From The Fred Hutchinson Cancer Research Center and
the Howard Hughes Medical Institute, Seattle, Washington 98109 and the
§ Institute for Systems Biology, Seattle, Washington
98105
Received for publication, October 19, 2000, and in revised form, November 20, 2000
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ABSTRACT |
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The yeast Mediator complex is required for
transcription by RNA polymerase II (pol II) in vivo and
in vitro. This complex of over 20 polypeptides associates
with pol II and is recruited to transcription complexes at promoters.
Previous isolations of yeast Mediator-containing complexes in different
laboratories have identified several distinct complexes. To identify
the major forms of Mediator in yeast, Mediator was isolated from
nuclear extracts using a two-step chromatographic procedure, avoiding ion exchange chromatography and high salt conditions to prevent dissociation of subunits during purification. Components of the Mediator complexes were identified by mass spectrometry and Western analysis. The major form of Mediator, termed pol II·Med, contained pol II and Mediator, including the Srb8-11 module. A second lower molecular size complex was also identified, termed Mediator core (Medc), which lacked pol II, Srb8-11, Rox3, Nut1, and the Rgr1 module.
Both of these complexes were active in transcription in vitro, although the Medc complex had significantly lower activity and could compete with the activity of the pol II·Med complex in vitro.
Transcription of protein-coding genes requires RNA polymerase II
(pol II)1 as well as six
general transcription factors termed TFIIA, TFIIB, TFIID, TFIIE, TFIIF,
and TFIIH. Together these factors comprise the minimal pol II
transcription machinery and can promote accurate transcription
initiation from a core promoter (1, 2). However, additional factors
termed coactivators are required for response to transcriptional
regulatory signals. These factors include the chromatin remodeling
machinery, histone acetylases, the TBP-associated factors, and
the Mediator complex (1, 3-7). The Mediator complex was first
discovered in yeast and consists of more than 20 polypeptides. The
Srb2, -4, -5, and -6 Mediator subunit genes were identified as dominant suppressors
of deletions in the C-terminal domain (CTD) of the largest pol II
subunit (8). Likewise, the Srb7-11 subunits were identified as
recessive suppressors of CTD deletions. The remaining subunits were
identified by biochemical fractionation and include proteins previously
identified as having positive and/or negative roles in gene regulation
(Hrs1/Pgd1, Gal11, Sin4, Nut1, Nut2, Rox3, and Rgr1) and other subunits
not previously known to be involved in transcription (Med1, -2, -4, -6, -7, -8, -9, and -11) (9-11).
In vivo, and in crude nuclear transcription extracts, the
yeast Mediator behaves functionally as a general transcription factor. Heat shock of a strain containing a temperature-sensitive Srb4 allele
results in a global decrease in pol II transcription equivalent to a
mutation in pol II (12, 13). Likewise, transcription in
vitro is highly dependent on Srb2, -4, and -6 in nuclear extracts (14, 15). In this nuclear extract system, mutation of Srb2, -4, or -6 blocks recruitment of factors except TFIIA and TFIID to the
preinitiation complex (PIC) (15). After transcription initiation,
Mediator along with TFIIA, TFIID, TFIIH, and TFIIE remain at the
promoter forming a scaffold for recruitment of pol II and other missing
factors required for transcription reinitiation (16). Specific
activators can stabilize this scaffold complex (16). In highly purified
in vitro systems, Mediator stimulates both basal and
activated transcription as well as phosphorylation of the pol II CTD
(17-20). In these highly purified systems, Mediator isolated from
strains carrying deletions of nonessential subunits is defective in
response to specific transcriptional activators.
In the past several years, human homologs of the yeast Mediator complex
have been identified by affinity purification, interaction with
specific activators, and on the basis of coactivator activity. At least
eight different human and mouse Mediator complexes have been identified
(1, 3, 21). These complexes differ in their exact polypeptide
composition but contain a common set of polypeptides, including
homologs to yeast Rgr1 and Med7. Interestingly, even though Srb2, -4, -5, and -6 are crucial to yeast Mediator function, no homologs to these
factors have yet been identified in any organism other than yeasts.
Because both yeast and human Mediator subunits interact with specific
transcription activators, a current model for activator function is
that interaction between specific activators and specific Mediator
subunits aids in recruitment of the transcription machinery to
promoters (1, 3). Furthermore, transcription reinitiation may be
facilitated by interactions between activators and Mediator subunits
that may stabilize the scaffold complex (16).
Several groups have reported biochemical fractionation of yeast
Mediator-containing complexes from whole cell extracts. Young and
coworkers followed purification of Mediator subunits by Western blot
assay and isolated a complex termed RNA pol II holoenzyme. This complex
contains pol II, Mediator (including the Srb8-11 subunits), TFIIB,
TFIIF, TFIIH, and SWI/SNF (18, 22, 23). In contrast,
fractionation by Kornberg, Kim, and coworkers found Mediator (lacking
the Srb8-11 subunits) associated with pol II and sometimes TFIIF in a
complex termed holopolymerase or holoenzyme (10, 11, 17). The reason
for these differences in Mediator complexes is not clear, because
similar methods involving ion exchange chromatography were used by all
three groups (24, 25). Finally, using a different purification method,
Jaehning and coworkers (26) have isolated a complex termed Paf1 or
alternative holoenzyme, which contains pol II, TFIIB, TFIIF, Cdc73,
Paf1, Ccr4, Hpr1, and the Mediator subunits Gal11 and Sin4.
Both biochemical and genetic studies have suggested that the Mediator
is composed of stable subcomplexes or modules. One module contains the
dominant CTD suppressors Srb2, -4, -5, and -6 as well as Med6 and Rox3
(27, 28). This subcomplex is stable in 1 M urea. A second
module consists of Rgr1, Gal11, Sin4, Hrs1/Pgd1, and Med2. Evidence for
this subcomplex is that pol II·Med with a C-terminal deletion of Rgr1
lacks Gal11, Sin4, and Hrs1 (20, 29). Also, deletion of Gal11 causes
loss of the Hrs1 subunit and vice versa (20). Deletion of
the Sin4 subunit causes loss of both Med2 and Hrs1 and deletion of Med2
causes loss of the Hrs1 subunit (19). Another distinct module consists
of Srb8-11, which acts genetically as a repressor of pol II (30).
Deletion of any one of these Srb genes suppresses deletions
in the pol II CTD, and deletion of the Srb8 subunit gene
causes loss of Srb10 and -11 in the holoenzyme (30). Whole genome array
analysis showed that deletion of Srb10 derepressed transcription from a subset of yeast genes (13).
In light of the different Mediator-containing complexes isolated by
biochemical fractionation and the distinct modules of Mediator subunits
identified by biochemical and genetic studies, we have investigated the
different forms of Mediator complexes in yeast. To avoid high salt
conditions and multiple ion-exchange chromatographic steps used
previously that could have caused dissociation of subunits during
isolation, affinity chromatography and gel filtration were used to
isolate yeast Mediator complexes under mild conditions. Using these
methods, two major yeast Mediator-containing complexes were identified
and found to be distinct from those complexes previously identified.
One complex is similar to the holoenzyme isolated by Kornberg, Kim, and
colleagues, but in addition contains the Srb8-11 module. The other
complex consists of a subset of Mediator components, and based
on other work, appears to be the core Mediator complex.
Construction of FLAG-tagged Yeast Strains--
A
PCR-mediated epitope-tagging method (31) was used to
C-terminally tag the yeast Srb5, Srb4, and Rgr1 open reading frames. In
brief, plasmid p3FLAG-KanMX (32), which contains a tagging cassette
(three copies of the FLAG epitope upstream from the KanMX6 marker) was
used as the PCR template. Each pair of PCR primers contained 45 bases
homologous to the target gene and 18 bases complementary to the tagging
cassette. PCR products were transformed to yeast, and the tagging
cassette was introduced into the target gene by homologous
recombination. Correct colonies were selected on G418 plates and
identified by colony PCR. FLAG-tagged proteins were confirmed by
Western blotting.
Extract Preparation and Purification of Protein
Complexes--
The C-terminal FLAG-tagged strains were grown in YPD
media, and nuclear extracts were prepared as described previously (33). 100 mg of nuclear extract (20-40 mg/ml of protein) was diluted to 30 ml with buffer A (20 mM HEPES-KOH, 10% glycerol, 0.01%
Nonidet P-40, 2 mM DTT, and proteinase inhibitors)
containing 0.25 M KOAc and incubated with 1 ml of anti-FLAG
M2-agarose beads (Sigma) at 4 °C for 4 h. After ten 30-ml
washes with buffer A containing 0.3 M KOAc, proteins were
eluted twice from the beads by incubation at 4 °C for 1 h with
buffer A containing 0.3 M KOAc and 0.5 mg/ml FLAG peptide
(Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys) (synthesized by Research Genetics).
The eluted proteins were concentrated by Centricon YM-30 (Millipore)
and applied to a TSK-G4000SWXL (TosoHaas) gel filtration column at 0.37 ml/min equilibrated in buffer B (20 mM HEPES-KOH, pH 7.5, 0.25 mM EDTA, 0.3 M KOAc, 20% glycerol, 0.01% Nonidet P-40, 1 mM DTT, and proteinase inhibitors). 135-µl fractions were
collected, and 5 µl was used in Western blot analysis. Fractions
containing protein complex were combined and concentrated by Microcon
YM-10 (Millipore) for use in the transcription assay and immobilized
template assay. For the estimation of molecular size of protein
complexes, blue dextran (2000 kDa), thyroglobulin (669 kDa),
apoferritin (443 kDa), and bovine albumin (66 kDa) (Sigma) were loaded
onto the same column as molecular size markers.
Western Blotting--
All Western blots were performed as
described (15). Proteins were detected with the following antibodies:
anti-Srb2, Rpb3, TFIIB, TBP (15), anti-Srb4, Srb10, Med6, Tfg2 (J. Movius and K. Coachman, Fred Hutchinson Cancer Research Center),
anti-Sin4 (D. Stillman, University of Utah), anti-Gal11 (H. Sakurai,
Kanazawa University, Japan), anti-Kin28, Rpb1 (Berkeley Antibody Co.), and anti-FLAG M2 (Sigma). Quantitative Western blots were performed as
described (15). Serial dilutions of recombinant proteins overexpressed
and purified from Escherichia coli (rSrb2, rSrb4, rSrb10,
rRpb3, rTFIIB, and Tfg2) were used as standards. Band intensities were
determined by densitometry using IQMAC v1.2 software (Molecular Dynamics).
Mass Spectrometry Analysis of the pol II·Med and Medc
Complexes--
Peak fractions corresponding to pol II·Med or Medc
from the gel filtration column were pooled and concentrated using
Microcon YM-10 (Millipore). During concentration, the buffer was
exchanged by repeated dilution with 100 mM
NH4HCO3 with 8 M urea. Potential disulfide bonds were reduced and alkylated by incubation with 1 mM DTT at 37 °C for 20 min followed by 10 mM
iodoacetamide for 20 min at room temperature. Protein mixtures were
digested with 100 ng of endoprotease Lys-C (Roche Molecular
Biochemicals). After 3 h at 37 °C, the urea concentration was
adjusted to 2 M by dilution with 100 mM
NH4HCO3, and CaCl2 was added to 5 mM. Digestion was continued by addition of modified trypsin
(Promega) to a final substrate-to-enzyme ratio of 25:1 (w/w) and
incubated for 16 h at 37 °C. After concentrating the samples to
50 µl and adjusting the pH to 3 with trifluoroacetic acid, the
peptides were desalted using C18 Ziptips (Millipore). Samples were
lyophilized and resuspended in 5 µl of buffer C (5% acetonitrile,
0.5% acetic acid, 0.005% heptafluorobutyric acid).
Identification of proteins was accomplished by microcolumn high
performance liquid chromatography coupled to electrospray ionization
tandem mass spectrometry and data base searching (34). A 100- by
365-µm fused silica capillary (Polymetrics Inc.) with a tapered tip
was packed to a length of 10 cm with a 5-µm C18 reverse phase resin
(Monitor). The sample was directly loaded onto the microcolumn by
helium pressurization of the sample in a stainless-steel bomb. The
mobile phase for high performance liquid chromatography (HPLC)
consisted of buffer C and buffer D (99.5% acetonitrile, 0.5% acetic
acid, and 0.005% heptafluorobutyric acid). A precolumn split was used
to deliver a flow rate of 300 nl/min through the column. The HPLC pump
was programmed to ramp solvent B from 10 to 38% in ~70 min.
Electrospray ionization was done at a voltage of 1.8 kV. A Finnigan LCQ
ion trap mass spectrometer was used to automatically acquire tandem
mass spectra during the entire gradient run. Tandem mass spectra were
searched against the Saccharomyces cerevisiae genome data
base obtained from Stanford University with the SEQUEST program. Each
high scoring peptide sequence and the corresponding tandem mass
spectrum was manually inspected to ensure the match was correct.
In Vitro Transcription Assay--
In vitro
transcription assay and primer extension were performed as
described (33).2 The
plasmid pSH515 was used as the template, which contains the HIS4 core
promoter and a Gal4-binding site (15). Gal4-VP16 was prepared as
described (15).
Immobilized Template Assay--
Immobilized template derived
from plasmid pSH515 was prepared as described previously (15).
Preinitiation complex (PIC) formation experiments were performed
essentially as described (15).2 Briefly, 2.5-µl
immobilized templates were preincubated with 48 ng of Gal4-VP16 for 10 min at room temperature. At the same time, nuclear extract mix was
prepared by adding 180 µg of nuclear extract and purified proteins to
a transcription mix containing 0.05% Nonidet P-40. Then, the
immobilized template and 0.5 µg of HaeIII-digested
E. coli competitor DNA were added to the nuclear extract mix
to a final volume of 50 µl. Reactions were incubated at room
temperature for 40 min to form PICs. After washing three times with
transcription buffer containing 0.05% Nonidet P-40 and 2.5 mM DTT, the immobilized template was digested with 60 units
of PstI at 37 °C for 30 min. The liberated mixture was
fractionated on a 4-12% NuPAGE gel (Invitrogen), and the composition
of PICs was analyzed by quantitative Western blotting.
Purification and Identification of Mediator
Complexes--
Previous biochemical fractionation of
Mediator-containing complexes from yeast whole cell extracts have
resulted in isolation of several complexes: the holoenzyme containing
pol II, Srb8-11, and the general transcription factors TFIIB, TFIIF,
TFIIH (18), the holoenzyme lacking Srb8-11 and all general
transcription factors (11), the holoenzyme including a single general
transcription factor TFIIF (17), and free Mediator (lacking both
Srb8-11 and pol II) (36). These fractionation methods have all used
multiple ion exchange chromatography steps and high ionic strength
buffers. It is possible that some of the differences in
Mediator-containing complexes isolated by different laboratories arise
from dissociation of subunits under these conditions. For example, it
has been shown that a fraction of Mediator can be separated from pol II
by ion exchange chromatography (36). To isolate Mediator complexes under gentler conditions, we first epitope-tagged the genomic copy of
the subunits Srb5, Srb6, or Rgr1 with three copies of the FLAG epitope
at the C terminus of these genes. As judged by cellular growth rates
compared with wild-type strains, and by in vitro
transcription using extracts made from these strains, the FLAG epitopes
did not affect the function of any of these Mediator subunits. As a
source for Mediator purification, we used yeast nuclear extracts,
because these extracts have a much higher specific transcription
activity compared with whole cell extracts used previously in
holoenzyme and Mediator purification (data not shown). In addition, the
nuclear extract system is clearly Mediator-dependent and
has been used extensively for biochemical study of transcription
initiation, reinitiation, and activation (15, 16, 37).
The first step in Mediator isolation was affinity purification on
anti-FLAG M2-agarose beads followed by peptide elution. Fig.
1 shows the small-scale affinity
purification results from three yeast strains containing C-terminal
FLAG-tagged Srb5, Srb6, and Rgr1, respectively. Recovery was measured
by probing Western blots for Mediator subunits, pol II subunit (Rpb1),
and several general transcription factors. Similar recoveries were
obtained for all the strains: ~70% of Mediator subunits, 5-10% of
pol II and <1% of both TFIIB and TFIIF were recovered. TFIIH and TBP were undetectable in the affinity-purified fractions (Table
I). These results indicate that our
FLAG-tag-based affinity purification is specific and efficient. A
large-scale affinity-purified fraction from the FLAG-tagged Srb5 strain
was then fractionated using gel filtration chromatography. In this
step, we monitored each fraction by Western blotting using antibodies
against Mediator subunits and general transcription factors (Fig.
2). The results revealed that the
Mediator subunits existed in two major forms: one large complex
(fractions 39-47) corresponding to an apparent size of ~1.9 MDa, and
one smaller complex (fractions 63-69) corresponding to an apparent
size of ~0.55 MDa. As shown below, the large complex is a form of pol
II·Mediator complex (termed pol II·Med), and the small complex
consists of a core subset of Mediator subunits termed Mediator core
(Medc).
The molar amounts of factors in the gel filtration fractions were
determined by comparison with recombinant protein standards for Srb2,
Srb4, Srb10, Rpb3 (pol II subunit), TFIIB, and Tfg1 (TFIIF subunit)
(Fig. 2; data not shown; Table
II). The pol II·Med complex
principally contains pol II, and Mediator, including the Srb8-11
complex. As quantitated by Western blot of the pol II subunit Rpb3, pol
II is estimated to be present at about one-half the molar ratio as
compared with Srb2, -4, and -10. As shown below from comparison of the
ratio of Mediator subunits to Rpb3 in preinitiation complexes (PICs),
we believe that pol II is substoichiometric in the pol II·Med
fractions compared with PICs. In contrast, a significantly lower amount
of the general factors TFIIB and TFIIF was present in these fractions
compared with Mediator subunits and pol II. We estimate that these
factors are present at a level of 0.15-0.05 compared with other
Mediator subunits. From these results, we conclude that the pol
II·Med fractions contain primarily the pol II·Mediator complex,
including Srb8-11 and a much lower amount of larger complexes
containing TFIIB and/or TFIIF. In contrast to the holoenzyme complex
isolated by Young and colleagues (18, 23), we do not detect TFIIH or
SWI/SNF in these fractions (not shown).
In contrast to the pol II·Med fractions, the Medc fractions lack a
subset of Mediator subunits (Fig. 2; Table II). Both the Gal11 and
Srb10 Mediator subunits were missing from these fractions. In addition,
Western blot assay of the Medc fraction with anti-Rgr1 and Sin4 showed
the absence of these subunits (not shown).These results suggest the
absence of both the Rgr1 and Srb8-11 modules. These modules have been
implicated in transcription regulation but may not be essential for
transcription, because most of these subunits are not encoded by
essential genes. Therefore, the Medc fraction likely represents a core
subcomplex of yeast Mediator. Probing of the gel filtration fractions
for the Rpb3 subunit of pol II showed that a fraction of pol II
dissociated from either the pol II·Med or Medc complexes during the
purification and eluted at the position expected for pol II enzyme.
The gel filtration chromatography of affinity-purified Mediator was
repeated using buffer containing 0.5 M potassium acetate, compared with the results shown in Fig. 2 using 0.3 M
potassium acetate (data not shown). Under these higher salt conditions, all of pol II dissociated from the Mediator complexes. However, the
amount of the Medc fraction remained constant compared with the amount
of intact Mediator. In addition, when gel filtration was performed
using affinity-purified fractions derived from the Rgr1-FLAG strain,
only the pol II·Med complex was detected (not shown). These results
together suggest that the Medc complex is not derived from dissociation
of the intact Mediator complex during purification but is rather a
pre-existing complex in the nuclear extract. Most likely the Mediator
core subunits are in excess over other Mediator subunits in the extract.
To more fully characterize the composition of pol II·Med and Medc, we
employed microcolumn HPLC-electrospray ionization tandem mass
spectrometry (LC/MS/MS) (38) (Table
III). This method uses liquid
chromatography and tandem mass spectrometry to separate and fragment
peptides in a complex mixture, thus offering the ability to identify
>100 proteins in a single run without use of SDS-PAGE. Analysis of the
pol II·Med fractions identified all 20 Mediator subunits, 9 of 12 pol
II subunits, and 3 of 4 subunits of the Srb8-11 module (Table
IIIA). For the Medc complex, we identified 13 Mediator
subunits, with 7 Mediator subunits (Rgr1, Gal11, Hrs1, Med2, Sin4,
Rox3, and Nut1) not detected (Table IIIB). Specifically, the
first five undetected subunits in Medc are all subunits of Rgr1 module,
which was previously implicated in both positive and negative control
of yeast transcription. Mass spectrometry analysis of the two fractions
also identified ribosomal subunits as contaminants in the pol II·Med
and Medc fractions. Specifically, many of the proteins of the large
ribosomal subunit were identified in the pol II·Med fraction. Probing
the FLAG affinity-purified fractions for TCM1 (RPL3), a protein in the
large ribosomal subunit, indicated that <0.2% of total TCM1 in the
starting extract contaminated the affinity-purified material (not
shown). However, because ribosomal subunits are present at levels at
least 500-fold over Mediator subunits (39), they represent a
significant source of contamination. Probing of the gel filtration
fractions with the TCM1 antisera showed near coelution of the large
ribosomal subunit and pol II·Med, indicating the close similarity in
molecular size of these two complexes (not shown).
Although the more sensitive Western blot assays showed detectable
amounts of TFIIB and TFIIF in the pol II·Med fractions, these factors
were not detectable in the mass spectrometry analysis, probably because
they are quite substoichiometric compared with the other polypeptide
subunits. A human homolog to the yeast Soh1 protein has been found in
the thyroid hormone receptor-associated proteins-SRB/MED cofactor
complex and PC2 Mediator complexes (3). However, yeast Soh1 was
not found in either the pol II·Med or Medc complexes. It is possible
that some subunits were not detected because they were
substoichiometric or simply missed due to the complexity of the samples.
In Vitro Transcription Assay of Mediator-containing
Complexes--
To investigate the role of the two Mediator-containing
complexes in transcription, in vitro transcription was
performed using the activator Gal4-VP16 and the yeast HIS4 promoter.
Transcription was assayed by complementation of yeast nuclear extracts
made from strains containing deletions in either the SRB2 or
MED9/CSE2 genes (Fig. 3). Both
extracts give very low levels of transcription in the absence of any
other factors (lanes 1 and 11). Addition of the
pol II·Med fraction to the Composition Analysis of PICs and the pol II·Med Complex--
The
results above show that the pol II·Med fraction is nearly equivalent
to the holoenzyme purified by Kornberg and Kim with the exception that
our fraction contains Srb8-11. Srb8-11 has been shown to negatively
regulate transcription under some conditions, and it was proposed that
the repressive activity of some human Mediator complexes is due to the
presence of Srb10 and Srb11 homologs (Cdk8 and cyclin C) (13, 40, 41).
Because our pol II·Med fraction was shown to be active in
transcription, we tested whether Srb8-11 was recruited to PICs under
these conditions. For this assay, we used the HIS4 promoter immobilized
to magnetic beads (15, 16). In this system, PICs are formed on the
immobilized template using nuclear extracts, washed, and then eluted
from the beads by digestion of the DNA with PstI which cuts
just upstream of the single Gal4 binding site at the promoter. Factors
bound to the template were assayed by Western blotting. As shown in Fig. 4, both the
When both the
To compare the relative amounts of subunits in the PICs to the ones in
the purified pol II·Med fraction, we also analyzed the pol II·Med
fraction in the same Western blot (Fig. 4, lane 7). This
analysis shows that about 2-fold more Rpb3 was present on PICs than in
the pol II·Med fraction. This suggests that pol II is somewhat
unstable in the Mediator complex and dissociates upon fractionation by
either affinity chromatography and/or gel filtration. In the
complementation experiment, pol II·Med fractions containing pol II
may be selectively recruited to the promoter, or the Mediator complex
lacking pol II may associate with free pol II in the extract. In
contrast to this behavior, Srb10 was substoichiometric in PICs compared
with the pol II·Med complex (about a 2-fold difference between PICs
and pol II·Med); (Fig. 4, compare lane 7 with lanes
2, 6, 8, 9). One possible
explanation for this result is that the Srb8-11 module may be unstable
in the PIC and a fraction of it dissociates upon washing and isolation of the PICs.
The yeast Mediator complex is critical for both basal and
activated transcription in vitro using a crude transcription
system. In vivo, Mediator is necessary for nearly all pol II
transcription with only a few reported exceptions (42, 43). Related
Mediator complexes have been found in mammalian cells where they may be central to integration of gene control signals. In both yeast and
mammalian cells, Mediator complexes have been isolated in multiple
forms. Whether these multiple forms are pre-existing distinct complexes
in vivo or whether they arise during purification is not known.
Here, we attempted to address the nature of the yeast
Mediator-containing complexes using affinity purification and gel
filtration chromatography, which avoids the high salt and ion exchange
methods, used previously. As a starting material, we used yeast nuclear extracts, which have a higher specific activity in transcription than
the previously used whole cell extracts. Using these methods, we found
two major Mediator-containing complexes in nuclear extracts. The first
complex, termed pol II·Med, migrates in gel filtration similarly to
the large ribosomal subunit and contains principally pol II and
Mediator, including the Srb8-11 module. This fraction also contains
detectable amounts of TFIIB and TFIIF, but at levels 1/7 to 1/20 the
levels of other Mediator subunits analyzed. Because the gel filtration
cannot separate large complexes of this size differing by only a few
polypeptides, we propose that this pol II·Med fraction contains
mainly a complex of pol II and Mediator (Fig.
5). Additionally, a much smaller amount
of complexes containing either TFIIB and/or TFIIF coexist in this
fraction. Therefore, this complex is nearly identical to the holoenzyme
isolated by Kornberg, Kim, and coworkers with the exception that it
includes the Srb8-11 module. It is likely that this latter module was
lost during their ion exchange purification. However, unlike the
results reported by Young and colleagues, we find no detectable TFIIH or SWI/SNF in this fraction.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Affinity purification of Mediator-containing
complexes from flag-tagged Srb5, Srb6 and Rgr1 strains. Nuclear
extracts (NE) made from flag-tagged Srb5, Srb6, and Rgr1
strains were incubated with anti-FLAG M2-agarose. Bound proteins were
washed and eluted twice with FLAG peptide. 1/130 NE, 1/130 flow-through
(FT), 1/3.6 elution 1 (E1), 1/2.4 elution 2 (E2) were loaded onto a 4-12% NuPAGE gel and analyzed by
Western blotting with antibodies directed against the proteins
indicated. The difference of TFIIB signals in elutions from the three
extracts is not significant owing to the poor linearity of the TFIIB
antibody signal.
Recovery of Mediator, pol II, and GTFs during affinity purification
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Fig. 2.
Gel filtration chromatography of FLAG-tagged
Srb5-associated proteins. The affinity-purified proteins derived
from the Srb5-tagged nuclear extract were subjected to gel filtration
chromatography on a TSK-G4000SWXL column. Fractions were separated by
SDS-PAGE in a 4-12% NuPAGE gel and analyzed by Western blotting with
antibodies directed against the proteins indicated. The migration
positions of molecular size markers were indicated as
arrows. Serial dilutions of recombinant proteins were loaded
as protein standards (S1-S4) in the following amounts: 120, 40, 13.3, and 4.4 fmol of rSrb4 and rRpb3; 200, 66.7, 22.2, and 7.4 fmol of rSrb2; 15, 5, 1.7, and 0.6 fmol of rTFIIB.
Quantitation results of the pol II · Med and Medc complexes
Identification of the pol II · Med and Medc components by
mass spectrometry
Srb2 extract increased transcription up
to 18-fold (lanes 3-6) compared with 14-fold with addition of recombinant Srb2 (lane 2). In contrast, addition of the
Medc complex increased transcription by up to 4-fold (lanes
8-10). Similar results were seen upon addition of these complexes
to the
Med9 extract (lanes 12-15). When both the pol
II·Med and Medc fractions were added to the
Srb2 extract, the Medc
complex inhibited the activity of pol II·Med (lane 7).
From this result, we conclude that these complexes compete for binding
to some common transcription factor, or compete for binding to the
promoter, and that the Medc complex has inherently lower transcription
activity. As discussed below, one possibility is that the Medc complex
may function efficiently at only a subset of yeast promoters.
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Fig. 3.
In vitro transcription activity of
the pol II·Med and Medc complexes. Nuclear extract made from
either Srb2 or
Med9 strains was complemented with recombinant
protein or Mediator complexes to test for their ability to synthesize
specific transcripts. For each reactions, 90 µg of
Srb2 nuclear
extract (lanes 1-10) or
Med9 nuclear extract
(lanes 11-15), 24 ng of Gal4-VP16 activator and the
following factors were added: Lane 2, 50 ng of rSrb2 (~2
pmol); lanes 3-6, 0.1, 0.3, 0.5, 1 µl of pol II·Med
(~0.2 pmol of Mediator/µl); lane 7, 1 µl of pol
II·Med + 1 µl of Medc; lanes 8-10, 0.1, 0.3, 0.5 µl
of Medc (~0.4 pmol of Mediator/µl); lanes 12 and
13, 1 and 1.5 µl of pol II·Med; and lanes 14 and 15, 0.5 and 1 µl of Medc. The transcription signal was
quantitated by using a PhosphorImager.
Med9 and
Srb2
extracts have a severe defect in recruitment of pol II, TFIIB, and
Mediator subunits to the promoter compared with wild-type extracts
(Fig. 4; compare lanes 1 and 4 with lanes
8 and 9). PIC formation was restored to
wild-type levels by addition of the pol II·Med complex (lanes
2 and 6). Srb10 was clearly a component of the PICs
formed in vitro both with wild-type extracts and extracts
supplemented with the pol II·Med fraction.
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Fig. 4.
Composition of PICs and the pol
II·Med. PICs were formed using the Immobilized Template Method.
PICs and the pol II·Med complex were resolved by SDS-PAGE and
analyzed by Western blotting using antibodies directed against the
proteins indicated. Nuclear extracts were made from Med9,
Srb2,
Srb5-FLAG, and wild-type (WT) strains. For each reaction,
180 µg of nuclear extract was incubated with immobilized templates
for 40 min, along with recombinant rSrb2 (100 ng) or pol II·Med (2 µl) where indicated, to form PICs. The PICs were then isolated and
analyzed as described under "Materials and Methods." Lanes
1-6, 8, and 9, PICs formed on immobilized
template; lane 3, nonspecific binding to the Dynabeads as a
control; lane 7, 0.13 µl of pol II·Med complex was
loaded on the same gel for comparison with PICs.
Med9 and
Srb2 extracts were supplemented with the
pol II·Med fraction, equivalent levels of Srb5-FLAG were seen in PICs
compared with the levels seen when nuclear extracts made from the
Srb5-flag strain were used to form PICs (compare lanes 2,
6, and 8). This suggests that the purified pol
II·Med complex is stable when added to the extracts and that subunits do not exchange between this complex and other Mediator subunits preexisting in the extracts. This nonexchange likely explains why the
Medc complex has low activity when added to nuclear extracts containing
the remaining Mediator subunits.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 5.
Schematic diagrams of Mediator complexes pol
II·Med (A) and Medc (B).
A novel subcomplex of Mediator was also identified in our work, termed
Medc. This complex contains no pol II subunits and also lacks subunits
of the Rgr1 module (Hrs1/Pgd1, Med2, Gal11, Sin4, Nut1, and Rgr1) as
well as Rox3, Nut1, and the Srb8-11 module. We estimate the molar
amount of Medc to be 20-30% of pol II·Med. The composition of the
Medc complex is generally consistent with previous biochemical and
genetic analysis of Mediator core subunits. These core subunits
contains most essential Mediator genes and have global functions in
transcription (3). They lack the Rgr1 regulatory module, which can have
a selective effect on regulated transcription (3). The Medc complex is
apparently pre-existing in the extract, because high salt buffers,
which dissociate pol II from pol II·Med, do not increase the relative
amount of this complex. Also, the Medc complex appears stable and does
not appear to exchange subunits when added to extracts lacking either
Srb2 or Med9. Although this Medc complex promotes transcription to a
weaker extent than the pol II·Med complex, it is possible that this
complex functions on only a subset of yeast genes in vivo. For example, the missing Mediator subunits with the exception of Rgr1
are all nonessential subunits (33). Another possibility is that this
complex functions specifically in repression of certain genes,
similarly to a proposed function for mammalian NAT complex (40).
Further genetic and biochemical studies will be required to determine
the function of this core Mediator complex.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank W. Reeves, N. Yudkovsky, and L. Hoskins for the Srb2 and
Med9 nuclear extracts. We thank T. Tsukiyama for p3FLAG-KanMX plasmid, J. Warner for the TCM1 antibody, D. Stillman for the Sin4 antibody, N. Thompson for the Rpb3 antibody, H. Sakurai for the Gal11 antibody, Y. J. Kim for the Rgr1 antibody,
J. Movius and K. Coachman for providing antibodies and recombinant
proteins, and R. Reeder, T. Tsukiyama, N. Yudkovsky, and W. Reeves for
comments on the manuscript.
![]() |
FOOTNOTES |
---|
* This work was supported by grants from the Natinal Institutes of Health (to S. H. and J. A. R.).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.
¶ An associate investigator of the Howard Hughes Medical Institute. To whom correspondence should be addressed: Basic Sciences Division, The Fred Hutchinson Cancer Research Center and the Howard Hughes Medical Institute, 1100 Fairview Ave. N, Mailstop A1-162, P. O. Box 19024, Seattle, Washington 98109-1024. Tel.: 206-667-5261; Fax: 206-667-6497; E-mail: shahn@fhcrc.org.
Published, JBC Papers in Press, December 11, 2000, DOI 10.1074/jbc.M009586200
2 Available from the S. Hahn laboratory on the web.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: pol II, RNA polymerase II; TF, transcription factor; CTD, C-terminal domain; PIC, preinitiation complex; pol II·Med, pol II·Mediator complex; Medc, Mediator core complex; HPLC, high performance liquid chromatography; LC/MS/MS/, microcolumn HPLC-electrospray ionization tandem mass spectrometry; PCR, polymerase chain reaction; DTT, dithiothreitol; PAGE, polyacrylamide gel electrophoresis; GTF, general transcription factor; TBP, TATA-binding protein.
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