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INTRODUCTION |
The TATA-binding protein
(TBP)1 is a universal
transcription factor that nucleates the assembly of transcription
preinitiation complexes on genes transcribed by all three RNA
polymerases (1). In cell extracts, TBP is associated with several
polypeptides, the so-called TBP-associated factors (TAFs) (2, 3). The main complex of TBP and TAFs (TAFIs) required for transcription by RNA
polymerase I is called SL1; however, a different complex (TAFIIs) is
required for transcription by RNA polymerase II and is called TFIID.
TBP also associates with RNA polymerase III-specific TAFs (TAFIIIs)
forming the TFIIIB transcription factor.
TBP is one of the most conserved proteins during the eukaryotic
evolution and consists of two direct repeats encompassing the
C-terminal two-thirds of the polypeptide which are highly conserved in
all known TBPs (4, 5). For long time it was thought that eukaryotic
cells contain a single TBP that mediates transcription by all three RNA
polymerases. However, a few years ago a Drosophila gene
product highly homologous to TBP was cloned that has been called
TBP-related factor (dTRF) (6). Biochemical analyses have shown that TRF
can form a stable complex with TFIIA and TFIIB on a TATA-containing
promoter and substitute for TBP in directing transcription in
vitro by RNA polymerase II (7). In cell extracts TRF is associated
with a novel set of TAFs (nTAFs) forming large multiprotein complexes.
In situ hybridization experiments have revealed that TRF
mRNA is expressed in the central nervous system and in primary
spermatocytes in adults (6). Immunostaining experiments using anti-TRF
antibodies showed that TRF protein is expressed in most of the early
embryo (until developmental stage 13); however, during embryonic
development becomes restricted to cells of the nervous system and
gonads. Immunofluorescence of Drosophila polytene
chromosomes has shown that TRF protein is associated with a few sites
on the chromosome (7).
In an effort to analyze whether mammalian cells contain homologues of
TBP, we have identified and cloned a mammalian TBP-related factor
(mTRF) from mouse embryos and human cells that shows considerable amino
acid sequence similarity to TBP and Drosophila TRF.
Recombinant mTRF can bind to the TATA box and substitute for TBP in
directing transcription by RNA polymerase II.
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EXPERIMENTAL PROCEDURES |
Cloning of mTRF--
To identify homologues to TBP, we searched
the expressed sequence tag (EST) data base of GenBankTM by
using the TBLASTN program and the amino acid sequence of human TBP and
Schizosaccharomyces pombe TBP as a probe (8). Several ESTs
were identified that encode different portions of a protein with
significant homology to the C-terminal portion of human TBP and
Drosophila TRF. The human ESTs encoding the different
portions of the polypeptide homologous to TBP (accession numbers
AA281228, W26331, AA448144, and AA412574) were overlapped to obtain a
complete open reading frame (ORF). EST AA412574 was obtained from the
IMAGE consortium through Research Genetics and sequenced further. The
ORF contained in the cDNA was identified using the ORF Finder
program at the National Center for Biotechnology Information. ESTs of
mouse origin (accession numbers AA798230, AA840611, AA821478, and
W89738) encoding a similar polypeptide were overlapped, and the ORF was
predicted as described above.
Expression and Purification of Recombinant mTRF--
The ORF of
mTRF was amplified by polymerase chain reaction using oligonucleotides
designed from the sequence obtained from the data base. The
oligonucleotide primer encoding the N terminus of the protein contained
a NdeI restriction site and the one encoding the C terminus
contained a BamHI site. The human ORF was amplified from
cDNA purified from a HeLa cDNA library in Lambda ZAP
(Stratagene), and the mouse ORF was amplified from embryo cDNA
(Marathon ready cDNA, CLONTECH). The amplified
fragment was digested with NdeI and BamHI
restriction enzymes and cloned in-frame into the NdeI and
BamHI sites of a bacterial expression vector (pET15b,
Novagen), which adds a His6 tag at the N terminus of the
polypeptide. Positive clones were sequenced using the Sequenase version
2 kit (U. S. Biochemical Corp.). All our attempts to express human
TRF in bacteria were negative, and only mouse TRF could be expressed.
Mouse TRF in pET15b was expressed in BL21(DE3). Bacteria were grown in
Luria-Bertoni medium at 37 °C to an A600 of
0.6, and the production of recombinant protein was induced with 0.5 mM IPTG and grown for an additional period of 4 h.
Bacteria were harvested by centrifugation and lysed by mild sonication
at 4 °C in 20 mM HEPES (pH 7.9), 500 mM KCl,
0.1% (v/v) Nonidet P-40, 10% (v/v) glycerol, 2 mM
imidazole, and 0.1 mM phenylmethylsulfonyl fluoride. The
lysate was cleared by centrifugation and loaded onto a 1-ml column of
nitriloacetic-acid-agarose containing immobilized Ni2+ ions
(Ni2+-NTA-agarose, Qiagen). The column was washed with 10 column volumes of lysis buffer supplemented with 20 mM
imidazole and eluted with 10-ml linear gradient of imidazole (0.02-0.3
M) in lysis buffer. Fractions containing recombinant mouse
TRF were detected by Western blot with monoclonal antibodies against
the His6 tag (CLONTECH), pooled, and
dialyzed against 20 mM HEPES (pH 7.9), 100 mM
KCl, 10% (v/v) glycerol, 1 mM dithiothreitol, 0.1 M EDTA, and 0.1 mM phenylmethylsulfonyl fluoride.
Specific Transcription Reactions--
Transcription reactions
and product analyses were performed as described previously (9).
Reactions mixtures (40 µl) were incubated at 30 °C for 45 min and
contained 20 mM HEPES-KOH (pH 7.9), 8 mM
MgCl2, 50 mM KCl, 10 mM ammonium
sulfate, 12% (v/v) glycerol, 5 mM 2-mercaptoethanol, 2%
(w/v) polyethylene glycol 20,000, RNase T1 (2 units), 0.6 mM ATP, 0.6 mM CTP, and 15 µM [
-32P]UTP, and 0.3 µg of supercoiled pML(C2AT)
template DNA, which contains the MLP promoter fused to a G-less
cassette (10). Transcription factors added to the reactions were:
recombinant TFIIA (30 ng), recombinant TFIIB (20 ng), recombinant TFIIF
(30 ng), recombinant TFIIE (20 ng), TFIIH purified from HeLa nuclear
extract, and affinity purified RNA polymerase II (50 ng). The
transcription factors were purified as described (11). Mouse TRF or
recombinant TBP was added as indicated in the figure legends. The
reaction products were separated on a 4% polyacrylamide/urea gel. Gels
were dried and exposed overnight to Kodak MS x-ray films.
DNA Binding Assays--
DNA binding assays were performed as
described (9). The protein components, as indicated in the figure
legends, were incubated with 0.1-1 ng (approximately 5000 cpm) of
labeled DNA probe containing the Ad-MLP sequences from
40 to +20 for
30-45 min at 30 °C. The complexes formed were separated by
electrophoresis through a 4% polyacrylamide gel containing Tris
borate-EDTA buffer (1× TBE; pH 8.2, 40 mM Tris, 40 mM boric acid, 1 mM EDTA) supplemented with 4%
(v/v) of glycerol. Electrophoresis was performed at 100 V until the
bromphenol blue dye reached the bottom of the gel. The gels were dried
and exposed overnight to x-ray films.
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RESULTS |
Cloning and Expression of mTRF--
To identify mammalian genes
encoding proteins homologous to TBP, we used the BLAST server at the
National Center for Biotechnology Information to screen the
nonredundant GenBankTM EST data base by querying with the
amino acid sequence of human TBP. Several ESTs from mouse and human
encoding different portions a protein with homology to TBP were found.
The nucleotide sequence of the ESTs contained a ORF of 558 base pairs
that encodes a polypeptide of 186 amino acids with a calculated
molecular mass of 20.5 kDa. The polypeptide shows significant homology
to the amino acid sequence of the C-terminal domain of all the TBP
proteins that have been cloned at the present (64% similar and 41%
identical) and to Drosophila TRF (60% similar and 38%
identical; Fig. 1). Both ORFs from mouse and human encode a identical protein (Fig. 1, compare mTRF and hTRF).

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Fig. 1.
Sequence alignments of mTRF, dTRF, and human
and yeast TBP. The alignment was performed using the Clustal W
program at the PBIL in Lyon, France. Asterisks indicate
identical amino acids, and double dots indicate conserved
amino acids. The numbers indicate the amino acid position of
human TBP. The dashes represent spaces or no amino
acid.
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To test the biochemical activities of mTRF, we inserted the amplified
mouse ORF into an expression vector, and the recombinant protein was
produced in bacteria. Fig. 2A
shows that a protein of Mr 25,000 is produced
upon induction with IPTG (lanes 2 and 3). The
molecular mass of the recombinant protein is slightly higher than the
predicted size. This protein was purified by
Ni2+-NTA-agarose chromatography (Fig. 2B,
lane 3), and its identity was confirmed by Western blot
analysis using antibodies against the His6 tag epitope
(Fig. 2C, lane 2).

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Fig. 2.
Expression and purification of mTRF. The
mouse ORF encoding TRF was cloned in-frame into the bacterial
expression vector pET15b, and the protein was induced and purified from
BL21 (DE3) bacteria. A, a 12% Coomassie Blue-stained
SDS-polyacrylamide gel of bacterial extracts (5 µg) prepared after
induction with IPTG (lanes 2 and 3). The lane
labeled negative is from a control extract prepared from cells that
contain pET15b without the insert (lane 1). B, a
15% Coomassie Blue-stained SDS-polyacrylamide gel showing mTRF (1 µg) purified by Ni2+-NTA-agarose chromatography
(lane 2). The lane labeled Negative (lane
1) is a fraction prepared from cells that contain pET15b without
the insert. C, Western blot analysis of the fractions showed
in B. The blot was incubated with
anti-His6 tag monoclonal antibody and detected using
anti-mouse IgG conjugated to alkaline phosphatase.
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Mammalian TRF Binds to the TATA Box and Forms Stable Complexes with
TFIIB--
One of the characteristics of TBP is to bind to the TATA
boxes and form stable complexes with TFIIA and TFIIB on the promoter (4, 9, 12-14). To investigate the possibility that mTRF could bind the
TATA box of the promoter, we performed gel retardation experiments
using the Ad-MLP TATA box. Fig.
3A (lanes 2 and
3) shows that yeast or human TBP form a stable complex on
the TATA box in the presence of recombinant human TFIIA. As reported
earlier (9) human TBP (Fig. 3A, lane 5) or yeast
TBP (data not shown) does not form a stable complex in TBE gels in the
absence of TFIIA. However, in contrast to TBP, mTRF binds strongly to
the TATA box in the absence of TFIIA (Fig. 3A, lane
4), and its binding is not enhanced further by TFIIA (lanes
7 and 8). The binding of TRF is specific for the TATA
box, because it can be competed by an excess of unlabeled
oligonucleotide containing the TATA box (data not shown).

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Fig. 3.
Analysis of binding activity of mTRF to the
TATA box. Labeled promoter DNA containing the Ad-MLP TATA box was
incubated with different combinations of proteins, as indicated at the
bottom of each panel. Recombinant human TFIIA, human TFIIB,
yeast TBP, and human TBP and mTRF were expressed in bacteria and
purified to homogeneity. The products of the binding reaction were
analyzed on 5% acrylamide gels that contained 0.5× TBE buffer.
A, analysis of the binding activity of mTRF to the TATA box.
The labeled DNA probe was incubated with different amounts and
combinations of factors, as indicated at the bottom. In
lane 1, the labeled probe was incubated with no protein.
B, analysis of the binding activity of mTRF to the TATA box
in the presence of TFIIB. Human TBP was used as a positive control
(lanes 2-4). Different amounts of mTRF, in either the
presence or the absence of TFIIB, were added in lanes
6-9.
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To analyze the effect of TFIIB on the binding of mTRF to the TATA
box, we carried out the gel retardation experiment described in Fig.
3B. Human TBP in the presence of TFIIB forms a complex (Fig.
3B, lanes 2, 3, and 4);
however, TFIIB by itself does not (Fig. 3B, lane
5). As indicated in Fig. 3A, mTRF by itself binds strongly to the TATA box (Fig. 3B, lane 6),
forming a high molecular weight complex. The complex was dependent on
the amount of mTRF, since a smaller amount of mTRF does not form a
complex (Fig. 3B, compare lanes 6 and
9). Mammalian TRF in the presence of TFIIB also forms
complexes (lanes 7 and 8), but in contrast to
human TBP, it forms at least two different complexes. Those complexes are dependent on the presence of TFIIB, because an equal amount of mTRF
alone does not produce a complex (lane 9).
Mammalian TRF Substitute for TBP in a Specific in Vitro
Transcription Assay--
It is known that the C-terminal domain of TBP
is sufficient for TATA binding and basal transcription initiation by
RNA polymerase II (4, 5). The great similarity between TBP and mTRF and the ability of mTRF to bind to the TATA box and to form complexes with
TFIIB on the promoter DNA suggest that mTRF can substitute for TBP in
in vitro transcription. To study this possibility, we
carried out transcription reactions reconstituted with affinity purified RNA polymerase II, recombinant transcription factors, and
either TBP or mTRF using as a template the Ad-MLP. Fig.
4 shows that our reconstituted system in
the absence of TBP gave no detectable transcription (lane
1); however, as expected, human or yeast TBP (lanes 2 and 5) can direct transcription from the Ad-MLP. When mTRF
was added in place of TBP (Fig. 4, lanes 3 and 4), we obtained a good level of transcription, although
slightly lower than that obtained with TBP. Those results strongly
suggest that mTRF can replace TBP in directing transcription by RNA
polymerase II from the Ad-MLP.

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Fig. 4.
In vitro transcriptional activity
of TBP and mTRF. In vitro transcription reactions were
performed using the Ad-MLP and purified recombinant TFIIA, TFIIB,
TFIIE, and TFIIF. RNA polymerase II was affinity purified, and TFIIH
was purified from HeLa cell nuclear extracts. In lane 1 no
TBP was added. Lanes 2 and 5 contain 10 ng of
human and yeast recombinant TBP, respectively. Lanes 3 and
4 contain 5 and 10 ng of mTRF.
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It is well known that TBP can support basal transcription initiation by
RNA polymerase II; however, it cannot support activated transcription
in the absence of TAFs and coactivators in an assay reconstituted with
recombinant or highly purified transcription factors and RNA polymerase
II. We have also examined the possibility that mTRF could support
activated transcription in the absence of TAFs. However, in our system
mTRF is not able to support activated transcription using recombinant
transcription factors, highly purified TFIIH, affinity purified RNA
polymerase II, and the acidic activator Gal4-VP16 (data not shown).
This analysis indicates that mTRF can support basal transcription
initiation, but it cannot support activated transcription in the
absence of TAFs or additional cofactors.
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DISCUSSION |
In this study, we report the cloning and expression of a protein
homologous to TBP that we called mTRF. Our results allow us to conclude
that mTRF binds to the TATA box, forms complexes with TFIIB, and is
able to substitute for TBP in directing basal transcription by RNA
polymerase II. Mammalian TRF is highly homologous to the C-terminal
domain of TBP and also shows the two-directed repeated domain
structure, and the high degree of homology suggests that it can fold
into the same structure as TBP (15, 16).
Mammalian TRF, like TBP, is unable to support activated transcription
by Gal4-VP16 in our reconstituted transcription system, which lacks
TAFs and coactivators. However, we cannot rule out the possibility that
mTRF could support activated transcription in the presence of TAFs,
coactivators, or mediator-like activities similar to those described in
the yeast and human RNA polymerase II holoenzyme (17-19)
Although it was thought that eukaryotic cells contain a single TBP, it
has recently been reported that Drosophila cells contain a
TBP-like molecule TRF (6). Drosophila TRF is highly
homologous to TBP, and based in amino acid sequence comparison it has
been proposed that both proteins most likely bind similar TATA
sequences on the promoter DNA (7, 16). Biochemical analyses of
Drosophila TRF have revealed that it interacts with both
TFIIA and TFIIB, binds to the TATA box of several promoters, and is
able to support transcription by RNA polymerase II in place of TBP (7).
In vivo, Drosophila TRF localizes in the central
nervous system and male reproductive organs (6). In polytene
chromosomes TRF is located in a small number of chromosomal sites (7).
In cell extracts TRF is complexed with its own set of novel TAFs
(designed nTAFs) (6, 7). Based on those results it has been postulated that Drosophila TRF is a cell-specific transcription factor
that directs the transcription of a subset of neuron-specific genes (7,
20).
The function of mTRF could be similar to that of Drosophila
TRF in directing transcription from a small subset of genes in a cell-
or tissue-specific fashion. Alternatively, it is also possible that
mTRF may play a more general role in the expression of cellular genes.
A scan from the available EST data base shows that mTRF is expressed in
tissues such as testis, brain, retina, mammary gland, placenta, liver,
spleen, and lung. It is also expressed in cells such macrophage,
B-cells, and HeLa cells. These data suggest that mTRF may have a more
general pattern of expression than dTRF. Mammalian TRF may also be
complexed with its own set of TAFs that can confer to mTRF-specific
promoter or activator functions, as it has been postulated for
Drosophila TRF. Our preliminary observations suggest that
mTRF in HeLa cell extracts is part of a multiprotein complex, because
it elutes from gel filtration columns with an apparent molecular mass
of greater than 200 kDa, as detected by Western blot
analysis.2 The molecular
cloning of mTRF will allow us to determine its biochemical composition
and functions. It will also allow us to determine whether it is
expressed in a cell- or tissue-specific fashion.
We do not rule out the possibility that mTRF, like TBP, may also play
role in RNA polymerase I and III transcription, or it could use a novel
set of basal factors for directing transcription from selected
promoters. Because homologues of TBP have been found, it seems
plausible that there could be homologues of the other general
transcription factors as well.