From the
The TATA-binding protein (TBP) plays a key role in transcription
initiation. Several negative cofactors (NC1, NC2, and Dr1) are known to
interact with TBP in a manner that prevents productive interactions of
transcription factors TFIIA and TFIIB with promoter-bound TBP. To gain
insights into the regulatory interplay on the surface of TBP, we have
employed mutant forms of TBP to identify amino acid residues important
for interactions with the negative regulatory cofactor NC2 and the
general factor TFIIB. The results show the involvement of distinct
domains of TBP in these interactions. Residues (Lys-133, Lys-145, and
Lys-151) in the basic repeat region are important for interactions with
NC2, as well as with TFIIA (Buratowski, S., and Zhou, H. (1992)
Science 255, 1130-1132; Lee, D. K., DeJong, J.,
Hashimoto, S., Horikoshi, M., and Roeder, R. G. (1992) Mol. Cell.
Biol. 12, 5189-5196), whereas a residue (Leu-189) in the
second stirrup-like loop spanning S2` and S3` is required for
interaction with TFIIB. In addition, we demonstrate that NC2 is
identical to the previously cloned negative cofactor Dr1. The
implications of these results for TBP structure and function are
discussed.
Transcription initiation on genes encoding eukaryotic mRNA
involves assembly of a functional preinitiation complex containing
general initiation factors (TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH,
and TFIIJ) and RNA polymerase II (reviewed in Refs. 3 and 4). The first
step is the binding of the TATA-binding subunit (TBP)
The conserved core domain of TBP is known to fold into a
highly compact and symmetrical structure
(33) . Analyses of the
TBP core domain have suggested that most of the described deletion
mutants of TBP, as well as chimeras constructed with yeast and human
TBP fragments, cannot adopt the proper compact three-dimensional fold
of TBP
(34) . Thus, in the present study, single point mutants
have been employed to define amino acid residues (or domains) within
TBP, which are important for interactions with the negative regulatory
cofactor NC2 and the general factor TFIIB. Since lysine or leucine
residues are distributed extensively in the conserved carboxyl-terminal
core region, we earlier generated and analyzed a series of TBP point
mutants containing either leucine to lysine or lysine to leucine
changes
(17, 31, 35) . With knowledge of the
three-dimensional structure of TBP
(33) , the present analysis
has focused on TBP mutants whose residues are at exposed positions that
would allow direct protein-protein interactions. Based on previous
studies
(17) , TBP mutants defective in both basal and activated
transcription are excluded because they could simply reflect gross
structural perturbations that disrupt all functions, and indeed most of
these mutations map to the hydrophobic core that would be expected to
disrupt the structure
(33) . Selected mutant and wild-type TBPs
were expressed as hexahistidine fusion proteins in bacteria, at
comparable levels (data not shown), and purified by nickel column
chromatography to about 80% purity (Fig. 1). Each purified TBP
protein was then employed in various in vitro assays.
We thank D. Reinberg for providing recombinant Dr1 and
anti-Dr1 antisera; D. B. Nikolov and S. K. Burley for the figures
showing the structure of TBP and S. Stevens for a critical reading of
the manuscript.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(
)
of TFIID to the TATA box of the promoter. This step may be
facilitated by TFIIA
(5) and results in a stable complex, which
subsequently promotes the stepwise assembly of TFIIB, TFIIF/Pol II,
TFIIE, TFIIH, and TFIIJ into the promoter complexes. Consistent with
the central role of TFIID (via TBP) in recruiting other general factors
to the promoter complex, both TBP and TBP-associated factors within
TFIID have been implicated as a target for regulatory factors (reviewed
in Ref. 6). As examples most relevant to the present study,
interactions of TBP (or TFIID) with promoter DNA
(7, 8, 9, 10, 11) , with TFIIA
(12, 13) , and subsequently with TFIIB
(14, 15, 16, 17) have been shown to be
regulated by various activators. Moreover, several negative cofactors
( e.g. NC1, NC2, Dr1, and ADI) may be involved in these
regulatory interactions as well. By competing with TFIIA and/or TFIIB
for binding to TBP, they can prevent formation of a functional
preinitiation complex
(18, 19, 20, 21, 22, 23) .
Thus, it is possible that some activators and repressors might regulate
transcription by directly modulating interactions of TBP with negative
cofactors versus TFIIA and/or TFIIB. To understand these
regulatory interactions within TBP, it is important to first identify
the domains (or residues) of TBP that carry out each of these
interactions. Here, we have used site-directed TBP mutants to define
binding sites on TBP for the negative cofactor NC2 and the general
factor TFIIB. These analyses, along with previous studies of TBP mutant
interactions with TFIIA
(1, 2) , indicate that both NC2
and TFIIA interact with the basic repeat region, whereas TFIIB
interacts with a distinct domain defined, at least in part, as the
second stirrup-like loop region of TBP. Furthermore, we demonstrate
that NC2 is identical to the factor described as Dr1.
Purification of Recombinant
Proteins
Dr1, Wild-type, and mutant yeast TBPs
(17) , as well as yeast and human TFIIBs
(24) , were
expressed from the T7 polymerase vector pET11d as hexahistidine fusion
proteins in bacteria and were purified by nickel chromatography
(25) . GAL4-VP16 was expressed in and purified from bacteria as
described
(26) .
Preparation of Extracts and Transcription
Factors
HeLa nuclear extracts were prepared according to
Dignam et al.(27) . General transcription factors
(TFIIA, TFIIB, TFIID, TFIIE, TFIIF, and TFIIH) and the USA cofactor
fraction were purified from HeLa extracts as described
(18) with some modifications
(28) . Sp1 was separated
from the general transcription factors and purified by affinity
chromatography
(18) . Purification of the negative cofactor NC1
was as described
(29) . The negative cofactor NC2 was purified
from HeLa cell S100 extracts through phosphocellulose (P11),
DEAE-cellulose (DE52), DEAE-Sephadex, S-Sepharose, Mono-S, and Mono-Q
chromatographic steps as described
(19) . Yeast extracts were
prepared according to Kim et al.(17, 30) .
Yeast basal transcription factors and mediator were prepared by
chromatography of yeast whole cell extracts on Bio-Rex 70 (Bio-Rad) and
DE52 (Whatman). Fraction C was prepared from yeast nuclear extracts as
described
(17) .
Electrophoretic Mobility Shift (EMSA) and Immunoblot
Assays
The DNA fragment used for the EMSA contained the
TATA box and flanking regions of the adenovirus major late promoter
(positions -136 to +46). The formation of TBP-TFIIB-DNA
(21) and TBP-NC2-DNA
(19) complexes was determined by
EMSA as described. Dephosphorylation of purified NC2 was performed as
described
(20) . Immunoblotting was done following the
manufacturer's instructions (Amersham Corp.).
Human and Yeast in Vitro Transcription
Assays
Human factor assays (25 µl) contained 1 µl
of TFIIA (Mono-Q fraction, 0.5 mg/ml), 10 ng of recombinant TFIIB, 1
µl of TFIID (Mono-S fraction, 0.5 mg/ml), 1 µl of TFIIE/F/H
(Mono-S fraction, 0.3 mg/ml), 0.5 µl of RNA polymerase II (Mono-Q
fraction, 0.3 mg/ml), 1 µl of USA (heparin-Sepharose fraction, 0.3
mg/ml), and appropriate DNA templates (pGHMC
AT,
pMHIVMT, and pML
53) in the presence or absence of other tested
components and were analyzed as described
(28) . Yeast
transcription assays were performed with reconstituted fractions and
DNA template (pGAL4
6) and analyzed as described before
(17, 30, 31, 32) .
Figure 1:
Purified TBP mutants, yeast TFIIB,
human TFIIB, and human NC2. Individual purified proteins were resolved
by SDS-polyacrylamide gel electrophoresis and visualized by Coomassie
Brilliant Blue staining. Positions of protein molecular mass markers
are indicated as kDa.
Basic Repeat Region of TBP Is Important for Interaction
with NC2
To determine the domain of TBP important for
interaction with the negative cofactor NC2, we utilized TBP point
mutants and NC2 purified from HeLa cell nuclear extracts (Fig. 1)
to analyze TBP-NC2 complex formation on promoter DNA by EMSA
(19) . An analysis of 13 mutants showed that three TBP mutants
(K133L, K145L, and K151L) were defective in TBP-NC2-DNA complex
formation (Fig. 2) while maintaining full activity in TBP-DNA
complex (Fig. 2) and in TBP-TFIIB-DNA complex (see Fig. 6)
formation. The ability of these mutants to form normal levels of
TBP-DNA complexes but not NC2-TBP-DNA complexes is more evident in the
dose-response analysis of Fig. 3. Thus, three individual point
mutations specifically abolished TBP interactions with NC2 on the
promoter DNA. The mutated residues (Lys-133, Lys-145, and Lys-151) map
to the basic repeat region in the three-dimensional structure of TBP
(33) , indicating the importance of this domain for direct
interactions with NC2 (Fig. 8). Other mutants with changes
outside the basic repeat region showed normal TBP-NC2-DNA complex
formation (Fig. 2). In support of the idea that interaction of
NC2 with the basic repeat region of TBP is of functional relevance,
mutants with alterations in several different basic residues (Lys-145
and Lys-151 as well as Arg-141, which is also important for NC2
interactions) were resistant to transcriptional repression by NC2 (data
not shown).
Figure 2:
TBP mutants defective in interactions with
the negative cofactor NC2. Electrophoretic mobility shift assays
contained the radiolabeled DNA probe and proteins as indicated.
Specific TBP- and TBP-NC2-promoter complexes are indicated. Since
slightly variable amounts of TBP were used in the reactions, the minor
variations in band densities (other than those in lanes10, 13, and 14) do not reflect varying
abilities of TBP mutants to form TBP-NC2-DNA complexes. In particular,
to confirm defective NC2 interactions in three TBP mutants (K133L,
K145L, and K151L), we employed approximately 3.5-fold higher TBP
concentrations (3.5 ng) in lanes10, 13, and
14, as compared with wild-type TBP (1.0
ng).
Figure 6:
TBP mutants defective in interactions with
yeast TFIIB (yTFIIB). Electrophoretic mobility shift assays were
performed with radiolabeled DNA probe, wild-type or mutant TBPs, and
yeast TFIIB as indicated. The specific TBP-yTFIIB-DNA complexes are
indicated on the side. Under these conditions, stable TBP
binding requires the presence of other factors such as TFIIB
(21).
Figure 3:
Defective NC2 interactions in TBP mutants
K133L, K145L, and K151L. Electrophoretic mobility shift assays
contained the radiolabeled DNA probe and proteins as indicated.
Equivalent amounts of mutant or wild-type TBP (1.5 ng) were incubated
alone ( lanes2, 5, 8, 11)
or in increasing concentrations of NC2 (in ng 10). The
positions of the resulting protein-promoter complexes are
indicated.
Figure 8:
Schematic drawing of selected TBP point
mutants. TBP is a highly compact and symmetric structure, resembling a
saddle (33). The -carbon backbone is shown as a solidwhiteline. Also shown are the side chains of
positively charged lysine residues (K133L, K138L, K145L, K151L, and
K156L) in the basic repeat region and of the residue (Leu-189) located
in the second stirrup-like loop spanning S2` and S3`. Three mutants
(K133L, K145L, and K151L) are defective in interactions with NC2,
whereas the L189K mutant is defective in TFIIB interactions.
Previously, mutants K138L and K145L were shown to be defective in
interactions with TFIIA (1, 2) and a yeast ATP-dependent inhibitor of
TBP binding to DNA (21). In addition, mutations K156L and K138L were
reported to specifically affect transcription by RNA polymerases I and
III, respectively (31).
TFIIA Competes with NC2 for Binding to the Basic
Repeat Region of TBP
To examine the specificity of
interactions between purified NC2 and TBP, we challenged TBP-NC2-DNA
complexes with TFIIA. As shown in Fig. 4 a, addition of
increasing concentrations of TFIIA to a preformed TBP-NC2-DNA complex
transformed the latter into a TBP-TFIIA-DNA complex ( lanes4-8). These observations are in good agreement with
previous results
(19, 20) and support the view that NC2
and TFIIA interact with identical or closely overlapping domains of
TBP. The fact that both NC2 (above) and TFIIA
(1, 2) interact with the basic repeat region of TBP is consistent
with this possibility. Interestingly, the present (Fig. 2) and
previous
(2) analyses of TBP point mutants indicate that some
basic repeat residues are specifically important for interaction with
NC2 (K133L and K151L) or TFIIA (K138L), with one residue (K145L)
identified as being involved in interactions with both NC2 and TFIIA.
This suggests that the basic repeat region may contain distinct but
overlapping sites for interactions with NC2 and TFIIA.
Figure 4:
Effect of TFIIA on NC2-TBP interactions
and identity of NC2 and Dr1. a, electrophoretic mobility shift
assays with TBP, NC2, and TFIIA. TBP (1.0 ng) and NC2 (20 ng) were
incubated alone ( lane1), in the presence of 2 units
of calf intestine phosphatase ( lane2), 20 µg of
bovine serum albumin ( lane3), or increasing
concentrations of TFIIA (31.3, 62.5, 125, 250, and 500 ng of total
protein in lanes 4, 5, 6, 7, and
8, respectively). For the competition analyses in lanes4-8, TFIIA was added after preincubation of TBP and
NC2. The positions of the resulting protein-promoter complexes are
indicated. b, immunoblotting analysis of NC2 and Dr1. NC1 and
NC2 were prepared as described under ``Experimental
Procedures.'' A fraction eluted at 0.5 M KCl from
phosphocellulose and subsequently at 0.3 M KCl from
DEAE-cellulose (P11/0.5-DE/0.3) was used as a control. Proteins were
separated by 15% SDS-polyacrylamide gel electrophoresis, transferred to
nitrocellulose membranes, and probed with anti-Dr1 antisera. Positions
of protein molecular mass markers are indicated as kDa. c,
electrophoretic mobility shift analysis of TBP-NC2-promoter complexes
with anti-Dr1 antisera. Reactions contained radiolabeled DNA probe and
TBP ( lane1) or TBP plus NC2 ( lanes2-4). For the analysis in lanes3 and 4, preimmune serum or anti-Dr1 antiserum was
preincubated with NC2 before testing for DNA binding with TBP. Specific
TBP- and TBP-NC2-promoter complexes are indicated on the
side.
NC2 Is Identical to Dr1
The initial
description of NC2
(19) as a transcriptional inhibitor that
blocks association of TFIIA with TBP reflects closely the situation
reported later for Dr1
(20) . The latter study also demonstrated
that phosphorylation of Dr1 affected its interaction with TBP on the
DNA
(20) . Based on these properties, the effect of NC2
dephosphorylation on its binding with TBP was determined. As shown in
Fig. 4a, treatment of NC2 with calf intestine
phosphatase ( lane2versuslane1) but not with bovine serum albumin ( lane3) abolished its ability to interact with TBP. Consistent
with this suggestion that NC2 might have an activity similar to Dr1,
immunoblotting analyses showed that highly purified NC2 but not the
negative cofactor NC1 contains a protein that is recognized by anti-Dr1
antisera (Fig. 4 b, lane2). This
polypeptide corresponds to the dominant band in the SDS-polyacrylamide
gel electrophoresis analysis of NC2 shown in Fig. 1and its
molecular weight appears almost identical to that of the major
recombinant Dr1 polypeptide (Fig. 4 b, compare lane2 with lane1). Furthermore,
NC2-TBP-DNA complex formation was specifically abolished by anti-Dr1
antisera (Fig. 4 c, lane4) but not by
preimmune sera ( lane3). We also observed that the
NC2-mediated transcriptional repression (below) can be relieved by
anti-Dr1 antisera (data not shown). Altogether, these results suggest
that the NC2 and Dr1 might be identical proteins, although it cannot be
excluded that NC2 contains a functional polypeptide(s) in addition to
that which corresponds to the recombinant Dr1. The apparently different
mobilities of NC2-TBP
(19) and Dr1-TBP
(20) complexes,
when compared with that of TFIIA-TBP complexes
(Fig. 4 a), may be due to variations in the efficiency of
formation of multimerized complexes of NC2 (Dr1) under different
reaction conditions
(19, 20) . At the same time, we
cannot exclude the possibility that the NC2 and Dr1 preparations differ
in some way ( e.g. in the level of phosphorylation) that alters
the efficiency of multimerization, especially since phosphorylation is
known to be essential for formation of the higher order complexes
(20) . Related to this, even under our reaction condition
(19) some NC2-TBP complexes run slower than TFIIA-TBP complexes,
as reported for Dr1-TBP complexes
(20) , when a much higher
concentration of NC2 is used (data not shown).
NC2 Represses Transcription Both in the Yeast and the
Human in Vitro Systems
We next analyzed the effects of NC2
on basal and activated transcription in both yeast and human in
vitro systems. As shown in Fig. 5, the addition of
increasing amounts of NC2 to the yeast in vitro reconstituted
system resulted in inhibition of both basal and activator
(VP16)-dependent transcription. Similar repressive effects of NC2 were
observed for both basal (pML53 template) and activated
transcription by GAL4(1-147)-VP16
(pG
HMC
AT template) and Sp1 (pMHIVMT template)
in the human system reconstituted with highly purified factors
(Fig. 5, b and c). The previous result
(19) of a selective effect of NC2 on basal transcription may
reflect the use of a less purified system in which another factor(s)
may facilitate the observed discrimination. In the present study,
repression was observed with various TATA box-containing promoters in
both yeast and human systems. Thus, it seems likely that repression by
NC2 is a general mechanism, possibly competing with general factors
TFIIA and/or TFIIB to inhibit formation of functional preinitiation
complexes.
Figure 5:
Repression of transcription by NC2 in
yeast and human in vitro systems. a, yeast in
vitro reconstituted systems containing pGAL4X6 templates were
incubated with variable amounts of NC2 (20 and 100 ng of total protein)
in the absence ( lanes2 and 3) or presence
( lanes5 and 6) of GAL4(1-147)-VP16 as
indicated. b, reactions reconstituted with human transcription
factors and pGHMC
AT templates were incubated
with variable amounts of NC2 (10 and 50 ng of total protein) in the
absence ( lanes1-3) or presence ( lanes4-6) of GAL4(1-147)-VP16 as indicated.
c, reactions reconstituted with human transcription factors
were incubated with pMHIVMT (containing three Sp1 sites) and pML
53
(adenovirus major late core promoter) templates in the presence of Sp1.
Transcription reaction conditions were the same as in
b.
Distinct Regions of TBP Are Involved in Interactions
with TFIIB and NC2
Apart from their ability to prevent or
reverse formation of a TBP-TFIIA-promoter complex, NC2
(19) and
Dr1
(20) can also displace TFIIB from TBP-TFIIB-promoter
complexes. Hence, it seemed possible that TFIIB and NC2 also might
interact with the same or overlapping surface(s) of TBP. To define the
region of TBP important for interaction with TFIIB, we analyzed
TBP-TFIIB-DNA complex formation with TBP mutants and TFIIB. Under these
particular conditions, TBP alone failed to form a DNA complex, but a
TBP-TFIIB-promoter complex
(21, 36) was readily
observed with TFIIB addition (Figs. 6 and 7). These analyses
demonstrated that all but one of the tested TBP mutants, including
K133L, K145L, and K151L, form TBP-yeast TFIIB-DNA complexes comparable
with that displayed by wild-type TBP (Fig. 6). In contrast,
mutant L189K was defective in TBP-yeast TFIIB-DNA complex formation
(Fig. 6, lane16; Ref. 17). A similarly
defective TBP interaction was observed with human TFIIB on the promoter
DNA by electrophoretic mobility shift assays (Fig. 7). Since the
L189K mutant can interact with DNA under different conditions
(35) and since it can form TBP-TFIIA-DNA
(2) and
TBP-NC2-DNA (see Fig. 2) complexes as efficiently as does
wild-type TBP, this mutation appears to specifically affect
interactions with TFIIB. The failure of the L189K mutant to interact
with both yeast and human TFIIB suggests that the same (or overlapping)
regions of both yeast and human TBP containing a Leu-189 residue are
involved in interactions with TFIIB. In the three-dimensional structure
of TBP, the L189K mutation is found within the loop connecting strands
S2` and S3` (Fig. 8). This loop region is at a highly exposed
position that may be accessible for direct protein-protein
interactions. Despite the involvement of distinct TBP regions in
interactions with TFIIB and NC2, NC2 has been shown to dissociate TFIIB
from the TBP-TFIIB promoter complexes. This suggests that interaction
of NC2 with TBP might result in a TBP conformational change that
affects TBP-TFIIB interactions. Related to the possibility of dynamic
changes in TBP-promoter complexes during preinitiation complex
assembly, the region containing Leu-189 has been demonstrated to be
important for an activation-specific TBP-TFIIB complex, which is
induced (or stabilized) by acidic activators in the yeast in vitro transcription system
(17) . Recently, we have shown using a
yeast in vitro system that L189K supports basal but not
activated transcription, while K133L, K145L, and K151L, which are
important for NC2 interactions (see Figs. 2 and 3), can support normal
levels of both basal and activated transcription
(17) . The fact
that the L189K mutant shows normal basal transcription activities,
despite its deficiency in forming TBP-TFIIB complexes, must reflect the
presence of compensatory stabilizing interactions with other basal
factors under transcription conditions
(17) .
Figure 7:
TBP
mutants defective in interactions with human TFIIB (hTFIIB).
Electrophoretic mobility shift assays were performed with radiolabeled
DNA probe and wild-type or mutant (L189K) TBPs in the absence or
presence of hTFIIB. The arrow indicates the position of the
specific TBP-hTFIIB-DNA complex.
In contrast to
the situation with TFIIB, NC2 and TFIIA interact with the TBP basic
repeat region, which represents the convex surface of TBP-spanning
-helix H2 and
-strand S1` (Fig. 8). In addition, the
basic repeat region has been shown to be important for interactions
with several other regulatory factors (reviewed in Ref. 37), which
include the adenovirus E1a activator
(38) . Interestingly, E1a
is capable of preventing and/or reversing the TBP-Dr1 (NC2) interaction
and the corresponding repression of transcription
(20, 39) . These results (see also Ref. 19), along with
the present data, suggest that certain activators and/or positive
cofactors could, in some situations, reverse the action of negative
cofactors such as NC2 (Dr1) and thus increase promoter activity.
Furthermore, the present results indicate that the basic repeat domain
of TBP may be directly involved, at least in part, in the complex
interplay of different sets of transcriptional regulatory factors.
Recently, this region has been demonstrated to be important for
transcription by RNA polymerases I and III in addition to RNA
polymerase II (Ref. 31 and references therein). Clearly, the mutants
described here, along with previously identified TBP mutants (reviewed
in Ref. 37), will be useful for a further dissection of the functional
interactions of transcription factors on the surface of TBP. As
suggested by the present results and given the somewhat limited protein
binding surface of TBP, many of the TBP interaction sites may be
overlapping, and competition for regulatory factors could influence
transcription. At the same time, TBP structural changes induced by
interacting factors might modulate its ability to form preinitiation
complexes. Therefore, elucidation of the interplay of various
regulatory factors with TBP and their specific effect(s) on
TBP-promoter complexes during preinitiation complex assembly will be
critical for understanding transcriptional regulatory mechanisms.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.