Báo cáo khoa học: Mediator is required for activated transcription in a Schizosaccharomyces pombe in vitro system potx

Mediator is required for activated transcription in a
Schizosaccharomyces pombe in vitro
system
Evelyn Tamayo, Giuliano Bernal, Ursula Teno and Edio Maldonado
Programa de Biologia Celular y Molecular, Facultad de Medicina, Instituto de Ciencias Biomedicas, Universidad de Chile,
Santiago, Chile
RNA polymerase II (RNAPII) requires a set of general
transcription factors ) TFIIA, TFIIB, TFIID, TFIIE,
TFIIF and TFIIH ) to initiate transcription from a gene
promoter in vitro. General transcription factors have been
isolated from Saccharomyces cerevisiae, rat, human and
Drosophila, and their corresponding cDNAs have been
cloned. In this report, we describe a reconstituted in vitro
transcription system that consists of the following prepara-
tions of factors from the yeast Schizosaccharomyces pombe:
affinity-purified RNAPII, TFIIH, and recombinant TBP,
TFIIB, TFIIE and TFIIF. We show that this system can
support basal transcription from the adenovirus major late
promoter when purified RNAPII is used and activated
transcription when the RNAPII holoenzyme (RNAPII plus
the Mediator proteins) is included in the reaction. In con-
trast, the TATA binding protein-associated factors had no
effect on transcriptional activation in our Sc. pombe system.
These results indicate that Sc. pombe uses the same set of
general transcription factors as other eukaryotes and that
the Mediator is involved in activated transcription.
The ability to achieve basal levels of transcription from
protein-encoding genes in eukaryotes requires RNA
polymerase II (RNAPII) and a set of additional proteins
called general transcription factors (GTFs). The GTFs have
been purified to homogeneity from HeLa cells, rat liver,

Drosophila and the yeast Saccharomyces cerevisiae,and
have been named TFIIA, TFIID, TFIIE, TFIIF, TFIIB
and TFIIH [1]. The cDNAs that encode these factors have
been isolated, and their amino acid sequences show a high
degree of evolutionary conservation. These findings indicate
that the transcriptional machinery is highly conserved
among eukaryotes.
An in vitro transcription assay that consists of purified
RNAPII and recombinant GTFs can carry out basal
transcription but cannot respond to gene-specific transcrip-
tional activators. Activated transcription requires additional
multiprotein complexes named coactivators. The main
coactivators required for activated transcription in in vitro
systems are the TFIID complex and the Mediator [2].
Recent work suggests that the TFIID complex, which
contains the TATA binding protein (TBP) and other TBP-
associated factors (TAFs), plays an important role in
facilitating activation by gene-specific transcription factors
as wells as in recognition of the TATA box and other core
promoter sequences (necessary for both basal and activated
transcription) [3]. Mediator is a large multiprotein complex
that is brought to promoters by DNA-bound, gene-specific
transcriptional regulatory proteins and helps these proteins
to communicate with factors bound to the core promoter.
Mediator is required for transcription in vivo and for
optimal levels of both basal and activated transcription
in vitro in nuclear extracts from human cells [4,5]. Compo-
nents of both the TFIID complex and Mediator are
conserved from yeast to humans.
The yeast Schizosaccharomyces pombe can be genetically

manipulated and has served as an excellent model for the
study of cell division cycle control and DNA repair and
recombination. The Sc. pombe genome has been fully
sequenced and annotated and contains the smallest number
of protein-coding genes [4,] of any eukaryotic genome
sequenced to date [6]. Evidence suggests that the mechanism
of transcription initiation by Sc. pombe RNAPII is
more similar to that of higher eukaryotes than that of
S. cerevisiae. In Sc. pombe, transcription initiation occurs
25–30 bp downstream from the TATA box region of the
core promoter, whereas in S. cerevisiae, it occurs 40–120 bp
downstream from the TATA box. Also, transcription
initiation from mammalian promoters that have been
introduced into Sc. pombe occurs at the same sites as it
does in mammalian cells [7].
On the basis of these observations, we have begun to
study mechanisms of transcriptional activation in Sc. pombe
in order to compare these processes with those of higher
eukaryotes. Given the observed similarities, findings from
the Sc. pombe system can probably be extrapolated to
higher eukaryotes. Studies of RNAPII transcription in
Sc. pombe has been hampered by the lack of a reconstituted
in vitro transcription system that responds to transcriptional
activator proteins. Here we describe an in vitro transcription
system that contains the following preparations from
Sc. pombe: affinity-purified RNAPII, highly purified
TFIIH, and recombinant TBP, TFIIB, TFIIE and TFIIF.
Correspondence to E. Maldonado, Programa de Biologia Celular y
Molecular, Facultad de Medicina, Instituto de Ciencias Biomedicas,
Universidad de Chile, Santiago, Chile. Fax: + 56 2735 5580,

Tel.: + 56 2678 6207, E-mail:
Abbreviations: RNAPII, RNA polymerase II; GTF, general
transcription factor; TBP, TATA binding protein;
TAF, TBP-associated factor; CTD, C-terminal domain;
Ad-MLP, adenovirus major late promoter.
(Received 8 February 2004, revised 7 April 2004,
accepted 26 April 2004)
Eur. J. Biochem. 271, 2561–2572 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04187.x
We have also purified the RNAPII holoenzyme RNAPII
plus the Mediator complex from Sc. pombe and demon-
strated that it is able to stimulate basal transcription and
support activated transcription in the absence of TAFs.
Materials and methods
Cloning of
Sc. pombe
TFIIE, TFIIF, TFIIB and the p52
subunit of TFIIH
To identify homologs of TFIIE, TFIIF, TFIIB and TFIIH,
we searched the Sc. pombe genome sequence using the
BLASTP
program and the amino acid sequences of human
and S. cerevisiae TFIIE, TFIIF and TFIIB, and of the p62
subunit of human TFIIH. We identified homologs of
both subunits of TFIIE, TFIIF and TFIIB, and of the Tfb1
subunit of TFIIH. The cDNAs encoding both subunits of
TFIIE, the smallest subunit of TFIIF, and Tfb1 subunit
of TFIIH were amplified by PCR from a cDNA library of
Sc. pombe. The oligonucleotides used in the PCR amplifi-
cations were designed from the nucleotide sequences
obtained from the Sc. pombe genome sequence database.

The primer that was complementary to the N-terminal
sequences of the various genes contained an NdeI recogni-
tion site, and the primer that was complementary to the
C-terminal sequences contained a BamHI recognition site.
The PCR products were digested with NdeIandBamHIand
cloned in-frame into the NdeIandBamHIsitesofthe
bacterial expression vector pET15b (Novagen), which adds
a hexahistidine tag at the N terminus of the protein. Positive
clones were sequenced using the Sequenase version 2 kit
(USB). The cDNA encoding the largest subunit of TFIIF
was synthesized by the GeneScript Corporation and cloned
in-frame into the NcoIandBamHI sites of the bacterial
expression vector pET19b (Novagen).
Expression and purification of
Sc. pombe
TFIIE, TFIIF,
TFIIB, TBP and the Tfb1 subunit of TFIIH
TFIIB, TFIIEa, TFIIEb, TFIIFa, TFIIFb and the Tfb1
subunit of TFIIH were expressed in Escherichia coli strain
BL21 (DE3 pLysS). The bacteria were grown in TB medium
(500mL)at37°CtoD
600
¼ 0.8. Production of the proteins
was then induced with 0.5 m
M
isopropyl thio-b-
D
-galacto-
side, and the culture was incubated an additional 4 h at
37 °C. Bacteria were harvested by centrifugation and lysed

with mild sonication at 4 °C in buffer A [20 m
M
Hepes
pH 7.9, 500 m
M
KCl, 0.1% (v/v) NP-40, 0.1 m
M
phenyl-
methanesulfonyl fluoride]. The lysate was cleared by
centrifugation, and the insoluble pellet containing the
recombinant protein was washed twice with buffer A
containing 0.05% (v/v) sodium deoxycholate and 0.1%
(v/v) Triton X-100. Recombinant proteins were extracted
from the pellet by incubation for 8 h at 4 °Cin20mL
20 m
M
Hepes pH 7.9, 6
M
guanidium hydrochloride,
0.1 m
M
phenylmethanesulfonyl fluoride. The mixture was
then centrifuged to pellet the cell debris, and the soluble
protein was diluted six times with dialysis buffer [20 m
M
Hepes pH 7.9, 10% (v/v) glycerol, 2 m
M
dithiothreitol,
1m
M

EDTA, 100 m
M
potassium acetate, 0.1 m
M
phenyl-
methanesulfonyl fluoride]. The recombinant protein was
incubated overnight at 4 °C, dialysed against 100 vols
dialysis buffer, and centrifuged to eliminate any precipitated
material. The TFIIFa (250 lg) and TFIIFb (250 lg)
preparations were mixed and incubated for 1 h at 4 °C.
The mixture was loaded onto a 10-mL gel filtration column
made of ACA22 resin (Sigma) and eluted from the column
with dialysis buffer; 200-lL fractions were collected. Both
subunits were detected in the column eluate by Western
blotting with antibodies to the His-tag (Clontech). Fractions
containing the TFIIF heterotetramer were used in in vitro
transcription experiments. TFIIEa and TFIIEb were mixed
and purified by the same procedure as described for TFIIF.
Sc. pombe TBP was a gift from D. Reinberg (Department
of Biochemistry, HHMI-UMDNJ, Piscataway, NJ, USA).
Purified proteins were subjected to electrophoresis in an 8%
polyacrylamide gel followed by Coomassie blue staining.
Purification of TFIIH, RNAPII and the RNAPII holoenzyme
TFIIH and RNAPII were purified from Sc. pombe whole-
cell extracts that were prepared from the wild-type strain
972h. Cells were grown to D
600
¼ 2inYPDmediaand
harvested by centrifugation (10 000 g,4°C, 30 min). The cell
pellet was washed twice with distilled water and introduced

into liquid nitrogen. Cell extracts were prepared from 500 g
yeast cells (wet weight) that were obtained from 200 L
culture. The cells were mixed with liquid nitrogen in a 4-l
Waring blender and blended four times for 5 min each at
maximum speed. The blending was repeated until 95% of the
cells were broken as judged by viewing the cells under a light
microscope. The broken cells were mixed with 750 mL buffer
B (100 m
M
Hepes pH 7.9, 250 m
M
KCl, 5 m
M
EGTA,
10 m
M
EDTA, 2.5 m
M
dithiothreitol, 1 m
M
phenyl-
methanesulfonyl fluoride, 0.5 lgÆmL
)1
Pepstatin A), and
the mixture was centrifugated in a Sorvall rotor at 30 000 g,
4 °C, for 45 min. The supernatant was pooled and precipi-
tated with ammonium sulfate at 40% saturation, and the
precipitates were recovered by centrifugation at 30 000 g,
4 °C in a Sorvall rotor. The precipitate was resuspended in
400 mL buffer C (20 mM Hepes pH 7.9, 5 mM EGTA,

1 mM EDTA, 2 mM dithiothreitol, 20% (v/v) glycerol,
1 mM phenylmethanesulfonyl fluoride], dialysed against 4 L
of the same buffer. The extract was stored at )80 °C until use.
TFIIH was prepared from the yeast, cell extracts as
described by Li et al. [8]. The TFIIH-containing fraction
(500 lg protein) from the heparin–agarose column was
loaded onto a 10 mL ACA22 gel filtration column, and
eluted with dialysis buffer (200 lL fractions were collected).
The ACA22 column fractions were assayed by Western
blotting with antibodies to Tfb1, and the TFIIH-containing
fractions were pooled and used in the transcription assays.
TFIIH was free of RNAPII, GTFs, and Mediator as judged
by Western blotting with antibodies to TBP, the smallest
subunit of TFIIF, TFIIE, RNAPII and Srb4 (part of the
Mediator complex).
RNAPII was purified from the yeast cell extracts (1 g) by
chromatography on a DEAE-52 column (100 mL wet resin)
(Whatman) that had been equilibrated with buffer C. The
column was eluted with a 10-column volume gradient of
ammonium sulfate (0.1–0.5
M
in buffer C). The elution of
RNAPII was detected by Western blotting with monoclonal
antibodies to the C-terminal domain (CTD) of the largest
subunit of RNAPII (SWG16). Fractions containing
2562 E. Tamayo et al. (Eur. J. Biochem. 271) Ó FEBS 2004
RNAPII were pooled and dialysed against buffer C
containing 500 m
M
potassium acetate and 0.01% (v/v)

NP-40. Two milliliters of the RNAPII preparation
(1 mgÆmL
)1
protein) were incubated for 2 h at 4 °Cwith
500 lL of anti-CTD (SWG16) mAb that had been cross-
linked to protein A–agarose. After the incubation, the resin
was washed with buffer C containing 500 m
M
potassium
acetate and 0.01% (v/v) NP-40 and eluted with 500 lL
CTD peptide (YSPTSPS) at 1 mgÆmL
)1
in buffer C. The
fraction containing pure RNAPII was dialysed against
buffer C and used for the transcription experiments.
RNAPII holoenzyme was prepared from TAP-SpMed7
(a gift from C. Gustafsson, Karolinska Institute, Novum,
Sweden) cell extracts. Extracts were prepared as described
above from TAP-SpMed7 and dialysed against 20 m
M
Hepes pH 7.5, 200 m
M
potassium acetate, 10% (v/v)
glycerol, 1 m
M
dithiothreitol, 0.1% (v/v) NP-40 and
0.5 m
M
phenylmethanesulfonyl fluoride. After dialysis,
2 mL of extract was incubated with 500 lL IgG beads

(Amersham Biosciences) for 1 h at 4 °C. The IgG beads
were loaded into a column and washed with dialysis buffer.
After washing with 10 mL tobacco etch virus (TEV)
protease buffer, the beads were resuspended in 1 mL TEV
protease buffer and incubated at 30 °Cfor1hwith300U
TEV protease. Eluates were collected and dialysed against
dialysis buffer. TEV protease was eliminated from the
eluates for passage onto a Ni-NTA–agarose column. The
RNAPII holoenzyme was further purified on a heparin–
Sepharose column to separate the RNAPII holoenzyme
from TRAP240 complex.
Purification of the TFIID complex
We used two methods to purify the TFIID complex, which
consists of TBP and the TAFs. First, 500 lLofproteinA–
agarose containing crosslinked anti-TAF110 Igs were mixed
with 2 mL cell extract in buffer containing 20 m
M
Hepes
pH 7.5, 100 m
M
potassium acetate, 10% (v/v) glycerol,
1m
M
dithiothreitol, 0.1% (v/v) NP-40, 1 m
M
phenyl-
methanesulfonyl fluoride, 0.1 m
M
EDTA and incubated
at 4 °C for 1 h. The resin was then washed with the same

Hepes buffer, and protein was eluted with 500 lL6
M
urea,
20 m
M
Hepes pH 7.5, 0.1 m
M
EDTA, 1 m
M
dithiothreitol.
The eluate was dialysed against incubation buffer and
concentrated fivefold with a Centricon.
The native TFIID complex was also purified from whole-
cell extracts using conventional chromatographic meth-
ods (hydroxyapatite, phosphocellulose, DE52, Mono S,
Mono Q, Phenyl-Superose and ACA22 gel filtration col-
umns). We purified the TAF-containing complex until we
obtained the same set of polypeptides that were present in
the complex that was affinity purified with anti-TAF110.
Both, the affinity and chromatographic purified TAF-
containing complex (TFIID) were free of RNAPII, Medi-
ator and GTFs.
Purification of the Gal4 DNA binding domain, Gal4-AP2,
Gal4-VP16 and GAL4-CTF
The gene that encodes the Gal4-AP2 transcriptional
activator, which contains the Gal4 DNA binding domain
and an activation domain from the AP2 transcription
factor, was obtained from a Sc. pombe expression vector
(a gift from J. E. Remacle, Department of Cell Growth,
University of Leuven, Belgium) and cloned into pET15b

(Novagen) by PCR. Gal4-AP2 was then expressed in
bacteria and purified under native conditions on Ni-
NTA–agarose. Gal4-CTF and Gal4-Sp1 were purified by
the same method. Purified Gal4 and Gal4-VP16 were a
gift from D. Reinberg.
Preparation of antibodies to TFIIFb, Srb4, Tfb1, TAF110,
and TAF72
Antibodies were prepared by injecting rabbits with
500 lg of either purified recombinant TFIIFb,Srb4
and Tfb1, or TAF110 and TAF72, each mixed in
Complete Freund’s adjuvant according to NIH guide-
lines. Fifty days after the first injection, another injection
was given in Incomplete Freund’s adjuvant. After
3 weeks, blood was obtained from each rabbit and
serum was prepared. The antibodies were purified from
the serum by protein A–agarose chromatography and
used for Western blots.
Specific transcription reactions
The conditions for the transcription reactions were based
on those reported by Lue et al.[9].TheDNAtemplate
contained the adenovirus major late promoter (Ad-
MLP) promoter fused to the 377 bp G-minus cassette of
Sawadogo and Roeder [10]. The template contained five
Gal4 binding sites located 30 bp upstream from the
TATA box. Reactions mixtures (30 lL) contained
proteins and template as indicated in the legend of
each figure. Transcription reactions were performed in
50 m
M
Hepes pH 7.8, 50 m

M
potassium glutamate,
15 m
M
magnesium acetate, 2.5 m
M
dithiothreitol, 1 U
ribonuclease inhibitor (Promega), 10% (v/v) glycerol,
2% (w/v) polyethylene glycol, 0.4 m
M
ATP, 0.4 m
M
CTP, 0.5 lCi [
32
P]dUTP[aP], 4 m
M
phosphoenolpyru-
vate. After 30 min at 25 °C, the transcription reaction
was stopped by the addition of 100 lL stop buffer
(10 m
M
Tris/HCl pH 5.7, 300 m
M
NaCl, 5 m
M
EDTA,
10 U RNAaseT1) and incubated at 37 °C for 15 min.
The reaction was treated with 10 lL10%SDSand
100 lg proteinase K for 15 min at 37 °C. The tran-
scripts were precipitated with 3 vols ethanol and ana-

lysed by electrophoresis in a 6% polyacrylamide/7
M
urea gel in Tris/borate/EDTA buffer. The gels were
dried and analysed by autoradiography.
Purification of DNA templates
Supercoiled DNA templates were purified with Wizard
columns (Promega) followed by polyethylene glycol preci-
pitation. Relaxed DNA templates were purified on Qiagen
columns, and the preparation contained only 5% super-
coiled templates. However, using the Wizard columns we
obtained  95% supercoiled templates. Linear templates
were obtained by cutting the template with a restriction
enzyme (EcoRI).
Ó FEBS 2004 Sc. pombe requires Mediator for activated transcription (Eur. J. Biochem. 271) 2563
Results
Identification and purification of TFIIE, TFIIF, TFIIB, TBP
and the Tfb1 subunit from
Sc. pombe
Using the NCBI
BLASTP
program, we identified Sc. pombe
homologs of TFIIE, TFIIF, TFIIB and Tfb1 by querying
with the amino acid sequences of the homologous human
and S. cerevisiae factors. The genes that encode the various
factors where then cloned from Sc. pombe and expressed
and purified as described in Materials and methods.
After purification, the factors were at least 90% pure as
judged by SDS/PAGE followed by Coomassie blue staining
(Fig. 1A–D). Sc. pombe TFIIE is composed of two sub-
units, a (CAC32853) and b (CAC204446). The Sc. pombe a

subunit contains 434 amino acids and shares 26% amino
acid sequence identity with its S. cerevisiae homolog and
21% amino acid sequence identity with human TFIIEa.
Sc. pombe TFIIEb (CAA20446) contains 285 amino acids
and shares 38% amino acid sequence identy with its
S. cerevisiae homolog and 30% amino acid sequence
identity with its human homolog. Sc. pombe TFIIF is also
composed of two subunits, a (CAA22493) and b
(NP595082). Sc. pombe TFIIFa contains 490 amino acids
and shares 33% amino acid sequence identity with its
S. cerevisiae homolog. However, no homology with human
TFIIFa was detected. Sc. pombe TFIIFb has 301 amino
acids and displays 27% amino acid sequence identity with
human TFIIFb and 37% identity with S. cerevisiae
TFIIFb.TheSc. pombe Tfb1 subunit of TFIIH has 457
amino acids and shares 28% amino acid sequence identity
with the human p62 subunit of TFIIH. Tfb1 also displays
29% identity with its S. cerevisiae homolog.
Purification of RNAPII and RNAPII holoenzyme
from
Sc. pombe
RNAPII and the RNAPII holoenzyme were purified from
Sc. pombe cell extracts as described in Materials and
methods. RNAPII was purified by affinity chromatography
with antibodies against the CTD. The RNAPII holoenzyme
was purified by subjecting cell extracts made with the TAP-
SpMed7 Sc. pombe strain to affinity chromatography on
IgG beads. The purity of both preparations was evaluated
by SDS/PAGE followed by silver staining. As shown in
Fig. 2A, all of the RNAPII subunits were present in the

RNAPII holoenzyme preparation, and the holoenzyme
preparation also contained additional polypeptides, some of
which were identified by N-terminal amino acid microsequ-
encing. Furthermore, Srb4 and the smallest subunit of
TFIIF were present in the RNAPII holoenzyme as detected
by Western blot analysis (Fig. 2B). The smallest subunit of
TFIIF and Srb4 were not detected in the affinity purified
RNAPII. We did not detect TBP, TFIIB, TFIIE, TFIIH,
Fig. 1. Purification of recombinant factors. The various factors were amplified by PCR and cloned in the vector pET-15b. The plasmids containing
the cDNA encoding the various factors were transformed into BL21-(DE3) cells and the expression of the protein was induced with isopropyl thio-
b-
D
-galactoside. The various factors were purified as described Materials and methods. Five micrograms of each factor were analysed by SDS/
PAGE (8% acrylamide) followed by staining with Coomassie blue. (A) TFIIE; (B) TFIIF; (C) TFIIB; (D) pTBP. Migration positions of molecular
size standards are indicated to the left of the panels.
2564 E. Tamayo et al. (Eur. J. Biochem. 271) Ó FEBS 2004
TAF72 or TAF110 in preparations of the Sc. pombe
RNAPII holoenzyme, as measured by Western blot analysis
(data not shown). We estimated that affinity-purified
RNAPII preparation contained twofold more RNAPII
than did the affinity-purified RNAPII holoenzyme prepar-
ation, as measured by Western blotting with antibodies to
the CTD (Fig. 2C). TFIIH was purified from cell extracts as
described in Materials and methods, and its elution from
the ACA22 column was followed by Western blotting with
antibodies to Tfb1 and by inclusion in in vitro transcription
assays. A very precise coelution of Tfb1 and transcription
activity can be seen in Fig. 3A. The TFIIH preparation
appeared to contain only a few polypeptides and some of
them were identified by N-terminal amino acid microsequ-

encing (Fig. 3B).
Reconstitution of transcription
in vitro
using purified
recombinant factors from
Sc. pombe
To reconstitute transcription in vitro, we used purified
recombinant Sc. pombe TBP, TFIIF, TFIIB, TFIIE,
affinity-purified RNAPII, and TFIIH. We used the G-
minus template described above, which contained the Ad-
MLP promoter and five Gal4 binding sites upstream from
the TATA box. The preparation of DNA template was a
relaxed plasmid. This promoter has been used by Korn-
berg’s laboratory to study transcription in Sc. pombe [6].
As shown in Fig. 4A, strong transcription from the Ad-
MLP was detected when all the factors were present in the
reaction (lanes 1 and 8). However, the omission of TBP
(lane 2), TFIIH (lane 3), TFIIF (lane 4), TFIIE (lane 5),
TFIIB (lane 6) or RNAPII (lane 7) resulted in no
detectable transcription from the Ad-MLP promoter. The
transcripts were produced by RNAPII, as their pro-
duction was sensitive to 5 lgÆmL
)1
and 10 lgÆmL
)1
a-amanitin (lanes 9 and 10). Furthermore, the assay is
completely dependent on the addition of Sc. pombe
RNAPII (lane 7). These results indicate that RNAPII
and the GTFs are necessary and sufficient to reconstitute
Sc. pombe transcription in vitro.Furthermore,transcrip-

tion was initiated at the same position as it was in whole-
cell extracts as measured by primer extension (data not
shown). We also used the Sc. pombe adh promoter in the
in vitro transcription assay. The adh promoter displayed
essentially the same requirements for transcription as did
the Ad-MLP promoter, although transcription levels were
lowerwiththeadhpromoterthanwiththeAd-MLP
promoter (Fig. 4B, compare lanes 1 and 2 with lanes 3
and 4).
It has been reported that transcription driven by the adh
promoter is partially dependent on TFIIH [11]. To address
this discrepancy between the previous data and the data
reported here we tested, in reconstituted in vitro transcrip-
tion assays, both supercoiled and linear purified DNA
templates that carried the Ad-MLP (Fig. 4C). The super-
coiled Ad-MLP was transcribed in the absence of TFIIH at
levels similar to those obtained in the presence of TFIIH
(Fig. 4D, compare lanes 1 and 2). However, the transcrip-
tion of linear Ad-MLP templates required TFIIH (compare
lanes 3 and 4). We also tested the requirements of the
Sc. pombe adhpromoterinreconstitutedin vitro transcrip-
tion assays. Similar to the results of Sphar et al.[11],we
found that the supercoiled templates with the adh promoter
can be transcribed in the absence of TFIIH, although
transcription was less efficient under these conditions than it
was with the Ad-MLP (the minus-TFIIH assay yielded 50%
of the amount of transcripts produced in the presence of
TFIIH; Fig. 4E).
Fig. 2. Purification of RNAPII and the RNAPII holoenzyme. RNAPII was purified from Sc. pombe whole-cell extracts using affinity chroma-
tography with antibodies to the CTD as described in Material and methods. The RNAPII holoenzyme was purified from the Sc. pombe TAP-

SpMed7 strain by affinity chromatography on IgG beads. Some of the subunits of the Mediator and RNAPII were identified by N-terminal
microsequencing of proteins eluted from a preparative SDS/polyacrylamide gel. (A) Analysis of 200 ng of affinity-purified RNAPII (right panel)
and 300 ng of the RNAPII holoenzyme (left panel, TAP-sp. MED7). These analyses were performed by electrophoresis in a 4–15% SDS/
polyacrylamide gradient gel followed by silver staining. (B) Analysis of affinity-purified RNAPII (200 ng; left panel, RNAPII) and the RNAPII
holoenzyme (500 ng; right panel, TAP-SpMed7) by Western blotting with antibodies to Srb4, TFIIFb and the CTD (labelled Sp Rbp1). (C)
Comparison of varying amounts of the RNAPII largest subunit in the affinity-purified RNAPII and RNAPII holoenzyme (TAP-SpMed7)
preparations by Western blotting with antibodies to the CTD. The amounts (ng) are indicated at the top of the figure.
Ó FEBS 2004 Sc. pombe requires Mediator for activated transcription (Eur. J. Biochem. 271) 2565
The RNAPII holoenzyme stimulates basal and activated
transcription in a reconstituted transcription system
Next, we tested whether the RNAPII holoenzyme, which
contains the Mediator complex, is able to stimulate basal
transcription and support activated transcription in our
reconstituted system. As shown in Fig. 5A, no detectable
levels of basal transcription were observed in the presence of
the RNAPII holoenzyme if TBP (lane 1), TFIIB (lane 2),
TFIIH (lane 3) or TFIIE (lane 4) was omitted from the
transcription assay. And, as expected, omitting the RNAPII
holoenzyme from the reaction completely eliminated basal
transcription (lane 5). When TFIIF was omitted, detectable
levels of transcription were observed (lane 6), indicating
that the RNAPII holoenzyme preparation contains TFIIF
(as detected by Western blotting; Fig. 2B). The addition of
TFIIF did not augment transcription, which indicates that
TFIIF is in saturating amounts in the RNAPII holoenzyme
preparation (lane 7).
The RNAPII holoenzyme was able to carry out basal
transcription at a level that was twofold greater than that
observed with affinity-purified RNAPII (Fig. 2B, compare
lanes 6 and 7 with lanes 8 and 9). In this series of assays, we

used the same amount (in ng of protein) of the largest
subunit of RNAPII.
The RNAPII holoenzyme was also able to support
transcriptional activation (at least fivefold, as measured with
a Phosphoimager) by the mammalian Gal4-AP2 transcrip-
tional activator protein (lane 11), indicating that the
Mediator is involved in activated transcription. Neither
the inclusion of human recombinant TFIIA nor the Gal4
DNA binding domain had an effect on activated transcrip-
tion (lanes 12 and 10, respectively). We have not tested
Sc. pombe TFIIA in our reconstituted assay.
The levels of transcriptional activation observed in the
RNAPII holoenzyme assay were lower than those observed
with in whole-cell extracts (Fig. 5B). A 20-fold activation
of transcription was observed with whole-cell extracts plus
VP16 (lane 4), AP2 (lane 5) and CTF (lane 6) transcriptional
activator proteins. In the reconstituted assay with the
RNAPII holoenzyme, only a 10-, six-, and sixfold activation
of transcription was obtained with VP16 (lane 7), AP2 (lane
8), and CTF (lane 9), respectively. The same levels of
transcription were obtained in the whole-cell extract without
(lane 1) or with (lane 2) Gal4 DNA binding domain and in
the reconstituted assay with Gal4 binding domain (lane 3).
We also tested the ability of the acidic activator, Gal4-VP16,
to activate transcription in the presence of the RNAPII
holoenzyme in the reconstituted assay, and we obtained
essentially the same results as those observed with Gal4-
AP2 (Fig. 5C). However, the activation achieved with Gal4-
VP16 was 10-fold, which indicates that the VP16 transcrip-
tional activation domain is more potent than the AP2

domain.
We also investigated whether affinity-purified RNAPII
can support activated transcription. As shown in Fig. 5D,
affinity-purified Sc. pombe RNAPII was unable to support
activated transcription (lanes 3–5). The addition of human
TFIIA (lane 6) had no effect on basal transcription carried
out by the affinity-purified RNAPII; in fact, human TFIIA
inhibited basal transcription with the RNAPII, indicating
that its presence may be involved in squelching of the
function of transcription factors necessary for transcription.
RNAPII holoenzyme is at least twofold more potent in
directing basal transcriptioning of (compare lanes 1 and 2).
Also, we tested the effect of the transcriptional activation
domains of CTF and Sp1 in the reconstituted assay using
the RNAPII holoenzyme and found that Gal4-CTF is able
to activate basal transcription (Fig. 5E, compare lanes 1, 2,
and 3) whereas Gal4-SP1 is not (compare lanes 1, 4, and 5).
Antibodies against Srb4 inhibit activated
but not basal transcription
We next tested whether the ability of the RNAPII
holoenzyme to support activated transcription was the
Fig. 3. Analysis of the purification of TFIIH by gel filtration on an
ACA22 column. TFIIH was purified as described in Materials and
methods. (A) The active fraction from the heparin–agarose column
was further fractionated on an ACA22 gel filtration column, and the
fractions were analysed by Western blotting with antibodies to Tfb1
(top). The same fractions (5 lL each) were analysed for transcription
activity in a reconstituted in vitro assay that lacked TFIIH (bottom).
Fraction numbers are indicated at the bottom panel. Lane 1 represents
the imput from the heparin–agarose column. (B) The peak TFIIH

fraction (1 lg; lane 6) was analysed by SDS/PAGE (10% acrylamide)
and stained with colloidal Coomassie blue (Novex). The labelled
individual subunits were identified by N-terminal microsequencing.
Migration positions of molecular size standards are indicated to the left
of the panels.
2566 E. Tamayo et al. (Eur. J. Biochem. 271) Ó FEBS 2004
result of the presence of the Mediator. Antibodies to Srb4, a
component of the Mediator complex, were introduced into
the in vitro transcription reactions with the RNAPII
holoenzyme. As shown in Fig. 6A, preimmune serum or
antibodies to TAF110 did not inhibit the activation of basal
transcription by Gal4-VP16 from the Ad-MLP promoter
(Fig. 6, compare lane 2 with lanes 3 and 4). However,
antibodies to Srb4 inhibited activated transcription (with
the RNAPII holoenzyme and Gal4-VP16) (lanes 5 and 6)
but not basal transcription (with the RNAPII holoenzyme)
(compare lanes 2, 3 and 4 with lanes 5 and 6). Anti-Srb4 also
was not able to inhibit basal transcription when affinity-
purified RNAPII was used (lanes 7 and 8). Anti-CTD Igs
completely inhibited transcription.
Fig. 4. Analysis of in vitro transcription factor requirements. Transcription assays were performed as described in Materials and methods. Proteins
and 500 ng relaxed plasmid template DNA were added as indicated in each figure. (A) Transcription assays were performed with 300 ng affinity-
purified RNAPII, 300 ng TFIIH (ACA22 fraction), and 50 ng recombinant TFIIB, TBP, TFIIF, and TFIIE. Lanes 1 and 8, complete; lane 2,
without TBP; lane 3, without TFIIH; lane 4, without TFIIF; lane 5, without TFIIE; lane 6, without TFIIB; lane 7, without RNAPII. Lanes 9 and 10
show the complete assay in the presence of 5 and 10 lgÆmL
)1
a-amanitin. (B) Transcription from the Sc. pombe adh promoter (lane 1 and 2) and the
Ad-MLP promoter (lanes 3 and 4) was compared in the reconstituted assay (complete) and in whole-cell extracts (WCE). (C) Agarose gel showing
supercoiled and linear templates with the Ad-MLP promoter. (D) TFIIH requirement of Ad-MLP-driven transcription of supercoiled and linear
DNA templates. Proteins and DNA templates were added as indicated at the bottom of the figure. (E) TFIIH requirement of adh-driven

transcription of supercoiled and linear DNA templates. Templates and proteins were added as indicated at the bottom of the figure.
Ó FEBS 2004 Sc. pombe requires Mediator for activated transcription (Eur. J. Biochem. 271) 2567
The TAF-containing complex does not have an effect
on activated transcription
To test the effect of TAFs on activated transcription, we
purified the TAF complex by two methods and tested the
effect TAFs on activated transcription using the reconsti-
tuted in vitro transcription assay with the RNAPII holo-
enzyme, the activator Gal4-VP16, and the Ad-MLP. We
used affinity chromatography with antibodies to TAF110 as
well as conventional chromatography to isolate the TAF
complex. The TAF complex isolated by conventional
chromatography was similar in its properties to the complex
purified by affinity chromatography. A precise coelution
from the ACA22 column was observed for TAF110,
TAF72 and TBP, as detected by Western blotting of the
column fractions with specific antibodies (Fig. 7A). Also,
when the affinity-purified and conventionally purified
preparations were subjected to gradient SDS/PAGE, the
same set of polypeptides was observed in both preparations
(Fig. 7B). Also, we observed that the same set of
polypeptides can be immunoprecipitated with antibodies
against TAF72 and TBP from a preparation that was
first immunoprecipitated with anti-TAF110 (lanes 2–4).
Furthermore, the complex purified by conventional
Fig. 5. Analysis of transcriptional activity under a variety of conditions using RNAPII and the RNAPII holoenzyme. Transcription assays were
performed as described in Materials and methods with 50 ng of the Ad-MLP promoter and the amounts of RNAPII holoenzyme, RNAPII,
activators, and GTFs indicated at the bottom of each panel. (A) Analysis of the amount of transcriptional activation by Gal4-AP2 in the
reconstituted assay using the RNAPII holoenzyme. (B) Comparison of the levels of transcriptional activation achieved with Gal4-VP16, Gal4-AP2,
and Gal4-CTF in reconstituted assays (Reconstituted) with the RNAPII holoenzyme and in whole-cell extracts (pWCE). (C) Analysis of the

amount of transcriptional activation by Gal4-VP16 in the reconstituted assay using RNAPII and the RNAPII holoenzyme. (D) Analysis of the
amount of transcriptional activation achieved by Gal4-VP16 with purified RNAPII and the RNAPII holoenzyme. (E) Analysis of the tran-
scriptional activation function of Gal4-CTF and Gal4-Sp1 in the reconstituted assay using the RNAPII holoenzyme.
2568 E. Tamayo et al. (Eur. J. Biochem. 271) Ó FEBS 2004
chromatography shows the same set of polypeptides as the
complex purified by affinity chromatography (lane 5) and
the same polypeptides are immunoprecipitated by anti-
TAF110 from the ACA22 column (lane 6).
Next, we investigated the role of the TAFs in activated
transcription. As seen in Fig. 7C, the addition of affinity
purified TAFs to in vitro transcription reactions containing
the RNAPII holoenzyme and Gal4-VP16 did not stimulate
activated transcription from the Ad-MLP, as the same
activation fold is seen in the presence and absence of TAFs
(compare lanes 1, 3, 4 and 5). We know that the TAF-
containing complex contains TBP, because it can replace
TBP, which is a required component, in the transcription
assay (lane 2).
Because we used harsh conditions to elute the TAF-
containing complex from the anti-TAF110 column, which
migh have affected TAF coactivator function, we purified
the TAF-containing complex under native conditions by
conventional chromatography. As shown in Fig. 7D, this
TAF-containing complex did not stimulate basal transcrip-
tion directed by the RNAPII holoenzyme (compare lanes 1
and 2) either in the absence (lane 2) or presence of TFIIA
(lane 3). Likewise, TAFs were not able to stimulate basal
transcription directed by purified RNAPII (lanes 4–7) either
in the absence or presence of TFIIA (lane 5). TAFs were
also unable to stimulate activated transcription (compare

lanes 9, 10 and 11) even in the presence of TFIIA (lane 11).
TBP is contained in the conventionally purified TAF-
containing complex, because it can replace TBP in the
reconstituted assay (lane 12), which is dependent on the
presence of TBP (lane 13).
Discussion
In this report, we have described a reconstituted in vitro
transcription assay that contains components from
Sc. pombe: purified recombinant TBP, TFIIF, TFIIB and
TFIIE, affinity-purified RNAPII, and TFIIH. These are the
same components required for basal transcription in in vitro
assays with factors from human, Drosophila, rat, and
S. cerevisiae. Thus there is notable conservation between the
GTFs of Sc. pombe and other species, which indicates that
the mechanisms of basal transcription have been conserved
throughout evolution.
We also demonstrated that, in the reconstituted assay, the
RNAPII holoenzyme was able to stimulate basal transcrip-
tion and support activated transcription through the
associated Mediator complex. TAFs had no effect on
in vitro transcription carried out by the RNAPII
holoenzyme. Ours is the first reported reconstituted in vitro
transcription assay from Sc. pombe that can support
activated transcription in the presence of the Mediator.
This reconstituted assay could serve as a tool to study the
mechanisms of transcription in Sc. pombe,anorganismthat
is widely used to study other biological process.
We have reconstituted in vitro transcription by Sc. pombe
RNAPII using two different promoters, the Ad-MLP and
the adh promoter. We found that the two promoters have

the same factor requirements, but that the level of basal
transcription is lower with adh than Ad-MLP. The lower
levels of transcription of from the adh promoter is not a
result of the absence of adh promoter-specific factors,
because the levels of transcription from the adh promoter
are the same in whole-cell extracts and in the reconstituted
system. We believe that the difference is due to promoter
strength, because the Ad-MLP possesses a strong TATA
box and Initiator, and the Initiator appears not to be present
in the adh promoter. When we use circular relaxed
templates in our assay, transcription is absolutely dependent
on TFIIH. It has been reported by Sphar et al. [11] that
in vitro transcription from the adh promoter is partially
independent on TFIIH when supercoiled templates are
used. We investigated this issue using supercoiled and linear
templates with the Ad-MLP and adh promoters. We found
that supercoiled templates with either promoter are tran-
scribed in the reconstituted transcription assay without
TFIIH, whereas, like the transcription of circular relaxed
templates, transcription of linear templates is absolutely
dependent on TFIIH. Because the level of transcription
from the adh promoter falls by 50% in the absence of
TFIIH, while the amount of transcription from the
Ad-MLP is the same under both conditions, we believe
that transcription from the adh promoter is more dependent
on the presence of TFIIH. The difference in the requirement
of TFIIH for transcription from the adh and Ad-MLP
promoters could be explained by the presence of an Initiator
in the Ad-MLP promoter, which can nucleate preinitiation
complexes more efficiently than the adh promoter.

The Mediator is a multiprotein complex that contains
several polypeptides involved in transcriptional activation.
Mediator binds to the CTD of RNAPII to produce the
RNAPII holoenzyme and has been purified from S. cere-
visiae and several mammals [12–20] A Mediator complex
also has been identified in Sc. pombe [21–23] and contains
10 essential gene products that are homologs of the
Mediator subunits of S. cerevisiae as well as three non-
essential gene products that do not have homologs in other
organisms [22]. Recently, a Mediator complex that con-
tained the spsrb8, sptrap240, spsrb10 and spsrb11 subunits
was isolated from Sc. pombe [23] in a free form, devoid of
RNAPII. It has been suggested that spsrb8 and spsrb9 are
the Sc. pombe homologs of mammalian TRAP230/
ARC240 and TRAP240/ARC250, respectively.
Fig. 6. Antibodies to Srb4 inhibit transcriptional activation by Gal4-
VP16. Transcription reactions were performed as described in Mate-
rials and methods using 50 ng of the Ad-MLP. Proteins were added as
indicated at the bottom of the figure.
Ó FEBS 2004 Sc. pombe requires Mediator for activated transcription (Eur. J. Biochem. 271) 2569
We isolated the RNAPII holoenzyme from Sc. pombe
using the TAP-SpMed7 strain [23] and purified the holo-
enzyme further using heparin Sepharose chromatography
to separate small amounts of the TRAP240 complex, which
copurifies with the RNAPII holoenzyme. The RNAPII
holoenzyme was more active in our in vitro basal transcrip-
tion assay than was RNAPII, in agreement with the results
of Sphar et al. [11]. Our holoenzyme preparation also
supported activated transcription with two types of tran-
scriptional activators ) acidic (VP16) and proline-rich (AP2

Fig. 7. Effect of the TAF-containing complex on transcriptional activation by the Gal4-VP16 activator. Affinity-purified and chromatographically
purified TAF-containing complexes were prepared and transcription reactions were performed with 50 ng of the Ad-MLP template as described in
Materials and methods. Proteins were added as described at the bottom of each figure. The fold activation of transcription was calculated using a
Phosphoimager (Bio-Rad). (A) Western blot analysis of fractions from the ACA22 column step in the purification of the TAF-containing complex.
Antibodies to TAF110, TAF72, and TBP were used in the immunoblotting. (B) SDS/6–15% polyacrylamide gradient gel electrophoresis of the
various TAF-containing complexes purified by affinity and conventional chromatography. Lane 1, Immunoprecipitation with preimmune sera;
lane 2, immunoprecipitation with anti-TAF110; lanes 3 and 4, immunoprecipitation with anti-TAF72 and anti-TBP of fractions that were first
purified by anti-TAF110; lane 5, fraction from the ACA22 column; lane 6, immunoprecipitation of the ACA22 column with anti-TAF110. A 300-
ng aliquot of each factor were analysed by electrophoresis on an SDS/6–15% polyacrylamide gradient gel that was then stained with colloidal
Comassie blue (Novex). (C) The effect of the affinity-purified TAF-containing complex on the activation of transcription by Gal4-VP16 using the
RNAPII holoenzyme. (D) The effect of the conventionally purified TAF-containing complex on the activation of transcription by Gal-VP16 using
the RNAPII holoenzyme.
2570 E. Tamayo et al. (Eur. J. Biochem. 271) Ó FEBS 2004
and CTF) ) but it did not support transcriptional activa-
tion by Sp1. These results strongly suggest that the RNA
polymerase holoenzyme contains the Mediator subunits
required for activated transcription. These results are in
agreement with in vivo data showing that the activation
domains of VP16, AP2, and CTF can activate transcription
in Sc. pombe [24]. However, the in vivo targets of these
activators were not identified in this report. Therefore, we
extend these previous observations by demonstrating that
these activation domains function via the Mediator.
Because AP2 and CTF do not activate transcription in
S. cerevisiae, our results suggest that the transcriptional
activation process in Sc. pombe is more similar to that of
humans than to that of S. cerevisiae.
In our experiments, we observed that activated transcrip-
tion levels were higher in whole-cell extracts than in our
reconstituted Sc. pombe assay, which indicates that addi-

tional factors are required for more efficient transcription
activation in our reconstituted system. These missing factors
could be proteins that are not part of the Mediator such as
PC4 or it could be that some key subunits of the Mediator
are dissociated from the RNAPII holoenzyme during
purification. A homolog of human PC4 is present in
Sc. pombe [6]. Also, we found that the RNAPII holoenzyme
isolated by us contains TFIIF, a transcription factor that
was not found in the preparations of the RNAPII holoen-
zyme isolated by Spahr [11]. We believe that this difference
could result from the method used to detect TFIIF: we used
immunoblotting and Spahr used MALDI-TOF. An alter-
native explanation is that TFIIF is merely a contaminant of
the RNAPII holoenzyme preparation.
Antibodies to the Mediator component Srb4 were able to
inhibit transcriptional activation by the Gal-VP16 activator.
However, these antibodies had no effect on basal transcrip-
tion. This strongly suggests that the Mediator is involved in
transcriptional activation in Sc. pombe. The fact that anti-
Srb4 did not inhibit basal transcription directed by the
RNAPII holoenzyme is surprising. This result can be
explained by the fact that Srb4 contacts RNAPII directly
[22] and the anti-Srb4 antibodies can dissociate the Medi-
ator from RNAPII, but the enzyme is still able to transcribe.
TAFs are another type of transcriptional coactivator [25].
Although TAFs were shown not to be required for the
activation of transcription in a human nuclear extract [26], a
later report from the same group showed that the TAFs and
Mediator can act jointly to achieve activated transcription
[4]. In vivo studies in S. cerevisiae suggest that TAFs are not

essential for activated transcription; rather, the TAFs
participate in transcription by conferring a dependence on
core promoter elements [27–29]. We have purified from
Sc. pombe a TAF-containing complex that appears to be the
homolog of TFIID in higher eukaryotes, as it possesses
TAF110, which is the homolog of human TAFII250. Also,
TAF72 of Sc. pombe is the homolog of human TAFII100
(isoform 1). For these reasons, we believe that the TAF-
containing complex that we purified from Sc. pombe is the
homolog of human TFIID. We assessed the role of the
Sc. pombe TAFs in our reconstituted in vitro transcription
assay; the results strongly suggest that TAFs are not required
for basal or activated transcription. It is important to note
that Sc. pombe TFIIA was not included in the reactions, and
human TFIIA had no effect on the results. However, we do
not know whether human TFIIA can functionally replace
Sc. pombe TFIIA in in vitro transcription assays. We do
know that human TFIIA can stimulate the binding of
Sc. pombe TBP to the TATA box. For this reason, and
considering that human TFIIA and Sc. pombe TFIIA have a
high degree of homology at the amino acid sequence level, we
believe that human TFIIA is likely to be able to replace
Sc. pombe TFIIA in in vitro transcription assays. The results
we obtained with TAFs are in agreement with other reports
that indicate that TAFs are not required for transcriptional
activation in vivo in S. cerevisiae [27–29]. However, we do not
rule out the following two possibilities: (a) for certain
promoters in Sc. pombe, TAFs play a role in transcriptional
activation; and (b) another Sc. pombe TAF-containing
complex that is different from the homolog of human TFIID

can activate transcription in an Sc. pombe in vitro system.
Acknowledgements
We thank C. C. Allende for critical reading of the manuscript and
C. Gustafsson for the TAP-SpMed7 strain. We also thanks J. E.
Remacle for the Sc. pombe Gal4-AP2 expression vector. This work was
supported by Grant #1010824 from the Fondo de Desarrollo Cientı
´
fico
y Tecnolo
´
gico-Chile (FONDECYT-Chile).
References
1. Orphanides, G., Lagrange, T. & Reinberg, D. (1995) The General
transcription factors of RNA polymerase II. Genes Dev. 10, 2657–
2683.
2. Lewis, B.A. & Reinberg, D. (2003) The mediator coactivator
complex: functional and physical roles in transcriptional regula-
tion. J. Cell Sci. 116, 3667–3675.
3. Albright, S.R. & Tjian, R. (2000) TAFs revisited: more data reveal
new twists and confirm old ideas. Gene 242, 1–13.
4. Baek, H.J., Malik, S., Qin, J. & Roeder, R.G. (2002) Requirement
of TRAP/mediator for both activator-independent and activator-
dependent transcription in conjunction with TFIID-associated
TAF(II)s. Mol. Cell. Biol. 22, 2842–2852.
5. Mittler, G., Kremmer, E., Timmers, H.T. & Meisterernst, M.
(2001) Novel critical role of a human Mediator complex for basal
RNA polymerase II transcription. EMBO Rep. 2, 808–813.
6. Wood, V., Gwilliam, R., Rajandream, M.A., Lyne M., Lyne R.,
Stewart A., et al. (2002) The genome sequence of Schizosaccha-
romyces pombe. Nature 415, 871–880.

7. Toyama, R. & Okayama, H. (1990) Human chorionic gonado-
tropin alpha and human cytomegalovirus promoters are extremely
active in the fission yeast Schizosaccharomyces pombe. FEBS Lett.
268, 217–221.
8. Li, Y., Flanagan, P.M., Tschachner, H. & Kornberg, R.D. (1994)
RNA polymerase II initiation factors interactions and transcrip-
tion start site selection. Science 263, 805–807.
9.Lue,N.F.,Flanagan,P.M.,Sugimoto,K.&Kornberg,R.D.
(1989) Initiation by yeast RNA polymerase II at the adenoviral
major late promoter in vitro. Science 246, 661–664.
10. Sawadogo, M. & Roeder, R.G. (1985) Factors involved in specific
transcription by human RNA polymerase II: analysis by a rapid
and quantitative in vitro assay. Proc. Natl Acad. Sci. USA 82,
4394–4398.
11. Spahr, H., Khorosjutina, O., Baraznenok, V., Linder, T., Samu-
elsen, C.O., Hermand, D., Makela, T.P., Holmberg, S. & Gus-
tafsson, C.M. (2003) Mediator influences Schizosaccharomyces
pombe RNA polymerase II-dependent transcription in vitro.
J. Biol. Chem. 278, 51301–51306.
Ó FEBS 2004 Sc. pombe requires Mediator for activated transcription (Eur. J. Biochem. 271) 2571
12. Kim, Y J., Bjorklund, S., Li, Y., Sayre, M.H. & Kornberg, R.D.
(1994) A multiprotein mediator of transcriptional activation and
its interaction with the C-terminal repeat domain of RNA poly-
merase II. Cell 77, 599–608.
13. Koleske, A.J. & Young, R.A. (1994) An RNA polymerase
holoenzyme responsive to activators. Nature 368, 466–469.
14. Chao, D.M., Gadbois, E.L., Murray, P.J., Anderson, S.F., Sonu,
M.S., Parvin, J.D. & Young, R.A. (1996) A mammalian SRB
proteinassociatedwithanRNApolymeraseIIholoenzyme.
Nature 380, 82–85.

15. Maldonado, E, Shiekhattar, R., Sheldon, M., Cho, H., Drapkin,
R., Rickert, P., et al. (1996) A human RNA polymerase II com-
plex associated with SRB and DNA-repair proteins. Nature 381,
86–89.
16. Rachez, C., Suldan, Z., Ward, J., Chang, C.P., Burakov, D.,
Erdjument-Bromage,H.,Tempst,P.&Freedman,L.P.(1998)A
novel protein complex that interacts with the vitamin D3 receptor
in a ligand-dependent manner and enhances VDR transactivation
in a cell-free system. Genes Dev. 12, 1787–1800.
17. Naar, A.M., Beaurang, P.A., Zhou, S., Abraham, S., Solomon,
W. & Tjian, R. (1999) Composite co-activator ARC mediates
chromatin-directed transcriptional activation. Nature 398,832.
18. Naar, A.M., Taatjes, D.J., Zhai, W., Nogales, E. & Tjian, R.
(2002) Human CRSP interacts with RNA polymerase II CTD
and adopts a specific CTD-bound conformation. Genes Dev. 16,
1339–1344.
19. Malik, S., Gu, W., Wu, W., Qin, J. & Roeder, R.G. (2000) The
USA-derived transcriptional coactivator PC2 is a submodule of
TRAP/SMCC and acts synergistically with other PCs. Mol. Cell 5,
753–760.
20. Sun, X., Zhang, Y., Cho, H., Rickert, P., Lees, E., Lane, W. &
Reinberg, D. (1998) NAT, a human complex containing Srb
polypeptides that functions as a negative regulator of activated
transcription. Mol. Cell 2, 213–222.
21. Spahr, H., Be
`
ve, J., Larsson, T., Bergtro
¨
m, J., Karlsson, K A. &
Gustaffson, C.M. (2000) Purification and characterization of

RNA polymerase II holoenzyme from S. pombe. J. Biol. Chem.
275, 1351–1356.
22. Spahr, H., Samuelsen, C.O., Baraznenok, V., Ernest, I.,
Huylebroeck, D., Remacle, J.E., Samuelsson, T., Kieselbach, T.,
Holmberg, S. & Gustafsson, C.M. (2001) Analysis of Schizo-
saccharomyces pombe mediator reveals a set of essential subunits
conserved between yeast and metazoan cells. Proc. Natl Acad. Sci.
USA 98, 11985–11990.
23. Samuelsen, C.O., Baraznenok, V., Khorosjutina, O., Spahr, H.,
Kieselbach, T., Holmberg, S. & Gustafsson, C.M. (2003)
TRAP230/ARC240 and TRAP240/ARC250 Mediator subunits
are functionally conserved through evolution. Proc.NatlAcad.
Sci. USA 100, 6422–6427.
24. Remacle, J.E., Albrecht, G., Brys, R., Braus, G.H. &
Huylebroeck, D. (1997) Three classes of mammalian transcription
activation domain stimulate transcription in Schizosaccharomyces
pombe. EMBO J. 16, 5722–5729.
25. Featherstone, M. (2002) Coactivators in transcription initiation:
here are your orders. Curr. Opin. Gen. Dev. 12, 149–155.
26. Oelgeschlager, T., Tao, Y., Kang, Y.K. & Roeder, R.G. (1998)
Transcription activation via enhanced preinitiation complex
assembly in a human cell-free system lacking TAFIIs. Mol. Cell 1,
925–931.
27. Moqtaderi, Z., Bai, Y., Poon, D., Weil, P.A. & Struhl, K. (1996)
TBP-associated factors are not generally required for transcrip-
tional activation in yeast. Nature 383, 188–190.
28. Walker, S.S., Reese, J.C., Apone, L.M. & Green, M.R. (1996)
Transcription activation in cells lacking TAFIIs. Nature 383,
185–188.
29. Shen, W.C. & Green, M.R. (1997) Yeast TAF (II), 145 functions

as a core promoter selectivity factor, not a general coactivator. Cell
90, 615–624.
2572 E. Tamayo et al. (Eur. J. Biochem. 271) Ó FEBS 2004