From the School of Health Sciences, Faculty of
Medicine, Kanazawa University, 5-11-80 Kodatsuno, Kanazawa, Ishikawa
920-0942, Japan and the ¶ Kazusa DNA Research Institute, 1532-3 Yana, Kisarazu, Chiba 292-0812, Japan
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ABSTRACT |
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Saccharomyces cerevisiae Gal11, a component of the holoenzyme of RNA polymerase II, interacts through its functional domains A and B with the small (Tfa2) and large (Tfa1) subunits of the general transcription factor (TF) IIE, respectively. We have recently suggested that Gal11 functions through a common pathway with TFIIE in transcriptional regulation (Sakurai, H., and Fukasawa, T. (1997) J. Biol. Chem. 272, 32663-32669). Here, we report that the activity of the TFIIH-associated kinase, responsible for phosphorylation of the largest subunit of RNA polymerase II at the carboxyl-terminal domain (CTD), is enhanced cooperatively by Gal11 and TFIIE. The enhancement of CTD phosphorylation was observed in the holoenzyme of RNA polymerase II, but not in its core enzyme. The stimulatory effect was completely abolished in the absence of either domain B of Gal11 or the Tfa1 subunit of TFIIE, suggesting that the domain B-Tfa1 interaction is necessary, if not sufficient, for an extensive phosphorylation of the CTD by TFIIH. Stimulation of basal transcription by Gal11 was coupled with enhancement of TFIIH-catalyzed CTD phosphorylation in a cell-free transcription system, suggesting that Gal11 activates transcription by stimulating the CTD phosphorylation in the cell.
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INTRODUCTION |
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In eukaryotes, RNA polymerase II (RNAPII)1 and a set of general transcription factors (TFs) including TATA-binding protein (TBP), TFIIB, TFIIE, TFIIF, and TFIIH assemble to form the preinitiation complex on the core promoter to initiate transcription from an accurate start site (1-4). The largest subunit (Rpb1) of RNAPII contains a repeated heptapeptide sequence at the carboxyl terminus, which is referred to as the carboxyl-terminal domain (CTD) (for review, see Ref. 5). The CTD is required for transcription of most, if not all, of the genes in vivo (5) as well as for mRNA processing (6). During the transcription cycle, the CTD is phosphorylated by a kinase present within TFIIH (4, 7, 8). Formation of the preinitiation complex requires RNAPII with unphosphorylated CTD, whereas elongation of transcripts is accomplished by RNAPII with phosphorylated CTD (4). These observations suggest that phosphorylation of the CTD by the TFIIH-associated kinase is an obligatory step in the transcription process from initiation to elongation.
In the yeast Saccharomyces cerevisiae, partial truncation of the CTD causes defects in expression of various genes (5, 9, 10). Genetic screening for suppressors of truncation mutations of the CTD by Nonet and Young (10) led to identification of a class of genes called SRB. Subsequent biochemical analyses suggested that nine Srb proteins (Srb2 and Srb4-11) form a complex, which is tightly associated with RNAPII at the CTD (11-14). The Srb-RNAPII complex also contains the general transcription factors TFIIB, TFIIF, and TFIIH and the global transcription regulators Gal11 and Swi/Snf. The whole complex has been termed RNAPII holoenzyme since it has been implicated to be a preformed initiation subcomplex (12-17). Another form of RNAPII holoenzyme was isolated by Kornberg and co-workers (18) as a "mediator"-RNAPII complex. The mediator was fractionated from whole cell extracts for the capacity that confers core RNAPII responsiveness to DNA sequence-specific activators in the presence of the general transcription factors in vitro (18). The mediator fraction, also associated with RNAPII at the CTD, contains TFIIF and global transcription regulators including Srb2, Srb4, Srb5, Srb6, Gal11, Sin4, Rgr1, Rox3, and Med6 (18-21). Thus, TFIIF, some of the Srb proteins, and Gal11 are common components in both forms of the holoenzyme, whereas the presence of the other components is still controversial. The holoenzyme possesses properties distinct from those of core RNAPII: stimulation of basal as well as activator-induced transcription, interaction with activators, and enhancement of CTD phosphorylation by TFIIH in vitro (12, 13, 18, 21).
Loss-of-function mutations of GAL11 result in a wide variety
of mutant phenotypes, including inefficient utilization of galactose or
nonfermentable carbon sources and temperature-sensitive growth on rich
media (22). Purified or recombinant Gal11 stimulates basal
transcription not only in cell-free systems consisting of nuclear or
whole cell extracts (23), but also in a reconstituted transcription
system (22, 24). Gal11 contains two domains (designated A and B) that
are essential for Gal11 function in the cell. Domain A, comprising
amino acid residues 866-929, is involved in binding to the small
subunit of TFIIE, whereas domain B sequences (from 116 to 255) bind to
the large subunit of TFIIE (24). Recently, we constructed a mutant form
of TFIIE (TFIIE-C) that fails to interact with Gal11 and found that
the TFIIE-
C mutant shows phenotypes quite similar to
those of gal11 null mutations (22). Furthermore, combination
of TFIIE-
C with a gal11 null mutation did not
result in an enhanced effect compared with the respective single
mutations. Based on these findings, we have suggested that TFIIE
and Gal11 function in a common regulatory pathway of
transcription (22).
In this work, we addressed the question of how Gal11 and TFIIE regulate transcription in the light of recent findings concerning functional interactions between TFIIE and TFIIH in yeast (25, 26) as well as in mammalian cells (4, 8). Mammalian TFIIE has been shown to regulate the enzymatic activities of TFIIH such as CTD kinase, DNA helicase, and ATPase (27-31). Here, we show that Gal11 stimulates TFIIH-catalyzed phosphorylation of the CTD in the presence of TFIIE only when holo-RNAPII is used as substrate. We further demonstrate that the enhanced phosphorylation of the CTD by Gal11 is associated with stimulation of transcription in a cell-free transcription reaction. In light of these results, the role of Gal11 in the transcription process is discussed.
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EXPERIMENTAL PROCEDURES |
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Plasmids
Plasmid pSK720 contains polyhistidine-tagged full-length
GAL11 in the pQE32 vector (QIAGEN Inc.) (22). Expression
constructs of Gal11-A (pSK723) or Gal11-
B (pSK724) were created
by removal of domain A (amino acids 866-929) or domain B (amino acids
48-326) of Gal11 (24) from pSK720, respectively.
Protein Purification
TFIIH purified from yeast (Mono Q fraction) was a gift from Drs. Jesper Svejstrup and Roger Kornberg (32). Holo-RNAPII prepared from a GAL11 wild-type or a gal11 null yeast, core RNAPII, recombinant TBP, and recombinant TFIIB were gifts from Drs. Young-Joon Kim and Roger Kornberg (18). Recombinant proteins (full-length Gal11, Tfa1, and Tfa2) were expressed in Escherichia coli and purified as described (22, 33).
Both Gal11-A and Gal11-
B were expressed in E. coli
JM109 cells harboring pSK723 and pSK724, respectively. The respective extract was adsorbed on Ni2+-nitrilotriacetic acid-agarose
(QIAGEN Inc.) as described (22), and the slurry was washed with buffer
F (0.1 M Hepes-KOH, pH 7.6, 10% glycerol, 0.1 M potassium acetate, and 0.1% Nonidet P-40) containing 20 mM imidazole.
Gal11-A Purification--
After washing the resin with buffer
F containing 100 mM imidazole, Gal11-
A was eluted with
buffer F containing 200 mM imidazole and 0.5 M
potassium acetate. The pooled fraction was loaded onto a Sephadex G-25
column (Amersham Pharmacia Biotech) equilibrated with buffer B (22)
containing 0.1 M potassium acetate (buffer B-0.1) and
eluted with the same buffer. Proteins were then loaded onto an
S-Sepharose column (Amersham Pharmacia Biotech) equilibrated with the
same buffer. After washing with buffer B-0.2, the proteins were eluted
with buffer B-0.35. The pooled fraction was diluted with buffer B to
give a potassium acetate concentration of 0.1 M and
fractionated on a Q-Sepharose column (Amersham Pharmacia Biotech).
Gal11-
A was eluted with buffer B-0.35 after washing with buffer
B-0.2.
Gal11-B Purification--
Gal11-
B was eluted from
Ni2+-nitrilotriacetic acid-agarose with buffer F containing
100 mM imidazole. The pooled fraction was loaded onto a
Q-Sepharose column equilibrated with buffer B-0.1. The flow-through
fraction was applied to a HiTrap heparin column (Amersham Pharmacia
Biotech), and Gal11-
B was eluted with buffer B-1.0. The yield of
Gal11-
A and Gal11-
B each was ~0.2 mg/liter of starting
culture.
CTD Kinase Assay
The reaction mixture (10 µl) contained TFIIH (30 ng) and
either holo-RNAPII (100 ng) or its core polymerase (50 ng) in a buffer containing 10 mM Hepes-KOH, pH 7.6, 0.1 M
potassium acetate, 5 mM MgSO4, 2 mM
dithiothreitol, 0.02% Nonidet P-40, 5% glycerol, 50 µg/ml bovine
serum albumin, 10 µM ATP, and 1 µCi of
[-32P]ATP. The reaction was carried out at 24 °C
for 40 min and terminated by the addition of SDS-containing loading
buffer. After heating at 94 °C for 7 min, the sample was loaded on
an SDS-polyacrylamide gel. Labeled proteins were visualized by
autoradiography and quantified by a BAS-1000 imaging analyzer (Fuji
Film). All experiments were repeated at least three times, and similar
results were obtained.
In Vitro Transcription Assay
Yeast nuclear extract was prepared from a gal11 null
strain (23). A transcription assay (20 µl) was carried out using the GAL7 gene (pSK164, 40 ng) as template as described (23),
except that concentrations of nucleoside triphosphates were 0.1 mM each CTP, GTP, and UTP; 20 µM ATP; and 10 µCi of [-32P]ATP. After incubation at 24 °C for
1 h, the mixture was divided into two portions. One was subjected
to primer extension to analyze transcripts (23), whereas the other was
used for analysis of CTD phosphorylation. The latter sample was
incubated at 45 °C for 10 min and then mixed with 200 ng of an
anti-CTD antibody (8WG16) and 10 µl of protein A-Sepharose (Amersham
Pharmacia Biotech) in 100 µl of a buffer containing 20 mM
Tris-Cl, pH 7.6, 150 mM NaCl, and 0.1% Nonidet P-40. After
incubation at 4 °C for 3 h on a rotating wheel, the resin was
washed three times with the same buffer. Bound proteins were extracted
with SDS loading buffer and fractionated on an SDS-polyacrylamide gel,
and 32P-labeled proteins were visualized by
autoradiography.
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RESULTS |
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Enhancement of TFIIH-catalyzed Phosphorylation of the CTD in the
Presence of Both Gal11 and TFIIE--
First, the effect of TFIIE on
the TFIIH-associated CTD kinase activity was determined by using
holo-RNAPII purified by the method of Kornberg and co-workers (18) as
substrate, which contains TFIIF and transcription regulators including
Gal11. A holo-RNAPII preparation from a GAL11 wild-type
yeast was incubated with TFIIH in the presence of
[-32P]ATP, and the phosphorylated proteins
were fractionated on an SDS-polyacrylamide gel and visualized by
autoradiography. As shown in Fig.
1A, a band with an approximate
molecular mass of 205 kDa, corresponding to that of Rpb1 (see Refs. 5
and 7), was phosphorylated by TFIIH (lanes 1 and
2). When TFIIE was added to the reaction mixture, the
phosphorylation of Rpb1 was enhanced by a factor of 5.4 ± 0.6 (compare lanes 2 and 3). Further addition of
Gal11 did not significantly affect Rpb1 phosphorylation (lanes 4 and 5). By contrast, when holo-RNAPII from a
gal11 null yeast was used as substrate (lanes
6-9), TFIIE alone could not stimulate Rpb1 phosphorylation
(compare lanes 6 and 8), and a high level of the
phosphorylation (11.3 ± 2.1-fold stimulation) was attained only
in the presence of both Gal11 and TFIIE (lane 9). The
successful stimulation of Rpb1 phosphorylation by TFIIE alone observed
in holo-RNAPII from the wild-type yeast (lane 3) was
therefore attributed to endogenous Gal11 in the holoenzyme preparation.
From these results, we concluded that Gal11 and TFIIE cooperatively
enhanced Rpb1 phosphorylation by TFIIH.
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Tfa1 Subunit of TFIIE Is Required for Enhancement of CTD Phosphorylation-- To determine which subunit of yeast TFIIE, Tfa1 or Tfa2 (33), was responsible for the enhancement of CTD phosphorylation by TFIIH, the respective subunit was added to a reaction mixture containing holo-RNAPII prepared from a gal11 null yeast. As shown in Fig. 2A, neither Tfa1 nor Tfa2 affected CTD phosphorylation if Gal11 was not added to the reaction (lanes 3 and 5). The addition of Gal11 enhanced phosphorylation of the CTD when Tfa1 was incorporated into the reaction (12.0 ± 2.5-fold stimulation), whereas no enhancement of phosphorylation was seen when Tfa2 was added (lanes 4 and 6). The presence of both Tfa1 and Tfa2 also caused a significant stimulation of CTD phosphorylation if Gal11 was also added to the reaction (lanes 7 and 8). We therefore concluded that Tfa1 of the TFIIE subunits was responsible for the stimulation of TFIIH-catalyzed CTD phosphorylation in cooperation with Gal11; the conclusion is comparable to the previous finding in mammalian systems that the large subunit of TFIIE is sufficient to activate phosphorylation of the CTD by TFIIH (29-31).
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Effect of Gal11 Mutants on Enhancement of CTD
Phosphorylation--
To further assess the cooperative function of
Gal11 and TFIIE, Gal11 derivatives with deletions of domain A or B,
known to be responsible for the interaction with Tfa2 or Tfa1 of TFIIE, respectively (22, 24), were employed (Fig. 2B). In the
presence of TFIIE, a Gal11 derivative lacking domain A (Gal11-A)
stimulated CTD phosphorylation, but only 3.0 ± 0.6-fold over the
control without Gal11, whereas full-length Gal11 did so more than
10-fold (compare lanes 1-3). Deletion of domain B
(Gal11-
B) resulted in total loss of the enhancement of CTD
phosphorylation (lane 4). These results suggested that the
interaction between domain B and Tfa1 was essential in the cooperative
function of Gal11 and TFIIE, but might not be sufficient (see
"Discussion").
CTD Phosphorylation in the Preinitiation Complex--
To study CTD
phosphorylation in holo-RNAPII as incorporated into the preinitiation
complex, the kinase reaction mixture contained TBP, TFIIB, TFIIE,
TFIIH, holo-RNAPII (which contains TFIIF (18)), and DNA encompassing
the promoter region of GAL7 from positions 93 to +43 (34).
The latter provided the site of assembly for the preinitiation complex.
Aliquots were withdrawn from the reactions at the indicated times for
analysis of phosphorylated proteins (Fig.
3A). At all the time points,
Rpb1 was two times more efficiently phosphorylated in the mixture in
which the preinitiation complex was supposed to be formed than in the
mixture in which the complex was not formed. Moreover, phosphorylated
Rpb1 migrated slightly more slowly when the preinitiation complex was
formed than when it was not formed, presumably due to an extensive
phosphorylation of the CTD at multiple sites in the former (compare
odd- and even- numbered lanes). An
electrophoretic mobility shift of Rpb1 due to an extensive
phosphorylation of the CTD at multiple sites by TFIIH was previously
observed using core RNAPII as substrate (7). However, in our assay
using holo-RNAPII, a mobility shift of Rpb1 was induced when it was
integrated in the preinitiation complex. Phosphorylation of the CTD
accompanying the mobility shift of the Rpb1 band was observed even in
Gal11-lacking holo-RNAPII (Fig. 3B, compare lanes
1-3), suggesting that Gal11 is not absolutely required for the
extensive phosphorylation of the CTD in the preinitiation complex.
However, the addition of Gal11 resulted in a further increase in CTD
phosphorylation by a factor of ~4.1 ± 0.3 (compare lanes
3 and 4), indicating that Gal11 induced further
phosphorylation of the CTD. In agreement with the results shown in Fig.
2B, Gal11-
A stimulated CTD phosphorylation, but only
2.0 ± 0.2-fold over the background level (Fig. 3B,
compare lanes 3 and 5), whereas Gal11-
B showed
no obvious effect (compare lanes 3 and 6).
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Phosphorylation of the CTD during Transcription--
To elucidate
the relationship of the Gal11/TFIIE-enhanced CTD phosphorylation to
transcription, transcripts were analyzed in parallel with
phosphorylated Rpb1 during the course of transcription in
vitro. Li and Kornberg (35) reported that a close correlation of
CTD phosphorylation with transcription was observed in a crude nuclear
extract system, but not in the basal system comprising purified
components. Thus, dependence of transcription on CTD phosphorylation
was observed only in the nuclear extract system. We therefore prepared
a nuclear extract from a gal11 null strain in which
transcription occurred on a template DNA containing the promoter region
of GAL7 in the presence of [-32P]ATP. After
the reaction was completed, transcripts were analyzed by primer
extension, while Rpb1 precipitated with the anti-CTD antibody was
subjected to SDS-polyacrylamide gel electrophoresis followed by
autoradiography (Fig. 4). In accordance
with Li and Kornberg (35), the addition of the protein kinase inhibitor H-8 to the reaction resulted in a complete arrest of both transcription of GAL7 and CTD phosphorylation of Rpb1 (compare lanes
1 and 2), suggesting that phosphorylation of the CTD is
necessary for transcription in the nuclear extract. The addition of
Gal11 enhanced transcription as well as CTD phosphorylation by factors
of 3.6 ± 0.4 and 2.0 ± 0.2 over the background levels,
respectively (compare lanes 1 and 3). The
stimulatory effect of Gal11 on CTD phosphorylation appeared rather
small compared with that on transcription. This may well be due to the
presence of nonspecific protein kinases other than TFIIH-associated
kinase and free core RNAPII in the nuclear extract, both of which would
lower the apparent effect of Gal11 on the CTD phosphorylation. Neither
Gal11-
A nor Gal11-
B exerted appreciable effects on both
transcription and CTD phosphorylation (lanes 4 and
5).
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DISCUSSION |
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Using yeast RNAPII holoenzyme as substrate, we have demonstrated
that CTD phosphorylation catalyzed by TFIIH is significantly enhanced
by a cooperative function of Gal11 and TFIIE. The observed stimulatory
effect depends on both domain B of Gal11 and the subunit Tfa1 of TFIIE.
Since Tfa1 binds both domain B (22, 24) and TFIIH (26), the domain
B-Tfa1-TFIIH interaction may be essential for phosphorylation of the
CTD in holo-RNAPII. A Gal11 derivative lacking domain A (Gal11-A)
stimulated CTD phosphorylation, but less efficiently than wild-type
Gal11. Neither Gal11 nor TFIIE led to a significant effect on
TFIIH-mediated CTD phosphorylation with core RNAPII as substrate. Since
domain A of Gal11 is known to encompass the region required for
interaction with holo-RNAPII as well (15), it is reasonable to suggest
that the lowered stimulatory ability of Gal11-
A is due to its weak
association with holo-RNAPII and that a tight association of Gal11 with
holo-RNAPII is a prerequisite for efficient stimulation of CTD
phosphorylation by TFIIH. Recent studies by Svejstrup
et al. (36) have suggested that phosphorylation of the CTD
causes dissociation of holo-RNAPII into the mediator and core RNAPII
and that the dissociated core polymerase travels along the template to
elongate the transcript. Taken all together, we suggest that the
Gal11-TFIIE-TFIIH interaction is involved in regulation of the
transition of holo-RNAPII to an elongation-competent complex in
yeast.
The present experiments have demonstrated that neither of the mutant
Gal11 proteins (Gal11-A and Gal11-
B) is capable of enhancing
transcription in a nuclear extract. These results are consistent with
those of in vivo analyses showing that neither mutant is
able to induce the expression of GAL7-lacZ (24). Since phosphorylation of the CTD is a key step in the transcription reaction
(4), we assume that the observed enhancement of CTD phosphorylation
would account for Gal11-dependent stimulation of
transcription in the cell. Previous studies have strongly suggested that Gal11 reinforces interactions between TFIIE and holo-RNAPII (24).
We therefore speculate that Gal11 in holo-RNAPII has at least two roles
in transcription stimulation: recruitment of TFIIE to holo-RNAPII,
which contributes to the formation of the preinitiation complex, and
enhancement of CTD phosphorylation in cooperation with TFIIE, which
triggers formation of the elongation complex.
We previously demonstrated that Gal11 stimulates basal transcription in a system reconstituted with core RNAPII and the general transcription factors (24). However, we failed to show a significant effect of Gal11 on CTD phosphorylation in core RNAPII (Fig. 1A). On the other hand, Li and Kornberg (35) have shown that CTD phosphorylation is dispensable for transcription initiation in the reconstituted system. These observations led us to speculate that Gal11 also regulates other enzymatic activities such as ATPase and DNA helicase of TFIIH besides its CTD kinase activity through TFIIE function since mammalian TFIIE has been shown to regulate TFIIH ATPase and DNA helicase activities (28-30). Although many more experiments are required to clarify the exact role of Gal11 in transcription, this work has clearly documented functional interactions among Gal11, TFIIE, and TFIIH and consequently further supports a model where Gal11 is involved in the transition from initiation to elongation in the transcription process.
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ACKNOWLEDGEMENTS |
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We thank Drs. Roger D. Kornberg, William J. Feaver, Young-Joon Kim, and Jesper Q. Svejstrup for providing plasmids and yeast general transcription factors.
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FOOTNOTES |
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* This work was supported by grants-in-aid for scientific research (to H. S.) from the Ministry of Education, Science, Sports, and Culture of Japan.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.
§ To whom correspondence should be addressed: Tel.: 81-76-265-2588; Fax: 81-76-234-4360; E-mail: sakurai{at}kenroku.ipc.kanazawa-u.ac.jp.
1 The abbreviations used are: RNAPII, RNA polymerase II; TF, transcription factor; TBP, TATA-binding protein; CTD, carboxyl-terminal domain.
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REFERENCES |
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