors including TFIIA (p55, p35, p19, and p12), TFIIB, TBP, TFIIEIn Vitro Transcription--
In vitro transcription
was typically carried out in a 25-µl reaction mixture containing 50 ng of pG5HMC2AT, 20 ng of pML53, 42 ng of
renatured TFIIA (35 ng of p55 and 7 ng of p12), 10 ng of TFIIB, 10 ng
of TBP, or 1 µl of FLAG-tagged TFIID (which contains approximately 1 ng of TBP as judged by Western blotting), 20 ng each of TFIIE
and
TFIIE
, 28 ng of renatured TFIIF (20 ng of RAP74 and 8 ng of RAP30),
1 µl (~7.5 ng) of FLAG-tagged TFIIH, and 0.6 µl (~18 ng) of
core-pol II or f:pol II using the conditions as described previously
(17). For activator-dependent transcription, 100 ng of PC4
and 50 ng of Gal4-VP16 were also included as specified. In the minimal
transcription system, 3 µl (~90 ng) of f:pol II was used in
conjunction with either 2 ng of TBP or 2 µl of FLAG-tagged TFIID. The
amount of FLAG-tagged Gal4 fusion proteins used in the minimal
transcription reaction was: 3 ng of Gal4 (1-94), 6 ng of Gal4-Pro, 6 ng of Gal4-Gln, and 30 ng of Gal4 (1-147). Reactions were then
performed and analyzed as described (36).
Template Challenge Experiments--
A two-step incubation
procedure described by Kaiser et al. (21) was used with a
minor modification. Briefly, 50 ng of pG5HMC2AT and 20 ng of pML53 were preincubated with either 2 µl of
FLAG-tagged TFIID (or 2 ng of TBP) or 3 µl of f:pol II at 30 °C
for 50 min. Ribonucleoside triphosphates (NTPs) and the other component
required for transcription (f:pol II or TFIID or TBP) were added after the preincubation period to initiate transcription. Reactions were
continued at 30 °C for 60 min and then analyzed for RNA formation (36). Gal4-VP16 and PC4, when added, were used at 50 and 100 ng,
respectively. The challenge template (500 ng of
pG5HMC2AT or 500 ng of pML
53) was included
either at the beginning or at the end of the preincubation period as
depicted in Fig. 3A.
Sarkosyl Disruption of Preinitiation Complex Formation--
In
this experiment, 0.015% Sarkosyl was added either at the beginning or
at the end of the preincubation period, which was performed in the
presence of TBP, f:pol II, and both pG5HMC2AT and pML53 DNA templates as described in the template challenge experiments. Ribonucleoside triphosphates were then added to initiate transcription. One hundred nanograms of PC4 were added at various time
points as outlined at the bottom of Fig. 4.
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RESULTS |
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Isolation of a TFIID-deficient Human Pol II Complex-- A human pol II complex was purified from a clonal HeLa-derived cell line (hRPB9-3) that conditionally expresses the FLAG-tagged RPB9 subunit of human pol II (see "Experimental Procedures"). The purified FLAG-tagged pol II complex (f:pol II) contains not only pol II subunits as detected by Western blotting with antibodies against RPB1, RPB2, RPB6, RPB8, and RPB92 but also a subset of GTFs including TFIIB, TFIIE, TFIIF, and TFIIH (Fig. 1A). The f:pol II complex contains stoichiometric amounts of TFIIB and TFIIF but substoichiometric quantities of TFIIE and TFIIH (Fig. 1A, compare relative signals detected in nuclear extracts and f:pol II), as also evidenced by quantitative Western blotting using purified recombinant proteins as standards.3 TFIID and TFIIA were not detected in f:pol II at a sensitivity of 1 ng with anti-TBP and anti-TAFII55 antibodies and at a sensitivity of 0.1 ng with anti-TFIIA p35 antibodies (Fig. 1A).2 Our f:pol II, enriched approximately 200-fold after immunoaffinity purification, did not seem to contain other transcriptional cofactors such as PC4 and Dr1 and transcriptional activators including Sp1, YY1, USF, p53, pRB, and the p50 subunit of NFkB.2
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f:pol II Is Functional in a Highly Purified in Vitro Transcription
System--
Recombinant human TFIIA, TFIIB, TFIIE, TFIIF, and
FLAG-tagged TFIID and FLAG-tagged TFIIH were used in conjunction with
either core-pol II (i.e. traditionally defined pol II) or
immunoaffinity-purified f:pol II for transcriptional analysis. Both
core-pol II and f:pol II, normalized by the content of RPB2 in the
purified complexes, showed comparable levels of transcriptional
activities irrespective of whether TBP or TFIID was used as the
TATA-binding factor (Fig. 1B, top and
bottom panels, lanes 1 and
9). The pG5HMC2AT template contains
5 Gal4-binding sites preceding the HIV-1 TATA box and the adenovirus
major late (ML) initiator element in front of a G-less cassette of
approximately 380 nucleotides, whereas pML53, which lacks the
activator-binding sites, has a shorter G-less cassette (~280
nucleotides) driven only by the major late promoter TATA and initiator
elements. In our transcription system, TFIIB, TFIIF, and a TATA binding
activity (either TBP or TFIID) were essential for transcription by
core-pol II (Fig. 1B, lanes 3, 4, and
6), whereas TFIIE and TFIIH, although not necessary for transcription from supercoiled DNA templates (44-46) (Fig.
1B, lanes 5 and 7), are required for
transcription from linearized DNA molecules.2
Interestingly, transcription from different promoter elements seem to
require differential amounts of TFIIE and TFIIH, as leaving out TFIIE
and TFIIH affected transcription from pG5HMC2AT
more dramatically than from pML
53 (Fig. 1B, lanes
5 and 7). In contrast, transcription by f:pol II
required only a TATA-binding factor (Fig. 1B, lanes
9-16), although leaving out TFIIE significantly reduced basal
transcription from both DNA templates. The transcription data not only
functionally confirm the identities of GTFs detected in our purified
f:pol II (Fig. 1, A and B) but further suggest that a two-component pathway comprised of preassembled f:pol II and a
TATA-binding factor is probably sufficient for the assembly of a
functional preinitiation complex (see below). Obviously, TFIIA and TAFs
were not needed for basal transcription from either DNA template (Fig.
1B, compare top and bottom
panels, lanes 1 versus
2 and lanes 9 versus
10).
TAF-independent and TAF-dependent Transcriptional
Activation--
To investigate the role of TBP and TAFs in
activator-dependent transcription, we compared in parallel
the transcriptional activities of TBP and TFIID in a transcription
system comprised of f:pol II, TBP or TFIID, PC4, and Gal4-VP16. In this
assay, TFIID was the only source of TAFs, which were not found in f:pol II as judged by Western blotting (Fig. 1A) and
transcriptional assays (Fig. 1B, lanes 12 in
upper and lower panels, and Fig. 1C,
lane 14). As expected, no transcription could be detected in
the absence of a TATA binding activity provided by either TBP or TFIID
(Fig. 2A, lanes 1 and 6). When TBP or TFIID, containing an equivalent amount
of TBP, was also included, basal transcription could be detected from
both pG5HMC2AT and pML53 DNA templates (Fig.
2A, lanes 2 and 7). If Gal4-VP16 was
added to the system without PC4, only minor if any enhancement of
transcription was observed (Fig. 2A, compare lanes
2 and 3, and 7 and 8), confirming the importance of additional cofactors other than TAFs in mediating activator function (17, 36). Surprisingly, the coactivator PC4 in the
absence of an activator acts as a repressor to suppress basal
transcription mediated by TBP (Fig. 2A, compare lanes
2 and 4). PC4 repression was not obvious in the case of
TFIID (Fig. 2A, compare lanes 7 and
9), indicating that TAFs can overcome PC4 repression.
Surprisingly, when Gal4-VP16 was also provided, we observed
transcriptional activation mediated by both TBP and TFIID (Fig.
2A, compare lanes 2 and 5, and
7 and 10). These data suggest that human TBP, in
the absence of TFIID TAFs, can also mediate transcriptional activation
in a mammalian cell-free transcription system, as previously shown in
yeast (5). The presence of TAFs, however, help overcome PC4 repression
and further enhance the level of activation mediated by TBP (Fig.
2A, compare lanes 7 and 9, and
7 and 10).
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PC4 Repression Could Also Be Alleviated by Preincubating TBP and
f:pol II with the DNA Template--
Since PC4 is a nonspecific
DNA-binding protein (20-22), it may inhibit transcription by blocking
the access of protein factors to the promoter region. If so, PC4
repression should be alleviated by preincubating components of the
general transcription machinery with the DNA template. To test this
hypothesis, we first examined the requirement of protein factors for
template commitment in our minimal transcription system reconstituted
with TBP (or TFIID) and f:pol II. Template commitment is usually the
rate-limiting step for the assembly of a functional preinitiation
complex (48) and is likely to be regulated by various transcription
factors and cofactors. As outlined in Fig.
3A, both
pG5HMC2AT and pML53 DNA templates were
preincubated with TBP (or TFIID) or f:pol II, individually or
simultaneously, for 50 min. The other transcription components and
ribonucleoside triphosphates were then added to initiate transcription.
Reactions were continued for an hour before they were analyzed for RNA
formation. In this experiment, a 10-fold excess of
pG5HMC2AT template was added either during or
after the preincubation period to test the stability of the protein-DNA complex. Demarcating the transcription reaction into two separate steps
via order-of-addition did not change the overall yield of transcripts
(Fig. 3, B and C, lanes 1 and
2). When excess pG5HMC2AT was added
during the preincubation period, transcription from pML
53 was
reduced because less protein became available to the pML
53 DNA
template (Fig. 3, B and C, compare lanes
2 and 3, lanes 5 and 6, and
lanes 8 and 9). If TFIID was preincubated with
DNA templates before template challenge, no reduction of pML
53
transcription was observed (Fig. 3B, compare lanes
2 and 4). This result was consistent with previous
observations that TFIID could stably bind to the promoter region once
committed (48-51). The same results were obtained by using pML
53 as
the challenge template.2 In contrast, preincubation of TBP
or f:pol II did not resist template challenge (Fig. 3B,
compare lanes 5 and 7, and Fig. 3C, compare lanes 2 and 4, and lanes 5 and
7), indicating that TBP or f:pol II alone could not stably
bind to the promoter region. This result is also consistent with the
observation that TBP, in the absence of other general transcription
components, could not commit transcription to a particular template
(51). Interestingly, when TBP and f:pol II were both present during
preincubation, this promoter-bound complex became stable and was thus
resistant to template challenge (Fig. 3C, compare
lanes 8 and 10). We conclude from these template
challenge experiments that in our two-component transcription system
only TFIID, but not TBP or f:pol II alone, can stably bind to the
promoter region and thereby commit to the transcription process,
consistent with previous results performed either with nuclear extracts
or with complete transcription systems reconstituted with partially
purified components (48-51).
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PC4 Inhibits the Assembly of a Functional Preinitiation Complex-- To further define the mechanism of PC4 repression, we divided the transcription reactions into multiple steps and performed an order-of-addition experiment with Sarkosyl challenge. In previous studies (52, 53), Sarkosyl was used to prevent the formation of a functional preinitiation complex. At a concentration of 0.015%, Sarkosyl would inhibit the formation of a preinitiation complex but not the preformed complex in transcription systems reconstituted either with partially purified GTFs (52) or with TBP and f:pol II.2 Therefore, only a single round of transcription should occur, if 0.015% Sarkosyl was added after formation of the preinitiation complex. Comparison of transcription signals obtained in the absence and presence of Sarkosyl (added at t50 after preincubation, see the time course outlined at the bottom of Fig. 4) indicated that there are approximately two to three rounds of transcription in our two-component system (Fig. 4, compare lanes 3 and 7). If Sarkosyl was included during the preincubation period (t0), transcription was abolished (Fig. 4, compare lanes 3 and 5) as no functional preinitiation complex could be formed in the presence of 0.015% Sarkosyl. We then asked if PC4 would also inhibit later steps of transcription, such as elongation and reinitiation, by adding PC4 at different time points after the assembly of the preinitiation complex. Consistent with the template challenge experiment (Fig. 3D), PC4 repression could be alleviated if transcription components were allowed to assemble on the promoter region before transcription started (Fig. 4, compare lanes 2, 4, and 10). However, the transcription level in the presence of PC4 was not restored to the original level without PC4 even after preincubation of TBP and f:pol II (Fig. 4, compare lanes 3 and 4, 9 and 10), indicating that PC4 might also affect elongation or reinitiation. When Sarkosyl was added to score for a single round of transcription, no inhibition by PC4 was observed (Fig. 4, compare lanes 11 and 12, lanes 15 and 16, lanes 19 and 20). These results suggest that PC4 also inhibits reinitiation but not elongation, in agreement with its role in preventing the assembly of a functional preinitiation complex.
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DISCUSSION |
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The isolation of a TFIID-deficient f:pol II complex provides us with a unique opportunity to investigate the role of TAFs in the transcriptional process via a two-component transcription system reconstituted with f:pol II and either TBP or TFIID. The observations that TAFs indeed contribute to a high level of activation and that TBP can also mediate activation in our transcription system (Fig. 2) indicate that the previous discrepancy between in vivo yeast studies and in vitro mammalian cell-free transcription systems are not due to species variation. It is likely that some components of the general transcription machinery may affect TBP-mediated activation. Indeed we have recently observed that human TFIIH has a significant effect on TBP-mediated activation,4 which can also be recapitulated in a transcription system reconstituted with individually purified general transcription factors. In addition to GTFs, our f:pol II also contains minor amounts of human SRBs such as cyclin C and CDK8, and some chromatin remodeling factors including GCN5 and BRG1,2 suggesting that f:pol II may correspond to previously characterized pol II holoenzymes (5-13). The exact relationship, however, remains to be further defined.
In our two-component transcription system, PC4 plays a dual role in the transcription process. In the absence of an activator, PC4 acts as a negative cofactor to suppress basal transcription, whereas in the presence of an activator, PC4 acts as a coactivator to mediate transcriptional activation. While this paper was under review, the Roeder laboratory (54) also reported that PC4 could inhibit basal transcription in a system reconstituted with only TBP, TFIIB, TFIIF, and pol II. Although the double-stranded DNA binding activity of PC4 has been shown to be essential for its coactivator function (21), it is not clear which PC4 activity is responsible for repression of basal transcription in the absence of an activator. Since deletion of the N-terminal 21 amino acid residues did not affect PC4 repression of basal transcription, it seems that the N-terminal SEAC (serine/acidic-rich) domain is not involved in transcriptional repression by PC4.2 It is therefore likely that PC4, being a nonspecific DNA-binding protein, competes with TBP or TFIID for promoter binding. In the study of SV40 DNA replication, it was found that PC4 could inhibit the formation of RNA primers required for both leading and lagging strand DNA synthesis (23). This inhibition, however, could be partially reversed by the addition of excess HSSB (23), indicating that PC4 might repress RNA primer synthesis via its nonspecific DNA binding activity. The observations were consistent with our results that PC4 repression of basal transcription could be overcome by preincubation of transcriptional components with the DNA template (Figs. 3D and 4). Alternatively PC4 may interact with components of the basal transcription machinery, thereby titrating out critical protein factors needed for transcription. Indeed, we also observed that PC4 repression of basal transcription could be overcome by increasing amounts of TFIID, TFIIH, or f:pol II in the transcription reactions.2 It is likely that activators may help stabilize or enhance the formation of a functional preinitiation complex, thereby overcoming repression by negative factors. The activation domain plays an essential role in this process, since the Gal4 DNA-binding domain alone cannot alleviate repression (Fig. 2B). Despite a low level of activator-dependent transcription, which was detected from pG5HMC2AT in the absence of PC4 in the minimal transcription system containing only Gal4-VP16, f:pol II, and TBP or TFIID (Fig. 2A, compare lanes 2 and 3, and lanes 7 and 8), the presence of PC4 significantly increased the overall level of activation (Fig. 2A, compare lanes 2, 3, and 5, and lanes 7, 8, and 10). It remains to be investigated if SRBs found in f:pol II are responsible for this stimulation or if f:pol II may contain specific cofactors that potentiate the activation mediated by Gal4-VP16.
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ACKNOWLEDGEMENTS |
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We are grateful to E. Kershnar for providing FLAG-tagged human TFIIH, to Z. F. Burton for pET23d/RAP30 and pET23d/RAP74NspV, to J. DeJong for TFIIA expression plasmids and anti-TFIIA antibodies, to H. Ge for pET11a/PC4, pBn-Fo:14.5 and anti-PC4 and -Dr1 antibodies, to M. Meisterernst for the PC4 deletion clones, to D. Reinberg for anti-TFIIH antibodies, to R. G. Roeder for anti-TFIIB, -TBP, -TFIIE, -TFIIF, and -RPB6 antibodies, to N. Thompson and R. Burgess for anti-RPB1, -RPB2, and -RPB8 antibodies, and to P. Rickert and E. Lees for anti-cyclin C and -CDK8 antibodies. We also thank H. Ge for advice on PC4 purification, and R. Dodson, S. Hou, and D. Shapiro for comments on the manuscript.
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FOOTNOTES |
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* This work was supported in part by Research Grant 5-FY96-1196 from the March of Dimes Birth Defects Foundation and in part by Research Project Grant RPG-97-135-01-MBC from the American Cancer Society.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
A Pew Scholar in the Biomedical Sciences. To whom correspondence
should be addressed: Dept. of Biochemistry, 430 Roger Adams Laboratory,
University of Illinois, 600 South Mathews Ave., Urbana, IL 61801. Tel.:
217-244-3085; Fax: 217-244-5858; E-mail: c-chiang{at}uiuc.edu.
1 The abbreviations used are: GTF, general transcription factor; pol, polymerase; PC4, positive cofactor 4; SRB, suppressor of RNA polymerase B; TBP, TATA-binding protein; TAF, TBP-associated factor; TFIID, transcription factor IID; f:pol II, a FLAG-tagged RNA polymerase II complex; USA, upstream stimulatory activity; PIC, preinitiation complex; ss, single-stranded; HSSB, human single-stranded DNA-binding protein.
2 S.-Y. Wu and C.-M. Chiang, unpublished data.
3
The f:pol II complex contains approximately 50 fmol µl1 of pol II subunits, 70 fmol
µl
1 of TFIIB, 135 fmol µl
1 of TFIIF,
1.2 fmol µl
1 of TFIIE, and 3.2 fmol µl
1
of TFIIH.
4 S.-Y. Wu, E. Kershnar, and C.-M. Chiang, manuscript in preparation.
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REFERENCES |
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