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J Biol Chem, Vol. 273, Issue 47, 30851-30854, November 20, 1998
§,
,,,
, and
From the Department of Clinical Chemistry and
Transfusion Medicine, Göteborg University, Bruna Stråket 16, 413 45 Göteborg, Sweden, the ¶ Department of Structural Biology,
Stanford University School of Medicine, Stanford, California
94305-5400, and the ** Molecular Biology Program, Memorial
Sloan-Kettering Cancer Center, New York, New York 10021
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ABSTRACT |
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Abstract
Introduction
Procedures
Results
Discussion
References
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Mediator was isolated from yeast on the basis of
its requirement for transcriptional activation in a fully defined
system. We have now identified three new members of mediator in the low molecular mass range by peptide sequence determination. These are the
products of the NUT2, CSE2, and MED11 genes.
The product of the NUT1 gene is evidently a component of
mediator as well. NUT1 and NUT2 were earlier
identified as negative regulators of the HO promoter,
whereas mutations in CSE2 affect chromosome segregation. MED11 is a previously uncharacterized gene. The existence
of these proteins in the mediator complex was verified by
copurification and co-immunoprecipitation with RNA polymerase II holoenzyme.
An in vitro system reconstituted with pure general
transcription factors and RNA polymerase II from yeast can support
basal transcription but is not responsive to transcriptional activators (1). This observation prompted a search for coactivators, which could
support activated transcription in vitro. Such an activity was purified to homogeneity and demonstrated to be a holoenzyme form of
RNA polymerase II, made up of core polymerase and a mediator complex
(1). Mediator was subsequently purified as a discrete entity that not
only enables transcriptional activation but also stimulates both basal
transcription and phosphorylation of the C-terminal domain of the
polymerase (1, 2).
Earlier studies identified 16 different subunits in the mediator
(1-5). These can be divided into three subgroups. The SRB gene products were identified in a screen for RNA polymerase II C-terminal domain-interacting proteins and were subsequently isolated in a complex with RNA polymerase II, giving the first indication of the
existence of the RNA polymerase II holoenzyme (6). The mediator
subunits encoded by GAL11, RGR1, SIN4, PGD1/HRS1, and ROX3 were all found by earlier genetic studies to affect
both the positive and negative regulation of transcription (7). Biochemical evidence indicates that at least Gal11, Rgr1, Sin4, and
Pgd1/Hrs1 belong to a physically distinct mediator subassembly (3). The
third subgroup is comprised of the MED genes, which were all
previously uncharacterized, and whose products were identified through
peptide sequencing. Genetic and biochemical evidence indicates that
several members of this group of genes are essential for the function
of individual transcriptional activators, such as Gal4 and Gcn4, both
in vivo and in vitro
(5).1
The widespread effect of the mediator on transcription is reflected in
genetic studies that have characterized its subunits in a diverse array
of cellular activities (7). Here we report that the products of the
NUT2, CSE2, and MED11 genes are all components of
the mediator. We present evidence for the occurrence of the NUT1 gene product in mediator as well. Med11 is the product
of a previously uncharacterized open reading frame. The
characterization of NUT1 and NUT2, identified as
negative regulators of the HO gene (9) and CSE2, whose
mutations affect chromosome segregation (10), adds to the list of
different functions regulated through specific mediator subunits.
Protein Sequencing--
Peptides were generated from bands
corresponding to proteins of 12 and 21 kDa, bound to polyvinylidene
difluoride membrane by tryptic digestion in situ (11, 12),
and fractionated by reverse-phase
HPLC2 (13) with the use of a
1-mm Reliasil C18 column. Selected peak fractions were analyzed by a
combination of automated Edman chemical degradation (14) and
matrix-assisted laser-desorption ionization time-of-flight mass
spectrometry. Peptide sequences were compared with entries in the
Saccharomyces Genome Data Base (Stanford Genomic Resources, Stanford
University) with the use of the National Center for Biotechnology
Information BLAST program. Experimental masses of these and additional
peptides were compared with the theoretical average isotopic masses of
fragments expected to result from tryptic digestion of the identified
proteins (with the use of PeptideSearch software; Dr. Matthias Mann,
European Molecular Biology Laboratory, Heidelberg, Germany).
Antibodies and Immunoblot Analyses--
Recombinant Cse2 and
Med11 proteins fused to glutathione S-transferase were
overproduced in Escherichia coli BL21(DE3) pLysS cells and
subsequently purified using glutathione-Sepharose 4B (Amersham
Pharmacia Biotech) according to the supplier's recommendations. The
purified glutathione S-transferase fusion proteins and a
synthetic polypeptide corresponding to the 15 N-terminal amino acids of Nut2 were used to immunize rabbits. The antisera used in this study
were taken 10 days after the second booster injection (Antibody AB, Sweden).
Protein Purification--
Purification of the RNA polymerase II
holoenzyme was as described (15) with the following modifications.
After fractionation on hydroxyapatite, the mediator/holopolymerase peak
was loaded on a Mono Q 10/10 column (Amersham Pharmacia Biotech). After
washing with 40 ml of buffer Q-0.15 and 40 ml of buffer Q-0.5, the
column was developed with a linear gradient (112 ml) of buffer Q-0.5 to
Q-1.2, and 1.2-ml fractions were collected. Immunoblotting revealed a
peak of free mediator around Q-0.6 and a peak of holopolymerase eluting
around Q-0.8. Purification of free mediator was as described earlier
(2).
Immunoprecipitation--
Anti-Med2 and anti-Cse2 antibodies were
coupled to protein A-Sepharose beads and used to immunoprecipitate
purified RNA polymerase II holoenzyme as described (3) with the
following modifications. RNA polymerase II holopolymerase Mono Q
fraction 89 (10 µl) was diluted with 30 µl of buffer Q, 0.5 (25 mM Tris acetate, pH 7.8, 10% glycerol, 1 mM
EDTA, 0.5 M potassium acetate) and sedimented for 5 min at
13,000 rpm prior to incubation with the beads. Following incubation
with the diluted fraction for 10 h at 4 °C, the beads were
washed three times with 100 µl of IP-400 buffer (20 mM
Tris-Cl, pH 7.5, 10% glycerol, 0.1 mM EDTA, 0.1% Nonidet
P-40, 0.4 M potassium acetate) and eluted twice for 10 min
at room temperature with 50 µl of 5 M urea. To the
combined eluates was added 20 µl of 2× SDS loading buffer (20%
glycerol, 10% 2-mercaptoethanol, 4.6% SDS, 125 mM
Tris-Cl, pH 6.8, 0.1% (w/v) bromphenol blue) for SDS-PAGE and immunoblotting.
In previous work, free mediator complex was purified and 16 polypeptides in the size range between 14 and 130 kDa were described. Analysis of mediator by SDS-PAGE and Coomassie Blue staining indicated the presence of additional subunits in the molecular mass range below
21 kDa. To investigate further, mediator was analyzed by SDS-PAGE in a
15% gel containing 2% glycerol and staining with silver, leading to
the identification of five bands, ranging from 10 to 19 kDa (Fig.
1.). Two of these corresponded to the
previously reported mediator components, Srb6 and Srb7. In addition
there were two protein bands of about 19 kDa that we termed Med9 and Med10 and a protein band about 14 kDa that we termed Med11.
INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References
EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
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References
RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References
View larger version (71K):
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Fig. 1.
Polypeptide composition of free mediator in
the low molecular mass range. Peak fractions from
Bio-Sil SEC 400 were pooled an analyzed by SDS-PAGE in an 15% gel.
Proteins were revealed by staining with silver. Positions of molecular
mass markers are indicated on the left.
Purified mediator was separated by 12% SDS-PAGE and transferred electrophoretically to a polyvinylidene difluoride membrane. The Med9, Med10, and Med11 bands were cut out and subjected to tryptic digestion. Because the Med9 and Med10 bands migrated so closely together, these two bands were cut out together and analyzed simultaneously. The resulting peptides were fractionated by reverse-phase HPLC and sequenced. Perfect matches were found to sequences in the Saccharomyces cerevisiae genome data base (Table I), and the masses of peptides determined by mass spectrometry were in good agreement with the theoretical masses of the expected tryptic peptides for the individual gene products. The Med9 and Med10 bands proved to be the products of two previously characterized genes, NUT2 and CSE2. The Med11 band yielded peptide sequences from an uncharacterized open reading frame, denoted MED11. The Cse2 protein contained an earlier noted putative basic region leucine zipper motif (10), but otherwise we could not find any significant sequence features related to transcription for any of the these proteins.
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Cse2 and Med11 were expressed in recombinant form, and polyclonal antibodies were obtained. Polyclonal antibodies were also obtained against a 15-amino acid polypeptide corresponding to the N-terminal end of Nut2. The association of these proteins with RNA polymerase II holoenzyme was shown by copurification and co-immunoprecipitation. RNA polymerase II holoenzyme was purified from strain BJ926 by chromatography on Bio-Rex 70, DEAE-Sephacel, hydroxyapatite, and Mono Q. Immunoblot analysis showed coelution from Mono Q of Med4, Nut2, Cse2, Med11, and the polymerase subunit Rpb1 (Fig. 2).
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Immunoprecipitation was performed with anti-Cse2 antibodies coupled to protein A-Sepharose. Holopolymerase was incubated with the antibody Sepharose, followed by washing under stringent conditions. Immunoblot analyses revealed that Med2, Med4, Nut2, Cse2, and Med11 were almost entirely bound by the antibody Sepharose (Fig. 3). Immunoprecipitation with anti-Med2 gave the same result (data not shown). Antibodies generated against Nut2 and Med11 were not useful for immunoprecipitation. We conclude that Nut2, Cse2, and Med11 in the holopolymerase preparation were entirely associated with the holoenzyme.
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Reinvestigation of of the 130-kDa region of the purified mediator indicated that Nut1, genetically closely related to Nut2 might be a mediator component as well. Peptide mass fingerprinting revealed many tryptic fragments with amino acid sequences corresponding to Nut1 (data not shown). The genetic studies (9) and presence of Nut1 in a highly purified mediator fraction make a strong case for Nut1 being a mediator subunit, although its tight association with the complex and stoichiometry remain to be shown.
A BLAST similarity search of the GenBankTM data base revealed homologs of Nut2 in both Schizosaccharomyces pombe (spNut2) and Caenorhabditis elegans (ceNut2) (Fig. 4). The region of homology between the deduced amino acid sequences of Nut2 and spNut2 encompasses almost all of the S. pombe protein and 144 of 157 residues of the S. cerevisiae protein. The ceNut2 shared homology with Nut2 over a 132-amino acid region. In addition, several putative mouse and human homologs of the Nut2 protein were identified in a search of the National Center for Biotechnology Information data base of expressed sequence tags (data not shown).
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DISCUSSION |
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Top
Abstract
Introduction
Procedures
Results
Discussion
References
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Our results demonstrate the presence of Cse2, Nut2, and Med11 in mediator and RNA polymerase II holoenzyme. Immunoprecipitation experiments and copurification data establish that these three proteins are true components of the holoenzyme preparations. In addition, peptide mass fingerprinting indicated that Nut1 is likely to be a subunit of mediator as well.
The CSE2 gene was originally identified in a search for mutations affecting chromosome segregation in S. cerevisiae. Disruption of CSE2 results in an increase in chromosome missegregation, slower growth, and both a cold- and temperature-sensitive phenotype. It seems likely that the connection between CSE2 and chromosome segregation is indirect and involves transcription. CSE2 might, for example, be necessary for cell cycle-dependent activation of certain genes required for proper chromosome segregation. A high copy number suppressor of a CSE2 mutation has been isolated and identified as SCM2, a gene encoding a yeast tryptophan permease. SCM2 suppresses the cold-sensitive phenotype of the cse2 mutation but fails to suppress temperature sensitivity and chromosome missegregation. The function-specific suppression of the CSE2 disruption phenotype may result from the participation of this mediator subunit in the regulation of several different sets of genes. The 17-kDa Cse2 protein contains a putative basic region leucine zipper motif, indicating that it may possess a DNA binding activity. It will be of interest to learn whether mutations in this DNA binding domain give rise to a specific phenotype. Leucine zipper motifs have also been observed in the Med7 and Med8 subunits of the mediator, indicating that these motifs might serve a role in the organization of the mediator.
HO transcription is dependent on Swi4p and Swi6p for relief
of repression by the URS2 region upstream of the HO promoter (8). NUT1 and NUT2 were recently isolated in a screen
for mutants that would suppress the Swi4p/Swi6p dependence of a
synthetic reporter gene containing part of URS2 (9). The identification
of Nut2 as a component of the mediator therefore points to a role for the RNA polymerase II holoenzyme in transcriptional repression of the
HO promoter. Nut2, as well as other mediator subunits, may
play a specific role in cell cycle-dependent transcription, because the HO transcription only takes place at START, the
G1 to S phase transition. The same screen that revealed
Nut1 and Nut2 also found two genes coding for the previously identified mediator components, Sin4 and Rox3, as well as the SRB8,
SRB9, SRB10, and SRB11 genes. This
similar genetic profile further indicates that the association of Nut1
and Nut2 with mediator/holopolymerase is physiologically relevant and
not an artifact of biochemical isolation. Similarity searches revealed
NUT2 homologs in human, C. elegans and S. pombe genomes (Ref. 9 and data presented here). Five S. cerevisiae mediator components, Med6, Med7, Srb7, Rgr1, and Nut2,
now have homologs in human cells. The occurrence of an S. pombe homolog provides the first evidence for the existence of an
RNA polymerase II holoenzyme in this organism as well. The copurification of spNut2 and S. pombe RNA polymerase II
through several steps of fractionation of an S. pombe
extract provides further evidence for a mediator-like activity in
fission yeast.3
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FOOTNOTES |
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* The work in the Kornberg laboratory was supported by National Institutes of Health Grant GM36659.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.
§ Supported by grants from the Swedish Cancer Society (project 3947), the Magn. Bergvalls Foundation, Swedish Society for Medical Research, and Swedish Society of Medicine. To whom correspondence should be addressed. Tel.: 46-31-3424088; Fax: 46-31-828458; E-mail: cgustaff{at}ss.gu.se.
Recipient of the Cancer Research Fund of the Damon
Runyon-Walter Winchell Foundation Fellowship DRG-1361.
Supported by Grant DBI-940123 from the National Science
Foundation and by National Cancer Institutes Core Grant 5 P30 CA08748 to the Sloan-Kettering Sequencing Laboratory.
The abbreviations used are: HPLC, high pressure liquid chromatography; PAGE, polyacrylamide gel electrophoresis.
1 L. C. Myers, C. M. Gustafsson, K. C. Hayashibara, P. O. Brown, and R. D. Kornberg, manuscript in preparation.
3 H. Spåhr and C. M. Gustafsson, unpublished observations.
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Abstract
Introduction
Procedures
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Discussion
References
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