MOLECULAR AND CELLULAR BIOLOGY, Nov. 2005, p. 9406–9418
Copyright © 2005, American Society for Microbiology. All Rights Reserved.
Vol. 25, No. 21
Structure-Function Analysis of the Human TFIIB-Related Factor II
Protein Reveals an Essential Role for the C-Terminal Domain
in RNA Polymerase III Transcription
Ashish Saxena,1,2Beicong Ma,3Laura Schramm,2† and Nouria Hernandez2,3*
Genetics Program, Stony Brook University, Stony Brook, New York 117941; Cold Spring Harbor Laboratory,
Cold Spring Harbor, New York 117242; and Howard Hughes Medical Institute, Cold Spring Harbor
Laboratory, Cold Spring Harbor, New York 117243
Received 3 June 2005/Returned for modification 26 July 2005/Accepted 4 August 2005
The transcription factors TFIIB, Brf1, and Brf2 share related N-terminal zinc ribbon and core domains.
TFIIB bridges RNA polymerase II (Pol II) with the promoter-bound preinitiation complex, whereas Brf1
and Brf2 are involved, as part of activities also containing TBP and Bdp1 and referred to here as Brf1-TFIIIB
and Brf2-TFIIIB, in the recruitment of Pol III. Brf1-TFIIIB recruits Pol III to type 1 and 2 promoters and
Brf2-TFIIIB to type 3 promoters such as the human U6 promoter. Brf1 and Brf2 both have a C-terminal
extension absent in TFIIB, but their C-terminal extensions are unrelated. In yeast Brf1, the C-terminal
extension interacts with the TBP/TATA box complex and contributes to the recruitment of Bdp1. Here we have
tested truncated Brf2, as well as Brf2/TFIIB chimeric proteins for U6 transcription and for assembly of U6
preinitiation complexes. Our results characterize functions of various human Brf2 domains and reveal that the
C-terminal domain is required for efficient association of the protein with U6 promoter-bound TBP and SNAPc,
a type 3 promoter-specific transcription factor, and for efficient recruitment of Bdp1. This in turn suggests that
the C-terminal extensions in Brf1 and Brf2 are crucial to specific recruitment of Pol III over Pol II.
In eukaryotes, the job of transcribing nuclear genes is di-
vided among three different RNA polymerases: RNA polymer-
ase I (Pol I), RNA Pol II, and RNA Pol III. A common step in
transcription initiation by all three eukaryotic RNA poly-
merases is the recruitment of the polymerase to the proper
promoters by a specific factor or complex. This transcription
factor serves as a bridge between promoter-bound factors that
nucleate preinitiation complex assembly and the RNA poly-
merase enzyme itself. For Pol II and Pol III transcription, the
factors that accomplish this task are TFIIB and TFIIIB, re-
Although TFIIB is a single polypeptide, the TFIIIB activity
is minimally composed of three polypeptides (see references
11 and 33 for reviews). In human cells, two forms of the TFIIIB
complex have been characterized thus far: Brf1-TFIIIB, which
functions on type 1 and 2 Pol III promoters, and Brf2-TFIIIB,
which functions on type 3 Pol III promoters such as the human
U6 small nuclear RNA (snRNA) promoter. Brf1-TFIIIB and
Brf2-TFIIIB both contain the TATA-box binding protein TBP
and the SANT domain protein Bdp1. They differ by the pres-
ence of different members of a family of TFIIB-related factors:
Brf1 in Brf1-TFIIIB and Brf2 in Brf2-TFIIIB.
TFIIB, Brf1, and Brf2 each contain a Zn ribbon domain at
their N terminus, followed by a core domain consisting of two
imperfect repeats. In this region, human Brf2 is 20% identical
with human TFIIB and 18% identical with human Brf1. In
addition, Brf1 and Brf2 contain a C-terminal extension. In
Brf1, this C-terminal segment contains two regions called ho-
mology domains II and III, which are conserved among yeast
and human proteins. Moreover, a third region called homology
domain I is conserved among several different yeast species
(25, 28). Such domains have thus far not been characterized
Although all TFIIB family members share the function of
bridging promoter-bound factors and the Pol, TFIIB and yeast
Brf1 accomplish this function differently, even on TATA-box-
containing Pol II and Pol III promoters. Indeed, each TFIIB
family member can associate with a TBP/TATA box complex,
but TFIIB and Brf1 do so primarily through different regions.
TFIIB assembles with the TBP/TATA box complex through its
core domain, which interacts directly with a region within the
C-terminal stirrup of the TBP core domain (3, 12, 17, 30, 31,
42). The core domain of yeast Brf1 also interacts with the
TBP/TATA box complex, but the highest-affinity anchorage
point is between homology domain II within the C terminus of
Brf1 and surfaces on the N-terminal lobe of the TBP core
domain, on the face of the TBP/TATA box complex opposite
the TFIIB binding site (9, 19, 20, 22, 36).
TFIIB and Brf1 also recruit the RNA Pol differently. With
TFIIB, the Zn ribbon domain is crucial for the recruitment of
RNA Pol II: it interacts with the dock domain of Pol II and
allows the positioning of the TFIIB core domain on the poly-
merase (3, 5, 7, 8, 12, 17, 31, 42; see reference 13 for a review).
In contrast, with Brf1 the zinc ribbon is not necessary for
Pol III recruitment but instead plays an essential role at a later
stage in transcription initiation, that of promoter opening (14,
20, 23). It is likely that interactions between the C34 subunit of
Pol III and the Brf1 core and homology regions I and II (1, 25,
40, 41), as well as between the C17 subunit of Pol III and the
Brf1 core (10), are strong enough for recruitment of the poly-
* Corresponding author. Mailing address: Cold Spring Harbor Lab-
oratory, 1 Bungtown Rd., Cold Spring Harbor, NY 11724. Phone:
(516) 367-8421. Fax: (516) 367-6801. E-mail: Hernande@cshl.edu.
† Present address: Department of Biological Sciences, St. John’s
University, Jamaica, NY 11439.
merase even in the absence of any protein-protein contact
contributed by the zinc ribbon.
Like Brf1, Brf2 is a Pol III transcription factor, but it is
specifically required for transcription from type 3 promoters
such as the human U6 promoter. These promoters are located
upstream of the transcribed region and their core region con-
sists of a proximal sequence element (PSE) and a TATA box,
which are recognized by a multisubunit complex we call SNAPc
and by TBP, respectively. We have now examined the functions
of various regions of Brf2 for U6 transcription and for assem-
bly of a preinitiation complex containing SNAPc, TBP, Brf2,
and Bdp1, and we have tested the ability of Brf2/TFIIB chi-
meric proteins to perform these functions. The results suggest
that a common feature among TFIIB family members involved
in Pol III transcription is the use of their C-terminal extension
to associate efficiently with a TBP/TATA box complex and to
MATERIALS AND METHODS
Sources of proteins. Full-length Brf2 and TFIIB, as well as the Brf2 trunca-
tions and the four TFIIB/Brf2 chimeras, were all expressed in Escherichia coli
with the T7 system from (37). The proteins contained an N-terminal FLAG tag
and a C-terminal His tag, except for full-length Brf2, which had only the
C-terminal His tag. The proteins were purified via their His tags by Ni-nitrilo-
triacetic acid (NTA) agarose (QIAGEN) chromatography and analyzed by so-
dium dodecyl sulfate (SDS)–12.5% polyacrylamide gel electrophoresis. The gels
were stained with the GelCode Blue Stain reagent (Pierce) for 1 h according to
the manufacturer’s instructions and were scanned with an Odyssey Infrared
Imager (LI-COR) to measure the intensities of the appropriate bands. The
proteins were also analyzed by immunoblotting with the Odyssey Infrared Im-
aging System (LI-COR).
TBP was expressed in E. coli as a glutathione S-transferase (GST)-fusion
protein and bound to glutathione agarose beads. The beads were washed, and
TBP was released from the beads by thrombin cleavage at a site located just after
the GST tag. Bdp1 was expressed in E. coli and purified first through a
C-terminal His tag by Ni-NTA agarose chromatography and then through an
N-terminal FLAG tag by anti-FLAG Antibody M2 agarose (Sigma) chromatog-
raphy. Both TBP and Bdp1 were dialyzed against buffer D100(50 mM HEPES
[pH 7.9], 0.2 mM EDTA, 20% glycerol, 0.1% Tween 20, 100 mM KCl, 3 mM
dithiothreitol [DTT], 0.5 mM phenylmethylsulfonyl fluoride).
SNAPcwas expressed in Sf9 insect cells as described previously (15) and
purified via a His tag fused to the C terminus of SNAP190 by Ni-NTA agarose
(QIAGEN) chromatography. The RNA Pol III complex used in the reconsti-
tuted U6 transcription system was purified from a HeLa cell line expressing a
FLAG- and His-tagged RPC4 (RPC53) subunit of Pol III, as described previ-
For the GST pull-down assay, GST, GST-tagged PCMT, and GST-tagged TBP
were all expressed in E. coli with the T7 system from (37) and bound to gluta-
thione-agarose beads. The beads were then washed four times with HEMGN150
buffer (25 mM HEPES [pH 7.9], 150 mM KCl, 12.5 mM MgCl2, 0.1 mM EDTA,
10% glycerol, 0.1% NP-40, 1 mM phenylmethylsulfonyl fluoride, 2 mM DTT,
and protease inhibitors), resuspended in the same buffer, and stored at 4°C.
In vitro transcription. The reconstituted U6 transcription assay and RNA
analysis were performed as described by (18).
Electrophoretic mobility shift assays. The binding reactions contained the
proteins indicated in the figure legends and 9 ?g of fetal bovine serum, 25 ng of
poly(dG-dC)-poly(dG-dC), and 75 ng of pUC118 in a buffer containing 20 mM
HEPES (pH 7.9), 100 mM KCl, 10 mM MgCl2, 0.2 mM EDTA, 10% glycerol, 1
mM DTT, 0.07% Tween 20, and 0.15 mM ATP. The total reaction volume was
20 ?l. The mixtures were incubated at 4°C for 20 min before addition of the
radiolabeled probe and further incubation at 30°C for 30 min. The samples were
then fractionated on a 4% polyacrylamide gel (39:1 acrylamide-bisacrylamide) in
TGE buffer (50 mM Tris base, 380 mM glycine, 2 mM EDTA).
GST pull-down assay. Either full-length Brf2, full-length human TFIIB, the
B/2/? chimera, or the B/2/2 chimera were mixed with 10 ?l of glutathione-
agarose beads bound by either GST, GST-PCMT, or GST-TBP in HEMGN150
buffer containing 400 ?g of ethidium bromide/ml in a total reaction volume of
60 ?l. The reactions were incubated for 2 h at 4°C, rotating end over end. The
FIG. 1. Truncated Brf2 proteins lacking the regions N or C terminal
to the core domain are inactive for U6 transcription. (A) Structure of
human TFIIB, Brf1, and Brf2 and of the recombinant “full-length” (FL)
ing to the zinc ribbon structure determined by nuclear magnetic reso-
nance in Pyrococcus furiosus TFIIB (44) and those modeled in Saccharo-
myces cerevisiae TFIIB and Brf1 (14) are indicated in light blue. The
locations of the structured core domain of human TFIIB (2, 30) and the
corresponding regions of human Brf1 and Brf2 are indicated in light
purple. The histidine tag (HT) is indicated in red. (B) The full-length Brf2
protein (Brf2 FL) or the derivatives indicated above the lanes were pro-
duced in E. coli, purified on a nickel affinity column, fractionated on an
SDS-polyacrylamide gel, and visualized by staining with the GelCode
Blue Stain reagent (Pierce). (C) After normalization of the amounts, the
same proteins were visualized by immunoblot with an anti-Brf2 antibody
(CS1230, 1:5,000 dilution). (D) The transcription reactions contained
100 ng of TBP, 350 ng of Bdp1, 2 ?l of SNAPc, and 2 ?l of Pol III. They
were complemented with either buffer alone (lane 1) or 0.5, 1, and 2 ?g
of full-length Brf2 (lanes 2 to 4), Brf2(66-419) (lanes 5 to 7), or Brf2(2-
289) (lanes 8 to 10). U6, correctly initiated U6 RNA; IC, internal control
for RNA handling and recovery. In lane 11, only the internal control was
VOL. 25, 2005STRUCTURE-FUNCTION ANALYSIS OF Brf29407
9408SAXENA ET AL.MOL. CELL. BIOL.
beads were then washed two times with HEMGN150, two times with HEMGN300
(containing 300 mM KCl instead of 150 mM KCl), and two times once again with
HEMGN150, always in the presence of 400 ?g of ethidium bromide/ml. The
beads were then resuspended in 5? Laemmli buffer (325 mM Tris-HCl [pH 8],
10% SDS, 0.025% bromophenol blue, 50% glycerol, 500 mM ?-mercaptoetha-
nol) and boiled. The proteins eluted from the beads were then fractionated on a
10% SDS-polyacrylamide gel, transferred to a nitrocellulose membrane, and
visualized by immunoblot with an anti-His tag monoclonal antibody (QIAGEN)
at a 1:3,000 dilution. The immunoblots were developed with the Odyssey Infra-
red Imaging System (LI-COR).
Both the N- and the C-terminal regions of Brf2 are required
for U6 snRNA gene transcription. The top of Fig. 1A shows a
comparison of the structures of human TFIIB, Brf1, and Brf2.
These three proteins each contain a zinc-binding domain
(shown in blue) and a core domain (shown in purple). Brf1 and
Brf2 additionally contain unique C-terminal domains. To de-
termine the role of different parts of Brf2, we synthesized the
truncated Brf2 proteins illustrated in the lower part of Fig. 1A.
Brf2(66-419) lacks the region N-terminal to the core domain,
which includes the Zn ribbon motif. The second truncation,
Brf2(2-289), lacks the C-terminal domain of the protein. The
full-length and truncated proteins all contain a histidine tag at
their C terminus (indicated in red), which was used for puri-
fication after expression in E. coli. The resulting proteins are
shown in Fig. 1B and, as revealed by immunoblot with an
anti-Brf2 antibody after normalization of the amounts, in
Fig. 1C. These proteins were then tested for U6 transcription
in a minimal in vitro transcription system, which consists of
recombinant TBP, Bdp1, Brf2, and SNAPccombined with
highly purified Pol III from a HeLa cell line expressing tagged
RPC4 (RPC53) (18).
As seen in Fig. 1D, TBP, Bdp1, and SNAPccombined with
purified Pol III in the absence of Brf2 did not give rise to any
detectable U6 transcript in the reconstituted in vitro transcrip-
tion assay (lane 1). As expected, addition of recombinant full-
length Brf2 resulted in efficient U6 transcription (lanes 2 to 4).
However, when either Brf2(66-419) or Brf2(2-289) were used
in place of full-length Brf2, U6 transcripts were not detectable
(lanes 5 to 7 and lanes 8 to 10, respectively), suggesting that
both of the regions N and C terminal to the Brf2 core domain
are important for transcriptional activity.
Visualization by EMSA of the assembly of a U6 transcrip-
tion preinitiation complex. To enable us to study the functions
of the N- and C-terminal regions of Brf2 in the formation of
the transcription initiation complex, we set out to establish an
electrophoretic mobility shift assay (EMSA) in which we could
visualize the assembly of the various U6 transcription factors
on the U6 promoter. We first focused on the Brf2-TFIIIB
components. As shown in Fig. 2A, unlike Brf2 or Bdp1, TBP
on its own was able to form a protein-DNA complex on a
probe containing the wild-type U6 PSE and TATA box (lanes
2 to 4). As observed previously, the addition of both TBP and
Brf2 resulted in the formation of a prominent complex (hence-
forth referred to as the “two-factor complex,” labeled 2 in
lane 5; see also Fig. 2E, which summarizes the nomenclature
used for some of the complexes), reflecting the cooperative
binding of these two factors (6, 27, 43). With TBP and Bdp1, a
weak complex migrating much more slowly than the TBP com-
plex was obtained (lane 6, complex labeled TBP/Bdp1). Since
Bdp1 on its own does not bind detectably to the probe (lane 4),
this suggests protein-protein interactions between Bdp1 and
TBP on the DNA. With TBP, Brf2, and Bdp1, a robust com-
plex migrating even more slowly than the TBP/Bdp1 complex
was obtained (henceforth referred to as the “three-factor com-
plex,” labeled 3 in lane 7; see Fig. 2E). This complex was
supershifted by anti-Bdp1 and anti-TBP antibodies (which as
expected did not bind to the probe on their own, see lanes 12
and 13), confirming that it contains Bdp1 and TBP (compare
lanes 8 to 11 to lane 7). On the other hand, we did not observe
a supershift with any of our anti-Brf2 antibodies (data not
shown). Nevertheless, since the three-factor complex was not
observed in the absence of Brf2 and migrated more slowly than
the TBP/Bdp1 complex (compare lanes 6 and 7), it is highly
likely that it indeed contains Brf2 but that the Brf2 epitopes
recognized by the antibodies we tested are masked. None of
FIG. 2. Assembly of the U6 preinitiation complex. (A) Assembly of the Brf2-TFIIIB complex on the U6 TATA box. Either a probe with a
wild-type mouse U6 PSE and human U6 TATA box (lanes 1 to 13) or a wild-type mouse PSE and a mutant TATA box (lanes 14 to 17) was used.
Lanes 1 and 14 show the probes alone. We added 10.5 ng of TBP, 75 ng of Brf2, and 113 ng of Bdp1 to the binding reactions as indicated above
the lanes, as well as 1 ?l of 1:3-diluted (lane 8) and 1 ?l of undiluted (lanes 9 and 12) purified rabbit polyclonal anti-Bdp1 antibody (CS913) and
1 ?l of 1:3 diluted (lane 10) and 1 ?l of undiluted (lanes 11 and 13) purified mouse monoclonal anti-TBP antibody (SL30b). The locations of
protein/DNA complexes containing TBP only, TBP and Bdp1, TBP and Brf2 (complex 2), or TBP, Brf2, and Bdp1 (complex 3) are indicated, as
well as are the supershifts obtained after antibody addition. (B) Assembly of a four-factor complex. The probe contained a wild-type human PSE
and a wild-type human TATA box. The amounts of TBP, Brf2, and Bdp1 were as described in panel A. In lanes 2 to 6 and lanes 7 to 11, 1 ?l of
a 1:54, 1:27, 1:9, 1:3, or 1:1 dilution of SNAPcwas added. The locations of the complexes containing SNAPcalone, TBP and Brf2 (complex 2), TBP,
Brf2, and Bdp1 (complex 3), or TBP, Brf2, Bdp1, and SNAPc(complex 4) are indicated. (C) The four-factor complex is supershifted by anti-TBP,
anti-Bdp1, and anti-SNAP190 antibodies. The probe contained a wild-type human PSE and a wild-type human TATA box. The amounts of TBP,
Brf2, and Bdp1 were as described in panel A. The binding reactions contained, in addition, 1 ?l of a 1:2 dilution of SNAPc(lanes 2 to 8) and 1 ?l
of 1:3 diluted (lane 3) or 1 ?l of undiluted (lane 4) mouse monoclonal anti-TBP antibodies (SL30b), 0.5 and 1 ?l of rabbit polyclonal anti-Bdp1
antibodies (CS913, lanes 5 and 6), and 1 ?l of 1:3 diluted (lane 7) and 1 ?l of undiluted (lane 8) rabbit polyclonal anti-SNAP190 antibody (CS696).
The locations of the complexes containing SNAPcalone, TBP, Brf2, and Bdp1 (complex 3) or TBP, Brf2, Bdp1, and SNAPc(complex 4), as well
as the supershifts obtained after antibody addition, are indicated. The dotted white line indicates the position of complex 4 across the gel and serves
to reveal the very slight supershift obtained with the anti-TBP antibodies. (D) Subcomplexes of the four-factor complex. The probe contained a
wild-type human PSE and either a wild-type human TATA box (lanes 1 to 6) or a mutated TATA box (lanes 7 to 10). Where indicated above the
lanes, the binding reactions contained the same amounts of TBP, Brf2, and Bdp1 as in panel A and 1 ?l of 1:15 diluted SNAPcexcept for the
reaction in lane 9, which contained half this amount. The locations of complexes containing SNAPc, or TBP and Brf2 (complex 2), or TBP, Brf2,
and SNAPc(complex labeled with a white dot), or TBP, Brf2, and Bdp1 (complex 3), or TBP, Brf2, Bdp1, and SNAPc(complex 4) are indicated.
(E) Nomenclature used for some of the complexes.
VOL. 25, 2005STRUCTURE-FUNCTION ANALYSIS OF Brf29409
the complexes were observed with a probe containing a mu-
tated TATA box (lanes 15 to 17), indicating that they all
depend on TBP-TATA box interactions.
We next combined the Brf2-TFIIIB components with
SNAPc. As shown in Fig. 2B, as we titrated increasing amounts
of SNAPcinto a binding reaction containing the three Brf2-
TFIIIB components, we observed a gradual increase in the
formation of a large complex (henceforth referred to as the
“four-factor complex,” labeled 4 in lane 4; see Fig. 2E) migrat-
ing more slowly than the three-factor complex containing TBP,
Brf2, and Bdp1 (compare complex 4 in lanes 4 to 6 with
complex 3 in lane 1). This complex was not observed in the
absence of the Brf2-TFIIIB components (lanes 7 to 11) and
could be supershifted with anti-TBP, anti-Bdp1, and anti-
SNAP190 antibodies (Fig. 2C, compare lanes 3 to 8 with lane
2), confirming that it contains TBP, Bdp1, and SNAPc. Like
complex 3, complex 4 was not supershifted by the anti-Brf2
antibodies we tested (data not shown), most likely because
within the complex, the relevant Brf2 epitopes are not acces-
sible to the antibodies.
In addition to complex 4, binding reactions containing
SNAPcand Brf2-TFIIIB gave rise to four prominent smaller
complexes (see, for example, Fig. 2B, lane 5). As shown in
Fig. 2D, comparison with binding reactions containing various
subsets of the four components on either a wild-type probe or
a probe with a mutated TATA box indicated that, of these four
smaller complexes, the one migrating the fastest is the two-
factor complex containing TBP and Brf2 (compare lanes 3 and
6, complex 2, see also lanes 8 to 10), whereas the one above it
corresponds to just SNAPcbound to the DNA (compare lanes
1 and 6; see also lanes 4, 7, 9, and 10). The third complex from
the top in lane 6 comigrates with one formed by TBP, Brf2, and
SNAPc(compare lanes 4 and 6, complex labeled with a white
circle; see also lanes 9 and 10), whereas the complex just below
the four-factor complex in lane 6 corresponds to the three-
factor complex (compare lanes 5 and 6, see also lanes 8 to 10).
Thus, by EMSA we can visualize a complex containing the
basal transcription factors SNAPcand Brf2-TFIIIB, which are
known to be sufficient to reconstitute U6 transcription when
combined with a highly purified Pol III complex (18), as well as
visualize various subcomplexes.
The C-terminal region of Brf2, but not the N-terminal re-
gion, is required for binding with TBP and interacting with
SNAPcon the U6 snRNA gene promoter. We used the EMSA
system described above to determine whether the N- and
C-terminal regions of Brf2 might be involved in initiation com-
plex assembly. In the interest of space, all EMSA experiments
presented hereafter show only the region corresponding to the
upper half of the gel containing the various complexes. Lanes
1, 2, and 3 in Fig. 3A show the two-, three-, and four-factor
complexes (containing, respectively, TBP and Brf2; TBP, Brf2
and Bdp1; and TBP, Brf2, Bdp1 and SNAPc) obtained with
full-length Brf2 (complexes labeled 2, 3, and 4). With the
Brf2(66-419) truncated protein, the two-, three-, and four-
factor complexes were all observed (lanes 10 to 12), indicating
that the region N-terminal to the core domain is not required
for binding to the TBP-TATA box complex (lane 10), recruit-
ment of Bdp1 (lane 11), or assembly of the full complex (lane
12). In contrast, with a Brf2(66-289) truncated protein contain-
ing just the Brf2 core domain and with the Brf2(2-289) trun-
cated protein, none of these complexes were obtained (lanes 4
to 9). In particular, we did not observe a two-component com-
plex containing TBP and a truncated Brf2 (lanes 4 and 7). The
only complexes observed were those containing either just
SNAPc(compare lanes 6 and 9 with lane 16) or SNAPcand
TBP (compare lanes 6 and 9 with lane 13), a finding consistent
with previous observations that SNAPcand TBP bind cooper-
atively to the U6 promoter (27, 29). Thus, the Brf2 region
C-terminal of the core domain is required for detectable bind-
ing of Brf2 together with TBP to the U6 TATA box in the
EMSA. Moreover, it is required for the formation of all sub-
sequent complexes containing both Brf2 and TBP.
Brf2, which does not bind to DNA on its own, can be re-
cruited to the U6 promoter not only through protein-protein
interactions with TBP binding to the TATA box but also
FIG. 3. Activity of truncated versions of Brf2 in preinitiation com-
plex assembly. (A) The C-terminal region of Brf2 is required for
binding to the TBP/TATA box complex, whereas the N-terminal re-
gion is not required for preinitiation complex assembly. The probe
contained a wild-type human PSE and a wild-type human TATA box.
Where indicated above the lanes, the binding reactions contained
10.5 ng of TBP, 113 ng of Bdp1, 1 ?l of 1:2 diluted SNAPc, and 250 ng
of full-length Brf2 (Brf2 FL) or derivatives as indicated above the
lanes. The various complexes are labeled with the symbols indicated in
Fig. 2E on the left of each lane or with the factors they contain on the
sides of the panel. (B) The C-terminal region of Brf2 is required for
cooperative binding with SNAPc. The probe contained wild-type hu-
man U6 PSE and TATA box. Where indicated above the lanes, the
binding reactions contained the same amounts of TBP, SNAPc, and
Brf2 or Brf2 derivatives as in panel A. The complexes labeled with an
arrowhead on the left of lanes 4 and 5 contain SNAPcand Brf2.
9410 SAXENA ET AL.MOL. CELL. BIOL.
through protein-protein interactions with SNAPcbinding to
the PSE (16). However, with the truncated Brf2 proteins miss-
ing the C-terminal region, we did not observe novel complexes
that might contain Brf2 but lack TBP. We wondered, there-
fore, whether the C-terminal region of Brf2 might be required
for cooperative binding with SNAPc. As shown in Fig. 3B,
when only SNAPcand Brf2 were added to the U6 promoter
probe, we observed a weak complex migrating more slowly
than the SNAPc-only complex and slightly faster than the TBP/
SNAPccomplex (lane 4, complex labeled with a white arrow-
head, compare with lanes 1 and 3). This complex most likely
contains SNAPcand Brf2 and presumably forms due to inter-
actions between Brf2 and the PSE-bound SNAPc. Notably, a
similar, even darker complex was observed when full-length
Brf2 was replaced by Brf2(66-419) but not when it was re-
placed by Brf2(2-289) (compare lane 4 with lanes 5 and 6).
Thus, the C-terminal region of Brf2 is not only required for
cooperative binding with TBP bound to the TATA box but also
for cooperative binding with SNAPcbound to the PSE.
These results indicate that the C-terminal domain of Brf2 is
necessary for the protein to form a complex with TBP, as well
as with SNAPc, on a U6 promoter. Because of the failure to
form these initial complexes, higher-order complexes in turn
cannot form. Thus, the inability of Brf2(2-289) to direct U6
transcription (Fig. 1D above) can be ascribed to a failure of
transcription initiation complex assembly. On the other hand,
the inability of Brf2(66-419) to direct U6 transcription proba-
bly results from a defect at a step after transcription factor
assembly, such as Pol III recruitment or promoter opening.
TFIIB/Brf2 chimeras. As discussed above, Brf2 and TFIIB
have several features in common but they are used by different
RNA polymerases—TFIIB by Pol II and Brf2 by Pol III. To
learn more about the functions of the various Brf2 regions and
the determinants of RNA polymerase specificity, we con-
structed a set of TFIIB/Brf2 chimeric proteins, which are
shown in Fig. 4A. We divided Brf2 into core region and regions
N and C terminal to the core and TFIIB into core region and
region N terminal to the core and constructed four chimeras by
swapping these regions between the two proteins. The chime-
ras were named based on the origin of the regions from which
they were comprised. Thus, for example, 2/B/? consists of the
N-terminal region of Brf2, the core domain of TFIIB, and no
C-terminal domain, whereas B/2/2 consists of the N-terminal
region of TFIIB and the core and C-terminal domains of Brf2.
The four chimeras, as well as full-length Brf2 and TFIIB, were
expressed in E. coli and partially purified through a C-terminal
His tag. The resulting proteins are shown in Fig. 4B as visual-
ized by staining with the GelCode Blue Stain reagent (Pierce)
and in Fig. 4C as visualized by immunoblot with an anti-His tag
antibody after normalization of the amounts.
We then tested the four chimeras for U6 transcription in the
minimal in vitro transcription system, and the results are shown
in Fig. 4D. Combining full-length Brf2, but not full-length
TFIIB, with TBP, Bdp1, SNAPc, and purified Pol III reconsti-
tuted U6 transcription (compare lanes 2 to 4 to lanes 5 to 7 and
to lane 1), consistent with previous observations showing that
TFIIB is not active for Pol III-directed U6 transcription (26).
Both the 2/B/? and B/2/? chimeras were inactive in this assay
(data not shown) as was the B/2/2 chimera (lanes 8 to 10). The
2/B/2 chimera showed barely detectable activity (lanes 11 to
13). This prompted us to widen our titration of the 2/B/2
chimera, but in all cases the U6 signal was extremely weak
(lanes 15 to 19), although clearly detectable in the original film.
Thus, the chimeras are generally inactive for U6 transcription,
except for the 2/B/2 protein, which displays extremely weak
TFIIB cannot recruit Bdp1 to the U6 promoter. To under-
stand the defects of TFIIB in terms of U6 transcription activity,
as well as those of the chimeric proteins, we tested their ca-
pacity to assemble a U6 preinitiation complex by using EMSA.
Figure 5A, lanes 1 to 6, show the locations of the SNAPc
complex alone (lane 1), the two-, three-, and four-factor com-
plexes (lanes 3, 5, and 6) and the complexes containing TBP
and SNAPc(lane 4, complex labeled TBP/SNAPc) and TBP,
Brf2, and SNAPc(lane 4, complex labeled with a white dot on
the left). We first focused on TFIIB. As expected, TFIIB alone
was unable to bind to the probe but bound cooperatively with
TBP to the U6 TATA box (lanes 7 and 8). However, upon
addition of Bdp1, no additional complex was observed (lane 9),
indicating that the TBP/TFIIB complex cannot recruit Bdp1.
Interestingly, when TBP, TFIIB, Bdp1, and SNAPcwere pre-
sent in the binding reaction, we obtained a complex containing
TBP, TFIIB, and SNAPc(lane 10, complex labeled with a
white dot on the left). Thus, on the Pol III U6 promoter, TFIIB
can assemble efficiently with TBP and SNAPcbut is unable to
The B/2/? chimera does not bind with TBP to the U6 pro-
moter, whereas the 2/B/? chimera does not recruit Bdp1. We
next examined the B/2/? and 2/B/? chimeras. In Fig. 5B, the
locations of the two-, three- and four-factor complexes in the
EMSA are shown (lanes 1 to 3), as are those of the complexes
containing just SNAPc(lane 13); TBP and SNAPc(lane 10,
complex labeled TBP/SNAPcon the left of the panel); and
TBP, Brf2, and SNAPc(lane 11, complex labeled with a white
dot on the left). The B/2/? chimeric protein contains the core
of Brf2 but lacks its C-terminal region. Consistent with the
deletion results obtained above, which indicate that the Brf2
C-terminal region is necessary for efficient cooperative binding
with TBP, as well as with SNAPc(Fig. 3), the B/2/? chimera
was unable to form any of the Brf2-dependent complexes
(lanes 4 to 6). The only complexes visible were those contain-
ing either just SNAPcor both SNAPcand TBP (see lane 6).
With the 2/B/? chimeric protein, which contains the core do-
main of TFIIB, we observed a two-factor complex containing
TBP and 2/B/? (lane 7), a finding consistent with the known
ability of the TFIIB core to bind to a TBP-TATA box complex
(3, 12, 17, 42). This complex was, however, unable to recruit
Bdp1 (lane 8), although it could assemble with SNAPcto form
a complex containing TBP, 2/B/?, and SNAPc (lane 9, com-
plex labeled with a white dot). This is consistent with the
inability of full-length TFIIB to recruit Bdp1 (Fig. 5A). More-
over, the results obtained above with Brf2(66-419) (Fig. 3A)
indicate that the N-terminal region of Brf2 is not required for
Bdp1 recruitment but do not address the possibility that this
region is capable of recruiting Bdp1 in a manner redundant
with the core or C-terminal regions of Brf2. The results with
the 2/B/? chimera show that the N terminus of Brf2 cannot
recruit Bdp1 when targeted to a TBP-TATA box complex.
Chimeric Brf2 proteins containing the N-terminal or core
region of TFIIB can support assembly of the four-factor com-
VOL. 25, 2005STRUCTURE-FUNCTION ANALYSIS OF Brf29411
plex. The activities of the B/2/? and 2/B/? chimeras in tran-
scription complex assembly indicated an essential role for the
Brf2 C-terminal domain in binding to the TBP-TATA box
complex and perhaps in recruiting Bdp1. We therefore tested
in the EMSA the B/2/2 and 2/B/2 chimeras, which differ from
B/2/? and 2/B/? only by addition of the Brf2 C-terminal do-
main. As shown in Fig. 5A, both proteins were capable of
forming a complex with TBP on the U6 TATA box (lanes 12
and 16, complex 2), although the complex formed with B/2/2
was weaker than that obtained with either Brf2 (lane 3) or
2/B/2. This is surprising since Brf2(66-419), which lacks the
N-terminal region of Brf2 altogether, binds as efficiently as
full-length Brf2 to a TBP/TATA box complex (Fig. 3). More-
over, as shown below, B/2/2 binds as efficiently as Brf2 to TBP
in a GST pull-down assay. These differences may indicate that
the presence of the N-terminal region of TFIIB in place of that
of Brf2 somehow reduces the B/2/2 chimera’s ability to assem-
ble with TBP on the U6 TATA box but not off the DNA. We
have not, however, pursued this observation further. For the
2/B/2 chimera, its binding to the TBP-TATA box complex may
be due to the TFIIB core region, the Brf2 C-terminal region, or
both since these two regions both contain TBP-binding activity.
However, as with B/2/2, the two-factor complex obtained with
2/B/2 migrated as a tight band close to the position observed
with full-length Brf2 (compare lanes 12 and 16 with lane 3) and
very different from the diffuse and slower-migrating complex
observed with TFIIB and TBP (lane 8). Although we do not
know what causes the TFIIB-TBP-TATA box complex to be
diffuse, this suggests that 2/B/2 binds to the TBP-TATA box
complex in a manner more similar to Brf2 than TFIIB.
The promoter-bound B/2/2 and 2/B/2 proteins could recruit
Bdp1 (Fig. 5A, lanes 13 and 17, complex 3) and SNAPc(lanes
14 and 18, complex 4), although the recruitment of SNAPcby
2/B/2 is not efficient (lane 18). The B/2/2 protein’s ability to
promote assembly of the four-factor complex is consistent with
the results above (Fig. 3) showing that the core and C-terminal
regions of Brf2 together are sufficient for interaction with TBP,
Bdp1, and SNAPcon the DNA. Its inactivity in U6 transcrip-
tion indicates that the essential function(s) of the N-terminal
region of Brf2 cannot be efficiently performed by the N-termi-
nal region of TFIIB. In the case of the 2/B/2 protein, its ability
to recruit Bdp1 contrasts with the inability of 2/B/? to perform
this same function and indicates that the Brf2 C-terminal re-
gion is required for Bdp1 binding. Moreover, the formation of
a four-factor complex with 2/B/2 indicates that when the
C-terminal region of Brf2 is present the core region of Brf2 can
be replaced with that of TFIIB. Indeed, the 2/B/2 chimera is
capable of directing a very low level of U6 transcription
(Fig. 4D). However, the efficiency of both four-factor complex
assembly and transcription is very low, indicating that either
the TFIIB core region is not placed optimally for full function
in the chimeric protein or that there is some function of the
Brf2 core region that cannot be fully replaced by the TFIIB
core region. One possibility is that the interactions between the
2/B/2 chimera and both Bdp1 and SNAPctogether may not be
stable enough to support efficient reconstituted U6 in vitro
transcription. Another possibility is that the core domain of
Brf2 is involved in Pol III recruitment, which was not assayed
in our EMSA experiments.
The C-terminal region of Brf2 is required for binding to
TBP off DNA. To test which Brf2 region is required to bind to
TBP off the promoter DNA, we performed the GST pull-down
experiment shown in Fig. 6. GST-TBP, as well as the nega-
tive controls GST and GST-protein carboxyl methyltransfer-
ase (GST-PCMT), were immobilized on glutathione-agarose
beads, and the binding of various histidine-tagged proteins to
these beads was visualized by immunoblotting with anti-HT
antibodies. Lanes 1 to 4 show the proteins tested, namely,
TFIIB, Brf2, B/2/?, and B/2/2. Both TFIIB and Brf2 bound
specifically to GST-TBP in this assay, with Brf2 binding even
more strongly than TFIIB (lanes 5 to 8). Strikingly, however,
the B/2/? chimera, which contains the Brf2 core but not the
Brf2 C-terminal region, bound very weakly to GST-TBP (lane
11), although the signal was clearly above the background seen
with both GST alone and GST-PCMT (lanes 9 and 10). With
the B/2/2 chimera, which contains the Brf2 C-terminal domain,
very efficient and specific binding to GST-TBP was observed
(lanes 12 to 14). These results provide further evidence for the
crucial role of the C-terminal region of Brf2 in TBP binding
and show that the region performs this function even off the
TFIIB, Brf1, and Brf2 are all members of a family of tran-
scription factors that share a number of common features, yet
they are used by different classes of promoters: TFIIB by Pol
II-recruiting promoters and Brf1 and Brf2 by Pol III-recruiting
promoters. We have analyzed here the functions of various
regions of Brf2 for U6 transcription complex assembly and in
vitro transcription, and we have tested the ability of Brf2/
TFIIB chimeric proteins to perform these functions. The re-
sults, which are summarized in Fig. 7, reveal an essential role
for the Brf2 C-terminal region and provide mechanistic in-
sights on how RNA polymerase specificity is determined at the
human snRNA promoters.
N-terminal region of Brf2. We find that the N-terminal re-
gion of Brf2 can be deleted or replaced by the TFIIB
N-terminal region without major effects on the ability of Brf2
to bind to a TBP/TATA box complex, recruit Bdp1, or interact
FIG. 4. Brf2-TFIIB chimeric proteins. (A) The structures of the various chimeras are shown. The numbers under the boxes correspond to
amino acids. The C-terminal His tags are indicated in red, and the N-terminal FLAG tags are indicated in blue. (B) The chimeras indicated above
the lanes were produced in E. coli, purified on a nickel affinity column, fractionated on an SDS-polyacrylamide gel, and visualized by staining with
the GelCode Blue Stain reagent (Pierce). (C) After normalization of the amounts, the same proteins were visualized by immunoblotting with a
mouse monoclonal anti-His tag antibody (QIAGEN) at a 1:3,000 dilution. (D) The transcription reactions contained 100 ng of TBP, 350 ng of
Bdp1, 2 ?l of SNAPc, and 2 ?l of Pol III. They were complemented with either buffer alone (lane 1); 1, 2, or 4 ?g of the proteins indicated above
the lanes (lanes 2 to 13); 1 ?g of Brf2 (lane 14); or 12, 37, 110, 330, or 1,000 ng of 2/B/2 (lanes 15 to 19). U6, correctly initiated U6 RNA; IC, internal
control for RNA handling and recovery.
VOL. 25, 2005STRUCTURE-FUNCTION ANALYSIS OF Brf29413
with SNAPc(Fig. 7). The N-terminal region is, however, re-
quired for U6 transcription, and for this function it cannot be
replaced by the TFIIB N-terminal domain. These results are
consistent with what is known about the roles of the N-terminal
domains of human and yeast TFIIB and yeast Brf1. Indeed, in
these proteins, the N-terminal domain is dispensable for asso-
ciation with a TBP-TATA box complex and, in the case of
yeast Brf1, for recruitment of Bdp1, but plays essential roles at
later stages (9, 20).
In TFIIB, the N-terminal domain associates tightly with
Pol II and is required for Pol II recruitment (3, 4, 12, 17, 31,
42). The crystal structure of a Pol II/TFIIB complex (5) and
site-specific photo-cross-linking studies of both a Pol II/TFIIB
complex and a complete preinitiation complex (7, 8; see ref-
erence 13 for a review) indicate that within the N-terminal
region of TFIIB, the zinc ribbon is located within the center of
the pocket formed by the clamp, wall, and dock domains of Pol
II, most likely making contacts with one surface of the dock,
whereas the B finger, which is located immediately down-
stream of the zinc ribbon on the linear TFIIB sequence, ex-
tends within the space from the RNA exit channel to the active
site of Pol II. The zinc ribbon and the B finger appear to form
the highest-affinity anchorage point between TFIIB and Pol II,
which then allows, with the help of other components of the
preinitiation complex, the proper positioning of the TFIIB
core region on the polymerase. This in turn likely orients the
promoter DNA over the central cleft of the polymerase (7, 8).
Thus, in the Pol II preinitiation complex, the N-terminal region
of TFIIB does not contribute essential contacts with TBP or
the promoter DNA but is intimately associated with the cata-
lytic core of the enzyme.
In yeast Brf1, the N-terminal domain is dispensable for re-
cruitment of Pol III but is required at a later stage, during
promoter opening (14, 20). In the case of human Brf2, the
function performed by its N-terminal domain is not known, but
FIG. 5. Activity of Brf2-TFIIB chimeras in U6 preinitiation com-
plex assembly. (A) Activity of TFIIB and chimeras containing the
C-terminal region of Brf2. The probe contained a wild-type human
PSE and a human TATA box. Where indicated above the lanes, the
binding reactions contained 10.5 ng of TBP, 113 ng of Bdp1, 1 ?l of
SNAPcdiluted 1:15, and 75 ng of Brf2, TFIIB, B/2/2, or 2/B/2, as
indicated. The various complexes are labeled with the symbols sum-
marized in Fig. 2E on the left of the lanes or with the factors that
they contain on the sides of the panel. (B) Activity of chimeras
lacking the C-terminal region of Brf2. The probe contained a wild-
type human PSE and a human TATA box. Where indicated above
the lanes, the binding reactions contained TBP, Bdp1, SNAPc, and
Brf2 or derivatives in similar amounts as in panel A. The various
complexes are labeled with the symbols summarized in Fig. 2E on
the left of the lanes or with the factors that they contain on the sides
of the panel.
FIG. 6. Binding of Brf2, TFIIB, and chimeric proteins to TBP off
DNA. GST alone, GST-TBP, or GST-PCMT were immobilized on
glutathione-agarose beads as indicated above lanes 5 to 14. 3 ?g of
TFIIB, Brf2, B/2/?, or B/2/2 were then mixed with 10 ?l of beads in the
presence of 400 ?g of ethidium bromide/ml. After incubation, the
beads were washed, resuspended in 5? Laemmli buffer, and boiled;
the eluted material was then loaded on an SDS-polyacrylamide gel.
The proteins were visualized with a mouse monoclonal anti-His tag
antibody (QIAGEN) at a 1:3,000 dilution. Lanes 1 to 4 show 20% of
the input material.
9414 SAXENA ET AL.MOL. CELL. BIOL.
it clearly cannot be performed by the TFIIB N-terminal region.
It is possible that the N-terminal regions of both Brf1 and Brf2
contribute in fact to specific recruitment of Pol III over Pol II.
The TFIIB, Brf1, and Brf2 zinc ribbon domains (but not the B
finger regions) are highly conserved (14, 34, 38), and so are the
Pol II and Pol III dock regions (see references 7), a finding
consistent with the Brf1 and Brf2 zinc ribbons associating with
Pol III in much the same way as the TFIIB zinc ribbon asso-
ciates with Pol II. Nevertheless, the specific amino acids in-
volved in the interactions may be different; indeed, it is striking
that, for example, the yeast TFIIB N-terminal region fails to
recruit human Pol II to the adenovirus 2 major late promoter,
even though both the yeast and human TFIIB N-terminal
regions can recruit yeast Pol II to this promoter (39). Similarly,
subtle differences within the zinc ribbons of TFIIB, Brf1, and
Brf2, as well as the lack of an obvious B finger in Brf1 and Brf2,
may contribute to specific recruitment of the correct RNA
polymerase. However, with Brf1, and perhaps Brf2, contacts
between the core and C-terminal domain and Pol III (1, 10, 25,
41) are clearly strong enough to allow recruitment of the poly-
merase even in the absence of the N-terminal region.
The N-terminal region of Brf2 may also be required, like
that of Brf1, for promoter opening, a function specific to
Pol III transcription initiation complexes which, unlike Pol II
transcription initiation complexes, do not require hydrolyzable
ATP to form the transcription bubble (21, 23, 24).
Core region of Brf2. In TFIIB, which does not contain a
C-terminal extension, the core region is sufficient for efficient
binding to the TBP/TATA box complex. This is different in
yeast Brf1, where the association of a truncated protein miss-
ing the C-terminal domain is so weak that it is only detected
after photochemical cross-linking (9, 20, 22, 35). Brf2 appears
to be very similar to Brf1. Deletion of either the core (6) or the
C-terminal domain (see Fig. 3 and 7) prevents association with
the TBP/TATA box complex as detected in an EMSA in the
absence of cross-linking, indicating that the core is required
but not sufficient for high-affinity association with the TBP/
TATA box complex.
How does the Brf2 core associate with TBP? In the case of
yeast Brf1, the weak association of a truncated protein corre-
sponding to the N-terminal half of the protein (including the
core domain) with TBP is prevented by radical mutations in
the TBP surface that interacts with TFIIB, suggesting that the
TFIIB and yeast Brf1 core domains contact human TBP in a
very similar manner (35). However, such mutations affect the
association of human full-length TFIIB family members differ-
ently. Radical mutations of TBP residues E284, E286, or L287
do not prevent association with full-length human Brf1 nor
Brf1-dependent transcription, but they reduce association with
TFIIB and change the mobility of the TBP/TFIIB/DNA com-
plex in an EMSA, suggesting that they somehow affect the
conformation of the complex (43). The different effects of TBP
mutations observed with truncated yeast Brf1 and full-length
human Brf1 may result from the ability of the Brf1 C-terminal
region to compensate for loss of contacts with TBP within the
Brf1 N-terminal region (35), or they may reflect different TBP
contacts with yeast and human Brf1 in truncated and full-
length proteins. Whichever the case, in the case of Brf2, radical
mutations of TBP residues E284 and E286 have an effect very
similar to that observed with TFIIB (43): reduction of the
intensity of the complex and change in conformation, as sug-
gested by a mobility change in the EMSA. Mutation of TBP
residue L287, on the other hand, has little effect, as it does with
Brf1 (43). Thus, the association of full-length Brf2 with TBP
appears to share some features with TFIIB and others
When we replaced the core region of Brf2 with the core
region of TFIIB, which has TBP/TATA box complex binding
activity, the resulting chimera (2/B/2) was only able to sustain
a barely detectable level of transcription, even though it was
capable of recruiting Bdp1 to the promoter. The very low levels
of transcription may be due to the very inefficient recruitment
of SNAPcby this chimeric protein (Fig. 5A). In addition, how-
ever, it may also result from inefficient recruitment of Pol III.
Indeed, the yeast Brf1 core domain associates with both the
C34 (1, 25) and the C17 (10) subunits of Pol III. The core
domain of Brf2 may be similarly involved in interactions with
Pol III-specific subunits.
C-terminal region of Brf2. In Brf1, the C-terminal region—
more specifically, the conserved region II within the C-termi-
nal region—provides the main anchor for binding to a TBP/
TATA box complex (1, 9, 22, 25). The structure of a yeast Brf1
region II/TBP/TATA box complex has been solved and shows
region II in Brf1 binding to the convex and lateral surfaces of
TBP (19). We find that removing the C-terminal region of Brf2
inhibits U6 transcription (Fig. 1D) and prevents Brf2 from
associating efficiently with a TBP/TATA box complex (Fig. 3A)
and with TBP in solution (Fig. 6). An alignment of the
FIG. 7. Functions of the N-terminal, core, and C-terminal regions
of Brf2. The functions identified in the present study are indicated. A
minus sign indicates that the region is dispensable for a given function;
plus signs indicate that the region is required (but not necessarily
sufficient). N.D, not determined; N.D (not sufficient), the function of
the region was not tested but the region is known to be insufficient for
function (for example, the contribution, if any, of the core domain of
Brf2 to binding to the TBP/TATA box complex was not tested directly,
but we know that the Brf2 core domain is insufficient for this function
because Brf2 truncated at position 290 did not bind to the TBP/TATA
VOL. 25, 2005STRUCTURE-FUNCTION ANALYSIS OF Brf2 9415
C-terminal domains of human Brf2 (HsBrf2) and putative Brf2
homologues from mouse (Mm), rat (Rn), zebra fish (Dr), and
fugu (Tr) is shown in Fig. 8A and reveals a striking concentra-
tion of conserved residues close to the end of the protein. Such
a high degree of sequence conservation between such varied
species suggests an important function and prompted us to
compare this segment to Brf1 conserved region II. One possi-
ble alignment is shown in Fig. 8B. The yeast and human Brf1
sequences are shown on top, together with the location of
?-helices as seen in the yeast Brf1 region II/TBP/TATA box
complex (19). Clearly, the Brf2 conserved sequence can be
aligned with the Brf1 conserved region II, with identical and
similar amino acids clustering in the region corresponding to
Brf1 ?-helices H22, H23, and the beginning of H24, which are
all involved in interactions with the convex and lateral surfaces
of TBP (19). This strongly suggests that the conserved Brf2
region near the end of the protein corresponds to the Brf1
conserved region II and provides the high-affinity contacts
interactions with SNAPcbecause it can be recruited to the U6
promoter by a subcomplex of SNAPcreferred to as mini-SNAPc
and consisting of SNAP50, SNAP43, and the N-terminal third of
SNAP190 (16). Although we have not previously observed re-
cruitment of Brf2 by mini-SNAPc, perhaps because the mini-
SNAPc/Brf2 complex was obscured by a comigrating complex
containing what appeared to be an alternative conformation of
mini-SNAPc(27), our present data indicate that the complete
SNAPccan recruit Brf2 to the U6 promoter and that this is
dependent on the C-terminal domain of Brf2 (Fig. 3B). Thus, the
C-terminal domain of Brf2 allows recruitment of the protein to
the U6 promoter, probably through direct contacts with SNAPc,
even in the absence of TBP.
In the absence of the entire TFIIB-related N-terminal half
of the protein, the Brf1 C-terminal domain retains some ability
to bind to a TATA box/TBP complex and to recruit Bdp1 (20).
Our results suggest that the C-terminal domain of Brf2 is
similarly involved in Bdp1 recruitment. Indeed, we find that
the chimeric protein 2/B/? binds strongly to the TBP/TATA
box complex but fails to incorporate Bdp1 (Fig. 5B). However,
when the C-terminal region of Brf2 is added to the 2/B/?
chimera to form 2/B/2, Bdp1 can then be recruited (Fig. 5A).
FIG. 8. Alignment of the human Brf2 sequence with Brf2 and Brf1 sequences from other vertebrate species. (A) Alignment of Brf2 sequences
from Homo sapiens (Hs; NP_060780), Mus musculus (Mm; NP_079962), Rattus norvegicus (Rn; XP_224944), Danio rerio (Dn; NP_001003536), and
Takifugu rubripes (Tr). This last protein sequence was assembled from expressed sequence tags. The alignment was performed with CLUSTAL W
with the default parameters. (B) Alignment of part of the S. cerevisiae (Sc) and H. sapiens (Hs) Brf1 conserved region II with a region near the
C terminus of H. sapiens (Hs), M. musculus (Mm), R. norvegicus (Rn), D. rerio (Dn), and T. rubripes (Tr) Brf2. The helical regions in the S. cerevisiae
Brf1 conserved region II, as determined in a cocrystal containing S. cerevisiae Brf1 conserved region II, the conserved region of TBP, and a TATA
box (19), are shown at the top. The first amino acids of each helix are indicated.
9416SAXENA ET AL.MOL. CELL. BIOL.
Thus, the C-terminal domain of Brf2 appears to play a crucial
role in the assembly of the preinitiation complex by providing
stabilizing interactions with TBP, SNAPc, and Bdp1.
Determinants of RNA polymerase specificity. RNA poly-
merase specificity is determined at two levels. The first level is
the arrangement of the promoter elements themselves. In the
human snRNA promoters, it is the presence or absence of a
TATA box downstream of the PSE that largely determines Pol
III or Pol II transcription, respectively. This in turn somehow
results in the specific recruitment of either TFIIB in the case of
Pol II snRNA promoters or Brf2 in the case of Pol III snRNA
promoters. Ironically, we do not yet understand how the pres-
ence of a TATA box specifies Brf2 recruitment over TFIIB
recruitment. Indeed, we show here that it is possible to assem-
ble efficiently a complex containing TBP, SNAPc, and TFIIB
(or the 2/B/? chimeric protein) on the U6 promoter. This is
somewhat reminiscent of the situation in the yeast U6 snRNA
promoter, which consists of both a TATA box upstream of the
transcription start site and a downstream binding site for the
factor TFIIIC. The yeast U6 upstream promoter has no spec-
ificity for recruitment of Brf1 over TFIIB. Rather, it is the
binding of TFIIIC to the downstream site that specifies Brf1-
TFIIIB recruitment and Pol III transcription (32). It seems
likely that, in human cells, as-yet-unidentified factors associ-
ated with either TFIIB, Brf1, TBP, or SNAPcprevent assembly
of TFIIB and/or favor assembly of Brf2 into the U6 initiation
The second level of RNA polymerase specificity determina-
tion is protein-protein interactions among factors recruited by
the promoter elements. Thus, even though the U6 promoter is
capable of assembling complexes containing either TBP,
SNAPc, and TFIIB or TBP, SNAPc, and Brf2, only the latter
complex (or complexes formed with TFIIB/Brf2 chimeras con-
taining the Brf2 C-terminal region) is capable of recruiting
Bdp1. Moreover, the C-terminal domain of Brf2 may also be
involved in the recruitment of Pol III. With yeast Brf1, the
C-terminal domain of the protein, and in particular homology
domains II and III, have been shown to associate with the yeast
Pol III subunit C34, which has no counterpart in Pol II (1, 25).
Thus, the C-terminal extensions present in the TFIIB family
members involved in RNA Pol III transcription, i.e., Brf1 and
Brf2, are key to favoring recruitment of Pol III over that of
Pol II because they allow recruitment of Bdp1 and, probably,
of Pol III itself through Pol III-specific subunits.
We thank Robert S. Haltiwanger, Patrick Hearing, and Winship
Herr for helpful comments during the course of this project. We also
thank Yuling Sun for generating the constructs expressing the TFIIB-
Brf2 chimeras and J. Duffy for artwork and photography.
This study was funded in part by NIH grant GM38810. N.H. and
B.M. are supported by the Howard Hughes Medical Institute.
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