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INTRODUCTION |
Cyclin-dependent kinase 7 (CDK7)1 was originally
isolated as the catalytic subunit of the trimeric cdk-activating kinase
(CAK) complex. This complex, consisting of CDK7, cyclin H, and MAT1, is
responsible for activation of the mitotic promoting factor in
vitro (1-3). The discovery that CDK7 was also a component of the
basal transcription repair factor IIH (TFIIH) implicated a dual role
for CDK7 in transcription as part of TFIIH and in the control of the
cell cycle as the trimeric CAK complex (4-7). TFIIH is a multisubunit
protein complex identified as a factor required for RNA polymerase II
(RNAPII)-catalyzed transcription (8-11), and subsequently this complex
was found to play a key role in nucleotide excision repair (12-14). At
least nine polypeptides with molecular masses of 89, 80, 62, 52, 44, 40, 37, 36, and 34 kDa co-purify with mammalian TFIIH. The cDNAs
encoding all of these subunits have now been cloned. p89 and p80 are
the gene products of ERCC3 (XPB) and ERCC2 (XPD), respectively
(13-15). p62 and p44 are the mammalian counterparts of the yeast TFB1
and SSL1 gene products that are required for DNA nucleotide excision repair (16). p34 exhibits partial sequence homology to p44 and also
contains zinc-finger motifs (17). The p40, p37, and p36 subunits of
TFIIH are identical to the vertebrate CAK complexes, CDK7, cyclin H,
and p36/MAT1, respectively (5-7). Two subcomplexes containing some
TFIIH polypeptides can also be isolated from extracts of HeLa cells
(18, 19): (i) a five-subunit core TFIIH complex that includes ERCC3
(XPB), p62, p52, p44, and p34 but is devoid of detectable levels of
ERCC2 (XPD) or CAK; (ii) an XPD·CAK complex that includes XPD and all
three CAK components (CDK7, cyclin H, and p36/MAT1). The addition of
XPD·CAK to the core TFIIH potently stimulates the TFIIH
transcriptional activity (18, 19). These observations suggest that core
TFIIH and XPD·CAK interact to form a complex that constitutes the
TFIIH holoenzyme (holo-TFIIH). Biochemical analysis has therefore
revealed that CDK7 is a component of at least three complexes, the
trimeric CAK complex (20-22), the quaternary complex with the XPD, and
the nine-subunit TFIIH complex (18, 19). In addition, a number of
studies have suggested that the trimeric CAK complex is the
CDK7-containing complex involved in cell cycle control (21, 23). These
studies were based on initial reports that identified the trimeric CAK
complex as the kinase responsible for activating a number of cdks
(1-3). However, in Saccharomyces cerevisiae, Kin28 and
Ccl1, the counterparts of CDK7 and cyclin H, associate with S. cerevisiae TFIIH, although they do not exhibit detectable CAK
activity (4, 24). The CAK activity of S. cerevisiae has
recently been identified as the gene product of
Cak1/Civ1 (25, 26). However, a recent report
indicates that CDK7 is essential for mitosis and cdk-activating kinase
activity in Drosophila melanogaster (27).
Here we show that the trimeric CAK complex exhibits an inhibitory
activity in RNAPII-dependent transcription. This inhibition results from the preclusion of TFIIF and RNAPII from the preinitiation complex. These studies reveal a novel role for the trimeric CAK in
regulation of transcription.
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EXPERIMENTAL PROCEDURES |
Purification of CAK from HeLa Nuclear Extract--
Trimeric CAK
was purified from 3 g of HeLa nuclear extract (Fig.
1A). Nuclear extract was loaded on a 1-liter column of
phosphocellulose (Whatman) and fractionated stepwise by the indicated
KCl concentrations in buffer A (20 mM Tris-HCl, pH 7.9, 0.2 mM EDTA, 10 mM 2-mercaptothanol, 20% glycerol,
0.2 mM phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, and 1 µg/ml pepstatin. The phosphocellulose 0.3 M KCl
fraction (400 mg) was dialyzed to 0.1 M KCl in buffer A and
loaded on a 100-ml DEAE-Sephacel column (Amersham Pharmacia Biotech).
The column was eluted with 0.5 M KCl in buffer A. The 0.5 M KCl elution (260 mg) was dialyzed to 100 mM
KCl in buffer A and loaded on a 100-ml Q-Sepharose column (Sigma). The
column was resolved using a linear 10-column volume gradient of
100-600 mM KCl. Fractions containing CDK7 (~200
mM KCl, 37 mg) were dialyzed to 10 mM potassium phosphate in buffer B (5 mM Hepes, pH 7.5, 1 mM
dithiothreitol, 0.2 mM phenylmethylsulfonyl fluoride, 10 µM CaCl2, 10% glycerol, 1 µg/ml leupeptin,
and 1 µg/ml pepstatin) and loaded on a 20-ml hydroxyapatite column
(American International Chemical). The column was resolved using a
linear 10-column volume gradient of 10-600 mM potassium
phosphate in buffer B. CDK7-containing fractions (100 mM
potassium phosphate, 8.6 mg) were dialyzed to 1 M ammonium sulfate in buffer C (20 mM Hepes, pH 7.9, 4 mM
dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 0.5 mM EDTA, 10% glycerol) and fractionated on a
phenyl-Superose HR 5/5 column (Amersham Pharmacia Biotech). The
phenyl-Superose column was resolved using a linear 10-column volume
gradient of 1 M to 0 mM ammonium sulfate in
buffer A. CDK7-containing fractions (~0.4 M ammonium
sulfate, 0.5 mg) were precipitated with 60% ammonium sulfate and
fractionated on a Superose 6 HR 10/30 column (Amersham Pharmacia
Biotech) equilibrated in 1 M KCl in buffer A.
Generation of Recombinant CAK Polypeptides in SF9 Cells--
We
generated and purified CAK using a detailed protocol for the production
of CAK in insect SF9 cells as described previously (21).
CTD Kinase Assay--
CTD kinase assays for phosphorylation of
the carboxyl-terminal domain of RNAPII were described previously
(7).
In Vitro Transcription--
Transcription assays were
reconstituted as described (7) using recombinant TBP (10 ng), TFIIB (10 ng), TFIIE (10 ng), TFIIF (10 ng), highly purified HeLa holo-TFIIH (7)
or highly purified rat TFIIH (6), and highly purified HeLa core RNAPII
(28). The rat somatostatin promoter (29), human T-cell leukemia virus, type 1 (HTLV-1) promoter (30), or Ad-MLP leading to a 390-nucleotide G-less cassette was used as the template (100 ng).
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RESULTS |
The Trimeric CAK Complex Purified from HeLa Cells Is a
Transcriptional Repressor--
The trimeric CAK complex from HeLa
nuclear extract was purified as described under "Experimental
Procedures" (Fig. 1A) using the CTD-kinase activity and Western blot analysis with antibodies against CDK7 and cyclin H. Nuclear extract was initially fractionated by phosphocellulose chromatography. The 0.3 M KCl step
elution contained approximately 40% of CDK7 immunoreactivity and was
devoid of TFIIH activity or ERCC3 immunoreactivity, which fractionated into the 0.5 M KCl step elution (data not shown). The 0.3 M KCl fraction was therefore further purified through five
additional steps (Fig. 1A). Analysis of the CTD kinase
activity of CAK on the sizing column (Superose 6) revealed the peak of
activity eluting at ~200 kDa, consistent with the previously reported
size for the trimeric CAK complex (21). We estimate that our
purification resulted in ~2000-fold enrichment in CAK. Analysis of
column fractions in a transcription system reconstituted with
recombinant TBP, TFIIB, TFIIE, TFIIF, highly purified HeLa or rat
holo-TFIIH, and highly purified HeLa core RNAPII revealed an inhibitory
activity copurifing with the CDK7 complex throughout the purification
(Fig. 1B). The transcriptional inhibitory activity was also
observed in the last two steps of the purification
(phenyl-Superose and Superose 6) (Fig. 1, C and
D). Highest concentrations of the CAK complex tested
resulted in about 95% inhibition in basal transcription.

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Fig. 1.
Isolation of the trimeric CAK.
A, HeLa nuclear extract was fractionated by chromatography
as described under "Experimental Procedures." The
horizontal and diagonal lines indicate stepwise
and gradient elution, respectively. Salt concentrations for protein
elutions are given in millimolars. Also shown is a Western blot
analysis of fractions derived from the phenyl-Superose with the
antibodies indicated on the left and a CTD kinase analysis
of fractions derived from the Superose 6 column. B,
hydroxyapatite column fractions (3 µl) were analyzed using
transcription reactions reconstituted with the full set of basal
factors using linear Ad-MLP (100 ng) and Western blot analysis (20 µl
of column fractions) using anti-CDK7 antibodies (Oncogene Research
Products). C, transcription reactions were reconstituted
with the full set of factors using a supercoiled HTLV-1 promoter as
described under "Experimental Procedures," 1X represents
2 µl of the pool from the CDK7 containing fractions from a
phenyl-Superose column. D is the same as B except
6 µl of the pool from the phenyl-Superose or Superose 6 columns were
used. nt, nucleotide.
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Recombinant Trimeric CAK Displays Transcriptional Inhibitory
Activity--
To determine if the inhibitory activity associated with
the HeLa fractions is the CAK complex, we expressed either the trimeric (CDK7-cyclin H-MAT1) or the dimeric CAK complex (CDK7-cyclin H) in
insect SF9 cells. These complexes were purified as previously reported
(21) (Fig. 2A). Moreover, as
the highly purified holo-TFIIH complex also contains CAK, we sought to
determine the action of the trimeric CAK complex in an assay free of
TFIIH. Similar to a previous report (31), reconstituted transcription
using the rat supercoiled somatostatin promoter is independent of TFIIH or TFIIE but is highly stimulated by these factors (Fig.
2B). Analysis of recombinant trimeric CAK complex in
reconstituted transcription assay either in the presence (Fig.
2C, lanes 1 and 2) or the absence
(lanes 3-5) of TFIIH revealed that this complex is a potent
inhibitor of transcription. The inhibitory effect of CAK is achieved at
roughly a 1:1 stoichiometry (0.25 pmol) with other basal factors.

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Fig. 2.
Recombinant CAK inhibits transcription.
A, SDS-polyacrylamide gel electrophoresis followed by either
staining with Coomassie Brilliant Blue R-250 for recombinant dimeric
(lane 1) or silver for trimeric (lane 2) CAK
complexes. Note that in the trimeric CAK complex the p36/MAT1 subunit
contained a six-histidine tag and therefore displayed a larger
molecular mass than that of cyclin H. The asterisk depicts
the position of a contaminating polypeptide. B,
transcription reactions were reconstituted using a supercoiled rat
somatostatin promoter and the full set of factors. Lane 1 represents the complete reaction. The other reactions were identical
except for the omission of the indicated protein. C,
transcription reactions in the presence or absence of CAK(3) were
carried out with a complete set of factors as in panel B
(lanes 1 and 2) or in the absence of holo-TFIIH (lanes
3-5). 1X represents approximately 50 ng of recombinant
CAK. Approximately 30 ng of holo-TFIIH was added in lanes 1 and 2. Lanes 1 and 2 employed a supercoiled
HTLV-1 promoter, and lanes 3-5 employed a supercoiled rat
somatostatin promoter. nt, nucleotide.
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The p36/MAT1 Subunit of the Trimeric CAK Complex Is Required for
the Inhibitory Activity--
To further ascertain the role of the
p36/MAT1 subunit of the CDK7 complex in transcriptional repression, we
compared the recombinant dimeric and trimeric CAK complexes. Although
the two CAK complexes displayed similar kinase activity as ascertained
by phosphorylation of a CTD peptide (Fig.
3A), analysis of the dimeric
CAK in transcription revealed that p36/MAT1 is required for the
inhibitory activity of the CAK complex (Fig. 3B). In
contrast to the trimeric CAK, which inhibited transcription driven from
either the supercoiled somatostatin (lanes 2 and
3) or supercoiled HTLV-1 (lanes 8 and 9) promoters, the dimeric CAK was devoid of any inhibitory
activity with either promoter (lanes 4-6 or 10 and 11). These data indicate that the p36/MAT1 subunit of
the CAK complex is required for inhibition of transcription and that
the CAK-mediated inhibition is independent of the promoter used.

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Fig. 3.
p36/MAT1 is required for the repressing
activity of CAK. A, CTD kinase activity of dimeric (50 ng, lane 1), trimeric (50 ng, lane 2), and a
trimeric kinase-deficient mutant of CAK (50 ng, lane 3) was
measured using a heptapeptide repeat of the RNAPII-CTD. B,
transcription was reconstituted using either a supercoiled rat
somatostatin promoter (lanes 1-6) or a supercoiled HTLV-1
promoter (lanes 7-11). Reactions were carried out in the
absence of holo-TFIIH with the addition of the indicated proteins.
1X represents 50 ng for each protein preparation.
C, transcription was reconstituted using a supercoiled
HTLV-1 promoter and a full set of basal factors in the absence of
holo-TFIIH (lane 1). The other reactions were identical to
lane 1 except for the addition of proteins indicated
(lanes 3-5). 1X represents 50 ng of p36/MAT1 or
50 ng of CAK. D, transcription was reconstituted as in
C. 50 ng of each factor was added to each transcription
reaction. nt, nucleotide.
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p36/MAT1 Is Not Sufficient for Transcriptional Inhibition--
To
determine whether the p36/MAT1 subunit of the CAK complex is sufficient
for transcriptional repression, the p36/MAT1 subunit was expressed in
Escherichia coli, and the purified p36/MAT1 was analyzed for
its activity in transcription. In contrast to trimeric CAK (Fig.
3B, lane 2), the addition of p36/MAT1 not only
failed to inhibit transcription (lanes 3-5) but also
displayed a small stimulatory activity (compare lanes 1 and
3). These results demonstrate that although p36/MAT1 is
required for the inhibitory activity of the CAK complex, it is not
sufficient for inhibition.
Addition of p36/MAT1 to Dimeric CAK Can Reconstitute
Transcriptional Repression--
The p36/MAT1 subunit of CAK was
produced in SF9 cells, and purified protein was tested for its ability
to confer repression when added to the dimeric CAK complex. As Fig.
3C indicates, neither the dimeric CAK (lane 2)
nor the p36/MAT1 protein alone were sufficient to mediate repression.
However, the addition of the p36/MAT1 subunit to dimeric CAK
reconstitutes the transcriptional repression observed with trimeric CAK
(lane 4).
The Trimeric CAK Inhibits Transcription by Precluding RNAPII and
TFIIF from the Preinitiation Complex--
To analyze which step during
the formation of the preinitiation complex the trimeric CAK may target
to repress transcription, we incubated the basal transcription factors
in a stepwise fashion with the DNA for 30 min before adding the
trimeric CAK complex (Fig.
4A). Preincubation of DNA with
either TBP (lane 2) or TBP and TFIIB (lane 3)
could not overcome the inhibitory effect of CAK, indicating that the
TBP·TFIIB complex (TB) formation is not the target of the CAK
complex. However, the addition of TFIIF to the preinitiation complex,
which results in the formation of the TBF complex, could partially
relieve the CAK repression (lane 4). Formation of the TBPolF
or TBPolFE complex by further preincubation with RNAPII or RNAPII and
TFIIE resulted in a complete recovery of transcription (lanes
5 and 6). These results indicate that trimeric CAK
precludes the entry of RNAPII and TFIIF into a competent preinitiation
complex, and a preformed preinitiation complex is refractory to the
action of trimeric CAK. This contention is further substantiated when
we analyzed whether the addition of excess TFIIF or RNAPII can overcome
the inhibitory activity of the trimeric CAK complex. As shown in Fig.
4B, the addition of increasing amounts of TFIIF partially
overcomes the CAK-mediated repression (lanes 3 and
4), whereas the addition of excess RNAPII could completely restore transcription (lane 5). We conclude that trimeric
CAKs repress transcription by precluding RNAPII and TFIIF entry into the preinitiation complex.

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Fig. 4.
CAK represses transcription by precluding
TFIIF and RNAPII entry into the pre-initiation complex.
A, transcription reactions were initiated by incubating the
factors shown on the right panel of the figure with a
supercoiled rat somatostatin DNA template for 30 min. The remainder of
the transcription factors, CAK, and the nucleotide mixture were then
added to the reaction for an additional 60 min. The numbers
to the right of the right panel correspond to the
lane numbers shown in the bottom of the left panel.
B, transcription reactions were reconstituted with the full
set of factors using a supercoiled HTLV-1 promoter in the absence of
holo-TFIIH (lane 1). Other reactions are as in lane
1 except for the addition of proteins indicated. 1X
represents 25 ng for TFIIF and 100 ng for RNAPII. CAK (50 ng) was added
to lanes 2-5. C, transcription reaction was
reconstituted as in B. 1X represents 70 ng of the
mutant trimeric kinase. D, transcription assays were
performed as in B and C except either increasing
concentrations of rat TFIIH (1X represents 20 ng) or human
CAK (phenyl-Superose; 1X represents 20 ng) were added to
each reaction. nt, nucleotide.
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The Mechanism of CAK-mediated Repression Is Independent of Its
Kinase Activity--
To address whether the kinase activity of CDK7
plays a role in CAK-mediated inhibition, we analyzed a kinase-deficient
mutant of CDK7, in which lysine 41 was replaced by alanine (Fig.
3A, lane 3). The purified dimeric CAK/K41A,
produced in insect cells, was mixed with the p36/MAT1 subunit, produced
in insect cells, and analyzed in a reconstituted transcription system.
As Fig. 4C indicates, the addition of kinase-deficient CAK
resulted in a potent inhibition of transcription (compare lane
1 to lanes 2 and 3). These results indicate
that CAK-mediated inhibition is not because of the kinase activity of
CAK and may result from CAK physically destabilizing the preinitiation complex.
Excess TFIIH Can Overcome the Inhibitory Effect of
CAK--
Because TFIIH displays a stimulatory activity in
transcription by stabilizing the preinitiation complex, we analyzed
whether increasing concentrations of TFIIH can relieve the CAK-mediated repression. As Fig. 4D reveals, the addition of excess TFIIH
can overcome the inhibitory activity of CAK (compare lanes
2-4 to 6-8). These results indicate that TFIIH and
CAK are in a competition for the preinitiation complex. Whereas TFIIH
stimulates transcription by stabilizing the preinitiation complex
formation, CAK exerts an inhibitory effect by disrupting its formation.
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DISCUSSION |
The novelty of this work lies in the following. First, it
demonstrates transcriptional inhibitory activity for trimeric CAK in a
fully defined system, comprised of essentially homogeneous basal
factors and RNA polymerase II. Second, it shows that CAK inhibits
transcription by preventing RNA polymerase II and TFIIF entry into the
preinitiation complex. Third, it presents evidence for the requirement
of p36/MAT1 in the CAK-mediated inhibitory effect. Finally, it
demonstrates that the inhibitory activity is independent of the kinase
activity of CDK7.
The trimeric CAK complex was initially identified as the kinase complex
responsible for phosphorylation and consequent activation of other
cyclin-dependent kinases from mammalian cells (1-3). It
was later discovered that CDK7, cyclin H, and MAT1 were also components
of the basal transcription factor TFIIH (4-7). Furthermore, it was
observed that TFIIH can be dissociated into two subcomplexes, one
containing the core TFIIH subunits (XPB, p62, p51, p44, and p34) and
the other containing XPD and the three CAK subunits (18, 19). We found
that the trimeric CAK purified from HeLa cells contained a
transcriptional inhibitory activity in a highly purified reconstituted
transcription system. The inhibitory activity associated with the HeLa
fractions was demonstrated to be mediated by the trimeric CAK complex,
because the recombinant trimeric CAK produced in insect cells inhibited
transcription. Interestingly, the p36/MAT1 subunit of the trimeric CAK
was required for the transcriptional inhibition. This observation lends
further support to the physiological relevance of CAK-mediated
inhibition, as the predominant form of CAK in mammalian extracts not
associated with TFIIH contains the p36/MAT1 subunit (19, 21). The
transcriptional inhibition by the CAK complex did not result from CDK7
kinase activity, because the kinase-deficient mutant of CAK is also a
potent inhibitor of transcription. Our studies revealed that CAK
inhibited transcription by preventing the formation of the
TBPolF complex. Therefore, either preforming the TBPolF
complex or the addition of excess TFIIF, RNAPII, or TFIIH was able to
stabilize the complex and to overcome the inhibitory effect of CAK.
A number of studies have concluded that the trimeric CAK complex
represents the form of CAK involved in cell cycle control (1-3,
21). Here we present evidence indicating that the trimeric CAK complex
displays a novel role in transcription distinct from that of its
function when associated with TFIIH. Our findings are a further support
for CAK as a regulator of transcription in addition to the function of
CAK in cell cycle control.