Direct Interaction between the Subunit RAP30 of Transcription
Factor IIF (TFIIF) and RNA Polymerase Subunit 5, Which
Contributes to the Association between TFIIF and RNA Polymerase
II*
Wenxiang
Wei,
Dorjbal
Dorjsuren,
Yong
Lin,
Weiping
Qin,
Takahiro
Nomura,
Naoyuki
Hayashi, and
Seishi
Murakami
From the Department of Molecular Oncology, Cancer Research
Institute, Kanazawa University, Takara-machi 13-1, Kanazawa 920-0934, Japan
Received for publication, October 23, 2000, and in revised form, December 19, 2000
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ABSTRACT |
The general transcription factor
IIF (TFIIF) assembled in the initiation complex, and RAP30 of TFIIF,
have been shown to associate with RNA polymerase II (pol II),
although it remains unclear which pol II subunit is responsible for the
interaction. We examined whether TFIIF interacts with RNA polymerase II
subunit 5 (RPB5), the exposed domain of which binds transcriptional
regulatory factors such as hepatitis B virus X protein and a
novel regulatory protein, RPB5-mediating protein. The results
demonstrated that RPB5 directly binds RAP30 in vitro using
purified recombinant proteins and in vivo in COS1 cells
transiently expressing recombinant RAP30 and RPB5. The RAP30-binding
region was mapped to the central region (amino acids (aa) 47-120) of
RPB5, which partly overlaps the hepatitis B virus X protein-binding
region. Although the middle part (aa 101-170) and the N-terminus (aa
1-100) of RAP30 independently bound RPB5, the latter was not involved
in the RPB5 binding when RAP30 was present in TFIIF complex. Scanning
of the middle part of RAP30 by clustered alanine substitutions and then
point alanine substitutions pinpointed two residues critical for the
RPB5 binding in in vitro and in vivo assays.
Wild type but not mutants Y124A and Q131A of RAP30 coexpressed
with FLAG-RAP74 efficiently recovered endogenous RPB5 to the
FLAG-RAP74-bound anti-FLAG M2 resin. The recovered endogenous RPB5 is
assembled in pol II as demonstrated immunologically. Interestingly,
coexpression of the central region of RPB5 and wild type RAP30
inhibited recovery of endogenous pol II to the FLAG-RAP74-bound M2
resin, strongly suggesting that the RAP30-binding region of RPB5
inhibited the association of TFIIF and pol II. The exposed domain of
RPB5 interacts with RAP30 of TFIIF and is important for the association
between pol II and TFIIF.
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INTRODUCTION |
RNA polymerase II (pol
II)1 synthesizes all
messenger RNA in eukaryotes. Promoter-specific transcription initiation
requires the concerted action of a complex array of factors including
the general transcriptional factors TFIID, TFIIA, TFIIB, TFIIE, TFIIF, and TFIIH. Transcriptional activators and repressors bind distal elements of the promoter and modulate transcription by interacting with
the components of preinitiation complex (1-4), and another group of
proteins, cofactors or mediators, affect transcription positively or
negatively by communicating with promoter-specific regulatory factors
and the transcriptional machinery. All transcriptional factors and
cofactors modulate positively or negatively initiation and/or
elongation of mRNA synthesis. Therefore, RNA polymerase subunits
may be additional targets for transcriptional regulators, because pol
II is the core enzyme of the transcription machinery of gene
expression. Several lines of evidence for communication between
transcriptional regulators and pol II subunits have accumulated (5-14).
Pol II consists of 12 subunits, among which RPB5, RPB6, and RPB8 are
commonly shared by pol I, II, and III (15). RPB1 and RPB2 of pol II are
responsible for most of the catalytic activities for RNA synthesis,
although the other subunits contribute to the integrity of the
supramolecular complex in structure and function. We have previously
found that HBx, a multifunctional viral regulator protein of hepatitis
B virus, directly interacts with RPB5 (5), and both RPB5 and HBx
communicate with TFIIB. The trimeric interaction of these three factors
may facilitate transcription and HBx acts as coactivator in activated
transcription (7, 8). Based on these observations, we proposed that
RPB5 is a communicating subunit of pol II that interacts with
transcriptional regulators. In support of the notion, we previously
identified a novel protein, RPB5-mediating protein (RMP), which
counteracts the coactivator function of HBx by competitively binding
RPB5 (6). In yeast, RPB5 is an essential subunit and cannot be
complemented by human RPB5. Miyao et al. (16) reported that
yeast RPB5 is important for activated transcription of some genes in
yeast. Recently, a backbone model of a 10-subunit yeast RNA polymerase
II revealed the relative position of each subunit. RPB5 has two parts,
the exposed domain (N-terminal two-thirds) and the embedded domain, the
former being a component of the jaw in close proximity to duplex DNA
downstream of the active site (17, 18). In this context, the exposed
domain of RPB5 may be needed to interact with factors for transcription regulation.
TFIIF is a unique general transcription factor that functions in
initiation, elongation, and probably recycling of transcription (19,
20). Eukaryotic TFIIF is a heteromeric tetramer of RAP30 and RAP74 (21,
22) and communicates with transcription regulator factors (23-28).
RAP30 and RAP74 have been isolated as RAPs (RNA polymerase
II-associated proteins) using an affinity
column containing immobilized pol II (29). The structure and function
of both the RAP30 and RAP74 subunits of TFIIF have been well defined
(19-20, 30-32). The N-terminus of RAP30 is proposed to bind RAP74 to
form the TFIIF complex and is necessary for transcription initiation (33, 34). The middle part of RAP30 is a
-homologous region, which is proposed to associate with pol II (35, 36) and is essential
for transcription elongation (37). The C-terminus of RAP30 has been
demonstrated to contain a cryptic DNA-binding domain, which is
suggested to be homologous to the DNA template binding of prokaryotic
factor (38, 39). RAP30 has been shown to be necessary for most, if
not all, preinitiation complex formation and gene transcription (37,
40-42).
RAP30 has been shown to directly bind pol II (41), but it remains
unclear which subunit of pol II is responsible for the binding with
RAP30. Therefore, we examined the possibility that TFIIF interacts with
RPB5. Here we show that RPB5 directly binds to the middle part of RAP30
(aa 101-170) in the presence of RAP74. The residues Tyr124
and Gln131 of RAP30 were identified as critical for the
RPB5 binding, and furthermore, these mutants of RAP30 eliminated the
efficient recruitment of pol II to RAP30, indicating that the direct
binding of RAP30 and RPB5 is important for the recruitment of pol II to
TFIIF.
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MATERIALS AND METHODS |
Plasmid Constructions--
The plasmids pNKFLAG and pNKGST,
derived from pSG5UTPL, are FLAG-tagged and GST-tagged mammalian
expression vectors, respectively, as reported (7, 43). The plasmids
pGENK1 and pGENKS are bacterial expression vectors for GST-fused
proteins as reported previously (6, 7). The full-length and truncated
GST-RPB5 expression plasmids have been described (7). The plasmids
pGST-RAP30 and His-ET-RAP74 were a gift from R. G. Roeder. The
RAP30 cDNA of pGST-RAP30 was used as template to amplify PCR
products of RAP30 with the primer set of CAGAATTCATGGCCGAGCGCGGGGAA and
GCAGATCTGTCACTCTTTTCTTC, generating an artificial EcoRI site
at the 5'-end and a BglII site at the 3'-end, respectively.
The PCR product of RAP74 was amplified with the primer set of
CAGAATTCATGGCGGCCCTAGGCCCT and GCGGATCCTCCTTGAGGGAGAAGTG, with
His-ET-RAP74 as template, generating an artificial EcoRI
site at the 5'-end and a BamHI site at the 3'-end,
respectively. The PCR products were digested and inserted into the
EcoRI and BamHI sites of pNKGST, pNKFLAG, pGENKS,
and pYFLAG to construct various expression vectors. The resultant plasmids were used as template to construct a truncated version of
RAP30 by a PCR-assisted method (43). The truncation mutants of RAP30
cDNA, RAP30/d1, d2, d3, d4, and d5, encode the initiation codon
followed by amino acids 2-100, 2-176, 177-249, 101-249, and
101-176, respectively. An alanine scanning was applied to construct
clustered or single alanine substitution mutants of the middle part of
RAP30 by a splicing PCR method using mutated oligonucleotide
primer sets (7, 43). All of the constructs were sequenced by the
dideoxy method using Taq sequencing kits and a DNA sequencer
(370A; Applied Biosystems Inc. Co. Ltd.).
Preparation of Recombinant Proteins--
GST-fused proteins were
expressed in Escherichia coli by induction with 0.4 mM isopropyl-D-thiogalactopyranoside at
30 °C for 3 h. The cells were harvested and sonicated in PBST
buffer (phosphate-buffered saline containing 1% Triton X-100) (6, 7).
After centrifugation, the extracts (supernatants) were collected and
stored at
80 °C. For purification, the extracts were incubated
with glutathione-Sepharose 4B (Amersham Pharmacia Biotech) at room
temperature for 1 h. The beads were precipitated, washed four
times with an excess amount of PBST buffer, and then eluted with 10 mM reduced glutathione in 50 mM Tris-HCl (pH
8.0). The eluted proteins were divided into aliquots and stored at
80 °C.
The His-tagged RAP74 protein was expressed in BL21 (DE3)/pLys using 0.4 mM isopropyl-D-thiogalactopyranoside for 3 h at 30 °C. The cells were harvested and sonicated in native binding
buffer (20 mM sodium phosphate, 500 mM NaCl, pH
7.8), and the His-tagged proteins were purified by incubating the
sonication supernatant with nickel-resin (Invitrogen). After extensive
washing, the bound proteins were eluted with imidazole elution buffer
(300 mM imidazole, 20 mM sodium phosphate, 500 mM NaCl, pH 6.3).
FLAG-tagged proteins were expressed in BL21 by induction with 0.4 mM isopropyl-D-thiogalactopyranoside at
30 °C for 3-6 h. The cells were harvested and sonicated in 50 mM Tris-HCl, pH 8.0, 150 mM NaCl, and 0.1%
Triton X-100. After centrifugation, the supernatant was stored at
80 °C. FLAG-tagged proteins were purified by incubating the
sonication supernatant with anti-FLAG M2 resin (Kodak Scientific
Imaging Systems) followed by several washes. The bound proteins were
eluted with buffer containing FLAG peptide (0.2 mg/ml FLAG peptide, 50 mM Tris-HCl, pH 8.0, 150 mM NaCl).
In Vitro Reconstitution of TFIIF--
The reconstitution of
TFIIF was carried out basically according to the method of Maldonado
(44) but with one additional step for purification. Equimolar amounts
of partially purified GST-RAP30 (1 mg) and His-RAP74 (2 mg) were
mixed in 1.2 ml of buffer A (20 mM HEPES-KOH, pH 7.5, 10%
glycerol, 2 mM dithiothreitol, 0.5 mM EDTA)
containing 4 M urea, 0.5 M KCl. The mixture was
dialyzed against 100 volumes of buffer A with 0.5 M
KCl for 3 h at 4 °C and then dialyzed against 100 volumes of buffer A with 0.1 M KCl for another 3 h.
The dialyzed solution was centrifuged at 15,000 rpm for 10 min at
4 °C, and the supernatant was collected and incubated with 60 µl
of nickel resin for 20 min. After being washed with a solution
containing 0.5 M NaCl, 50 mM imidazole, and 20 mM Tris-HCl (pH 8.0) three times, the nickel resin-bound
proteins were eluted with elution solution (0.5 M NaCl, 0.2 M imidazole, 20 mM Tris-HCl, pH 8.0). The
eluted proteins were then incubated with 40 µl of packed
glutathione-Sepharose 4B in 1.0 ml of PBST buffer for 30 min. After
extensive washings, the bound proteins were eluted with 10 mM of reduced glutathione in 50 mM Tris-HCl (pH
8.0). Ten µl of the eluate was fractionated by 12.5% SDS-PAGE and
stained with Coomassie Brilliant Blue.
Antibodies--
Anti-RPB5, anti-RPB3, and anti-GST polyclonal
antibodies were reported previously (5-7). Anti-RPB6 and anti-RPB9
antibodies were a gift from R. G. Roeder, and anti-CTD monoclonal
antibody (7G5) was kindly provided by M. Vigneron. Anti-FLAG M2
antibody was purchased commercially (Kodak Science Imaging Systems).
In Vitro GST Resin Pull-down Assays--
A GST resin pull-down
assay was carried out as reported (6, 7). Approximately 1 µg of GST
or GST-fused protein immobilized on 20 µl of glutathione-Sepharose 4B
preblocked in 0.5% nonfat milk and 0.05% bovine serum albumin was
incubated with 0.1 µg of FLAG-tagged proteins in 0.5 ml of modified
GBT buffer for 1-2 h on a rotator at 4 °C. After being washed four
times with modified GBT buffer, the bound proteins were eluted,
fractionated by 12.5% SDS-PAGE, and subjected to Western blot
analysis using anti-FLAG monoclonal antibody (M2).
Immunoprecipitation, in Vivo Pull-down Assay, and Western Blot
Analysis--
Transient transfection of COS1 cells was carried out as
reported previously (6, 7). The cells were harvested, washed, and
sonicated in LAC buffer (10% glycerol, 20 mM HEPES, pH
7.9, 50 mM KCl, 0.4 M NaCl, 1 mM
MgCl, 0.1 mM dithiothreitol, 0.1 mM EDTA, 9 mM CHAPS, 0.5 mM phenylmethylsulfonyl fluoride,
10 µg/ml aprotinin and leupeptin) and then centrifuged. The total
cell lysates were stored at
80 °C. Approximately 1.5 mg of protein of total cell lysates was diluted with 4 times the volume of TBST buffer (50 mM Tris-HCl, pH 7.5, 50 mM NaCl,
0.05% Tween 20) and incubated with 50 µl of packed Sepharose 4B for
30 min. The supernatant was then obtained by centrifugation.
Supernatants with FLAG-tagged proteins were immunoprecipitated with 20 µl of 50% anti-FLAG M2 resin, rotated for 2 h at 4 °C, and
washed four times with washing buffer (50 mM Tris-HCl, pH
7.5, 150 mM NaCl) (TBS). For in vivo GST
pull-down assay, COS1 cell lysates with GST or GST-fused protein were
incubated with 20 µl of glutathione-Sepharose 4B at 4 °C. After
being rotated for 2 h and washed four times with washing buffer
(20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.1%
SDS, 1 mM EDTA), the bound proteins were eluted,
fractionated by 12.5% SDS-PAGE, transferred onto nitrocellulose
membranes, and subjected to Western blot analysis with the antibody.
The proteins were visualized by enhanced chemiluminescence (ECL),
according to the manufacturer's instructions (Amersham Pharmacia Biotech).
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RESULTS |
RPR5 Binds TFIIF through RAP30 in Vitro and in Vivo--
Since
RPB5 and TFIIF seem to play roles in the initiation of transcription
and both have been reported to interact with TFIIB (7, 33), we examined
whether RPB5 interacts with TFIIF. The recombinant GST-RAP30 and
His-RAP74 were independently expressed in E. coli, partially
purified, mixed at an equimolar ratio in denaturating conditions, and
dialyzed to renature the proteins (Fig.
1A, lane
1). Finally, the reconstituted TFIIF of the two factors was
purified on two successive affinity columns, nickel resin and
glutathione resin, as described under "Materials and Methods" (Fig.
1B, lanes 2 and 3). The
two-step purification is better than the one-step purification of TFIIF
(data not shown). The reconstituted TFIIF (rTFIIF) or each
subunit of TFIIF (Fig. 1B) was incubated with the purified
bacterial recombinant FLAG-RPB5 and then subjected to GST resin
pull-down assay using glutathione resin. The reconstituted TFIIF and
GST-RAP30 bound FLAG-RPB5, while GST-RAP74 and GST alone did not
recover FLAG-RPB5. The result indicates the direct binding of FLAG-RPB5
and reconstituted TFIIF and indicates that RAP30 might be the subunit
responsible for the binding.

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Fig. 1.
RPB5 binds reconstituted TFIIF and the RAP30
subunit in vitro. A, in
vitro reconstitution of TFIIF was carried out as described under
"Materials and Methods." Lanes 1-3, the
mixed solution of partially purified recombinant His-RAP74 and
GST-RAP30, the renatured proteins eluted from nickel resin, and the
reconstituted TFIIF (rTFIIF) eluted from glutathione resin,
respectively. B, bacterially expressed GST-RAP30, GST-RAP74,
GST, and the reconstituted TFIIF were purified, fractionated by 12.5%
SDS-PAGE as described under "Materials and Methods," and then
visualized by Coomassie Brilliant Blue staining. The positions of
molecular mass markers are indicated in kDa in A and
B. C, GST resin pull-down assay. Approximately 1 µg of GST, GST-RAP30, or GST-RAP74 or 2 µg of the reconstituted
TFIIF immobilized on glutathione resin was incubated with 0.1 µg of
purified recombinant FLAG-RPB5 in GBT buffer for 2 h at 4 °C.
After extensive washing, bound proteins were eluted, fractionated by
12.5% SDS-PAGE, and subjected to Western blot analysis with anti-FLAG
M2 antibody.
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Next we confirmed the binding of RPB5 and TFIIF in mammalian cells,
COS1 cells. GST-RPB5 and both GST-RAP30 and FLAG-RAP74, or GST-RPB5 and
either subunit of TFIIF in the FLAG-tagged form, were transiently
overexpressed. All of the proteins were similarly expressed in the
cells as detected by anti-FLAG M2 and anti-GST antibodies (Fig.
2, B and C). The
supernatants of lysates were incubated with anti-FLAG M2 resin, washed
extensively, and subjected to Western detection using anti-GST antibody
(Fig. 2A). GST-RPB5 was efficiently recovered with
FLAG-RAP74 together with GST-RAP30, although it was not copurified when
GST-RAP30 was absent. Similar to the in vitro experiment,
RPB5 directly interacted with RAP30 but not with RAP74. The result
clearly indicates that RPB5 directly binds to RAP30 in the presence or
absence of RAP74.

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Fig. 2.
RPB5 binds RAP30 subunit of TFIIF in
vivo. A, COS1 cells were cotransfected with
the mammalian expression plasmids, pNKFLAG-RAP30 or pNKFLAG-RAP74,
together with pNKGST-RPB5 and pNKGST-RAP30 as indicated at the
top. Cell lysates were prepared as described under
"Materials and Methods." Total lysate (~1.5 mg of protein) was
immunoprecipitated with 20 µl of packed anti-FLAG M2 antibody-bound
resin. After washing, the bound proteins were eluted and then
fractionated by 12.5% SDS-PAGE and subjected to Western blot analysis
with anti-GST antibody. The total lysates (5% of the samples) were
directly subjected to Western blot analysis with anti-FLAG M2
(B) and anti-GST (C) antibodies.
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Mapping of the RAP30-binding Region, Which Covers the
HBx-binding Site in RPB5--
The RAP30-binding region of RPB5 was
delineated using various truncation mutants by which we previously
demonstrated the HBx and TFIIB bindings of RPB5 (7). A series of
truncation mutants of RPB5 in GST form were tested for activity to bind
FLAG-RAP30 (Fig. 3A). Similar
amounts of the bacterial GST fusion proteins immobilized on glutathione
resin (1 µg) (Fig. 3B) were incubated with FLAG-RAP30
in vitro and subjected to GST resin pull-down assay (Fig.
3C). Mutant d13 (aa 47-120), the central region of RPB5, is
the minimal binding region of RAP30, since neither R83 (aa 73-120),
the minimal region for the HBx-binding, nor RAD2 (aa 45-93) retained
the ability to bind FLAG-RAP30. Therefore, the RAP30-binding region was
mapped within aa 47-120, which covers the HBx-binding region (7).

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Fig. 3.
Delineation of the RAP30-binding region in
RPB5. A, schematic map of various RPB5 deletion
constructs. The expression plasmids were constructed previously (7).
RAP30-binding ability is summarized on the right.
B, various GST-fused RPB5 truncation proteins were expressed
in E. coli, purified, fractionated by 12.5% SDS-PAGE as
described under "Materials and Methods," and visualized by
Coomassie Brilliant Blue staining. The positions of molecular mass
markers are indicated on the left in kDa. C, GST
resin pull-down assay. Approximately 1 µg of GST or GST-fused RPB5
truncation proteins immobilized on glutathione resin was incubated with
0.1 µg of bacterially expressed FLAG-RAP30 in GBT buffer. Pull-down
assay and Western blot analysis were carried out with anti-BLAG M2
antibody as described in the legend to Fig. 1. Lane
9 shows 5% of the input of FLAG-RAP30 used for the
pull-down assay.
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RAP30 Has Two Independent Regions, the N-Terminus and the Middle
Part, to Bind RPB5, although the Former Is Masked by RAP74 in the TFIIF
Complex--
The RPB5-binding region of RAP30 was mapped using a
series of truncation mutants of RAP30 in GST-fused form both in
vitro and in vivo (Fig.
4A). Bacterially expressed
proteins of wild type and truncation mutants of GST-RAP30 were purified
(Fig. 4B) and incubated with FLAG-RPB5 in vitro
(Fig. 4C). All proteins but mutant d3, the C-terminus of
RAP30 (aa 177-249), pulled down FLAG-RPB5. RAP30 seems to have two
separate regions to bind RPB5, since both d1 (aa 1-100) of N-terminus
and d5 (aa 101-176) of the middle part bound to RPB5. To confirm the
result of the in vitro pull-down assay, an in
vivo pull-down assay was carried out with the lysates of the COS1
cells coexpressing FLAG-RPB5 and wild type or mutant GST-RAP30. Neither
GST-RAP30/d3 nor GST alone could pull down FLAG-RPB5, confirming the
pull-down experiment in vitro (data not shown).

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Fig. 4.
RPB5 binds to both the N- terminus and the
middle part of RAP30 in vitro. A,
schematic representation of various RAP30 deletion constructs.
RAP30/d1, d2, d3, d4, and d5 contain the initiation codon followed by
aa 2-100, 2-176, 177-249,101-249, and 101-176, respectively.
RPB5-binding ability is summarized on the right.
B, bacterially expressed proteins of various GST-fused RAP30
deletion constructs were purified, fractionated by 12.5% SDS-PAGE, and
visualized by Coomassie Brilliant Blue staining. The positions of the
molecular mass markers are indicated. C, GST resin pull-down
assay. Approximately 1 µg of GST or GST-fused full-length and
truncated RAP30 immobilized on glutathione resin was incubated with 0.1 µg of bacterially expressed FLAG-RPB5 in GBT buffer for 2 h at
4 °C. Pull-down assay and Western blot analysis were carried out
with anti-FLAG M2 antibody as described under "Materials and
Methods."
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Since the N-terminus of RAP30 has been reported to be essential for the
complex formation with RAP74 (33, 34), we evaluated the RPB5 binding of
RAP30 in the presence of RAP74 in vitro and in
vivo. At first, the purified bacterial wild type and mutant RAP30
in GST-fused form were examined for binding to FLAG-RAP74. The
N-terminal RAP30 (d1, aa 1-100) as well as the full-length RAP30 could
pull-down FLAG-RAP74, but no binding was observed with d3 and d5
mutants of RAP30, confirming that the N-terminal region is critical for
the RAP74-binding (Fig. 5A,
lanes 1 and 4). Next we examined the
effect of RAP74 on the RPB5-binding of RAP30 in vitro. As
shown in Fig. 5B, the N-terminal RAP30 (d1) could not
recruit FLAG-RPB5 in the presence of FLAG-RAP74, while RAP30/d1 could
bind to FLAG-RPB5 in the absence of RAP74. In contrast, the middle part
of RAP30 (d5) could bind FLAG-RPB5 regardless of the presence of
FLAG-RAP74 (Fig. 5B, lanes 3 and
4). Since the full-size RAP30 bound FLAG-RPB5 even in the
presence of FLAG-RAP74 (Fig. 5B, lanes
5 and 6), it is strongly suggested that the
middle part of RAP30 is responsible for interacting with RPB5 in the TFIIF complex. The N-terminal RAP30 (d1) predominantly bound to FLAG-RAP74 when equal amounts of FLAG-RAP74 and FLAG-RPB5 were present.

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Fig. 5.
RAP74 binds to RAP30 and inhibits the
N-terminus of RAP30 from interacting with RPB5. A,
RAP74 binds to the N-terminus of RAP30 in vitro.
Approximately 1 µg of bacterial recombinant GST-RAP30 or the
GST-fused truncation protein of RAP30 was immobilized on glutathione
resin and incubated with 0.1 µg of FLAG-RAP74 in GBT buffer for
2 h at 4 °C. Pull-down assay and Western blot analysis were
carried out with anti-FLAG M2 antibody as in Fig. 1. B,
RAP74 inhibits the N-terminus of RAP30 from interacting with RPB5
in vitro. Approximately 1 µg of bacterial GST-RAP30 or the
RAP30 truncation mutants, was immobilized on glutathione resin
and incubated with 0.1 µg of FLAG-RPB5 in the presence of 0.1 µg of
FLAG-RAP74 (+) or bacterial lysate ( ) as shown at the top
for 2 h at 4 °C. Pull-down assay and Western blot analysis were
carried out with anti-FLAG M2 antibody as in A. C, RAP74 binds to the N-terminus of RAP30 in
vivo. COS1 cells were cotransfected with the mammalian expression
plasmids, pNKFLAG-RAP74 and pNKGST-RAP30, or the RAP30 truncation
mutant as indicated at the top. The total cell lysates (1.5 mg) of each sample were incubated with 20 µl of packed glatathione
resin for 2 h at 4 °C. In vivo pull-down assay and
Western blot analysis were carried out with anti-FLAG M2 antibody.
D, RAP74 inhibits the N-terminus of RAP30 from interacting
with RPB5 in vivo. COS1 cells were transfected with the
mammalian expression vectors, pNKFLAG-RPB5 and pNKGST-RAP30, or the
RAP30 truncation mutant d1 together with pNKFLAG-RAP74 (+) or vehicle
control pNKFLAG ( ) as indicated on the top. In vivo
pull-down assay and Western blot analysis were carried out as in
C. The positions of the molecular mass markers are indicated
in kDa on the right of B and D. About
5% of input was applied in the last lane of each
panel.
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To prove a similar role for the middle part of RAP30 in the RPB5
binding in TFIIF, lysates of COS1 cells coexpressing FLAG-RAP74 and
wild type or truncated RAP30 in GST-fused form were subjected to a GST
resin pull-down assay. The result confirmed that FLAG-RAP74 binds to
the N-terminus (d1) but not to other regions (d3 and d5) of RAP30 (Fig.
5C). Then the lysates of COS1 cells coexpressing FLAG-RAP74,
FLAG-RPB5, and wild type or d1 mutant of RAP30 in GST-fused form were
subjected to GST resin pull-down assay. RAP30/d1 recruited FLAG-RPB5 in
the absence of FLAG-RAP74, but no recruitment was observed when
FLAG-RAP74 was present (Fig. 5D, lanes
1 and 2). In contrast, the full-length RAP30
recruited FLAG-RPB5 even in the presence of FLAG-RAP74 (Fig.
5D, lanes 3 and 4). This
result is consistent with the result in vitro and indicates
that the middle part of RAP30 is the RPB5-binding region in the
TFIIF complex.
Amino Acid Residues of RAP30 Important for the RPB5
Binding--
To evaluate the relevance of the direct binding between
the middle part of RAP30 of the TFIIF complex and RPB5, we tried to identify the amino acid residue(s) that may be critical for the interaction between RPB5 and RAP30. Clustered alanine substitutions covering 3-7 amino acid residues in a row were introduced to the middle part of RAP30 (aa 101-176). A primary screening was carried out
to search for defective mutants by GST resin pull-down assay in
vitro using FLAG-RPB5 and mutants of RAP30/d4 (deleting the N-terminus of RAP30) in GST-fused form. All but two clustered mutants,
cm 124 (aa 124-126) and cm 131 (aa 130-132), retained the ability to
bind RPB5 (data not shown). Next, a point alanine substitution was
introduced to the six single-amino acid residues covering two cm
mutants defective in RPB5 binding.
Effects of the six single alanine-substitution mutations in the middle
part of RAP30 on the RPB5 binding in TFIIF complex were evaluated in
GST resin pull-down in vitro and in mammalian cells. TFIIF
was reconstituted with His-RAP74 and wild type or mutated GST-RAP30
in vitro and purified to examine the ability to bind
FLAG-RPB5. The single mutations in the middle part of RAP30 have no
inhibitory effect on the TFIIF complex formation, the same as the
clustered mutants. Only two mutants of RAP30 with point mutations at aa
124 and 131 were defective in RPB5-binding, although the wild type and
the other mutant proteins retained the binding ability (data not
shown). Next, the lysates of COS1 cells transiently coexpressing
GST-RPB5, FLAG-RAP74, and wild type or mutated RAP30 in GST-fused form
were prepared and subjected to coimmunoprecipitaton with anti-FLAG M2
antibody bound to resin. These recombinant proteins were well expressed
in the COS1 cells as detected by Western blotting with either anti-FLAG
M2 or anti-GST antibody (Fig.
6A). As shown in Fig.
6B, the two point mutations, m124 and m131, eliminated the
RPB5 binding but had no effect on the TFIIF complex formation in
vivo (Fig. 6C). These results indicate that two
residues, Tyr124 and Gln131, are critical for
the direct binding of RAP30 in the TFIIF complex to RPB5 in
vivo and in vitro.

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|
Fig. 6.
Amino acid residues of RAP30 critical for the
RPB5-binding in TFIIF form. A, COS1 cells were
transfected with the mammalian expression plasmids, pNKFLAG-RAP74,
pNKGST-RPB5, and pNKGST-RAP30, or the RAP30 mutants with a single amino
acid substitution as indicated at the top. Total lysates (60 µg) were fractionated and detected by Western blot analysis. The
positions of the molecular mass markers are indicated on the
left. B, the total lysate of each sample (1.5 mg
of protein) shown in A was immunoprecipitated with 20 µl
of packed anti-FLAG M2 antibody-bound resin. After washing, the bound
proteins were eluted, fractionated, and subjected to Western blot
analysis. C, the RAP30 mutants are intact for the
RAP74-binding. COS1 cells were transfected with the mammalian
expression plasmids, pNKFLAG-RAP74 and pNKGST-RAP30, or the RAP30
mutant with a single amino acid substitution. The total lysate (1.5 mg
of protein) was immunoprecepitated with anti-FLAG M2 antibody-bound
resin. The bound proteins were eluted, fractionated, and subjected to
Western blot analysis. Anti-FLAG M2 (upper
panels) or anti-GST (lower panels)
antibody was used in (A-C) for Western blot
detection.
|
|
RPB5 Is an Important Subunit of RNA Polymerase II for the
Association with TFIIF through Binding to RAP30--
The direct
interaction of RPB5 and TFIIF was demonstrated in vitro and
in vivo; however, all experiments were carried out with a
free RPB5 subunit instead of the assembled form of RPB5 in pol II.
Since the middle part of RAP30 has been proposed to be the pol
II-interacting region (35, 36), RPB5 may be the subunit of pol II
responsible for the interaction with TFIIF. To test this possibility,
GST-RAP30 and FLAG-RAP74 were transiently overexpressed in COS1 cells,
and immunoprecipitation was carried out with anti-FLAG M2
antibody-bound resin. The GST-RAP30 efficiently recruited endogenous
RPB5 (Fig. 7A, lane
1), although GST alone could not recover endogenous RPB5
(data not shown). The mutant RAP30, m124, or m131 in GST-fused form
could not recover endogenous RPB5, but mutant m132 recruited endogenous
RPB5 as the wild type RAP30 did. The result indicates that the binding
of RAP30 to RPB5 is critical for the association of TFIIF and pol II,
since the two mutants defective in the binding to free RPB5 have no
ability to recruit endogenous RPB5. In contrast, wild type RAP30 and
mutant m132 could recruit endogenous RPB5. To further confirm the
relevance of the direct binding of RPB5 and RAP30 to the recruitment of TFIIF to pol II, FLAG-RAP74 and GST-RAP30 were transiently
overexpressed in COS1 cells in the presence of GST-RPB5/d13, which
covers the RAP30-binding region but lacks the embedded domain into RPB1
and RPB2 (see "Discussion"). FLAG-RAP74 recruited GST-RAP30 but
could not recruit endogenous RPB5 or the other subunit of pol II when GST-RPB5/d13 was present. In contrast, FLAG-RAP74 efficiently pulled
down all of the examined subunits of pol II when the RAP30-binding region of RPB5, RPB5/d13, was absent. The endogenous RPB5 recruited to
TFIIF might be assembled in pol II, since the molar ratios of the pol
II subunits in the recruited fractions of wild type and m132 mutant of
RAP30 in TFIIF are similar to those detected in the unfractionated
sample (Fig. 7B, lanes 1,
5, and 6). m124 and m131 mutants of RAP30 were
defective in the recruiting of pol II subunits (Fig. 7B,
lanes 3 and 4). Taken together, these results clearly indicate that the direct binding of RPB5 and RAP30 in
TFIIF is critical for the recruitment of TFIIF to pol II and that two
residues, Tyr124 and Gln131, of RAP30 are vital
for the recruitment.

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|
Fig. 7.
TFIIF mutants defective in associating with
endogenous pol II. A, TFIIF mutants defective in
binding to endogenous RPB5. COS1 cells were transfected with
pNKFLAG-RAP74 and pNKGST-RAP30 or the RAP30 mutant as indicated at the
top. Immunoprecipitation was carried out with anti-FLAG M2
antibody-bound resin. The bound proteins were eluted, fractionated, and
subjected to Western blot with anti-GST (upper
panel) and anti-RPB5 (lower panel)
antibodies. Lane 5 shows 3% input of the total
lysate. B, TFIIF mutants defective in association with
endogenous pol II. COS1 cells were transfected with pNKFLAG-RAP74 and
pNKGST-RAP30 or the RAP30 mutant in the presence or the absence of
pNKGST-RPB5/d13. The total cell lysate (~2.5 mg of protein) of each
sample was immunoprecipitated with 20 µl of packed anti-FLAG M2
antibody-bound resin. After washing, the bound proteins were eluted,
fractionated, and subjected to Western blot analysis with antibodies
against RPB1, RPB3, RPB5, and RPB9 as indicated on the
right. Lane 6 shows 3% of the total
cell lysate used for lane 1. C, the
total lysates (60 µg) for use in B were subjected to
Western blot analysis with anti-FLAG M2 (upper
panel) and anti-GST (lower panel)
antibodies.
|
|
 |
DISCUSSION |
Although basal transcription in vitro requires TATA
binding protein (TBP) and TFIIB in addition to pol II
when the template is negatively supercoiled (45) and TFIIB is able to
bind RNA polymerase II (46), the complex formation in the absence of TFIIF seems to be rather weak, since TBP and TFIIB are not
able to recruit pol II into the preinitiation complex as detected by electrophoretic mobility shift assay. The addition of TFIIF or recombinant RAP30 alone was demonstrated to recruit the pol II to the
complex in the assay, indicating that RAP30 is necessary for the
recruitment of pol II in preinitiation complex (40, 42). The middle
part of RAP30 has been assessed to be important for recruitment of pol
II by the fact that pol II blocked the phosphorylation of serine
residues located in the middle part of RAP30 by protein kinase (35).
However, the subunit of pol II responsible for interacting with TFIIF
remains unknown.
In this report, we showed that RPB5 binds RAP30 but not RAP74 and
associates to TFIIF through the binding to RAP30. Although both the
N-terminus and the middle part of RAP30 independently bind RPB5 in the
absence of RAP74, the middle part but not the N-terminus of RAP30 can
interact with RPB5 in the presence of RAP74 in vivo and
in vitro. This result is consistent with the previous
reports of the interaction between RAP74 and the N-terminus of RAP30 in
the TFIIF complex (33, 34). This phenomenon seems to be similar to the
finding that RAP74 bound RAP30 and blocked TFIIB from binding to RAP30
(33), although RAP30 free from RAP74 actually functions during
transcription. We specified two residues, Tyr124 and
Gln131, of RAP30 as critical for the RPB5 binding. One
possibility is that the residues are involved in the direct interaction
between RPB5 and RAP30. Alternatively, the residues are critical for
the proper conformation of RAP30 and indirectly contribute to the interaction. In the latter case, the alanine substitutions may increase
the helical content of the region, although no gain-of-function of the
side chain is expected by the substitutions. The actual molecular
details of the interaction between RPB5 and RAP30 remain to be
elucidated. The two critical residues are within the
homology region of RAP30 (aa 111-152) and actually within the proposed sequence
of sigma factor (corresponding to aa 119-134 of RAP30), which
interacts with bacterial RNA polymerase (35). However, the clustered
and point alanine substitution mutants covering the homology region did
not exhibit any defect in RPB5 binding except those with the two
residues, strongly suggesting that the amino acid residues conserved
among
70 and RAP30 are not involved in the RPB5 binding.
Importantly, endogenous pol II was efficiently copurified with
overexpressed recombinant wild type RAP30 complexed with RAP74 but not
with the mutated RAP30 at aa 124 or 131, both of which are intact to
bind RAP74. Furthermore, the overexpressed central region of RPB5
efficiently inhibited the association between recombinant RAP30 and
endogenous pol II. These results strongly suggest that the binding
between RPB5 and RAP30 is the most important interaction for the
association of pol II and TFIIF even if it is not exclusive.
Crystal models of yeast RPB5 showed that it consists of an N-terminal
exposed domain (aa 1-141), a linker, and an evolutionary ancient
C-terminal domain conserved from archaea to humans (1, 2). Although
less than 60% of amino acid residues are conserved among humans and
Saccharomyces cerevisiae, the structural model of human RPB5
is quite close to that of yeast (by Insight II, data not shown) except
for a longer loop of yeast RPB5 around aa 67-75 (in aa number of yeast
RPB5). Within the exposed domain, the RAP30 binding region (aa 47-120)
is immediately downstream of TFIIB-binding regions (aa 21-47) and
overlaps partly the HBx-binding region. RPB5 and duplex DNA downstream
of the active site are very close in the crystal model, which is
consistent with the report that RPB5 is in the region from
5 to +15
of the promoter site in preinitiation complex (47). Although a crystal
model of RAP30 or TFIIF is not available at present, the TFIIF complex is supposed to make contact with promoter DNA in the region from
61
to +34 of DNA template (48). The direct binding of RAP30 and RPB5
described in this paper seems to be consistent with the previous
findings and may occur between pol II and TFIIF complex.
Our results raise several important questions. First, the close
location of the TFIIB-binding region and the RAP30-binding region of
RPB5 may imply that RAP30 and TFIIB can interact with each other on the
platform of RPB5. The crystal model may not support interaction between
TFIIB and RPB5 in the pol II complex. However, the possibility of
trimeric interaction among the three proteins should be examined, since
interaction between RAP30 and TFIIB has been reported, and a crystal
model of the full-length TFIIB complexed with pol II has not been
reported. A linker between the N-terminal domain and the core domain of
TFIIB may make the TFIIB molecule flexible. Second, the mutations of
RAP30 may affect basal or activated transcription in vitro.
In addition, HBx may affect the binding between RPB5 and RAP30 or
TFIIF, since HBx and RAP30 share a common sequence of RPB5 for the
bindings. Study of the interaction of TFIIF and pol II through the same
region of RPB5 may improve our understanding of the transcription
modulation mechanism of HBx.
 |
ACKNOWLEDGEMENTS |
We thank Dr. R. G. Roeder for anti-RPB6
antibody and plasmids of His-pET-RAP74 and pGST-RAP30. We are grateful
to Dr. M. Vigneron for anti-CTD monoclonal antibody; to Dr. K. Masutomi
for the construction of pNKGST, pNKFLAG, and pGENK plasmids; and to F. Momoshima, M. Yasukawa, and K. Kuwabara for technical assistance. We
also thank Dr. K. Ikeda for encouraging discussions.
 |
FOOTNOTES |
*
This work was partly supported by Grants-in-aid for
Scientific Research on Priority Area C and Grants-in-aid for Scientific Research B of the Ministry of Education, Sciences, Sports and Culture
of Japan.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 Molecular
Oncology, Cancer Research Institute, Kanazawa University, Takara-machi 13-1, Kanazawa 920-0934, Japan. Tel.: 81-76-265-2731; Fax:
81-76-234-4501; E-mail: semuraka@kenroku.ipc.kanazawa-u.ac.jp.
Published, JBC Papers in Press, January 22, 2001, DOI 10.1074/jbc.M009634200
 |
ABBREVIATIONS |
The abbreviations used are:
pol I, II, and III,
RNA polymerase I, II, and III, respectively;
TFIIF, transcription
factor;
HBx, hepatitis B virus X protein;
RPB, RNA polymerase II
subunit;
GST, glutathione S-transferase;
PAGE, polyacrylamide gel electrophoresis;
RMP, RPB5-mediating protein;
PCR, polymerase chain reaction;
CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid.
 |
REFERENCES |
1.
|
Ge, H.,
and Roeder, R. G.
(1994)
Cell
78,
513-523[Medline]
[Order article via Infotrieve]
|
2.
|
Goodrich, J. A.,
Hoey, T.,
Thut, C. J.,
Admon, A.,
and Tjian, R.
(1993)
Cell
75,
519-530[Medline]
[Order article via Infotrieve]
|
3.
|
Inostroza, J. A.,
Mermelstein, F. H.,
Ha, I.,
Lane, W. S.,
and Reinberg, D.
(1992)
Cell
70,
477-489[Medline]
[Order article via Infotrieve]
|
4.
|
Meisterernst, M.,
and Roeder, R. G.
(1991)
Cell
67,
557-567[Medline]
[Order article via Infotrieve]
|
5.
|
Cheong, J. H.,
Yi, M.,
Lin, Y.,
and Murakami, S.
(1995)
EMBO J.
14,
143-150[Abstract]
|
6.
|
Dorjsuren, D.,
Lin, Y.,
Wei, W.,
Yamashita, T.,
Nomura, T.,
Hayashi, N.,
and Murakami, S.
(1998)
Mol. Cell. Biol.
18,
7546-7555[Abstract/Free Full Text]
|
7.
|
Lin, Y.,
Nomura, T.,
Cheong, J.,
Dorjsuren, D.,
Iida, K.,
and Murakami, S.
(1997)
J. Biol. Chem.
272,
7132-7139[Abstract/Free Full Text]
|
8.
|
Lin, Y.,
Tang, H.,
Nomura, T.,
Dorjsuren, D.,
Hayashi, N.,
Wei, W.,
Ohta, T.,
Roeder, R.,
and Murakami, S.
(1998)
J. Biol. Chem.
273,
27097-27103[Abstract/Free Full Text]
|
9.
|
Lin, Y.,
Nomura, T.,
Yamashita, T.,
Dorjsuren, D.,
Tang, H.,
and Murakami, S.
(1997)
Cancer Res.
57,
5137-5142[Abstract]
|
10.
|
Petermann, R.,
Mossier, B. M.,
Aryee, D. N.,
Khazak, V.,
Golemis, E. A.,
and Kovar, H.
(1998)
Oncogene
17,
603-610[CrossRef][Medline]
[Order article via Infotrieve]
|
11.
|
Schlegel, B. P.,
Green, V. J.,
Ladias, J. A.,
and Parvin, J. D.
(2000)
Proc. Natl. Acad. Sci. U. S. A.
97,
3148-3153[Abstract/Free Full Text]
|
12.
|
Tan, Q.,
Linask, K. L.,
Ebright, R. H.,
and Woychik, N. A.
(2000)
Genes Dev.
14,
339-348[Abstract/Free Full Text]
|
13.
|
Koleske, A. J.,
and Young, R. A.
(1994)
Nature
368,
466-469[CrossRef][Medline]
[Order article via Infotrieve]
|
14.
|
Murakami, S.
(1999)
Intervirology
42,
81-99[CrossRef][Medline]
[Order article via Infotrieve]
|
15.
|
Woychik, N. A.,
Liao, S. M.,
Kolodziej, P. A.,
and Young, R. A.
(1990)
Genes Dev.
4,
313-323[Abstract]
|
16.
|
Miyao, T.,
and Woychik, N. A.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
15281-15286[Abstract/Free Full Text]
|
17.
|
Todone, F.,
Weinzierl, R. O.,
Brick, P.,
and Onesti, S.
(2000)
Proc. Natl. Acad. Sci. U. S. A.
97,
6306-6310[Abstract/Free Full Text]
|
18.
|
Cramer, P.,
Bushnell, D. A.,
Fu, J.,
Gnatt, A. L.,
Maier-Davis, B.,
Thompson, N. E.,
Burgess, R. R.,
Edwards, A. M.,
David, P. R.,
and Kornberg, R. D.
(2000)
Science
288,
640-649[Abstract/Free Full Text]
|
19.
|
Roeder, R. G.
(1996)
Trends Biochem. Sci.
21,
327-335[CrossRef][Medline]
[Order article via Infotrieve]
|
20.
|
Nikolov, D. B.,
and Burley, S. K.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
15-22[Abstract/Free Full Text]
|
21.
|
Conaway, J. W.,
and Conaway, R. C.
(1989)
J. Biol. Chem.
264,
2357-2362[Abstract/Free Full Text]
|
22.
|
Flores, O.,
Ha, I.,
and Reinberg, D.
(1990)
J. Biol. Chem.
265,
5629-5634[Abstract/Free Full Text]
|
23.
|
Kim, J. B.,
Yamaguchi, Y.,
Wada, T.,
Handa, H.,
and Sharp, P. A.
(1999)
Mol. Cell. Biol.
19,
5960-5968[Abstract/Free Full Text]
|
24.
|
Joliot, V.,
Demma, M.,
and Prywes, R.
(1995)
Nature
373,
632-635[CrossRef][Medline]
[Order article via Infotrieve]
|
25.
|
Lipinski, K. S.,
Esche, H.,
and Brockmann, D.
(1998)
Virus Res.
54,
99-106[CrossRef][Medline]
[Order article via Infotrieve]
|
26.
|
Dubrovskaya, V.,
Lavigne, A. C.,
Davidson, I.,
Acker, J.,
Staub, A.,
and Tora, L.
(1996)
EMBO J.
15,
3702-3712[Medline]
[Order article via Infotrieve]
|
27.
|
Martin, M. L.,
Lieberman, P. M.,
and Curran, T.
(1996)
Mol. Cell. Biol.
16,
2110-2118[Abstract]
|
28.
|
McEwan, I. J.,
and Gustafsson, J.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
8485-8490[Abstract/Free Full Text]
|
29.
|
Sopta, M.,
Carthew, R. W.,
and Greenblatt, J.
(1985)
J. Biol. Chem.
260,
10353-10360[Abstract/Free Full Text]
|
30.
|
Ren, D.,
Lei, L.,
and Burton, Z. F.
(1999)
Mol. Cell. Biol.
19,
7377-7387[Abstract/Free Full Text]
|
31.
|
Tan, S.,
Aso, T.,
Conaway, R. C.,
and Conaway, J. W.
(1994)
J. Biol. Chem.
269,
25684-25691[Abstract/Free Full Text]
|
32.
|
Chang, C.,
Kostrub, C. F.,
and Burton, Z. F.
(1993)
J. Biol. Chem.
268,
20482-20489[Abstract/Free Full Text]
|
33.
|
Fang, S. M.,
and Burton, Z. F.
(1996)
J. Biol. Chem.
271,
11703-11709[Abstract/Free Full Text]
|
34.
|
Yonaha, M.,
Aso, T.,
Kobayashi, Y.,
Vasavada, H.,
Yasukochi, Y.,
Weissman, S. M.,
and Kitajima, S.
(1993)
Nucleic Acids Res.
21,
273-279[Abstract]
|
35.
|
McCracken, S.,
and Greenblatt, J.
(1991)
Science
253,
900-902[Medline]
[Order article via Infotrieve]
|
36.
|
Sopta, M.,
Burton, Z. F.,
and Greenblatt, J.
(1989)
Nature
341,
410-414[CrossRef][Medline]
[Order article via Infotrieve]
|
37.
|
Tan, S.,
Conaway, R. C.,
and Conaway, J. W.
(1995)
Proc. Natl. Acad. Sci. U. S. A.
92,
6042-6046[Abstract/Free Full Text]
|
38.
|
Tan, S.,
Garrett, K. P.,
Conaway, R. C.,
and Conaway, J. W.
(1994)
Proc. Natl. Acad. Sci. U. S. A.
91,
9808-9812[Abstract/Free Full Text]
|
39.
|
Garrett, K. P.,
Serizawa, H.,
Hanley, J. P.,
Bradsher, J. N.,
Tsuboi, A.,
Arai, N.,
Yokota, T.,
Arai, K.,
Conaway, R. C.,
and Conaway, J. W.
(1992)
J. Biol. Chem.
267,
23942-23949[Abstract/Free Full Text]
|
40.
|
Flores, O.,
Lu, H.,
Killeen, M.,
Greenblatt, J.,
Burton, Z. F.,
and Reinberg, D.
(1991)
Proc. Natl. Acad. Sci. U. S. A.
88,
9999-10003[Abstract]
|
41.
|
Killeen, M. T.,
and Greenblatt, J. F.
(1992)
Mol. Cell. Biol.
12,
30-37[Abstract]
|
42.
|
Killeen, M.,
Coulombe, B.,
and Greenblatt, J.
(1992)
J. Biol. Chem.
267,
9463-9466[Abstract/Free Full Text]
|
43.
|
Murakami, S.,
Cheong, J. H.,
and Kaneko, S.
(1994)
J. Biol. Chem.
269,
15118-15123[Abstract/Free Full Text]
|
44.
|
Maldonado, E.,
Drapkin, R.,
and Reinberg, D.
(1996)
Methods Enzymol.
274,
72-100[Medline]
[Order article via Infotrieve]
|
45.
|
Parvin, J. D.,
and Sharp, P. A.
(1993)
Cell
73,
533-540[Medline]
[Order article via Infotrieve]
|
46.
|
Malik, S.,
Lee, D. K.,
and Roeder, R. G.
(1993)
Mol. Cell. Biol.
13,
6253-6259[Abstract]
|
47.
|
Kim, T. K.,
Lagrange, T.,
Wang, Y. H.,
Griffith, J. D.,
Reinberg, D.,
and Ebright, R. H.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
12268-12273[Abstract/Free Full Text]
|
48.
|
Robert, F.,
Douziech, M.,
Forget, D.,
Egly, J. M.,
Greenblatt, J.,
Burton, Z. F.,
and Coulombe, B.
(1998)
Mol. Cell
2,
341-351[Medline]
[Order article via Infotrieve]
|
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