The Small Nuclear RNA-activating Protein 190 Myb DNA
Binding Domain Stimulates TATA Box-binding Protein-TATA Box
Recognition*
Craig S.
Hinkley
,
Heather A.
Hirsch§,
Liping
Gu
,
Brandon
LaMere¶, and
R.
William
Henry
§
From the ¶ Biochemistry Research Training Program,
§ Cell and Molecular Biology Program, and
Department of Biochemistry and Molecular Biology,
Michigan State University, East Lansing, Michigan 48824
Received for publication, May 1, 2002, and in revised form, March 5, 2003
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ABSTRACT |
Human U6 small nuclear RNA (snRNA) gene
transcription by RNA polymerase III requires cooperative promoter
binding involving the snRNA-activating protein complex
(SNAPc) and the TATA-box binding protein (TBP). To
investigate the role of SNAPc for TBP function at U6
promoters, TBP recruitment assays were performed using full-length TBP
and a mini-SNAPc containing SNAP43, SNAP50, and a truncated
SNAP190. Mini-SNAPc efficiently recruits TBP to the U6 TATA
box, and two SNAPc subunits, SNAP43 and SNAP190, directly interact with the TBP DNA binding domain. Truncated SNAP190
containing only the Myb DNA binding domain is sufficient for TBP
recruitment to the TATA box. Therefore, the SNAP190 Myb domain
functions both to specifically recognize the proximal sequence element
present in the core promoters of human snRNA genes and to stimulate TBP recognition of the neighboring TATA box present in human U6 snRNA promoters. The SNAP190 Myb domain also stimulates complex assembly with
TBP and Brf2, a subunit of a snRNA-specific TFIIIB complex. Thus, interactions between the DNA binding domains of SNAP190 and TBP
at juxtaposed promoter elements define the assembly of a RNA polymerase
III-specific preinitiation complex.
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INTRODUCTION |
Transcription in eukaryotic organisms occurs by three different
RNA polymerases that transcribe different classes of genes. The
recruitment of a specific RNA polymerase to a given promoter is
dictated by the nature of the preinitiation complex (1-4); however,
the molecular determinants for RNA polymerase specificity are not
known. One possibility is that recruitment of
TBP1 complexes containing
distinct cadres of TBP-associated factors (TAFs) may confer polymerase
specificity. For example, the TBP complexes SL1, TFIID, and TFIIIB are
multiprotein TBP·TAF complexes that are required for transcription by
RNA polymerases I, II, and III, respectively (for review, see Refs.
5-7). These various TBP·TAF complexes play crucial roles by serving
as targets for regulatory proteins and providing specific promoter
recognition functions. Once recruited to a promoter, TBP·TAF
complexes further provide an important structural façade during
preinitiation complex assembly to recruit other general transcription
factors dedicated to transcription by a specific RNA polymerase.
In contrast to the well characterized TBP complexes that function for
transcription of most genes, how TBP is recruited to human small
nuclear (sn) RNA gene promoters is understood less well. None of the
aforementioned TBP·TAF complexes appears to function at these genes
(8, 9). Human snRNA genes are unusual because they have similar
promoters, and yet some snRNA genes are transcribed by RNA polymerase
II (e.g. U1) and others by RNA polymerase III
(e.g. U6) (for review, see Refs. 10 and 11). Thus, these
genes serve as an important model for understanding the mechanisms of
polymerase specificity (12). Each gene contains a proximal sequence
element (PSE) in the core promoter which recruits the general
transcription factor SNAPc (9), which is also known as
proximal sequence element transcription factor (13) or proximal sequence element binding protein (14). Substoichiometric levels of TBP
copurify with SNAPc during the biochemical fractionation of
SNAPc from HeLa cell nuclear extracts (15), and fractions enriched for SNAPc restore U1 snRNA transcription by RNA
polymerase II to HeLa cell extracts that have been immunodepleted of
endogenous TBP (9). Thus, SNAPc may play an important role
in recruiting TBP to the TATA-less promoters of RNA polymerase
II-transcribed snRNA genes. In contrast, the TBP present in the
SNAPc-enriched fractions does not support U6 transcription
by RNA polymerase III (9). One key difference between RNA polymerase
II- and III-transcribed snRNA genes is the presence of a TATA box in
the core promoter of RNA polymerase III-transcribed snRNA genes. TBP binds to this TATA box only poorly but is stimulated by
SNAPc. Furthermore, the combination of the PSE and TATA box
directs transcription by RNA polymerase III, and mutation of either
promoter element switches transcription to RNA polymerase II (16, 17).
These observations suggest that SNAPc-mediated recruitment
of TBP or TBP complexes acting at the TATA box is a key step in
assembling the specific preinitiation complex that preferentially
recruits RNA polymerase III. Thus, depending upon the promoter context, SNAPc may recruit a different TBP complex that determines
the type of RNA polymerase recruited to a snRNA promoter.
For most RNA polymerase III-transcribed genes, the TFIIIB complex,
which is composed of TBP with the tightly associated TFIIB-related factor (Brf1) and a loosely associated factor called Bdp1 (originally named B") (18), nucleates preinitiation complex assembly (19-26). Human U6 gene transcription by RNA polymerase III similarly requires both TBP and Bdp1; however, these genes do not require Brf1 (9, 24,
27-30). In contrast, transcription of these genes depends upon other
TFIIIB-like complexes that contain Brf1-related factors. Two different
Brf-like factors have been proposed to function for human snRNA gene
transcription by RNA polymerase III. One Brf-related factor, called
Brf2 (originally named BRFU/TFIIIB50) (18), shows limited
similarity to Brf1, including the zinc ribbon domain and the
TFIIB-related repeats, and functions specifically for transcription of
human U6 genes (30, 31). Another related factor, called Brf1_v2
(originally named Brf2) (18), is an alternatively spliced form
of Brf1 which contains the TFIIB-related repeat 2 and additional
carboxyl-terminal sequences that are unique to Brf1_v2 and also acts to
support human U6 transcription (32). Both Brf2 (33) and Brf1_v2
(32) can interact with TBP through the TFIIB-related repeat 2. Neither
Brf2 nor Brf1_v2 appears to be tightly associated with TBP or
Bdp1 in the absence of DNA binding by TBP (30, 32). However,
Brf2 does stimulate TBP binding to the TATA box in
electrophoretic mobility shift assays (EMSAs), and it may make DNA
contacts surrounding the TATA box (33). The circumstances whereby
Brf2, Brf1_v2, or both factors are used for human U6
transcription are currently unclear.
To understand how TBP is specifically recruited to human U6 snRNA gene
promoters, we have examined those proteins within SNAPc which are important for interactions with TBP and for cooperative DNA
binding of the U6 promoters by both SNAPc and TBP. We find that three subunits of SNAPc (SNAP43, SNAP45, and SNAP190)
can interact individually with the TBP DNA binding domain. Thus,
SNAPc may make extensive contacts with TBP. Further
analysis of SNAP43 and SNAP190 indicate that the SNAP190 Myb DNA
binding domain plays a pivotal role in TBP recruitment and stimulates
assembly of a complex containing TBP and the TFIIIB factor Brf2.
The juxtaposition of the PSE and TATA elements at human U6 promoters
suggests that the DNA binding domains of SNAP190 and TBP are well
positioned to facilitate these interactions during the early stages of
preinitiation complex assembly. The unique architecture of SNAP190 and
TBP at human U6 promoters thus may define whether RNA polymerase
III-specific TFIIIB factors are specifically recruited to these genes.
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EXPERIMENTAL PROCEDURES |
Plasmid Construction--
Inserts encoding amino- or
carboxyl-terminal truncations of SNAPc proteins or human
TBP were generated by PCR amplification and cloned into pET11c-derived
vectors. GST-SNAP190 RaRbRcRd contains amino acids 283-518, and
GST-SNAP190 RcRd contains amino acids 390-518. Internal deletions were
generated using the QuikChange site-directed mutagenesis protocol from Stratagene.
Protein Expression and Purification--
Proteins used in the
EMSAs were expressed as GST fusions in Escherichia coli
BL21-CodonPlus-RIL cells (Stratagene). Proteins were purified from
crude lysates on glutathione-Sepharose beads (Amersham Biosciences),
and if necessary, the GST moiety was removed by digestion with
thrombin. Proteins were purified further by ion exchange
chromatography. Brf2 was expressed and purified as described
previously (30). For Fig. 6, mini-SNAPc was assembled and
purified by ion exchange chromatography prior to use in EMSA. Purified
proteins were dialyzed against buffer D80 containing 20 mM
HEPES pH 7.9, 20% glycerol, 80 mM KCl, 0.2 mM
EDTA, 0.1% Tween 20, 5 mM MgCl2, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 1 mM benzamidine, 1 mM sodium
metabisulfite, and 1 µM pepstatin A.
GST Pulldown Assays--
GST pulldown assays were done
essentially as described previously (34).
EMSAs--
Mini-SNAPc was assembled for 2 h at
room temperature using ~200 ng of each individual wild-type or mutant
SNAP43, SNAP50, and SNAP190 (1-505) protein. Approximately 3 µg of
SNAP19 was included in reactions to assemble complexes containing
SNAP190 (1-505) proteins with internal deletions. Approximately
one-third of each assembly reaction was then used in EMSA using DNA
probes containing a wild-type or mutant mouse U6 PSE with a wild-type or mutant human U6 TATA box as described previously (9, 35). All DNA
binding reactions were performed in a 20-µl total volume. DNA binding
reactions using only SNAPc were performed in a buffer containing 60 mM KCl, 20 mM HEPES pH 7.9, 5 mM MgCl2, 0.2 mM EDTA, 10%
glycerol, 0.5 µg of poly(dI-dC), and 0.5 µg of pUC119 plasmid. Reactions were incubated for 20 min at room temperature after which
5,000 cpm of probe was added, and reactions were incubated an
additional 20 min. Samples were fractionated on a 5% nondenaturing polyacrylamide gel (39:1) in TGE running buffer (50 mM
Tris, 380 mM glycine, 2 mM EDTA). Reactions
containing both SNAPc and TBP were performed in a buffer
containing 100 mM KCl, 20 mM HEPES pH 7.9, 5 mM MgCl2, 0.2 mM EDTA, 10%
glycerol, 1 mM dithiothreitol, 0.07% Tween 20, 0.2 µg of
poly(dG-dC), and 0.2 µg of pUC119 plasmid. Reactions with SNAP190
RcRd and TBP also contained 0.5 µl of fetal bovine serum and 50 mM NaF but lacked KCl. The samples were fractionated on a
5% nondenaturing polyacrylamide gel (39:1) in TGEM running buffer (50 mM Tris, 380 mM glycine, 2 mM EDTA,
5 mM MgCl2). Reactions also containing
Brf2 were performed in a buffer containing 10 mM
HEPES pH 7.9, 20 mM Tris pH 8.4, 60 mM KCl, 7.5 mM MgCl2, 10% glycerol, 6 mM
-mercaptoethanol, 12 mM dithiothreitol, 0.2 µg of
poly(dG-dC), and 0.2 µg of pUC119 plasmid. Reactions were incubated for 20 min on ice after which 5,000 cpm of probe was added, and reactions were incubated for an additional 30 min at 30 °C. The samples were fractionated on a 4% nondenaturing polyacrylamide gel
(39:1) containing 1× TBE (90 mM Tris, 90 mM
boric acid, 2 mM EDTA) and 2.5% glycerol in 0.5 × TBE running buffer.
 |
RESULTS |
Mini-SNAPc Recruits TBP to the U6 snRNA TATA
Box--
Recruitment of TBP to the TATA box is a crucial early step in
preinitiation complex assembly at human U6 snRNA promoters (for review,
see Refs. 12 and 36). This step is facilitated by SNAPc,
which has two important roles in this process. First, interactions between SNAPc and TBP relieve negative regulation by the
amino-terminal domain of TBP and allow the carboxyl-terminal DNA
binding domain of TBP to bind efficiently to the TATA box. Second,
cooperative interactions between SNAPc and TBP increase
promoter occupancy by both SNAPc and TBP (35). Naturally
occurring SNAPc contains at least five proteins SNAP19,
SNAP43, SNAP45, SNAP50, and SNAP190 (15, 37-42). However, a
baculovirus-expressed mini-SNAPc with only three proteins,
SNAP43, SNAP50, and SNAP190 containing amino acids 1-514 is sufficient
for recruiting TBP to a U6 snRNA TATA box (43). To investigate further
the role of SNAPc for TBP recruitment to human U6
promoters, mini-SNAPc containing SNAP43, SNAP50, and SNAP190 (1-505) was assembled from bacterially expressed proteins and
tested for DNA binding ability and recruitment of TBP to the U6 TATA
box. As shown in Fig. 1A, none
of the individual bacterially expressed SNAPc proteins
could effectively bind to DNA containing a wild-type PSE (lanes
2-4). Similarly, little DNA binding activity was observed for
reactions containing pairwise combinations of SNAP50 plus SNAP190
(1-505), SNAP43 plus SNAP190 (1-505), or SNAP43 plus SNAP50
(lanes 5, 9, and 13, respectively).
However, as increasing amounts of SNAP43 (lanes 6-8),
SNAP50 (lanes 10-12), or SNAP190 (1-505) (lanes
14-16) were added to reactions containing the remaining two
proteins, significant levels of DNA binding activity were observed. The
minicomplex specifically recognizes the PSE because no DNA binding
activity was observed on DNA probes containing a mutant PSE (lane
17). Therefore, efficient PSE-specific DNA binding activity
requires all three proteins, SNAP43, SNAP50, and SNAP190 (1-505).

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Fig. 1.
A mini-SNAP complex containing the SNAP43,
SNAP50, and SNAP190 (1-505) proteins is sufficient to recruit human
TBP to the U6 TATA box. A, EMSAs were performed using
~63 ng of each SNAPc protein either individually
(lanes 2-4) or in pairwise combination (lanes 5,
9, and 13) with a DNA probe containing a high
affinity mouse U6 PSE. Approximately 7, 21, and 63 ng of SNAP43
(lanes 6-8), SNAP50 (lanes 10-12), or SNAP190
(1-505) (lanes 14-16) was added to reactions containing 63 ng each of the remaining two proteins as indicated. The positions of
free probe and mini-SNAPc bound to DNA are indicated.
Lane 1 contains the probe alone, and lane 17 contains a probe with a mutant PSE but is otherwise identical to
lane 16. B, EMSA using individual
SNAPc proteins (lanes 3-5, 10-12,
and 19-21), pairwise combinations of SNAPc
proteins (lanes 6-8, 13-15, and
22-24), or mini-SNAPc (lanes 9,
16, and 25) performed in the absence (lanes
3-9) or presence (lanes 10-25) of recombinant human
TBP. DNA binding reactions contained about 30 ng of each
SNAPc protein and 100 ng of TBP. The DNA probes used
contain a high affinity mouse U6 snRNA PSE and either a wild-type
(lanes 1-16) or mutant (lanes 17-25) human U6
snRNA TATA box. Lanes 1 and 17 contain DNA probes
alone, and lanes 2 and 18 contain probes with
recombinant TBP alone. The positions of the free probe,
mini-SNAPc, and mini-SNAPc·TBP complex are
indicated.
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To determine whether mini-SNAPc can efficiently recruit TBP
to the U6 TATA box, EMSAs were performed with mini-SNAPc
and full-length human TBP (Fig. 1B). As expected,
full-length human TBP alone did not bind appreciably to a probe
containing both a PSE and a TATA box (lane 2) or to a
similar probe containing mutations in the TATA box (lane
18). None of the SNAPc proteins either alone (lanes 3-5) or in pairwise combination (lanes
6-8) showed significant DNA binding to a probe with a wild-type
PSE. The combination of all three proteins (labeled
mrSNAPc) resulted in significant DNA binding to the
wild-type probe (lane 9) but not to a probe containing mutations in the PSE (data not shown). Recombinant human TBP was then
added to these SNAPc proteins to examine their ability to recruit TBP to the U6 TATA box. When incubated with recombinant TBP,
none of the SNAPc proteins either alone or in pairwise
combination showed significant DNA binding to probes containing a
wild-type PSE and either a wild-type (lanes 10-15) or
mutant (lanes 19-24) TATA box. In contrast,
mini-SNAPc efficiently recruited human TBP to a probe
containing both a wild-type PSE and TATA box as evidenced by the
formation of a complex that migrated more slowly than that observed
with mini-SNAPc alone (compare lane 16 with lane 9). Recruitment of TBP by SNAPc was
cooperative as evidenced by the dramatic increase in DNA binding by TBP
in the presence of mini-SNAPc (compare the upper
complex in lane 16 with lane 2). Mutations in the
TATA box did not affect DNA binding by mini-SNAPc but
abolished formation of the mini-SNAPc·TBP·DNA complex
(lane 25). Thus, as demonstrated with recombinant
mini-SNAPc expressed in insect cells by baculovirus
infection (43), mini-SNAPc assembled from bacterially
expressed proteins is able to recruit TBP to the U6 snRNA TATA box, and
these interactions are dependent upon both the PSE and TATA box
promoter elements. Furthermore, these results show that SNAP19, SNAP45,
or other eukaryotic proteins that may associate with recombinant
SNAPc are not required for either PSE-specific binding or
TBP recruitment to the U6 TATA box.
Multiple SNAPc Subunits Can Interact with TBP--
As
a first step to determine which SNAPc proteins are required
for TBP recruitment, interactions between individual SNAPc proteins and human TBP were analyzed by GST pulldown assays.
Previously, it was demonstrated that the TBP COOH-terminal DNA binding
domain and two regions of the NH2 terminus are important
for cooperative DNA binding by SNAPc and TBP (35). These
regions from the NH2 terminus include amino acids 1-54 and
the Q-rich region containing amino acids 55-95 (Fig.
2A). Therefore, we also tested
interactions between SNAPc proteins and TBP proteins that
lacked these regions of the NH2 terminus. As shown in Fig.
2B, three SNAPc subunits, SNAP190 (1-719),
SNAP45, and SNAP43, interacted well with full-length GST-TBP (1-335)
in these assays (lane 2), and these interactions were
specific because no interaction was observed with GST protein (lane 5) or beads alone (lane 6). In similar
assays, full-length SNAP190 (1-1469) also interacted well with
full-length TBP (data not shown). Moreover, the DNA binding domain of
TBP was sufficient for interactions with all three SNAPc
proteins because no significant difference for interactions involving
full-length GST-TBP (1-335) (lane 2), GST-TBP (96-335)
(lane 3), or GST-TBP (159-335) (lane 4) was
observed. These results suggest that TBP makes extensive contacts with
SNAPc and that the COOH-terminal DNA binding domain of TBP
is important for interactions with SNAPc. Although
interactions involving both SNAP43 and SNAP45 with TBP have been
described previously (15, 40, 41), the current observations reveal that
a previously uncharacterized interaction between SNAP190 and TBP may
contribute to SNAPc function for human snRNA gene transcription.

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Fig. 2.
Three SNAPc proteins interact
directly with the TBP DNA binding domain. A, schematic
of human TBP. The nonconserved NH2 terminus that is divided
into three segments (I, II, and III)
and the highly conserved COOH terminus that contains the DNA binding
domain of TBP are indicated. Segment II contains a stretch of glutamine
residues (Q) from amino acids 55-95. B,
SNAPc proteins were labeled with
[35S]methionine and used in GST pulldown assays with
GST-TBP (1-335) (lane 2), GST-TBP (96-335) (lane
3), GST-TBP (159-335) (lane 4), GST protein alone
(lane 5), or glutathione-Sepharose beads alone (lane
6). The SNAP190 protein contains amino acids 1-719. 10% of the
total input for each protein is shown in lane 1.
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No Single Region of SNAP43 Is Required for TBP Recruitment to the
U6 TATA Box--
Of the proteins present within
mini-SNAPc, both SNAP43 and SNAP190 protein interacted well
with TBP (Fig. 2B). To characterize further the interactions
between SNAP43 and TBP, regions of SNAP43 which are important for
mediating interactions with TBP were mapped by GST pulldown assays.
SNAP43 proteins containing NH2- or COOH-terminal truncations were tested for interactions with GST-TBP (1-335) and also
with GST-SNAP50. The data for separate experiments testing interactions
between SNAP43 proteins and either GST-TBP or GST-SNAP50 are shown in
Fig. 3, A and B,
respectively, and a summary of the results is shown in Fig.
3C. As expected, significant interactions of full-length
SNAP43 (1-368) with both GST-TBP and GST-SNAP50 were observed.
Interestingly, SNAP43 containing only the NH2-terminal 168 amino acids (1-168) interacted well with SNAP50 but not with TBP. In
contrast, the COOH-terminal region of SNAP43 containing amino acids
169-368 interacted well with both TBP and SNAP50. These interactions
are specific because the SNAP43 proteins did not interact with GST
protein (Fig. 3, A and B, lanes 3) or
the beads alone (Fig. 3, A and B, lanes
4). Ma and Hernandez (44) did not detect an interaction between
SNAP50 and the COOH-terminal region of SNAP43 using
coimmunoprecipitation assays, suggesting that this interaction is weak.
These observations suggest that SNAP50 makes extensive interactions
with at least two separate regions of SNAP43. Furthermore, a major
region for TBP interaction with SNAP43 is contained within amino acids
169-368.

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Fig. 3.
No single region of SNAP43 is required for
TBP recruitment to the U6 TATA box. A, full-length
SNAP43 protein or truncated SNAP43 proteins containing amino acids
1-168 or 169-368 were used in GST pulldown assays with GST-TBP
(lane 2), GST (lane 3), or glutathione-Sepharose
beads alone (lane 4). 10% of the total input for each
protein is shown in lane 1. B, same as
A except GST-SNAP50 was used instead of GST-TBP.
C, summary of results for the GST pulldown assays between
the SNAP43 proteins and GST-TBP or GST-50. A (+) indicates that a
direct interaction was observed; ( ) indicates that no interaction was
detected. D, EMSA containing ~30 ng each of SNAP50,
SNAP190 (1-505), and either full-length SNAP43 (lanes
4-6), SNAP43 (1-168) (lanes 7-9), or SNAP43
(169-368) (lanes 10-12) in the absence (lanes
4, 7, and 10) or presence (lanes
5, 6, 8, 9, 11, and
12) of recombinant human TBP. The DNA probe used contains a
high affinity mouse U6 snRNA PSE and a human U6 snRNA TATA box. The
positions of free probe, mini-SNAPc, and
mini-SNAPc·TBP complex are indicated. Lane 1 contains DNA probe alone, and lanes 2 and 3 contain recombinant TBP alone.
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To determine the contribution of SNAP43 for TBP recruitment to the U6
TATA box we then took advantage of the observation that both halves of
SNAP43 could interact with SNAP50 and SNAP190 (1-505) (Fig.
3B and data not shown) and could therefore potentially form a mini-SNAPc. First, the ability of each half of SNAP43 to
form DNA-binding competent complexes with SNAP50 and SNAP190 (1-505) was tested using an EMSA, and the results are shown in Fig.
3D. As with full-length SNAP43 (1-368) (lane 4),
both SNAP43 (1-168) and SNAP43 (169-368) assembled into complexes
capable of efficient DNA binding (lanes 7 and 10,
respectively). In all cases, DNA binding by these complexes was
PSE-specific because no DNA binding was observed using a probe with a
mutant PSE (data not shown). These observations suggest that SNAP43 is
essential for DNA binding by mini-SNAPc, but no single
region of SNAP43 is critical. Thus, it appears that SNAP43 coordinates
DNA binding by SNAP50 and SNAP190, and either half of SNAP43 is
sufficient for this activity. In contrast to strong DNA binding by
mini-SNAPc, full-length human TBP (1-335) was unable to
bind DNA efficiently in these assays (lanes 2 and
3). As expected, TBP is recruited to DNA efficiently by
mini-SNAPc containing SNAP43 (1-368) (lanes 5 and 6). Complexes assembled with SNAP43 containing either
the NH2-terminal region (amino acids 1-168) or
COOH-terminal region (amino acids 169-368) were capable of recruiting
TBP to the U6 snRNA TATA box (lanes 8, 9 and
11, 12, respectively). This result indicates that
no single region of SNAP43 is essential for TBP recruitment to the TATA
box under the conditions of this assay. This was unexpected in light of
the results of the GST pulldown experiments in Fig. 3, A and
B, and suggests that interactions other than those involving SNAP43 are sufficient for TBP recruitment to the U6 snRNA TATA box.
The SNAP190 RcRd Myb Repeats Are Sufficient for TBP Recruitment to
the U6 TATA Box--
Both SNAP43 and SNAP190 are capable of
interacting with TBP; however, no single region of SNAP43 is essential
for recruitment of TBP to the U6 TATA box, and thus it seems possible
that interactions between SNAP190 and TBP are important for TBP
recruitment. First, to determine whether SNAP190 (1-505) can interact
with TBP, interactions between truncated SNAP190 proteins and GST-TBP
were tested in GST pulldown experiments. Significant interactions
between both SNAP190 (1-900) and SNAP190 (1-719) with GST-TBP
(1-335) were observed (Fig.
4A, lane 2).
Importantly, SNAP190 (1-505) also interacted well with TBP. This
interaction was reduced compared with that observed for longer versions
of SNAP190, suggesting that amino acids 506-719 influence TBP
recruitment, but it still was significantly greater than that observed
for SNAP190 (1-164) (compare lane 2 with lane 1 for each SNAP190 protein). As shown in Fig. 4B, SNAP190
(1-164) is capable of interacting with GST-SNAP19 (lane 2)
but is not capable of interacting with GST-TBP (1-335) (lane
3). In contrast, SNAP190 (1-900) interacts well with both GST-SNAP19 (lane 2) and GST-TBP (1-335) (lane
3). Together, these observations indicate that SNAP190 interacts
with TBP specifically, and the region of SNAP190 contained within amino
acids 164-505, which encompasses the SNAP190 Myb DNA binding domain,
is important for this interaction.

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Fig. 4.
SNAP190 (1-505) interacts directly with
human TBP. A, full-length or COOH-terminally truncated
SNAP190 proteins were used in GST pulldown assays with GST-TBP (1-335)
(lane 2), GST (lane 3), or glutathione-Sepharose
beads alone (lane 4). 10% of the input for each protein is
shown in lane 1. B, SNAP190 (1-900) and SNAP190
(1-164) were used in GST pulldown assays with GST-SNAP19 (lane
2), GST-TBP (1-335) (lane 3), GST (lane 4),
or glutathione-Sepharose beads alone (lane 5). 10% of the
total input for each protein is shown in lane 1.
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The above observations raise the interesting possibility that
interactions between the SNAP190 DNA binding domain and TBP may be
important for TBP recruitment to the U6 TATA box. To determine whether
SNAP190 contributes to TBP recruitment, SNAP190 (1-505) proteins with
internal deletions were assembled into a mini-SNAPc and
tested for both PSE-specific binding and TBP recruitment by EMSA. A
schematic representation of the mutant SNAP190 proteins tested is shown
in Fig. 5A. SNAP190 (1-505)
contains an NH2-terminal region located between amino acids
84 and 133, which is required for interactions with SNAP19 and SNAP43
(44), and an unusual Myb DNA binding domain containing four and
one-half Myb domain repeats located between amino acids 263 and 503. The SNAP190 Myb repeats are referred to as Rh, Ra, Rb, Rc, and Rd (38).
When tested with SNAP43 and SNAP50, complexes containing the mutant SNAP190 (1-505) proteins were either unable to bind (
165-259,
a, and
b) or bound poorly (
h) to DNA (data not shown).
However, it was shown previously that mini-SNAPc containing
SNAP190 with deletions of amino acids 1-84 and 134-262 failed to bind
DNA, but binding was stimulated by the addition of SNAP19 (44).
Therefore, mini-SNAPc was assembled in the presence of
SNAP19 and tested again for DNA binding. As shown in Fig.
5B, all of the mini-SNAP complexes containing SNAP190
(1-505) proteins with internal deletions were able to bind DNA
efficiently in the presence of the SNAP19 (lanes 3,
5, 7, and 9). Binding was PSE-specific
and was not observed on DNA with mutant PSE sites (data not shown).
These complexes were also all able to recruit TBP to a TATA box
(lanes 4, 6, 8, and 10).
Therefore, no single region of SNAP190 between amino acids 164 and the
RcRd Myb repeats is essential for TBP recruitment to the U6 TATA box
under these conditions, although amino acids in these regions may
contribute to efficient TBP recruitment by the full-length SNAP190.

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Fig. 5.
The SNAP190 RcRd Myb repeats are sufficient
to recruit TBP to the U6 TATA box. A, schematic of the
wild-type and mutant SNAP190 proteins. The region of SNAP190 required
for interaction with SNAP19 and SNAP43 and the Myb DNA binding domain
that contains four (Ra through Rd) and a half (Rh) Myb repeats are
indicated. B, EMSAs containing SNAP19, SNAP43, SNAP50, and
either SNAP190 (1-505) h (lanes 3 and 4),
SNAP190 (1-505) a (lanes 5 and 6), SNAP190
(1-505) b (lanes 7 and 8), or SNAP190
(1-505) 165-259 (lanes 9 and 10) in the
absence (lanes 3, 5, 7, and
9) or presence (lanes 4, 6,
8, and 10) of recombinant human TBP. The DNA
probe used contains a high affinity mouse U6 snRNA PSE and a human U6
snRNA TATA box. Lane 1 contains DNA probe alone, and
lane 2 contains probe with recombinant TBP alone.
C, EMSAs were performed with 60 ng of rTBP (lanes
2, 6, 10, and 14), 1 µg of
SNAP190 RcRd (lanes 3, 7, 11, and
15), or rTBP and SNAP190 RcRd (lanes 4,
8, 12, and 16). The DNA probes used
contain a high affinity mouse U6 snRNA PSE and a human U6 snRNA TATA
box (lanes 1-4), a wild-type PSE and mutant TATA box
(lanes 5-8), a mutant PSE and wild-type TATA box
(lanes 9-12), or a mutant PSE and mutant TATA box
(lanes 13-16). Lanes 1, 5,
9, and 13 contain probe DNA alone. The presence
(+) or absence ( ) of rTBP or SNAP190 RcRd is indicated above each
lane. The positions of free probe and SNAP190 RcRd and rTBP
complexes are indicated ( cd/TBP). D, human TBP
or SNAP19 proteins were used in GST pulldown assays with GST-SNAP190
(1-505) (lane 2), GST-SNAP190 RcRd (lane 3), GST
(lane 4), or glutathione-Sepharose beads alone (lane
5). 20% of the total input for each protein is shown in
lane 1.
|
|
Although the SNAP190 Rh, Ra, and Rb repeats are dispensable for DNA
binding, both SNAP190 Rc and Rd repeats are crucial for PSE-specific
binding (38, 43), and therefore mutant SNAP190 lacking RcRd could not
be tested directly in the above assays. However, even though SNAP190
(1-505) requires SNAP43 and SNAP50 for efficient DNA binding (see Fig.
1), the SNAP190 RcRd Myb repeats alone are sufficient for DNA binding,
but with reduced specificity (38, 45). To examine directly whether the
SNAP190 RcRd Myb repeats can recruit TBP to the U6 snRNA TATA box in
the absence of any other SNAPc protein, an EMSA was
performed (Fig. 5C). The DNA probes used in these
experiments contained a wild-type PSE and TATA box (lanes
1-4), a wild-type PSE and mutant TATA box (lanes
5-8), a mutant PSE and wild-type TATA box (lanes
9-12), or a mutant PSE and mutant TATA box (lanes
13-16).
Little binding to any of the probes was observed with either SNAP190
RcRd (lanes 3, 7, 11, and
15) or TBP (lanes 2, 6, 10, and 14) proteins alone. Lack of detectable binding by the
SNAP190 RcRd protein alone was somewhat surprising, but binding was
detectable on gels lacking MgCl2 (45, and data not shown).
Similarly, no binding was observed with both proteins when the PSE and
TATA box were both mutated (lane 16). Interestingly, the
SNAP190 RcRd protein was able to recruit TBP to the U6 snRNA TATA box
(compare lane 4 with lane 2). TBP was present on
the DNA because antibodies directed against TBP supershifted the
complex (data not shown). Recruitment of TBP was dependent on a
wild-type TATA box because the SNAP190 RcRd protein was unable to
recruit TBP when the TATA box was mutated (lanes 5-8).
SNAP190 RcRd and TBP bound to the DNA probe with a mutant PSE albeit
more weakly than to the probe containing a wild-type PSE (compare
lane 12 with lane 4), suggesting that TBP can
recruit SNAP190 RcRd in the absence of a strong PSE. Similar results
were obtained with a GST-tagged SNAP190 RcRd protein (data not shown).
Thus, the SNAP190 RcRd Myb DNA binding domain is sufficient for TBP
recruitment to the U6 promoter.
As shown above, the Myb DNA binding domain of SNAP190 can stimulate TBP
recruitment to the TATA box in DNA binding assays. To determine whether
the SNAP190 RcRd protein can interact with TBP, GST pulldown
experiments were performed. As shown in Fig. 5D, full-length
TBP interacts well with GST-SNAP190 (1-505) and with GST-SNAP190 RcRd
(lanes 2 and 3, respectively) but not with the
negative control GST or beads alone samples (lanes 4 and
5, respectively). To examine further the specificity of this
interaction, SNAP19 binding was also tested. As was observed with TBP,
SNAP19 interacts well with GST-SNAP190 (1-505). However, SNAP19 did
not interact with GST-SNAP190 RcRd (lane 3). This result was
expected because SNAP19 interacts with the NH2-terminal
region of SNAP190 (44). Importantly, this result supports the notion
that the SNAP190 RcRd interaction with TBP is specific.
SNAPc Stimulates TFIIIB Assembly at Human U6
Promoters--
SNAPc binding to the PSE is a crucial early
event during the assembly of a functional preinitiation complex at
human U6 promoters. As described, one function of SNAPc is
to stimulate TBP recruitment to the TATA box. However, additional
events are required, including the assembly of additional TFIIIB
components at these promoters. As a marker for TFIIIB assembly, we
followed the binding of Brf2 to human U6 promoter probes in
EMSAs. As shown in Fig. 6, neither TBP
alone (lanes 2, 10, 18, and
26) nor Brf2 alone (lanes 3,
11, 19, and 27) bound efficiently to
any of the U6 promoter probes. In contrast, mini-SNAPc
alone bound efficiently to wild-type PSE probes (lanes 4 and
20) but not to mutant PSE probes (lanes 12 and
28) as expected. When recombinant TBP was added to reactions containing mini-SNAPc, modest formation of a slower
migrating complex was observed (lane 5, labeled
mini-SNAPc + rTBP), and surprisingly,
complex formation was not affected by TATA box mutation (lane
21). This complex was inferred to contain both
mini-SNAPc and TBP because it was only observed in the
presence of both mini-SNAPc and TBP. SNAPc
stimulation of TBP binding was also less than that observed in the
experiments presented in Figs. 1 and 3 presumably because these
experiments were performed using different electrophoresis conditions.
Interestingly, a slower migrating complex was observed when Brf2
was added to reactions containing mini-SNAPc (lane
6, labeled mini-SNAPc + Brf2). This complex contained Brf2 because antibodies directed against the histidine tag on Brf2 were able to supershift this complex (data not shown). Mutation of the PSE severely debilitated SNAPc·Brf2 complex formation
(lane 14), whereas TATA box mutations did not affect this
complex (lane 22). Thus, mini-SNAPc can recruit
Brf2 to a U6 promoter in a PSE-dependent manner.
Next, the ability of SNAPc·Brf2 to recruit TBP was
tested. As increasing amounts of TBP were added to reactions containing mini-SNAPc and Brf2 (lanes 7-9),
formation of a new complex was observed (labeled
mini-SNAPc + Brf2 + rTBP). The
presence of Brf2 in this new complex was confirmed using
antibody supershift assays (data not shown). In these reactions, the
levels of the SNAPc·Brf2 complex was reduced
concomitantly with added TBP, whereas SNAPc binding was
unchanged, suggesting that TBP preferentially recognized the
SNAPc·Brf2 complex to form the
SNAPc·Brf2·TBP complex. Mutation of the PSE
severely impaired, but did not completely abrogate, the formation of
this new complex (lanes 15-17). This observation suggests
that TBP and Brf2 together can help stabilize SNAPc
binding to a weak PSE. In these reactions, a faster migrating
complex is also observed, which comigrates with the
SNAPc·Brf2 complex. However, this complex likely
corresponds to a complex containing Brf2 and TBP because the
SNAPc·Brf2 complex does not form on probes containing a mutated PSE (lane 14). Indeed, in subsequent
experiments the SNAPc·Brf2 and TBP·Brf2
complexes were observed to comigrate (data not shown). As expected,
when mutations were introduced into both the PSE and TATA box, DNA
binding by all factors was abolished (lanes 26-33).
Therefore, SNAPc can recruit Brf2 in a PSE-specific
manner, and together, these factors further stimulate TBP recruitment
to a human U6 promoter.

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Fig. 6.
Mini-SNAPc can recruit
Brf2 to a U6 snRNA promoter. EMSAs were performed with 1 µg of rTBP (lanes 2, 10, 18, and
26), 120 ng of Brf2 (lanes 3,
11, 19, and 27), 10 ng of
mini-SNAPc (lanes 4, 12,
20, and 28), rTBP and mini-SNAPc
(lanes 5, 13, 21, and 29),
Brf2 and mini-SNAPc (lanes 6,
14, 22, and 30), or Brf2,
mini-SNAPc, and increasing amounts of rTBP (0.1, 0.3 and
1.0 µg; lanes 7-9, 15-17, 23-25,
and 31-33, respectively). The DNA probes used contain a
high affinity mouse U6 snRNA PSE and a human U6 snRNA TATA box
(lanes 1-9), a mutant PSE and wild-type TATA box
(lanes 10-17), a wild-type PSE and mutant TATA box
(lanes 18-25), or a mutant PSE and mutant TATA box
(lanes 26-33). Lane 1 contains probe alone. The
presence (+) or absence ( ) of rTBP, Brf2, and
mini-SNAPc is indicated above each lane. The
positions of free probe and protein·DNA complexes are
indicated.
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|
The SNAP190 Myb Domain Stimulates Preinitiation Complex Assembly
with TFIIIB--
To determine whether the SNAP190 DNA binding domain
can facilitate TFIIIB assembly at human U6 promoters the ability of
GST-SNAP190 RcRd to form higher order complexes with TBP and
Brf2 was tested using an EMSA. As shown in Fig.
7, no significant DNA binding was
observed for TBP (lane 2), Brf2 (lane 3),
or GST-SNAP190 RcRd (lane 4) in reactions containing DNA
probes with wild-type PSE and TATA sequences. Similarly, no significant
DNA binding was observed in reactions containing GST-SNAP190 RcRd and
TBP (lane 5), which is consistent with the previous
observation that mini-SNAPc recruits TBP poorly under these
particular conditions (see Fig. 6). GST-SNAP190 RcRd also did not
facilitate efficient Brf2 binding (lane 6). This
result is distinct from that observed in Fig. 6 wherein
mini-SNAPc supported efficient Brf2 recruitment,
suggesting that an additional region of SNAP190 or other
SNAPc subunits is important for this function. In these
experiments, Brf2 and TBP bound DNA cooperatively as evidenced
by the greater DNA binding by both factors (lane 7) compared
with that observed with either factor alone. This result is consistent
with that described previously (33) and supports the notion that
Brf2 contributes to stable TBP binding to these promoters.
Interestingly, a significant increase in complex formation was observed
when GST-SNAP190 RcRd was added to reactions containing Brf2 and
TBP (compare lane 9 with lane 7), which suggests
that SNAP190 also cooperates with TBP and Brf2 for promoter
binding. This is specific for SNAP190 RcRd because comparable amounts
of GST did not stimulate TBP recruitment (lane 8). Mutations
in the TATA box completely abolished the formation of the
SNAP190·Brf2·TBP complex (lane 10), but
surprisingly, mutations in the PSE had only a modest effect on binding
(lane 11). The presence of GST-SNAP190 RcRd in this complex
was confirmed by antibody supershift experiments (data not shown).
Thus, it appears that Brf2 and TBP can help stabilize SNAP190
RcRd association with the promoter in the absence of strong DNA binding
by SNAP190. This result was unexpected but is consistent with the data
shown in Fig. 6 wherein mini-SNAPc·Brf2·TBP
complex formation was reduced, but not abolished, on probes containing
a wild-type TATA box with a mutant PSE. Altogether, these results
indicate that interactions between the SNAP190 Myb domain and TBP are
important for promoter recognition by TBP and for initial stages of
preinitiation complex assembly with TFIIIB.

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Fig. 7.
The SNAP190 RcRd Myb repeats stimulate
binding of TBP and Brf2 to the U6 promoter. The indicated
proteins individually (lanes 2-4), in pairwise combinations
(lanes 5-7), or rTBP, Brf2, and either GST
(lane 8) or GST-cd (lanes 9-12) were used in
EMSAs. Lane 1 contains probe alone. The DNA probes used are
represented schematically. The positions of free probe and
protein·DNA complexes are indicated.
|
|
 |
DISCUSSION |
Human U6 gene transcription depends upon a PSE and a TATA box that
together specify recruitment of RNA polymerase III to these promoters.
Earlier studies revealed that a strict spacing between the PSE and TATA
box is required for efficient transcription (46), which suggested that
proteins that bind to these sites interact with each other. Indeed,
SNAPc interacts cooperatively with TBP to bind to the U6
snRNA promoter (35). The data presented herein suggests that three
subunits, SNAP43, SNAP45, and SNAP190 interact directly with the
conserved COOH-terminal DNA binding domain of TBP. Thus,
SNAPc can make extensive contacts with TBP, and these interactions may be important for TBP function at human snRNA promoters. However, neither SNAP43 nor SNAP45 is critical for TBP
recruitment to a human U6 TATA box. One possibility is that these
factors participate in TBP function for RNA polymerase II transcription
of human snRNA genes that contain only a PSE and not a TATA box as is
present in human U1 genes, for example. This scenario seems more
plausible for SNAP43 than SNAP45 because SNAP45 is not essential for
SNAPc-dependent transcription in
vitro (43).
Coordinated Interactions of Juxtaposed SNAP190 and TBP DNA Binding
Domains--
SNAP190 interacts well with TBP via the TBP DNA binding
domain, and a TBP interaction region within SNAP190 was mapped to the
Myb DNA binding domain. Interestingly, the SNAP190 Myb domain alone is
sufficient to recruit TBP to a human U6 TATA box (see Fig.
5C). Full-length human TBP by itself does not bind well to a
TATA box because of an inhibitory effect of its
NH2-terminal domain. Therefore, the SNAP190 Myb RcRd DNA
binding domain is sufficient to overcome the inhibitory affect of the
TBP NH2 terminus on TBP-TATA box binding by the TBP DNA
binding domain. TBP binding to TATA box elements is a multistep
mechanism that involves initial binding to unbent DNA and conversion to
a more stable form containing bent DNA (47, 48). Recent experiments
suggest that the role of the NH2-terminal region of TBP is
to inhibit DNA bending rather than DNA binding (46). The function of
the NH2-terminal region is also linked to a surface-exposed
region of the TBP DNA binding domain called the inhibitory DNA binding
(IDP) surface. The IDP surface regulates both initial TATA box binding
and subsequent DNA bending, and it has been posited that the
NH2-terminal region directly contacts the IDP surface to
regulate negatively the transition to a TBP complex containing bent DNA
(47). Thus, an intriguing possibility is that the SNAP190 Myb domain
may directly contact the IDP surface to relieve negative regulation by
the TBP NH2 terminus and thus stimulate TBP conversion to a
stable complex containing bent DNA. In this context, the SNAP190 DNA
binding domain has two functions. One function is to recognize the PSE present in the core promoters of human snRNA genes, and a second function is to stimulate TBP recognition of the neighboring TATA box
present in human U6 snRNA gene promoters.
TBP Complex Recruitment to Human U6 snRNA Promoters--
In
addition to regulating TBP function at human U6 promoters, the SNAP190
Myb DNA binding domain might be sufficient to recruit a TBP-containing
complex that functions at these promoters. The observation that the
SNAP190 Myb domain facilitates formation of a complex containing TBP
and the TFIIIB factor Brf2 (see Fig. 7) is consistent with this
idea. One possibility is that the SNAP190 Myb RcRd repeats may function
through direct protein interactions with factors in the TFIIIB complex
other than TBP. However, the SNAP190 Myb RcRd domain alone does not
recruit Brf2 efficiently, suggesting that higher order complex
formation observed for SNAP190 RcRd with Brf2 and TBP is
mediated predominantly by the SNAP190-TBP interaction. It is
interesting that mini-SNAPc containing SNAP190 (1-505),
SNAP43, and SNAP50 can recruit Brf2 in a TATA box-independent fashion (see Fig. 6). This observation suggests that additional cross-talk between Brf2 and other regions of SNAPc
is likely important for efficient complex assembly with TFIIIB factors.
If so, these regions must reside within SNAP43, SNAP50, or SNAP190
(1-505) because these proteins are sufficient to reconstitute
SNAPc function in vitro. In addition, the strict
spacing arrangement between the PSE and TATA box may select which TBP
complex can bind to the U6 promoter. Because the two DNA elements must
be positioned close to each other to allow interactions between the DNA
binding domains of SNAP190 and TBP, their close proximity might present steric limitations to which TBP·TAF complex can recognize the U6 TATA
box. For example, the RNA polymerase II-specific TFIID complex that
contains TBP and at least 10 TAFs may be unable to contact the U6 TATA
box when SNAPc is bound to the PSE. In contrast, a smaller
complex such as one containing TBP, Bdp1, and a Brf protein might
present a better fit.
Assembly of an RNA Polymerase III Transcription Complex at Human U6
snRNA Promoters--
The upstream DSE in human U6 snRNA promoters
contains an octamer element that binds the transcription factor Oct-1.
Once bound to the octamer element the Oct-1 POU DNA binding
domain interacts directly with the SNAP190 protein to recruit
SNAPc to the U6 PSE (43, 49, 50). In the context of the
chromosome, SNAPc recruitment is dependent upon a
nucleosome positioned between the octamer site and the PSE (51, 52)
which effectively loops out the intervening DNA to juxtapose these
promoter elements. When the nucleosome is absent, the POU domain is
unable to recruit SNAPc probably because the distance
between the two sites is too great for the POU domain to stabilize
SNAPc binding (51). After or concomitant with
SNAPc binding to the PSE, an RNA polymerase III-specific TBP complex is recruited to the U6 TATA box. It seems unlikely that the
TATA box alone is sufficient to recruit a specific TBP complex. Indeed,
TBP can efficiently recruit Brf2 to a TATA box; however, this
recruitment is not specific for the U6 TATA box (33). In contrast,
SNAPc is sufficient to recruit and stabilize TBP binding to
the U6 TATA box (Fig. 1 and Ref. 43), and the SNAP190 Myb RcRd DNA
binding domain alone is sufficient for TBP and TBP·Brf2
recruitment via interactions with the TBP core region. Additional
interactions involving amino acids 34-83 of SNAP190 with TBP may
bolster TBP recruitment to the U6 TATA box (45). These observations
suggest a model for the SNAPc-mediated recruitment of a
snRNA-specific TFIIIB complex to the U6 snRNA promoter as shown in Fig.
8. Binding of SNAPc to the U6
PSE leads to recruitment of TBP through interactions with the SNAP190
Myb RcRd DNA binding domain and Brf2 through interactions with
other SNAPc proteins. Binding of TBP and Brf2 to the
TATA box further stabilizes SNAPc on the U6 promoter
leading to formation of a stable preinitiation complex. Thus, the
unique arrangement of core promoter elements at the U6 gene coordinates
interactions involving SNAPc, TBP, and Brf2 for
assembly of a RNA polymerase III-specific preinitiation complex.

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Fig. 8.
Schematic for promoter recognition by
SNAPc and TFIIIB. Efficient PSE recognition
by SNAPc is mediated by the SNAP190 Myb RcRd DNA binding
domain, which also functions to interact with the TBP DNA binding
domain to recruit a snRNA-specific TFIIIB complex. For simplicity, the
remaining SNAPc subunits are represented as a dotted
ellipse. Additional interactions between Brf2 and
SNAPc (dashed arrow) contribute to complex
stability at these promoters. Other TFIIIB components are not
shown.
|
|
 |
ACKNOWLEDGEMENTS |
We gratefully thank Laura Schramm and Nouria
Hernandez for Brf2 expression plasmids, Winship Herr for
anti-GST antibodies, and Grace Chen for technical assistance. We are
also indebted to Gauri Jawdekar, Nastya Gridasova, and Zakir Ullah for
helpful comments on the manuscript.
 |
FOOTNOTES |
*
This work was supported by National Institutes of Health
Grant R01 GM59805 and American Cancer Society Grant
RPG-00-263-01-GMC (to R. W. H.).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 should be addressed: Dept. of
Biochemistry and Molecular Biology, Michigan State University, 409 Biochemistry Bldg., Wilson Rd., East Lansing, MI 48824. Tel.:
517-353-3980; Fax: 517-353-9334; E-mail: henryrw@pilot.msu.edu.
Published, JBC Papers in Press, March 5, 2003, DOI 10.1074/jbc.M204247200
 |
ABBREVIATIONS |
The abbreviations used are:
TBP, TATA-box
binding protein;
EMSA, electrophoretic mobility shift assay;
IDP, inhibitory DNA binding;
PSE, proximal sequence element;
rTBP, recombinant TBP;
SNAP, snRNA-activating protein;
SNAPc, snRNA-activating protein complex;
snRNA, small
nuclear RNA;
TAF, TBP-associated factor;
Pou, Pit1/Oct1/Oct2/Unc86.
 |
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