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J. Biol. Chem., Vol. 277, Issue 30, 26831-26838, July 26, 2002
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abart
andFrom the Chemical Pathology Unit, Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, United Kingdom
Received for publication, April 1, 2002, and in revised form, May 13, 2002
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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In humans, transcription factor IIIB (TFIIIB)- Accurate initiation of transcription of
snRNA1 genes by RNA
polymerase III (pol III) requires the formation of a large,
multiprotein complex at the promoter (reviewed in Refs. 1-3). A
typical type 3 pol III core promoter, commonly represented by 7SK and
U6 genes, contains a proximal sequence element, covering ~15 bp and
located ~55 bp upstream of the transcription start (4) and TATA box at around Human B" is a large protein (1388 amino acid residues) that is similar
to yeast scB" over an ~300-amino acid residue stretch comprising the
SANT domain (43% identity), which is essential for transcription in
yeast, and N-terminal as well as C-terminal flanking segments
(~20% identity). The C-terminal domain consists of nine long
repeats of a 55-amino acid motif, each of which can be divided into two
divergent 26-28-amino acid parts with regularly spaced serine and
threonine residues that are potential phosphorylation sites. An
antibody raised against recombinant hB" identifies an ~160-kDa
protein in HeLa cells, and hB"-immunodepleted HeLa nuclear extract is
unable to support U6 and VA1 gene transcription, which can be both
restored with bacteria-expressed hB" (12). Thus, hB"/TFIIIB150 is also
involved, together with TBP and TFIIIB90 (12, 16, 17), in the other
form of human TFIIIB complex, (TFIIIB- It is generally accepted that binding of TBP to the TATA box nucleates
the formation of preinitiation complexes either through stepwise
assembly of TFIIB and other basal factors or through recruitment of a
holoenzyme (reviewed in Ref. 20). Here we report on the formation of a
minimal TFIIIB- Mutagenic selection, in vitro transcription, and binding
assays, in addition to cross-linking experiments using human factors, indicate that binding of TFIIB to mRNA pol II promoters is highly asymmetrical (21, 22) and dependent on a specific 7-bp TFIIB recognition element (BRE) immediately upstream of the TATA box (21).
Our previous study (14) suggests that the TFIIB-like factor BRFU makes
contacts with sequences flanking the TATA box, which may instead be
more extensive downstream. In the 7SK gene the sequence just downstream
from the TATA box is GC-rich and recognizably resembles a
"backward" BRE, suggesting it may play a role in BRFU·TBP·DNA
interaction. We have investigated this possibility by studying the
efficiency and accuracy of BRFU·TBP·DNA sub-complex formation and
their consequences for assembling of the whole TFIIIB- Our data indicate that sequences located downstream from the TATA box
can influence the assembly of TFIIIB- Recombinant Protein Purifications--
The plasmids encoding
C-terminal histidine-tagged human BRFU and B" proteins for both
in vitro transcription-translation and expression in
Escherichia coli cells have been described previously (12).
The plasmid encoding glutathione S-transferase TBP was derived from in vitro transcription-translation construct
pT Transcription Templates and Corresponding EMSA Probes--
The
7SKwt and 7SK TATA Electrophoretic Gel Mobility Assays--
Probes for EMSA were
prepared by end labeling double-stranded DNA with the Klenow enzyme and
The BRFU·TBP·DNA and B"·BRFU·TBP·DNA complexes were analyzed
using the "TBE gel system." Binding reactions contained 10 fmol of
probe, 0.2 µg of poly(dG-dC), 10 mM HEPES, pH 7.9, 30 mM Tris-HCl, pH 8.4, 60 mM KCl, 7.5 mM MgCl2, 10% glycerol, 6 mM
For detection of the TBP·DNA complex, the "TGEM gel system" was
employed. The untagged TBP was incubated with various probes for 40 min
at 30 °C in buffer containing 0.2 µg of poly(dG-dC), 10 mM HEPES, pH 7.9, 10 mM Tris-HCl, pH 8.0, 60 mM KCl, 5 mM MgCl2, 10% glycerol,
1 mM dithiothreitol, 0.01% Nonidet P-40, and 0.1 mg of
bovine serum albumin per ml. The binding reactions were resolved on 4%
polyacrylamide gel in 1× TGEM running buffer (50 mM Tris
base, 380 mM glycine, 2 mM EDTA, and 5 mM MgCl2) at 200 V.
Protein-Protein Interaction Assays--
The
[35S]Met-labeled full-length B", BRFU, and TBP were
produced using a TNT-coupled transcription-translation
system (Promega). GST-BRFU, GST-TBP, and GST proteins bound to
glutathione beads were rotated with 35S-labeled proteins in
the binding buffer (20 mM HEPES, pH 7.9, 100 mM
KCl, 10% glycerol, 0.5 mM MgCl2, 0.5% Nonidet
P-40, 0.2 mM EDTA, 0.4 mg of ethidium bromide per ml) at
4 °C for 3 h. Where indicated, binding reactions were
supplemented with 10 pmol of each E. coli expressed
full-length TBP or core TBP. The beads were washed five times with
binding buffer, boiled in SDS sample buffer, and then pelleted, and the
supernatant was loaded onto an SDS-10% polyacrylamide protein gel. The
35S-labeled proteins were detected by autoradiography or
analyzed on a PhosphorImager (Amersham Biosciences).
Transfections and S1 Nuclease Analysis--
Human embryonic
kidney 293 and cervical carcinoma HeLa cells were cotransfected with 5 µg of snRNA gene promoter reporter construct and 0.25 µg of VA1
plasmid using LipofectAMINE (Invitrogen) according to the
manufacturer's instructions. Cells were harvested 48 h after
transfection. Total RNA was isolated using the TRI Reagent® (Sigma)
and then analyzed by S1 assay (25). The sequences of S1
oligonucleotides for VA1, U2mTATA/7SK, and its BURE-deficient derivative were described previously (14).
In Vitro Transcription--
The nuclear extract was prepared
from HeLa cells as described previously (26). Twenty five-µl
transcription reactions were carried out as described by Murphy
et al. (27) with 1 µg/ml Human B" Loosely Associates with the DNA-bound TBP·BRFU
Complex--
We have shown previously (14) that the
snRNA-specific factor BRFU cooperates with TBP to create a relatively
strong complex on TATA box containing pol III snRNA gene promoters. TBP
and BRFU together with hB" constitute the
TATA-dependent snRNA-specific TFIIIB transcription
activity, designated as TFIIIB-
Human B" protein expressed in and purified from E. coli
(Fig. 1A) was first assayed
for DNA binding alone and in the presence of recombinant TBP (Fig.
1B). In this electromobility shift assay, we have used a
template derived from the TATA-containing, pol III-transcribed 7SK
gene. TBP alone can be detected binding to this template only when a
TGEM gel system is used but not in a TBE system (14). B" is unable to
form a stable complex with DNA in the TGEM system either alone or in
the presence of TBP (data not shown). Incubation of TBP, B", and DNA
did not result in the appearance of any B"-specific complex in the TBE
gel system (Fig. 1B, lanes 1 and 3).
We therefore tested the ability of B" to interact with a
BRFU·TBP·DNA complex. Indeed, in the presence of TBP, BRFU, and
template DNA, the addition of B" results in the appearance of a slowly
migrating complex on a TBE gel (Fig. 1B, compare lanes
5 and 4). To confirm this finding, we also used an
another probe, prepared from a pol III-transcribed snRNA gene construct
containing U2 promoter sequences with an introduced TATA box. Again, in
addition to the DNA·TBP·BRFU complex (Fig. 1B, compare
lanes 7 and 6) an additional slow migrating
complex appears in the presence of B" (lane 7). Taken
together, these results suggest that we detect formation of a
quaternary DNA·TBP·BRFU·B" complex. However, only a small amount
of the input B" appears to become associated with the
DNA·TBP·BRFU complex (compare intensities of upper
B"·BRFU·TBP·DNA band with the lower BRFU·TBP·DNA band in lanes 5 and 7; see also longer exposure in
lanes 5' and 7'). Thus, the quaternary complex
may be relatively unstable to the gel conditions used or B" may only be
loosely associated within the complex. We observed another complex just
above the DNA·TBP·BRFU complex (Fig. 1B, lanes
5 and 7, marked by asterisk). As shown in
Fig. 1A, hB" protein preparation contains an ~67-kDa
fragment, which was identified as a C terminus comprising part of B".
This B" fragment can also associate with the BRFU, TBP, and DNA to create a faster migrating complex. Inspection of the autoradiograms reveal 10% association of both B" protein forms with the
BRFU·TBP·DNA complex, from which full-length B" is responsible for
30% binding.
TBP Mediates the Assembly of Trimeric TFIIIB-
We have shown previously (14) that the conserved C-terminal core domain
of human TBP is sufficient for both interaction with BRFU and
recruitment of BRFU to the TATA box containing snRNA gene templates.
Therefore, we were interested to determine whether the C-terminal half
of TBP, like full-length TBP (flTBP), is capable of recruiting B" to
the immobilized BRFU. For this purpose, equimolar amounts of flTBP and
N-terminal truncated TBP
Together these data suggest that TBP is central for the assembly
BRFU with B", perhaps because TBP bridges, through its core domain,
these two proteins by direct interactions.
Deletion of the Zinc Ribbon Domain Reduces BRFU Binding to the TBP
and Allows Direct Interaction between BRFU and B" to Occur--
To
gain a better understanding of the architecture of the TFIIIB-
First, we compared the TBP binding abilities of equivalent amounts (as
determined by quantitation of Coomassie Blue-stained SDS-polyacrylamide
gel; data not shown) of GST-BRFUs that have been prebound to the
beads (Fig. 4B). The outcome of this GST pull-down study
confirms that 35S-TBP binds well to full-length BRFU (more
than 3-fold of 10% input) (Fig. 4B, lane 2).
Furthermore, deletion of repeat 1 (Fig. 4B, lane
4), repeat 2 (lane 5), and whole core (lane
6) resulted in an almost 50% reduction of BRFU·TBP binding,
relative to full-length BRFU. Surprisingly, BRFU missing the first 37 amino acid residues interacts poorly with TBP (below 10% input) (Fig.
4B, lane 3), suggesting that zinc ribbon plays an
important, if not decisive, role in BRFU·TBP interaction in solution.
Second, to address which of these BRFU mutants can recruit B", the same
amounts of GST-BRFUs used in the previous assay were incubated with
35S-B" in the absence or presence of TBP (Fig.
4C). Strikingly, whereas full-length as well as repeat 1, repeat 2, and whole core BRFU deletion mutants are capable of
recruiting B" only when TBP is present (Fig. 4C, compare
lanes 2, 6, 8, and
10 with lanes 3, 7, 9, and
11, respectively), the BRFU mutant lacking the zinc ribbon
appears to have B" binding affinity on its own (lane 4). This suggests that BRFU contains a built-in mechanism to down-regulate its direct binding to B", which is counteracted by TBP bridging.
A Novel GC-rich Regulatory Region "BURE" Downstream of the TATA
Box Is Responsible for Efficient DNA·TBP·BRFU Complex
Formation--
The snRNA promoters transcribed by RNA pol III (U6 and
7SK) contain the sequence TTTATATA located around
To investigate the role of this sequence, EMSA probes were constructed
from two transcription templates. As depicted in Fig. 5A, the minimal TATA element,
TTTATA, was inserted into the U2/7SK hybrid construct (14) (Fig.
5A, "wt"), and the 10-bp U2 region downstream
of TATA box was mutated from GGCGCGAAGG to CTTGTGCGCC (Fig.
5A, BURE Mutation of BURE Strongly Reduces pol III and Potentiates pol II
Transcription--
By having defined the BURE as an element required
in addition to the TATA box for efficient recruitment of the pol III
snRNA-specific TBP·BRFU sub-complex, we were interested to assess the
effect of mutation of the BURE on transcription in vivo. The
BURE mutant and parental U2mTATA/7SK constructs were transfected into
293 and HeLa cells, and total RNA was analyzed by S1 assay (Fig. 5, A and B). The pol III-dependent
adenovirus VA1 gene was included as an internal control in this and
subsequent transcription assays. The U2/7SK hybrid construct (14) gives
a substantial level of pol III snRNA gene transcription when a
TTTATATAT is present between
Quantitation of the reduction in formation of BRFU·TBP complexes on
the BURE mutant is shown in Fig. 5D. The data for GST- and
His-BRFU·TBP·DNA complex formation on "wt" and
BURE The Intact BURE in the 7SK Gene Promoter Contributes to Efficient
pol III Transcription--
To evaluate the importance of the BURE in
an authentic snRNA promoter recognized by RNA polymerase III, the 6-bp
GTGCGC block located downstream of the TATA box (Fig.
6A, wt) was
replaced by TTTTTT (Fig. 6A, BURE The BURE Is Required for Efficient Formation of the Trimeric
TFIIIB- Human TFIIIB can be chromatographically separated into two
distinct activities, DNA binding by TBP or TBP-containing complexes appears to be a
rate-limiting step for transcription initiation at various pol II (34)
and pol III promoters (35-37). Accordingly, many regulatory factors
have been described that interact directly with TBP or its associated
components to regulate transcription (11, 38, 39). In vitro,
interactions that affect the initial steps in preinitiation complex
formation can be readily monitored by electrophoretic mobility shift
and protein-protein pull-down assays. By using these approaches, we
show that a TBP·BRFU complex can recruit hB" both on and off DNA.
However, despite formation of a relatively strong DNA·TBP·BRFU
complex, association of hB" does not appear to be very stable to the
gel retardation conditions used. This resembles yeast TFIIIB, where TBP
and BRF also form a very stable complex (designated B') from which less
tightly associated scB" is separable by chromatography (40). In human HeLa cells, TFIIIB90/hBRF also forms an extremely strong complex with
TBP, whereas TFIIIB150/hB" seems to be either transiently or loosely
associated with the TBP·TFIIIB90 complex (13). Because TFIIIB- Mapping of protein-protein contacts within TFIIIB- An alternative factor to BRFU has also been implicated in transcription
of snRNA genes by pol III (41). This is a splice variant hBRF/TFIIIB90,
designated hBRF2, that is required for U6 gene transcription in
vitro but not for tRNA-type VA1 gene transcription. It is a small
protein (134 amino acid residues) that shares only one exon (encoding
the second TFIIB-related repeat and 32 adjoining amino acid residues)
with hBRF. Similarly to BRFU and BRF, BRF2 binds to TBP, albeit much
more weakly than BRF (41). Its possible interaction with hB" remains to
be determined. Also, it is not clear whether TFIIIB50/BRFU-associated
proteins, required for 7SK gene transcription (13), participate in
hB"/TFIIIB150 recruitment.
In mRNA gene promoters, the GC-rich BRE was originally identified
as an element that had a positive effect on transcription in
vitro (21, 42). This effect correlates with an increase in stable
TFIIB·TBP·DNA complex formation mediated by direct contact of the
BRE by TFIIB. Our functional studies on pol III-snRNA promoters indicate that the ~7-bp BRE-like segment contiguous with the
downstream boundary of the TATA box is important for efficient pol III
transcription both in vivo and in vitro.
Moreover, mutating this sequence causes a switch in polymerase
specificity when the TATA box is short, indicating that pol II-specific
preinitiation complexes are generated. The last finding is particularly
interesting and contributes to a model (Fig.
7) in which the composition and
architecture of the preinitiation complexes formed on snRNA gene TATA
promoters determine the selective recruitment of RNA pol III or II. Our results therefore establish the existence of a second core promoter element which, in addition to the TATA-element (5, 7), can modulate the
polymerase specificity for snRNA gene transcription. Gel retardation
assays suggest that this sequence, which is only modestly conserved,
can provide a preferential, albeit somewhat variable, binding surface
for BRFU. The observation that the GC-rich sequence, present
downstream of the adenovirus major late promoter "typical mRNA
gene" TATA box, still facilitates the DNA·TBP·BRFU complex
assembly and allows pol III machinery to initiate transcription (14)
illustrates the flexibility of this sequence. In the context of a
"weak" TATA box, it is clear that BRFU·BURE interaction is important for the creation of DNA·TBP·BRFU sub-complex itself. It
appears that a "strong" TATA box instead can overcome the BURE deficiency and direct DNA·TBP·BRFU sub-complex formation, but its
presumably altered conformation does not allow hB" to efficiently join
such a complex. In both cases, BRFU·BURE interaction
seems to play a substantial role in TFIIIB-
governs basal transcription from small nuclear RNA genes by RNA
polymerase III (pol III). One of the components of this complex,
BRFU/TFIIIB50, is specific for these promoters, whereas TATA-binding
protein (TBP) and hB" are required for pol III transcription from both gene external and internal promoters. We show that hB" is specifically recruited to a promoter-bound TBP·BRFU complex, which we have previously demonstrated as forming on TATA-containing templates. The
N-terminal region of BRFU, containing a zinc ribbon domain, acts as a
damper of hB" binding. TBP deactivates this negative mechanism through
protein-protein contacts with both BRFU and hB", which may then promote
their cooperative binding to form TFIIIB-. In addition, we have
identified a GC-rich sequence downstream from the TATA box (the BURE)
which, depending on the strength of TATA box, can either enhance BRFU
binding to the TBP·DNA complex or hB" association with the
BRFU·TBP·DNA complex, and subsequently stimulate pol III
transcription. Moreover, mutation of the BURE reduces pol III
transcription and induces transcription by RNA polymerase II from the
U2 gene promoter carrying a minimal TATA box.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
25 (5-7). The proximal sequence element is a binding site
for the multisubunit factor PBP/PTF/SNAPc (8-10).
TBP binds at the TATA box, and this interaction is enhanced by
PTF (11). Recently, we showed that TBP can also be recruited to
the TATA box by interaction with BRFU/TFIIIB50 (12, 13), which is a TFIIB- and BRF-related basal transcription factor, specifically required for U6/7SK transcription (12-14). BRFU/TFIIIB50 can be purified from human cells as part of a multisubunit complex, and functional data suggest that as yet unidentified additional
TFIIIB50-associated subunits are also required for an in
vitro 7SK transcription, reconstituted from partially purified
components. However, recombinant BRFU alone is sufficient to support
basal U6 transcription using a more defined and hence less complex
system (15). BRFU/TFIIIB50, together with TBP and hB"/TFIIIB150, may
constitute the minimal snRNA gene-specific TFIIIB activity (TFIIIB-)
that is required for transcription of U6 or 7SK genes in vertebrates
(12, 13).
) (18). This complex functions
instead on the tRNA-type Ad2 VA1 promoter (18, 19) that exemplifies a
class of pol III promoters whose essential elements are all located
within the gene coding region.
complex on TATA-containing snRNA promoters where hB"
can join a preassembled BRFU·TBP·DNA sub-complex. We find that the
N-terminal region of BRFU, containing a zinc ribbon, inhibits the
direct association of BRFU with hB" in solution. This autoinhibition is
deactivated by TBP bridging, which promotes cooperative assembly of
TFIIIB- from recombinant TBP, BRFU, and B" subunits. Such a
mechanism may ensure creation of the "proper" architecture of the
TFIIIB- complex.
complex on
snRNA promoters, transcribed by pol III, where this sequence has been
mutated. The outcome of this analysis provides evidence for requirement
of an intact GC-rich structure, designated as a BURE, for
BRFU·TBP·DNA assembly at the first level. At the second level, the
BURE influences the affinity of already assembled BRFU·TBP·DNA
sub-complex for hB". Transcription studies confirm the importance of
the BURE for efficient transcription both in vivo and
in vitro and for maintenance of polymerase specificity.
in two unique ways to regulate
the efficiency and specificity of basal pol III snRNA transcription.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
TBP (23) by PCR amplification of the TBP-coding region and placing into pGEX-2T (Amersham Biosciences) using BamHI and
EcoRI sites. Polyhistidine-tagged hBRFU and hB" were
purified by nickel chelate affinity chromatography as described
previously (14, 24). Immunoblot analysis of isolated B" with the
anti-His6 antibody (Roche Diagnostics) was performed by
using the enhanced chemiluminescence system (Amersham Biosciences) as
recommended by the manufacturers. Purification of GST-BRFU and its
deletion derivatives, 
1-37, 72-157, 169-266, and
72-266, GST-TBP, and GST on glutathione-agarose (Sigma) has been
described previously (14).
transcription constructs are identical
to O+P+ (9) and
O+P+T (5) construct,
respectively. In the 7SK BURE construct, the wild-type
sequence between 19 and 14 was mutated to TTTTTT. The U2mTATA/7SK
transcription construct contains U2 gene sequences 556 to +6 upstream
from sequences +1 to +458 of the marked 7SK gene (9) between the
EcoRI and PstI sites of pGEM4. The U2 sequence
between 32 and 27 was mutated to TTTATA, and thus a minimal TATA
box was introduced. In U2mTATA/BURE/7SK the sequence
between 23 and 14 was mutated to CTTGTGCGCC. The probes for EMSA
studies were made from the above characterized templates as described
previously (9). In the U2 TATA probe (14) the sequence between 26 and
18 was mutated to TTTATATAT as described by Lobo and Hernandez
(7).
-32P-labeled deoxynucleoside triphosphates. Two binding
and corresponding gel systems were used essentially as described
previously (14).
-mercaptoethanol, 20 mM dithiothreitol, and 0.1 mg of
bovine serum albumin per ml. Reactions were initiated by the addition
of proteins, and mixtures were incubated for 30 min at 30 °C. The
mixtures were electrophoresed on a 4% (37.5:1,
acrylamide-bisacrylamide) polyacrylamide gel, with 0.5× Tris
borate-EDTA (TBE) at 40 mA.
-amanitin, 250 ng of each 7SK
template, and 2 ng of VA1 construct as an internal control. Following
1 h of incubation at 30 °C, the transcripts were analyzed by S1
analysis as described for RNA collected from transfected cells. The S1
oligonucleotide sequence for 7SK and its derivatives has been published
previously (25).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(13). We therefore wondered whether
hB" could be selectively recruited to promoters by direct binding
to DNA, DNA·TBP, or DNA·TBP·BRFU sub-complexes.
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Fig. 1.
Human B" loosely associates with the
DNA-bound TBP·BRFU complex. A, a representative Coomassie
Blue-stained SDS-10% polyacrylamide gel of purified hB" protein
(fl B") used in this and subsequent DNA-protein binding
experiments. An asterisk marks the partial form of hB"
resulting from degradation (the identity was confirmed by
immunoblotting with an antibody against a stretch of six histidine
residues, which detects the histidine tag on C terminus of B").
M indicates protein marker standard above the
respective lane. B, mobility shift analysis of TBP·BRFU
and TBP·BRFU·B" binding to 7SK wild-type and U2 mutant probe with
an added TATA box. The type of probe used is indicated below the
lanes. Reaction mixtures contained 0.1 pmol of TBP (lanes
2-7), 1 pmol of BRFU (lanes 2 and 4-7),
and 0.2 pmol of full-length B" (lanes 1, 3,
5, and 7) and were electrophoresed on a TBE 3.5%
gel after incubation. The positions of B"·BRFU·TBP·DNA and
BRFU·TBP·DNA complexes and free DNA probe are indicated. The
asterisk indicates the complexes containing C-terminal
fragment of hB", BRFU, TBP, and DNA. Lanes 4'-7' show
longer exposure of binding reactions in lanes 4-7.
Complex Off the
Promoter--
We next explored whether a TBP·BRFU·B" (TFIIIB-)
complex can form in the absence of DNA. Because recruitment of B" to
the template is BRFU-dependent and BRFU recruitment to the
template is TBP-dependent (14), the possibility of direct
interactions between B" and BRFU and/or TBP were tested. For GST
pull-down assays, BRFU and TBP were produced in E. coli as GST-tagged proteins and immobilized, together with
GST alone, on glutathione-agarose beads (Fig.
2B). As no appreciable
interaction was observed between in vitro translated
[35S]methionine-labeled B" protein and GST-BRFU (Fig.
2A, lane 2), either conformational change of BRFU
or an additional interaction with TBP may be necessary for B"
recruitment. Indeed, when the assay was performed in the presence of
recombinant TBP, B" was retained on GST-BRFU beads (Fig. 2A,
lane 4). Ethidium bromide was added to preclude the
possibility that DNA might mediate interactions among these proteins.
To understand further the TBP-dependent mechanism through
which BRFU recruits B", the 35S-labeled B" was incubated
with the immobilized GST-TBP. As shown in Fig. 2A,
lane 7, direct interaction between B" and GST-TBP was
detected. BRFU and TBP have also been shown to associate directly in vitro (14). The interaction between TBP and BRFU was
confirmed in a reciprocal experiment in which 35S-BRFU was
selectively retained by immobilized GST-TBP (Fig. 2A, lane 10) but not GST alone (Fig. 2A, lane
11).
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Fig. 2.
TBP mediates the assembly of a trimeric
TFIIIB- complex from recombinant TBP, BRFU,
and B" subunits off the promoter. A, a GST-pull down
experiment performed with the immobilized GST fusion proteins noted
above the lanes. B" and BRFU were expressed
in vitro using rabbit reticulocyte lysates, and proteins
were labeled with [35S]methionine. Lanes 1,
6, and 9 contain 10% of the
35S-labeled proteins used as input (10% IN).
35S-B" was tested for interaction with GST-BRFU and GST in
the absence of TBP (lanes 2 and 3, respectively)
or in the presence of 10 pmol of TBP (lanes 4 and
5, respectively). GST-TBP and GST were incubated with
35S-B" (lanes 7 and 8) and
35S-BRFU (lanes 10 and 11). Proteins
retained after extensive washing were size-fractionated on SDS-10%
PAGE and visualized by autoradiography. The positions of
35S-B" and 35S-BRFU are indicated at the
right. B, SDS-PAGE analysis of GST-TBP
(lane 1), GST-BRFU (lane 2), and GST (lane
3) proteins used in these protein-protein binding reactions.
M indicates protein marker standard above the
respective lane.
1-154 (cTBP) were included into binding
reactions. As shown in Fig. 3,
35S-B" is brought to the GST-BRFU beads by cTBP as
efficiently as by flTBP (Fig. 3, compare lanes 4 and
2).
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Fig. 3.
The core domain of TBP bridges BRFU and B".
35S-Labeled B" was synthesized by in vitro
translation and incubated with immobilized GST-BRFU and GST in the
presence of 10 pmol of full-length TBP (lanes 2 and
3) or 180-amino acid C-terminal "core" TBP (lanes
4 and 5). The input (10% IN, lane
1) represents 10% of the 35S-B" protein used for this
pull-down experiment. Bound proteins were resolved by SDS-PAGE gel and
visualized by autoradiography.
complex, we focused our attention on the BRFU domain(s) responsible for
TBP binding and B" recruitment. We used a set of GST-BRFU protein
mutants where important TFIIB-like domains (Fig.
4A) were deleted (14).
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Fig. 4.
Deletion of the zinc ribbon domain reduces
BRFU binding to the TBP and allows direct interaction between BRFU and
B" to occur. A, scheme showing the
structural organization of BRFU. The locations of zinc ribbon and core
domains encompassing repeats 1 and 2 are indicated. In addition to
full-length protein (fl), mutated GST-tagged BRFU
derivatives were prepared: Zn ,
deletion of amino acids 1-37; Rep1, deletion
of amino acids 72-157; Rep2, deletion of
amino acids 169-266; Core, deletion of amino
acids 72-266. B, 35S-labeled TBP was
synthesized by in vitro translation, captured by GST-BRFU,
GST-BRFU derivatives, or GST alone (shown above the
lanes), and analyzed by SDS-PAGE. Lane 1, 10% of
the amount of 35S-TBP used in the pull-down assay. The
binding signals (lanes 2-7) were normalized against 10%
protein input (10% IN, lane 1), and the mean
values ± S.D. from three independent experiments are represented
graphically below the autoradiogram. C, GST-BRFU
or its derivatives, shown above the lanes, were
mixed with 35S-labeled B" (lanes 2,
4, 6, 8, and 10). The
corresponding binding reactions were each supplemented with 10 pmol of
TBP (lanes 3, 5, 7, 9, and
11). After being washed, bound proteins were separated by
SDS-PAGE. Lane 1 shows 10% of input 35S-B" used
in the interaction assay.
25 (27, 28), whereas the TATA box in mRNA promoters is often TATAAA. Thus, the
pol III snRNA-type TATA box appears to be inverted with respect to
mRNA promoters. A specific 7-bp TFIIB recognition element (BRE) is
located immediately upstream of "mRNA" TATA boxes in some cases and consists of a stretch of mostly G and C nucleotides (21). BRFU
shares sequence similarities with TFIIB (12) and also interacts with
TBP (14). We have noticed that a G/C-rich region that fits closely to
the BRE consensus is located downstream from the inverted TATA boxes in
the 7SK gene and fortuitously in the U2 TATA constructs (7)2 that are transcribed
efficiently by pol III. Thus, this sequence may play a role in either
BRFU recruitment and/or assembly of whole snRNA-specific TFIIIB and,
consequently, modulation of pol III transcription. As a potential
BRFU-binding site, we have tentatively named this region the
BRFU Recognition
Element (BURE).
). The "wt" construct
contains a region with remarkable similarity to an inverted ideal BRE:
(G/C)(G/C)(G/A)CGCC (21) just downstream of the TATA box (Fig.
5A, inv). The resulting "wild-type"
("wt") and mutant (BURE) probes were tested for their
potential to recruit two different TBP·BRFU complexes where the BRFU
is tagged with either GST or His. As shown in Fig. 5C, fewer
BRFU·TBP complexes were detected using the BURE probe
(lanes 4 and 6) than the "wt" probe
(lanes 3 and 5). Most important, this effect was
not due to a reduction of TBP binding to the BURE probe
as verified in Fig. 5C, lanes 1 and 2.
Interestingly, the BURE probe is able to retain even more
TBP in comparison to "wt" (Fig. 5C, lane 2 versus lane 1). Because no contacts outside the
8-bp TATA element have been observed in either structural or
biochemical studies of TBP·DNA interactions (29-31), we would not
expect the BURE sequence to simply modulate TBP binding. Similarly to
sequences flanking the TATA box on mRNA genes (32), the BURE may
rather influence TBP stability by altering its off rate rather than the on rate.
View larger version (39K):
[in a new window]
Fig. 5.
A novel GC-rich regulatory region "BURE"
downstream of the TATA box is responsible for efficient
DNA·TBP·BRFU complex formation and ensures efficient transcription
by pol III. Mutation of BURE strongly reduces pol III and
potentiates pol II transcription. A, schematic
representation of the U2/7SK gene promoter mutants. The
numbering indicates the distances from the pol III
transcription start site. The start sites and directions of the pol III
and pol II snRNA-specific transcriptions and the relative positions of
the protected transcripts by S1 probe are indicated above
the promoter structure. Below, in the sequence between 32
and 13, an introduced minimal TATA box is shaded and the
sequence that was then mutated is boxed
("wt"). The 3rd line shows the reverse
complement of this sequence with the BURE element
highlighted, and the TATA box shaded
(inv). Mutated nucleotides within the region enclosed by
dotted lines are shown in the 4th line. The
bottom line shows the resulting (BURE) mutant
in the original orientation. B, S1 nuclease analysis of
transcripts from U2mTATA/7SK and U2 BURE/mTATA/7SK hybrid
constructs (depicted in A) that were transfected into 293 (lanes 1 and 2) and HeLa cells (lanes
3 and 4). The U2mTATA/7SK template derivatives
transfected into each cell line are indicated above the
lanes. The positions of the pol III and pol II-specific
transcripts and the internal VA1 transcripts are indicated at the
left. An asterisk marks the undigested VA1 probe.
C, EMSAs were performed with the U2mTATA/7SK probes, shown
above the lanes, and TBP. In the left
panel, EMSA was conducted in the TGEM gel system in the absence of
BRFU (lanes 1 and 2), whereas the right
panel represents EMSA in the TBE gel system (see "Experimental
Procedures"), where GST-BRFU (lanes 3 and 4) or
His-BRFU (lanes 5 and 6) were added to the
reactions. The positions of GST-BRFU·TBP·DNA (arrow) and
His-BRFU·TBP·DNA (arrowhead) complexes are indicated at
the right. D, a, quantitation of the
TBP·DNA complexes (left panel of C) as a bar
graph is shown (mean ± S.D., n = 3).
D, b, the values shown for GST-BRFU·TBP·DNA
and His-BRFU·TBP·DNA complexes have been normalized to the mean
level of TBP·DNA complexes and are expressed relative to the values
obtained for the "wt" probe (arbitrarily designated 1.0). Each
bar represents the mean ± S.D. (n = 3).
26 and 18, both in vitro
(14) and in vivo.3
When a minimal TTTATA is present in the same U2/7SK construct, transcription that initiates at the same place as wild-type 7SK is
still detected (Fig. 5B, lanes 1 and
3; A and B, labeled pol III). When the BURE is mutated in "wt" (Fig. 5A,
BURE), the pol III transcript is practically
abolished (Fig. 5B, lanes 2 and 4).
The designated S1 oligonucleotide can also detect another transcript 6 nucleotides longer which, when transcribed from a TATA-less U2/7SK
template, originates at the U2 start site (Fig. 5A, labeled
pol II). This transcript has been identified previously as
-amanitin-sensitive and hence pol
II-dependent.3 Intriguingly, we find that
appearance of this transcript is responsive to BURE mutation and is in
the context of the intact minimal TATA box (Fig. 5B, labeled
pol II, lanes 2 and 4). These results
suggest that mutating BURE effectively reduces transcription by RNA
polymerase III from the U2mTATA/7SK template and allows pol II-type
transcription to occur.
U2mTATA/7SK probes (Fig. 5D,
b) was normalized to the corresponding binding of TBP to the
DNA (Fig. 5D, a). Mutation of the BURE reduces BRFU·TBP recruitment to ~28 and ~39%, respectively. Thus, the reduction in BRFU·TBP·DNA complex formation (Fig.
5D, b) correlates with the reduction in pol
III-specific transcription (Fig. 5B, lanes 2 and
4).
).
Fig. 6B demonstrates that this mutation decreases 7SK
transcription in both 293 cells and HeLa nuclear extract (Fig.
6B, compare lanes 1 and 3 with
lanes 2 and 4, respectively). Addition of a low
level of -amanitin to reconstituted transcription reactions (33) ensures that only polymerase III-specific transcription is detected. It
appears that for in vitro transcription, mutation of the
BURE has a smaller effect than the TATA box mutation (5) (Fig.
6B, compare lanes 4 and 5). We should
emphasize that the complete 7SK TATA box, TTTATATA, is able to recruit
TBP much more potently than the minimal TTTATA introduced into U2/7SK
(see short and long exposures of Figs. 6C and 5C,
lanes 1 and 2, respectively), and the strength of
binding of TBP on its own may well modulate the TBP·BRFU sub-complex
binding affinity to this class of promoter. Thus, the exact sequence of
the TATA box itself and the adjacent BURE may independently modulate
the level of transcription.
View larger version (39K):
[in a new window]
Fig. 6.
The intact BURE in the 7SK
gene contributes both to efficient pol III
transcription and formation of the trimeric TFIIIB-
complex at the promoter. A, the top
line shows 7SK promoter sequences from 31 to 13; the natural
TATA box is shaded, and the nucleotides that were then
mutated are boxed (wt). Below, the reverse
complement of the same sequence is shown with the BURE
highlighted and the TATA box shaded
(inv). The 3rd lane shows the nucleotides within
BURE that were mutated delimited by dotted lines. The
TTTTTT stretch that replaces these in the BURE deficient probe is shown
boxed (bottom line, marked
BURE). B, pol III transcription in
293 cells and HeLa nuclear extract, monitored by S1 analysis. 7SK
wild-type and 7SK-BURE mutant templates (depicted in A)
transfected into cells (lanes 1 and 2) or present
in each reaction (lanes 3 and 4) and 7SK TATA
box-deficient template (lane 5) are indicated above
the lanes. The positions of the 7SK and internal VA1 transcripts
are indicated at the right. C, EMSA conducted in
the TGEM gel system with a 7SK wild-type probe (lane 1) or a
7SK probe carrying BURE mutation (lane 2), in the presence
of TBP. D, EMSA performed in the TBE gel system with the 7SK
wt (wt) or the 7SK BURE mutant
(BURE) probe, and constant amounts of TBP and
BRFU, and either no B" (lanes 1 and 1') or
increasing amounts of B" (lanes 2-4 and 2'-4'), starting
from 0.05 to 1 pmol per reaction. The positions of BRFU·TBP·DNA
and B"·BRFU·TBP·DNA complexes are indicated at the
left.
Complex at the Promoter--
Our previous
observation that mutating the BURE strongly reduces pol III
transcription from two different snRNA-specific templates and the
results on impaired recruitment of TBP·BRFU sub-complexes suggest
that BURE can play a role in the assembly of TFIIIB- on the
promoter. Does it always function at the first step characterized by
TBP·BRFU recruitment? A demonstration of an additional molecular mechanism is shown in Fig. 6D. EMSA probes were derived from
7SK wt and 7SK BURE transcription constructs and
incubated with TBP, BRFU, and B". Strikingly, in the absence of B",
TBP·BRFU complex binding affinity to BURE probe is only
slightly reduced and parallels the binding of TBP alone (compare
lanes 1 and 1' in Fig. 6D with
lanes 1 and 2 in Fig. 6C). However,
increasing amounts of B" reveal that a larger complex is formed only on
the wt probe but not on the BURE probe (Fig.
6D, see lanes 3 and 3'). The highest
B" concentration used does not contribute to the enrichment of specific
DNA·TBP·BRFU·B" complex but, considering loss of lower
DNA·TBP·BRFU signal (Fig. 6D, lane 4), we
assumed larger than quaternary complexes are assembled using wt probe,
and these are incapable of entering the 3.5% non-denaturing gel. In contrast, the ternary DNA·TBP·BRFU complex is
prevalent on the BURE probe with little appearance of a
larger complex (Fig. 6D, lane 4'). The presented
data suggest that, in contrast to wt probe, conformation of the
TBP·BRFU sub-complex is altered in the absence of BURE, which causes
a reduction in B" recruitment and a subsequent low level of positioned
intact TFIIIB- complex for pol III transcription.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and (17, 18), required for transcription of snRNA genes and tRNA-type genes, respectively. Both types of promoter require TFIIIB components TBP and hB"/TFIIIB150 for activity, whereas specificity for snRNA promoters is given by the TFIIIB- component BRFU/TFIIIB50 (12, 13). Functional analysis of the BRFU/TFIIIB50 and hB"/TFIIIB150 requirements in a reconstituted U6/7SK
transcription system (12, 13), together with the demonstration that TBP
and BRFU are recruited coordinately to TATA box-containing promoters
(14), suggests that TFIIIB- is minimally a three-subunit complex. We
have therefore addressed the requirements for the assembly of this
complex in solution and for recruitment to snRNA promoters.
activity is not stably associated with TBP during purification (13), it
could be that TBP, BRFU, and DNA serve as mutually reinforcing and
interdependent components that together form a basis for hB"
recruitment. The presented data suggest that, unlike TFIIIB- that
depends on DNA-anchored TFIIIC (18), TFIIIB- can find its own way to
the promoter in naked DNA, which is initiated by a direct interaction
of TBP with a TATA box.
has shed some
light on the requirements for complex formation. The evidence that the
N terminus/zinc ribbon domain is primarily required for BRFU·TBP
association is surprising in the light of data on DNA·TBP·BRFU complex formation, where instead repeat 2 of the core was necessary (14). However, it is conceivable that the conformation of BRFU changes
when bound to DNA. In addition, radical deletions may alter the overall
conformation of the protein being analyzed. TBP has also been shown
here to bind independently to hB". Importantly, these interactions
allow association of both BRFU and hB", which does not occur in the
absence of TBP. Therefore, TBP is essential for mediating the assembly
of TFIIIB- de novo both on and off DNA. Moreover, the
C-terminal half of TBP, which binds BRFU, is sufficient to bridge BRFU
and hB". It was unexpected that deletion of the zinc ribbon domain
enables the direct association between BRFU and hB". How does this
region down-regulate BRFU binding to the hB"? It could perhaps mask
potential binding site(s) within BRFU. Because the N terminus is a
major contributor to the BRFU·TBP interaction, the rest of the
molecule is likely to be exposed for contact(s) with hB". An intriguing
possibility is that TBP, as well as interacting directly with hB",
recruits hB" indirectly through changes in the conformation of BRFU and
thus exposing its surface for binding to hB". Taken together, these
data indicate that the N-terminal region of BRFU acts as a damper of
hB" binding that is relieved by BRFU·TBP contacts. This would ensure
stable interactions in solution only when all three components are present.
complex formation as a whole and subsequently in determining the overall strength of the pol
III promoter. In agreement with the results of our gel retardation
analysis, hB" can be detected at the U6 promoter in vivo by
chromatin immunoprecipitation analysis indicating close contact with
the promoter (12). However, direct interaction between hB" and DNA
remains to be demonstrated.
View larger version (29K):
[in a new window]
Fig. 7.
A model for two-step modulation of
TFIIIB- complex assembly by the BURE.
Wild-type, BRFU contacts TBP and the BURE to form a strong ternary
complex which is joined by B". pol III snRNA gene transcription is then
favored. For BURE mutants, I, in the context of minimal TATA
box, BRFU is unable to recognize the debilitated BURE and therefore to
associate strongly with TBP·DNA complex. Pol III transcription is
then greatly diminished, and pol II transcription can occur, indicating
that TFIIB and the remaining pol II transcription machinery can now
assemble. For mutant II, in the context of complete TATA
box, TBP and BRFU form a relatively strong but altered complex, which
is not readily recognized by B". Unavailability of functional
TFIIIB- results in a reduction of pol III transcription.
The existence of such an additional promoter element would enable snRNA
promoters to encode a broad range of promoter strength and specificity
and increases the potential for regulation by transcriptional
activators and repressors.
| |
ACKNOWLEDGEMENTS |
|---|
We thank Nouria Hernandez for plasmids encoding His-tagged hBRFU and hB". Human recombinant untagged TBP was provided by Joost Zomerdijk. His-tagged core TBP was prepared by Diana Boyd.
| |
FOOTNOTES |
|---|
* This work was supported by Medical Research Council Senior Fellowship G117/309 (to S. M.).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.
To whom correspondence may be addressed. Tel.: 44-1865-275583;
Fax: 44-1865-275556; E-mail: pavel.cabart@path.ox.ac.uk.
§ To whom correspondence may be addressed. Tel.: 44-1865-275583; Fax: 44-1865-275556; E-mail: smurphy@molbiol.ox.ac.uk.
Published, JBC Papers in Press, May 16, 2002, DOI 10.1074/jbc.M203119200
2
P. abart and S. Murphy, unpublished results.
3 S. Murphy, unpublished observations.
| |
ABBREVIATIONS |
|---|
The abbreviations used are: snRNA, small nuclear RNA; pol III, RNA polymerase III; TFIIIB, transcription factor IIIB; TBP, TATA-binding protein; BRE, IIB recognition element; BURE, BRFU recognition element; GST, glutathione S-transferase; EMSA, electrophoretic mobility shift assay.
| |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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