From the Institute for Molecular and Cellular
Biology, Osaka University, Suita, Osaka 565-0871, Japan and the
§ Laboratory of Biochemistry and Molecular Biology, The
Rockefeller University, New York, New York 10021
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ABSTRACT |
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The general transcription factor TFIIE plays
important roles at two distinct but sequential steps in transcription
as follows: preinitiation complex formation and activation (open
complex formation), and the transition from initiation to elongation.
The large subunit of human TFIIE (TFIIE) binds to and facilitates
the enzymatic functions of TFIIH, but TFIIE also functions
independently from TFIIH. To determine functional roles of the small
subunit of human TFIIE (TFIIE
), deletion mutations were
systematically introduced into putative structural motifs and
characteristic sequences. Here we show that all of these structures
that lie within the central 227-amino acid region of TFIIE
are
necessary and sufficient for both basal and activated transcription. We
further demonstrate that two C-terminal basic regions are essential for
physical interaction with both TFIIE
and single-stranded DNA, as
well as with other transcription factors including the
Drosophila transcriptional regulator Krüppel. In
addition, we analyzed the effects of the TFIIE
deletion mutations on
TFIIH-dependent phosphorylation of the C-terminal domain of
RNA polymerase II and on wild type TFIIE
-driven basal transcription.
Both responsible regions also mapped within the essential 227-amino
acid region. Our results suggest that TFIIE engages in communication
with both transcription factors and promoter DNA via the TFIIE
subunit.
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INTRODUCTION |
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In eukaryotes productive transcription initiation by RNA polymerase II (Pol II)1 plays a key role in the regulation of gene expression in response to various developmental and environmental signals. Initiation by Pol II requires five general transcription factors (TFIIB, TFIID, TFIIE, TFIIF, and TFIIH) that act through core promoter elements and is regulated by tissue- and/or gene-specific factors that act through distal control elements (for reviews, see Refs. 1-5). Recent studies of preinitiation complex (PIC) formation have indicated the existence of the following alternative pathways: one involving the stepwise assembly of multiple general transcription factors, and one involving preassembly of a Pol II holoenzyme containing several general transcription factors and various coactivators (for reviews, see Refs. 6-8). Analysis of the PIC assembly pathway using isolated factors has revealed that this process begins with the binding of TBP (the TATA-binding protein) component of TFIID to the TATA box. This is then followed by the sequential interactions of TFIIB, a complex containing Pol II and TFIIF, TFIIE, and TFIIH. TFIIH, which is recruited through direct interaction with TFIIE, may stabilize and activate the PIC through its enzymatic activities, resulting in open complex formation.
In addition to their key roles in PIC formation and initiation, TFIIE
and TFIIH play important roles in the transition from initiation to
elongation (promoter clearance) (for reviews, see Refs. 1, 2, and 5).
Human TFIIE consists of 57-kDa () and 34-kDa (
) subunits and
forms an
2
2 heterotetramer with a
molecular mass of 180 kDa (9, 10). Human TFIIH consists of 9 subunits,
and surprisingly, some of these subunits have been implicated in
nucleotide excision repair and cell cycle regulation (for a review, see
Ref. 11). This multisubunit general transcription factor is quite
unique in that it contains the following three ATP-dependent catalytic activities: a kinase activity that
phosphorylates the CTD (C-terminal domain) of the largest subunit of
Pol II, a DNA-dependent ATPase, and a DNA helicase.
Importantly, TFIIE plays essential roles in the regulation of these
TFIIH activities; the CTD kinase and ATPase are positively regulated by
TFIIE whereas the DNA helicase activity is negatively regulated
(12-15). This coordinated regulation, as well as multiple interactions
of TFIIE and TFIIH with other transcription factors (reviewed in Ref.
5), probably provides a basis for the control of the two distinct steps
of transcription.
Human TFIIE, which is a highly acidic (pI 4.5) protein of 439 amino
acids, possesses several putative structural motifs and characteristic
sequences (10, 16). Previous studies demonstrated that the N-terminal
half of TFIIE
, which contains all the evolutionarily conserved
structural motifs, is essential for basal transcription, whereas the
C-terminal half is dispensable (15). A putative leucine zipper motif
and a similar hydrophobic repeat domain, which are separated by a
putative zinc finger motif, are important for heterodimerization with
the TFIIE
subunit. Furthermore, TFIIH binds to the C-terminal acidic
region of TFIIE
. This interaction may be important for recruiting
TFIIH into the PIC and for the modulation of the two functions of this
factor in transcription initiation and in the transition from
initiation to elongation.
Human TFIIE, which is a highly basic (pI 9.5) protein of 291 amino
acids, also possesses several conserved structural motifs and
characteristic sequences (10, 17, 18). Photocross-linking studies
revealed that TFIIE
binds to promoter DNA in the region between
positions
14 and
2 from the transcription initiation site (+1), a
property that distinguishes it from TFIIE
which cannot be
cross-linked with DNA (19). Two-dimensional crystallography of the
highly related yeast TFIIE with Pol II has confirmed these observations
by showing that TFIIE interacts with the active center of Pol II
relative to the transcription initiation site (20). Other recent
studies have demonstrated that the introduction of short mismatched
heteroduplex DNA regions around the initiation site (minimally from
positions
4 to +2) in topologically relaxed templates abolishes the
requirement for TFIIE, TFIIH, and
-
ATP hydrolysis (21-23).
Importantly, changes in the short mismatched region create differential
requirements for TFIIE and TFIIH with, most notably, a continued
requirement for TFIIE but lack of a requirement for the function of
TFIIH being observed (23). These results indicate two possibilities as
follows: first, that TFIIE and TFIIH play a role around the
transcription initiation site during open complex formation, and
second, that TFIIE has a unique function, possibly in promoter melting,
distinct from its role with TFIIH.
To elucidate further mechanisms of TFIIE function, and especially that
of TFIIE, we constructed a series of deletion mutants of TFIIE
.
We tested the ability of these mutants to support both basal and
activated transcription and to associate with other general
transcription factors and transcriptional regulatory factors, and we
examined their effects on CTD phosphorylation by TFIIH as well as their
dominant negative effects on basal transcription. In so doing, we have
succeeded in identifying a central core that is important for the
mediation of TFIIE function and which contains the C-terminal basic
regions essential for binding both to general transcription factors and
to single-stranded (ss) DNA. In addition, we provided a new clue to
approach that TFIIE is not only recruiting TFIIH into the PIC and
functioning as a bridge between Pol II and TFIIH for its CTD
phosphorylation but also playing an unidentified important role during
transcription initiation and in the transition from initiation to
elongation.
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EXPERIMENTAL PROCEDURES |
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DNA Templates--
For basal transcription assays, the plasmid
pML(C2AT)-50 containing the adenovirus type 2 major late
promoter was used as a template (24). To study transcriptional
activation, the plasmid pG5HM(C2AT) was used as the test
template (25), with the plasmid pML(C2AT)
-53Sh as the
base-line control (26). pG5HM(C2AT) contains five
GAL4-binding sites and the core promoter as described previously (15).
The two templates pML(C2AT)
-50 and
pG5HM(C2AT) give 390-nucleotide transcripts, and
pML(C2AT)
-53Sh gives a 290-nucleotide transcript.
Construction of Various Expression Vectors--
The isolated
plasmid p2EB contains the complete open reading frame of human TFIIE
(TFIIE
cDNA) cloned into pBluescript II SK(
) phagemid
(Stratagene) (17). This was first digested with XbaI and
ClaI, and the 1.7-kilobase pair cDNA fragment was
subcloned into the pGEM-7Zf(+) vector (Promega) to create a
BamHI site at the 3'-end of TFIIE
cDNA. The
oligonucleotide 5'-CCCTTCTCACTCAGCCATATGGACCCAAGCCTGTTG-3' was
then used to create an NdeI site at the first methionine
codon of TFIIE
cDNA and to disrupt a BamHI site
located just after the first methionine by site-directed mutagenesis
(27). Finally, the NdeI-BamHI fragment of this
cDNA clone (p2EBT) was subcloned into the 6HisT-pET11d vector to
construct the histidine (His)-tagged TFIIE
(6His-TFIIE
)
expression plasmid.
Construction of TFIIE Mutants--
Deletion mutants of
TFIIE
were constructed using plasmid p2EBT containing the wild type
TFIIE
cDNA and the described procedure of
oligonucleotide-mediated mutagenesis (27). A restriction site was
designed in each oligonucleotide to select for properly mutated
plasmids as described elsewhere (28), and the mutants were then checked
by sequencing. The NdeI-BamHI fragments of all mutants were subcloned into 6HisT-pET11d to create a His tag at the N
terminus. N-terminal and internal deletions were constructed by
deleting the indicated amino acid residues and C-terminal deletions by
creating termination codons at the residues shown in Fig. 1. (Because
of the large number of oligonucleotides used, we have refrained from
describing their exact sequences, but this information will be provided
on request.)
Expression and Purification of Recombinant
Proteins--
Recombinant proteins were expressed in E. coli BL21(DE3)pLysS by induction with
isopropyl--D-thiogalactopyranoside (34). For
purification, soluble bacterial lysates were used (17). His-tagged
proteins were purified on an Ni2+-nitrilotriacetic acid
column (Qiagen) by eluting with 100 mM imidazole HCl (pH
7.9). The large scale preparation of TFIIE
was as described before
(15) and resulted in >95% purity as judged by Coomassie Blue staining
of an SDS-polyacrylamide gel. All the deletion mutants of TFIIE
were
miniscale preparations. Lysates (1 ml) representing 50-100 ml of
culture were directly resuspended in Eppendorf tubes with 1 ml of
buffer B (20 mM Tris-HCl (pH 7.9 at 4 °C), 0.5 mM EGTA, 10% (v/v) glycerol, 1 mM
phenylmethylsulfonyl fluoride, 2 µg/ml antipain, 2 µg/ml aprotinin,
1 µg/ml leupeptin, 0.8 µg/ml pepstatin, 10 mM
2-mercaptoethanol) containing 500 mM NaCl (BB500) and 100 µl of Ni2+-nitrilotriacetic acid resin and incubated for
4 h at 4 °C. The resin samples were washed twice with 1 ml of
BB500, twice with 1 ml of buffer D (20 mM Tris-HCl (pH 7.9 at 4 °C), 20% (v/v) glycerol, 1 mM phenylmethylsulfonyl
fluoride, 10 mM 2-mercaptoethanol) containing 500 mM KCl (BD500), and twice with 500 µl of BD500 containing 20 mM imidazole HCl (pH 7.9). Bound proteins were eluted
twice with 300 µl of BD500 containing 100 mM imidazole
HCl (pH 7.9). Typical preparations were >75% pure.
In Vitro Transcription Assay--
General transcription factors
(TFIIF and TFIIH) were purified either from HeLa nuclear extracts
or from cytoplasmic S100 fractions as described previously (15). Pol II
was highly purified from HeLa nuclear pellets by DE52, A25, P11, and
high performance liquid chromatography-DEAE 5PW chromatography as
described elsewhere (35). In vitro transcription was carried
out as described (9, 15). To observe transcriptional activation, 20 ng
of TFIID containing flag-tagged TBP (25) was used instead of 20 ng of
TBP. As activators, 40 ng of either GAL4-VP16 (36) containing the
C-terminal acidic activation domain of VP16 (residues 413-490) fused
to GAL4-(1-94) (residues 1-94) or GAL4-CTF1 (37) containing the
C-terminal proline-rich activation domain of CTF1 (residues 399-499)
fused to GAL4-(1-94) was used. Autoradiography was performed at
80 °C with Fuji RX-U x-ray film. The incorporation of
[
-32P]CTP into transcripts was quantified using the
Fuji BAS2500 Bio-Imaging analyzer.
Generation of Antibody against TFIIE--
Two hundred
micrograms (100 µl) of purified 6His-TFIIE
(>99% pure) was mixed
with the same volume (100 µl) of complete Freund's adjuvant (Difco)
and injected into each rabbit. Two weeks after the first injection, a
second injection was carried out with 100 µg (100 µl) of purified
TFIIE
in 100 µl of incomplete Freund's adjuvant (Difco). A third
injection was carried out 2 weeks later using the same procedures as
described for the second injection. Blood was collected 8 days after
the third injection. The generated antibody recognized all of the
TFIIE
deletion mutants used in this study.3
Coimmunoprecipitation of TFIIE Mutants with
TFIIE
--
Polyclonal antisera against TFIIE
(0.01 µl) and 5 µl (packed volume) of protein G-agarose (Pierce) were incubated in
buffer C (20 mM Tris-HCl (pH 7.9 at 4 °C), 0.5% EDTA,
20% (v/v) glycerol, 0.5 mM phenylmethylsulfonyl fluoride,
10 mM 2-mercaptoethanol, 0.002% (v/v) Nonidet P-40)
containing 100 mM KCl (BC100) and 200 µg/ml bovine serum
albumin for 2 h at 4 °C with rotation. The protein G-agarose
beads were precipitated and washed with 500 µl of washing buffer I
(10 mM Tris-HCl (pH 7.9 at 4 °C), 500 mM NaCl, 0.1% Tween 20), twice with 500 µl of buffer C containing 1 M KCl (BC1000), and twice with 500 µl of BC100. Various
TFIIE
mutant proteins (200 ng) were incubated with TFIIE
(300 ng)
for 1 h at room temperature, and bound proteins were
coimmunoprecipitated after incubation with anti-TFIIE
-protein G
beads in a 500-µl reaction volume for 4 h at 4 °C with
rotation. The beads were washed twice with 500 µl of washing buffer
I, once with 500 µl of BC100, boiled in SDS sample buffer, and
analyzed by SDS-PAGE (12% acrylamide).
GST Pull Down Assays-- GST fusion proteins were used for protein interaction assays. Each tester protein (200 ng) was incubated with lysates containing 500 ng of GST proteins together with 5 µl (packed volume) of glutathione-Sepharose (Amersham Pharmacia Biotech) in a 500-µl reaction volume of BC100 with 200 µg/ml bovine serum albumin for 4 h at 4 °C with rotation. The glutathione-Sepharose resin was washed twice with 500 µl of buffer C containing 200 mM KCl (BC200), once with 500 µl of BC100, boiled in SDS sample buffer, and analyzed by SDS-PAGE. Pulled down tester proteins were detected by Western blotting as described in the coimmunoprecipitation assay.
Single-stranded DNA Binding Assay--
Four hundred nanograms of
His-tagged TFIIE deletion mutants were incubated with 5 µl (packed
volume) of ssDNA-agarose (Life Technologies, Inc.) in a 500-µl
reaction volume of BC100 with 200 µg/ml bovine serum albumin for
4 h at 4 °C with rotation. The ssDNA-agarose resin was washed
twice with 500 µl of buffer C containing 250 mM KCl
(BC250), once with 500 µl of BC100, boiled in SDS sample buffer, and
analyzed by SDS-PAGE (12% acrylamide). Bound mutants were detected by
Western blotting with anti-TFIIE
antisera (1:3000 dilution) as
described above.
Kinase Assay--
Assays were carried out essentially as
described (14, 15) with all general transcription factors in the
presence of Pol II and a DNA fragment containing the adenovirus type 2 major late promoter sequences from 39 to +29, except that 16 ng of
bacterially expressed recombinant TFIIF was used instead of 30 ng of
high performance liquid chromatography heparin-purified TFIIF from HeLa
nuclear extract. Phosphorylation reactions were done at 30 °C for
1 h and stopped by addition of 75 µl of phosphorylation stop solution (10 mM EDTA, 0.1% Nonidet P-40, and 0.05% SDS).
Phosphorylated proteins were trichloroacetic acid-precipitated,
analyzed by SDS-PAGE (5.5% acrylamide), and detected by
autoradiography performed at
80 °C with Fuji RX-U x-ray film.
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RESULTS |
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The Central Region of TFIIE Is Important for Both Basal and
Activator-dependent Transcription--
Human TFIIE
is
the small subunit of TFIIE and consists of 291 amino acid residues (10,
17). As judged from the predicted open reading frame of TFIIE
cDNA, it possesses several putative structural motifs and
characteristic sequences: a serine-rich sequence (residues 26-71), a
region similar to the Pol II binding region of RAP30 (TFIIF
)
(residues 79-111), a leucine repeat motif (residues 145-193), a
region similar to the bacterial sigma factor subdomain 3 (residues
163-193), a basic region-helix-loop-helix motif (residues 197-238),
and a region with basic region-helix-loop sequence (residues 258-291).
Except for the leucine repeat, these regions are different from the
putative motifs and sequences observed in TFIIE
(15). Based on these
sequences, a systematic series of N-terminal, C-terminal, and internal
deletion mutants were constructed (shown in Fig.
1, A and B).
Vectors encoding hexahistidine-tagged mutant and wild type TFIIE
proteins were expressed in bacteria, purified through nickel affinity
chromatography, and analyzed by SDS-PAGE (Fig.
2A and 3A). All
mutants were highly soluble in the bacterial cell extracts and were
easily purified, although some (
224-291,
96-119, and
117-153) were poorly expressed (Fig. 2A, lane 11; Fig.
3A, lanes 7 and 8,
respectively).
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The C-terminal Basic Regions of Human TFIIE Bind to Several
General Transcription Factors--
TFIIE functions in two distinct but
sequential steps, one during open complex formation and the other
during the transition from transcription initiation to elongation.
During both steps, the binding of TFIIE to various general
transcription factors and to Pol II is expected to be quite important.
In the present study, pull down assays (39) using GST fusion proteins
for each subunit of the general transcription factors and recombinant
TFIIE
showed that TFIIE
binds strongly to TFIIE
, TFIIB, and
TFIIF
(RAP30) and weakly to TFIIF
(RAP74) and TBP (Fig.
5A). Human TFIIE
also bound
to Pol II and strongly to itself.3 These results suggest
that TFIIE
plays essential roles in both steps of transcription by
binding to various general transcription factors. To better understand
these roles, we first identified the binding regions of TFIIE
for
three strongly associating factors TFIIE
, TFIIB, and TFIIF
.
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Two C-terminal Basic Regions of TFIIE Are Involved in Binding to
the Drosophila Transcriptional Repressor Krüppel--
Recently,
it has become apparent that vast numbers of transcriptional regulators
target various transcription factors, as well as histones, during the
multiple steps of gene transcription (for reviews, see Refs. 1 and 38).
Since TFIIE plays key roles in two of those steps, transcription
initiation and promoter clearance, it would be surprising if it were
not a target for transcriptional regulatory factors (for a review, see
Ref. 5). As analyzed both in tissue culture and in vitro,
the Drosophila segmentation gene product Krüppel (Kr),
which is a zinc finger protein, functions as a transcriptional
regulatory factor (31, 32). It has been demonstrated that monomeric Kr
acts as a transcriptional activator by binding to TFIIB and that the Kr
dimer, on the other hand, acts as a transcriptional repressor by
binding to TFIIE
(36, 40). Since this result was the first report of
the existence of transcriptional regulatory factor targeting general
transcription factor TFIIE, we analyzed Kr binding to TFIIE
.
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The C-terminal Basic Region of Basic Region-Helix-Loop Sequence
Binds to Single-stranded DNA--
As we reported previously (17), the
C-terminal basic region of the basic region-helix-loop (BR-HL) sequence
in TFIIE is similar to the basic regions of the basic
region-helix-loop-helix (BR-HLH) domains of the Myc-related family of
proteins (such as Myo-D1 and E12) (41-43). Thus, it was predicted that
this basic region may bind directly to single-stranded (ss) and/or
double-stranded (ds)DNA. Recent studies have lent further support to
this idea as follows: (i) photocross-linking studies revealed that
TFIIE
binds to a core promoter region (between
14 and
2) where
dsDNA is melted by transcription initiation (19); (ii) two-dimensional crystallography of yeast TFIIE (yTFIIE) with Pol II revealed that yTFIIE actually interacts with the active center of Pol II, which is
located near the transcription initiation site on DNA (20); (iii) short
mismatched heteroduplex DNA around the initiation site in topologically
relaxed templates abolishes the requirement for TFIIE, TFIIH, and ATP
(22, 23). Therefore, we tested whether TFIIE
can bind to DNA by
using both the gel retardation assay and the pull down binding assay
with ssDNA and dsDNA. While TFIIE
preferentially bound to ssDNA,
TFIIE
alone bound only weakly to ssDNA (less than 5% of the level
observed for wild type TFIIE
), and neither subunit alone was able to
bind to dsDNA. Surprisingly, however, binding to dsDNA was observed
when both subunits were mixed together to form active
TFIIE.3
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Effects of TFIIE Mutations on TFIIH-dependent CTD
Phosphorylation Correlate with Effects on Basal
Transcription--
Previous studies demonstrated that TFIIE strongly
stimulates CTD phosphorylation by TFIIH by itself and during formation
of the active initiation complex (12, 14) and that TFIIE
is essential for this stimulation which correlates well with the increase
in basal transcription activity (15). It was considered that TFIIE
might simply provide a bridge between TFIIH and Pol II to assist
TFIIH-mediated phosphorylation of Pol II, as TFIIH by itself had been
found not to interact well with Pol II. However, as described above,
there exists the possibility that TFIIE may have unique function(s) to
stabilize promoter melting by binding to the ssDNA region of the
promoter DNA (Fig. 8, Ref. 23).3 To check this novel
function of TFIIE, the effects of TFIIE
deletion mutations on CTD
phosphorylation were analyzed (Fig. 9).
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At Least Two Regions of TFIIE Are Essential for Productive
Transcription Initiation--
Structure-function analyses of TFIIE
revealed that except for the N-terminal serine-rich sequence, most of
the putative structural motifs and characteristic sequences are
functionally important for basal transcription (Figs. 2 and 3).
Importantly, the conserved basic and helix regions near the C terminus
are targets for the general transcription factors TFIIB and TFIIF
(RAP30), for the transcriptional regulator Kr (Figs. 6 and
7)3 as well as for ssDNA (Fig. 8). To get an insight to
proceed with the characterization of the regions in TFIIE
in
addition to the above-described C-terminal regions by demonstrating the
active effects of these regions on basal transcription, we employed the deletion mutants in a dominant negative competition assay (Fig. 10A). In this way, we found
that the deletion mutant (
257-291) lacking the C-terminal basic
region-helix-loop sequence actively suppressed basal transcription to
13% of the wild type level in a dose-dependent manner
(Fig. 10A, lanes 12-16). Two other deletion mutants
(
4-125 and
75-96), each of which lacks a region similar to the
Pol II binding region of RAP30 (TFIIF
) and the serine-rich sequence
in the case of mutant
4-125, also reduced basal transcription to
about 30% of the wild type level (Fig. 10A, lanes 7-11 and
17-21). On the other hand, the internal deletion mutant
(
197-232) lacking the TFIIE
binding region had no effect on
transcription even when added at a 64-fold excess over the amount of
wild type TFIIE
(Fig. 10A, lane 26 versus 22). The
230-255 mutant described above showed a modest negative effect on
basal transcription (56% of wild type transcription at a 64-fold
excess) although this mutant has so far failed to display an
interaction with other transcription factor or with DNA (Fig.
10A, lane 31 versus 27).
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DISCUSSION |
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Elucidation of the precise mechanisms involved in transcription
initiation by Pol II has been a long-standing issue in molecular biology. During stepwise assembly of the preinitiation complex (PIC),
TFIIE is essential for TFIIH recruitment and completion of PIC
formation, as well as for stabilization and activation of the PIC.
Recent studies have indicated that TFIIE, through interactions with
other factors and possibly with DNA, is localized near the
transcription initiation site (between positions 14 and
2) within
the PIC and near the active center of Pol II (19-21). It also appears
that TFIIE has a novel but unclear function during promoter melting
(23). Here, we have investigated the structure and function of the
small subunit of human TFIIE (TFIIE
). This included an examination
of the effects of TFIIE
deletion mutations on both basal and
activator-mediated transcription and identification and
characterization of TFIIE
interactions with the general
transcription factors TFIIE
, TFIIB, and TFIIF
(RAP30), the
Drosophila transcriptional repressor Kr, and ssDNA.
Structure-Function Relationships in TFIIE--
Analysis of
TFIIE
deletion mutants showed that a central 227-amino acid region
(residues 51-277) was sufficient to mediate both basal and activated
transcription. As summarized in Fig. 10B, this region
contains all of the previously noted structural motifs and
characteristic sequences with the exception of the N-terminal half of
the serine-rich sequence and the loop region of the basic
region-helix-loop sequence. All of the internal subdomains of the
central 227 amino acid region were essential for transcription activity
(Figs. 2B and 3B). Protein interaction studies
indicated that two basic regions and associated sequences located near
the C terminus (residues 197-210 and 258-270) are important for
direct TFIIE
interactions with various general transcription factors and transcriptional regulatory factors as well as with ssDNA (Figs. 5-8).3. These basic regions have similar primary
structures but may have different (context-dependent)
binding preferences, as summarized in Fig. 10B. Thus the
N-terminal half of the basic region-helix-loop-helix (BR-HLH) motif is
involved in TFIIE
interactions, whereas the second basic region and
the following N-terminal half of the helix region may be part of a
basic region-helix-loop (BR-HL) domain involved in interactions with
TFIIB and TFIIF
(RAP30) as well as with ssDNA. In contrast,
interaction of the Drosophila transcriptional repressor Kr
with TFIIE
appears to involve both regions.
Novel Functional Role(s) of TFIIE Mediated by Binding to
Single-stranded DNA--
Intriguingly, as described above, TFIIE
was found to bind to ssDNA through its second basic region (Fig. 8).
During the preparation for initiation, TFIIE joins the PIC after
recruitment of TFIIF and Pol II and becomes located near the active
center of Pol II through interactions with both Pol II and, most
likely, with promoter DNA in the region between positions
14 and
2,
resulting in the recruitment of TFIIH (for a review, see Refs. 1, 2,
and 5). In the step where Pol II is phosphorylated by TFIIH, TFIIE activates TFIIH by stimulating its kinase and ATPase activities (12,
14). However, the recent finding of differential requirements for TFIIE
and TFIIH when short mismatched heteroduplex DNAs are created around
the initiation site has raised the possibility that TFIIE has a
TFIIH-independent function during promoter melting (23). Our results
showing ssDNA binding activity by TFIIE suggest that TFIIE
may play
an additional role by binding to the single-stranded region present
within melted promoter DNA. Thus, although it is generally held that
TFIIE and TFIIH work together during the promoter clearance step
necessary for the transition from initiation to elongation (44), it
also is possible that TFIIE works independently of TFIIH to remove
certain general transcription factors from the initiation complex on
the promoter.
Functionally Important Regions of TFIIE in
Transcription--
In addition to identifying the regions important
for basal transcription, the effects of adding excess amounts of
TFIIE
mutant proteins on basal transcription assays containing wild
type TFIIE
were studied (Fig. 10A). This approach is
especially effective for both the identification of dominant negative
mutants that lack the active center of TFIIE
but maintain functional
interactions with other PIC components and, conversely, for the
identification of dominant negative mutants that have lost their
capacity to interact with other PIC components but still possess an
active center. The deletion mutant
257-291 of the C-terminal basic
region of the basic region-helix-loop sequence was the strongest
dominant negative mutant (13% of wild type transcription level when
added at a 64-fold excess) (Fig. 10A, lanes 12-16). Since
this basic region was identified as a target for the general
transcription factors TFIIB and TFIIF
(RAP30), as well as the
Drosophila transcriptional repressor Kr and ssDNA, it
appears to be quite important for TFIIE
function. Mutant
257-291
may still possess Pol II and TFIIE
binding regions, resulting in the
depletion of factors necessary for functional PIC formation. Two other
deletion mutants (
4-125 and
75-96) have similar dominant
negative effects (about 30% of wild type level when added at a 64-fold
excess) (Fig. 10A, lanes 7-11 and lanes 17-21).
The former mutant lacks the serine-rich sequence and the region similar
to the Pol II binding region of RAP30 (TFIIF
), whereas the latter
lacks only the region similar to the Pol II binding region of RAP30
(TFIIF
). However, because the first 50 amino acids of TFIIE
,
which contain the N-terminal half of the serine-rich sequence, are
dispensable for transcription, the former mutant,
4-125, may be
almost equivalent to the latter mutant,
75-96. Importantly, we
confirmed here that the region similar to the Pol II binding region of
RAP30 (TFIIF
) might also be essential for TFIIE
function. These
mutants may not be recruited to their proper position in the PIC
complexes because of a failure to bind to Pol II.
Functional Implications of TFIIE (and TFIIH) in Productive
Transcription--
In this study we have presented an initial
characterization of TFIIE structure and function, and we have
proposed novel function(s) for TFIIE during both transcription
initiation and in the transition from initiation to elongation. As
described above, three recent studies dealing with the role of TFIIE in
transcription initiation have suggested that TFIIE, as part of the PIC,
binds both to the core promoter just upstream from the transcription
initiation site (between positions
14 and
2) and to Pol II near its
active center (for a review, see Refs. 1, 2, and 5). In this way, in
conjunction with TFIIH, it would play an important role in promoter
opening, a step which can be circumvented by premelting the promoter
between positions
4 to +2 (19-23). Our results demonstrate that the
C-terminal basic region of TFIIE
binds to both TFIIB and TFIIF
(RAP30). These observations agree with photocross-linking results
showing that TFIIB and TFIIF
(RAP30) bind to the promoter DNA just
upstream of TFIIE
and stabilize the Pol II-TBP interaction at around
position
19 (19). Since TFIIB and TFIIF
(RAP30) may bind to
different surfaces on the promoter DNA (19) and since TFIIE
exists
as a dimer in the PIC (9, 10), we can envisage a model in which TFIIB
and TFIIF
(RAP30) bind to different TFIIE
subunits that are
docked to the DNA in parallel with their C termini facing upstream. As
mentioned above, TFIIE was able to bind to dsDNA, in contrast to the
TFIIE
or TFIIE
subunit which could not bind to dsDNA when added
individually.3 On the other hand, TFIIE
was able to bind
to ssDNA (Fig. 8).3 One possibility is that TFIIE is
recruited into the PIC and at that time TFIIE
assists TFIIE
to
bind to the promoter region (between
14 and
2). This result fits
well with the observation made by Coulombe and colleagues (19) that
TFIIE
could not bind to dsDNA but that TFIIE
could bind to dsDNA
with the assistance of TFIIE
.
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ACKNOWLEDGEMENTS |
---|
We thank Alexander Hoffmann, Sean Stevens,
and Kiyoe Ura for critical reading of the manuscript; Shigeru Hashimoto
and Masami Horikoshi for help in constructing expression vectors for
TFIIE deletion mutants; Zachary F. Burton for human TFIIF
(RAP74)
and TFIIF
(RAP30) clones; Jeff DeJong for human TFIIA
/
and
TFIIA
clones; and Frank Sauer and Herbert Jäckle for
Drosophila Krüppel cDNA clones. We also thank
Masayuki Yokoi and Takashi Nakamura for technical assistance, and our
colleagues for helpful discussion.
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FOOTNOTES |
---|
* This work was supported in part by fellowships from the Human Frontiers Scientific Program Organization (to T. O. and Y. O.), the Charles H. Revson Foundation (to Y. O.), by grants from the Ministry of Education, Science and Culture of Japan (to F. H. and Y. O), the Biodesign Reserch Program of the Institute of Physical and Chemical Reserch (RIKEN) (to F. H.), the Mitsubishi Foundation (to F. H.), the Terumo Life Science Foundation (to Y. O.), and the Yamanouchi Foundation for Research on Metabolic Disorders (to Y. O.). The work at the Rockefeller University was supported by National Institutes of Health Grants CA42567 and AI37327 (to R. G. 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.
¶ Present address: National Institute of Genetics, 1111 Yata, Mishima, Shizuoka 411-0801, Japan.
To whom correspondence should be addressed: Institute for
Molecular and Cellular Biology, Osaka University, 1-3 Yamada-oka, Suita, Osaka 565-0871, Japan. Tel.: 81-6-879-7978; Fax: 81-6-877-9382; E-mail: ohkumay{at}imcb.osaka-u.ac.jp.
1 The abbreviations used are: Pol II, RNA polymerase II; CTD, C-terminal domain; PIC, preinitiation complex; TBP, TATA-binding protein; HA, hemagglutinin; Kr, Krüppel; PAGE, polyacrylamide gel electrophoresis; ds, double-stranded; ss, single-stranded; GST, glutathione S-transferase; BR-HL, basic region-helix-loop; BR-HLH, basic region-helix-loop-helix.
2 A. Hoffmann and R. G. Roeder, unpublished data.
3 Y. Ohkuma, S. Yamamoto, F. Hanaoka, and Y. Ohkuma, unpublished data.
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