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(Received for publication, October 19,
1995; and in revised form, February 6, 1996) From the
A set of deletion mutants of human RNA polymerase II-associated
protein (RAP) 30, the small subunit of transcription factor IIF (TFIIF;
RAP30/74), was constructed to map functional domains. Mutants were
tested for accurate transcriptional activity, RAP74 binding, and TFIIB
binding. Transcription assays indicate the importance of both N- and
C-terminal sequences for RAP30 function. RAP74 binds to the N-terminal
region of RAP30 between amino acids 1 and 98. TFIIB binds to an
overlapping region of RAP30, localized to amino acids 1-176
(amino acids 27-152 comprise a minimal binding region). The
C-terminal region of RAP74 (amino acids 358-517) binds directly
and independently to TFIIB. Interestingly, RAP74 blocks TFIIB-RAP30
binding, both by binding TFIIB and by binding RAP30. When the TFIIF
complex is intact, therefore, TFIIB-TFIIF contact is maintained through
RAP74. If the TFIIB-RAP30 interaction is physiologically important, the
TFIIF complex must dissociate within some transcription complexes.
A minimal pathway for assembly of pre-initiation complexes on
RNA polymerase II-dependent promoters has been defined in
vitro(1, 2, 3, 4, 5) .
On promoters that include a TATA box, TBP ( TFIIB is a single polypeptide of 33 kDa (8, 9) that can be divided by sequence and mutational
analysis into N- and C-terminal regions. Near the N terminus is a
probable Zn TFIIF is a heteromeric factor of
28- (RAP30) and 58-kDa (RAP74)
subunits(19, 20, 21, 22) , but there
is some indication that in certain contexts these subunits may enter
complexes as separate factors(7, 23, 24) . Accurate initiation has been demonstrated from highly supercoiled
templates using a system consisting of RNA polymerase II, TFIIB, and
either TBP (25) or YY1, which is an initiator binding
protein(26) . One implication of these observations is that RNA
polymerase II and TFIIB might minimally suffice to select
transcriptional start sites. Consistent with this view, swapping Schizosaccharomyces pombe for Saccharomyces cerevisiae TFIIB and RNA polymerase II, in a system otherwise comprised of S. cerevisiae factors, shifts the position of the
transcriptional start to that characteristic of S.
pombe(27) . Also, some sua7 mutants in the S.
cerevisiae gene encoding TFIIB are altered for selection of
initiation sites(28) . sua8 mutants, in the gene
encoding the largest subunit of RNA polymerase II, affect
transcriptional starts in a very similar way to these sua7 mutants(29) . Interestingly, mutations in the S.
cerevisiae SSU71/TFG1 gene, which encodes the homologue of the
RAP74 subunit of human TFIIF, suppress abnormal start site selection in
a sua7ssu71 double mutant(30) . By itself the ssu71 mutant does not affect transcriptional starts, so TFIIF
may suppress the sua7 mutant indirectly through another
general factor. The RAP30 subunit of human TFIIF has been shown to
interact physically with the N-terminal region of TFIIB(12) ,
so the large subunit of TFIIF interacts genetically, and the small
subunit interacts physically, with TFIIB. The largest subunit of RNA
polymerase II has an interesting C-terminal domain (CTD) that consists
of 52 repeats of the consensus sequence
YSPTSPS(31, 32) . RNA polymerase II enters the
pre-initiation complex most efficiently in the dephosphorylated IIa
state. Within the complex the CTD is multiphosphorylated on the SP
serines, by a subunit of TFIIH and possibly other CTD kinases.
Elongating RNA polymerase II molecules are primarily in the highly
phosphorylated IIo state. So the level of CTD phosphorylation may
regulate elongation, and a CTD phosphatase may be important for
recycling dephosphorylated polymerase IIa to a promoter after
termination. A CTD phosphatase has recently been identified that
binds to RNA polymerase II(33, 34) . This phosphatase
interacts with a region of polymerase distinct from the CTD and
requires this contact for its dephosphorylation activity. The RAP74
subunit of TFIIF stimulates CTD phosphatase activity by binding RNA
polymerase II(34) . Consistent with this view, the C-terminal
region of RAP74, which binds RNA polymerase II, is required for
phosphatase stimulation. The C-terminal region of RAP74 is masked for
phosphatase stimulation (34) just as it is for RNA polymerase
II binding (35) and for TFIIB binding (this paper). Consistent
with functional interaction between TFIIB and TFIIF, TFIIB suppresses
stimulation of CTD phosphatase activity by RAP74(34) . In
this report, the region of RAP30 that binds RAP74 and the regions of
RAP30 and RAP74 that bind TFIIB were mapped. The region of RAP30 that
binds RAP74 overlaps with the region of RAP30 that binds TFIIB. RAP74
binding to TFIIB and RAP74 binding to RAP30 both block TFIIB-RAP30
binding.
RAP30-(1-249), RAP30-(1-227),
RAP30-(1-176), RAP30-(1-152), RAP30-(1-118),
RAP30-(1-98), RAP30-(27-249), and RAP30-(27-152)
accumulated within inclusion bodies and were purified by methods
previously described for full-length RAP30(37) .
RAP30-(51-249), RAP30-(110-249), and RAP30-(160-249)
were in the soluble fraction and were purified by
Ni RAP74 and RAP74 mutants were prepared
as described(35) . A production vector for human TFIIB and
instructions for its use were the kind gifts of D.
Reinberg(8) . RNA polymerase II was isolated from calf thymus (39) .
ELISA assays were done as described(40) .
Microtiter wells (Becton Dickinson; Pro-Bind) were coated overnight
with the protein to be immobilized (5 µg/ml) in 100 µl of 50
mM sodium borate, pH 9.0, at 4 °C. Wells were washed three
times with 200 µl of phosphate-buffered saline (PBS) containing
0.2% BSA and 0.05% Tween 20 (PBSBT) and blocked with 200 µl of
PBSBT for an hour at room temperature. Mobile proteins were added in 50
µl of SB 0.1 containing 0.2% BSA and incubated for 15 min at room
temperature. Glutaraldehyde was added to a final concentration of 1%
for 30 min to cross-link protein-protein interactions. Wells were
washed three times with 200 µl of PBSBT. 100 µl of rabbit
antiserum (1:1000 diluted) directed against the mobile phase protein
was added to each well and incubated 2 h at room temperature. Wells
were washed three times with PBSBT, and 100 µl of horseradish
peroxidase-conjugated goat anti-rabbit secondary antibody (Bio-Rad;
1:3000 diluted) was added and incubated for 1 h at room temperature.
Wells were washed 3 times with PBSBT. Color was developed with 100
µl of 50 mM sodium citrate, pH 4.0, 0.03%
H A set of RAP30 and RAP74 deletion mutants was constructed
with C-terminal histidine tags to aid in purification and binding
reactions (Fig. 1; (35) ). Each of the RAP30 mutants was
tested for the ability to stimulate accurate runoff transcription from
the adenovirus major late promoter (Fig. 2). Full-length
RAP30-(1-249) was much more active in this assay than any of the
deletion mutants. RAP30-(1-227) and -(1-176) supported a
much lower level of activity. RAP30-(1-152) had barely detectable
activity, and RAP30-(1-118) was inactive (data not shown). These
results are consistent with previously published data from the Conaway
laboratory (41, 42) that indicates that the C
terminus of RAP30 is involved in DNA binding and is important for
accurate initiation. Activity was also very sensitive to deletion of
N-terminal sequences. RAP30-(27-249) supported very weak activity (Fig. 2), but RAP30-(51-249) was inactive (data not
shown). Using a set of RAP30 mutants with short internal deletions,
Conaway and co-workers (43) have recently shown that amino acid
sequences between 16-30 and 136-210 are critical for
initiation of transcription. The extract system used by our laboratory
is more tolerant of C-terminal region mutations than the reconstituted
system, perhaps because of proteins present in the extract that are not
counted among the known general transcription factors.
Figure 1:
RAP30 and RAP74 deletion mutants.
RAP30-(1-249) is the histidine-tagged version of RAP30. Accurate
transcription (tx.) was determined from the adenovirus major
late promoter (Fig. 2). RAP74 binding to RAP30 was determined
using a Ni
Figure 2:
Transcriptional activities of RAP30
deletion mutants. An extract derived from human HeLa cells was depleted
of TFIIF by immunoprecipitation with anti-RAP30 antibodies. This
extract was supplemented with RAP74 (3.4 pmol) and the indicated
amounts of RAP30 or RAP30 mutant, and accurate transcription was
determined from the adenovirus major late promoter. Transcripts were
quantitated using a phosphorimager. Data are shown only for those
mutants for which stimulation of accurate transcription was detected.
Figure 3:
Interactions between TFIIF subunits and
TFIIB. A, both RAP30 and RAP74 bind directly and independently
to TFIIB. 300 pmol of RAP30 (30) (lanes 1 and 2) or RAP74 (74) (lanes 3 and 4) was
incubated with affinity beads containing covalently immobilized TFIIB (IIB) or no protein ligand (CT for control). After
washing with SB 0.25, bound protein was eluted and analyzed on an
SDS-PAGE gel developed with silver nitrate. 20-30% of input RAP30
and RAP74 was recovered from the beads. B, RAP30 sequences
between amino acids 1-176 are required for tight binding to
TFIIB. RAP30-(1-152) binds weakly to TFIIB. The binding assay was
done with TFIIB affinity beads and the indicated RAP30 mutants. C, a minimal TFIIB-binding region of RAP30 is localized
between amino acids 27-152. RAP30 or RAP30 mutants (500 ng) were
immobilized in the wells of a microtiter dish. After blocking,
increasing amounts of TFIIB were incubated in the wells. TFIIB binding
to RAP30 mutants was detected with anti-TFIIB antiserum. The A
Figure 4:
RAP74 blocks TFIIB-RAP30 binding by two
mechanisms. A, RAP74 blocks formation of a TFIIB-RAP30 complex (filled circles). TFIIB does not block formation of a TFIIF
(RAP30/RAP74) complex (open circles). RAP30 does not block
formation of a TFIIB-RAP74 complex (
Yonaha et
al.(44) demonstrated that RAP30 amino acids 1-110
were sufficient for binding to RAP74, and Tan et al.(43) demonstrated that amino acids between 16 and 20 were
critical for this interaction. Using an affinity bead procedure, we
have shown that RAP30 amino acids from 1 to 98 are sufficient for RAP74
binding (data not shown; results tabulated in Fig. 1). Taken
together with the results from accurate transcription assays (Fig. 2), these data indicate that most of the RAP30 deletion
mutants used in these studies have preserved at least some of the known
functions of RAP30 for supporting accurate transcription and/or RAP74
binding.
To demonstrate the specificity of TFIIB-TFIIF interactions
and to indicate their functional importance, the regions of RAP30 and
RAP74 required to bind TFIIB were mapped. In Fig. 3, B and C, RAP30 deletion mutants were tested for TFIIB
binding. Interaction was assayed using two methods that may differ in
sensitivity. For the experiment shown in Fig. 3B, TFIIB
was covalently immobilized on agarose beads and used as an affinity
adsorbent for RAP30 mutants. RAP30 and RAP30-(1-176) bound most
tightly to TFIIB (lanes 1 and 6). RAP30-(1-152)
also bound but much less efficiently (lane 7). None of the
other deletion mutants was observed to bind. Apparently, primary
sequence distributed over a significant portion of RAP30 contributes to
TFIIB binding. In Fig. 3C, an ELISA 10 plate test was
used to measure TFIIB-RAP30 binding. Microtiter plate wells were coated
with RAP30 or a RAP30 mutant. A control ELISA experiment developed with
anti-RAP30 antiserum demonstrated that immobilization of RAP30 mutants
was of comparable efficiency (data not shown). After blocking, TFIIB
was incubated with RAP30 mutants bound to the wells. After washing,
bound TFIIB was detected with anti-TFIIB antiserum, using a horseradish
peroxidase-linked second antibody and a colorimetric enzyme assay.
ELISA appears to provide a reliable measure of interaction and a
somewhat more sensitive detection of RAP30-TFIIB binding than the
affinity bead procedure. By ELISA, RAP30, with or without a C-terminal
histidine tag, and RAP30-(1-227), -(1-176),
-(27-249), and -(27-152) bound TFIIB most tightly.
RAP30-(1-152) and -(1-118) showed reduced affinity.
RAP30-(51-249) and -(110-249) were further reduced in
affinity, and RAP30-(1-98) was not observed to bind. Since
RAP30-(27-152) binds tightly to TFIIB by this analysis, most
TFIIB-RAP30 contacts appear to involve sequence within this region.
Since RAP30-(1-118) and -(110-249) bind TFIIB weakly, amino
acids between 27-118 and 110-176 (or 110-152) appear
to contribute to this interaction. The ELISA binding assay was done in
SB 0.1, and the affinity bead assay involved a washing step with SB
0.25, so a difference in salt concentration may account for slightly
different but consistent results using these two procedures. A set
of RAP74 mutants was also tested for binding to immobilized TFIIB (Fig. 3D). RAP74-(358-517) bound most tightly (lane 4). Full-length RAP74, RAP74-(87-517), and
-( Sequences within the N-terminal region and central region of RAP74
affect masking of the C-terminal domain for RNA polymerase II binding (35) and CTD phosphatase stimulation(34) . Since
RAP74-(358-517) appears to bind more tightly to TFIIB than
full-length RAP74, or RAP74-(87-517), -(207-517), and
-(
RAP74 could block TFIIB-RAP30 binding by two
mechanisms. 1) RAP74 could bind TFIIB to block the RAP30 binding site
on TFIIB. 2) RAP74 could bind RAP30 to block the TFIIB binding site on
RAP30. Both mechanisms contribute to blocking the TFIIB-RAP30
interaction (Fig. 4D). RAP74 mutants containing the
TFIIB binding region (RAP74 amino acids 358-517) but missing the
RAP30 binding region (RAP74 amino acids 1-172) block TFIIB-RAP30
binding (left panel). There is a close correlation between the
RAP74 mutants that bind TFIIB most tightly and the ability of these
mutants to compete the TFIIB-RAP30 interaction (compare Fig. 3D and 4D (left panel)). Also,
RAP74 mutants containing the RAP30 binding region but missing the TFIIB
binding region block TFIIB-RAP30 binding (right panel). Since the TFIIB-RAP74 interaction takes precedence over the
TFIIB-RAP30 interaction, one question that arises is whether the
TFIIB-RAP74 interaction is maintained in higher order complexes
containing RNA polymerase II. Although this question is difficult to
address directly, we have tested the effect of RNA polymerase II on
binding of TFIIB and RAP74 (Fig. 4E). Since both TFIIB
and RNA polymerase II bind to overlapping regions within the C-terminal
domain of RAP74 (amino acids 358-517 for TFIIB binding versus 363-444 for polymerase binding), these proteins might
compete or cooperate for binding. RNA polymerase II does not appear to
strongly compete or facilitate the TFIIB-RAP74 interaction. When RAP74
was immobilized and TFIIB was added as the binding partner, addition of
RNA polymerase II was not observed to affect formation of complexes
containing TFIIB and RAP74 (filled circles). When TFIIB was
immobilized and RAP74 added as the binding partner, addition of RNA
polymerase II caused a moderate reduction in the retention signal (open circles). Of course, RNA polymerase II is expected to
interact with both TFIIB and RAP74, and polymerase might interfere with
binding between anti-RAP74 antibody and RAP74. The slight apparent
inhibition of binding that is observed, in the experiment in which
TFIIB was immobilized (open circles), could be attributable to
antibody interference or distribution of TFIIB-polymerase and
RAP74-polymerase complexes. It may be difficult to exchange TFIIB and
RAP74 bound to separate polymerase molecules into a RNA polymerase
II-TFIIB-RAP74 complex, since this requires dissociation of either
TFIIB or RAP74 from polymerase. Consistent with this idea, when RNA
polymerase II was immobilized and competition for binding was between
RAP74 and TFIIB, a smaller reduction in the retention signal was
observed. This was true whether detection was for TFIIB
( There is mounting evidence for functional interaction between
TFIIF and TFIIB. These factors cooperate to bring RNA polymerase II
into the pre-initiation
complex(3, 4, 5, 6) .
Transcriptional start sites are selected by a complex that includes
TFIIF, TFIIB, and RNA polymerase II, and genetic experiments implicate
each of these factors in this
process(27, 28, 29, 30) . TFIIB and
TFIIF also can cooperate to control dephosphorylation of the
CTD(34) . RAP30 appears to consist of three functional
regions. The N-terminal region binds RAP74(43, 44) ,
the central region binds RNA polymerase II(45) , ( Analysis of sequence (46, 47) and
deletion mutants (35) indicates that RAP74 is also divided into
three functional regions. The RAP74 primary sequence can be divided
into an N-terminal basic region, a highly charged central region with
overall negative charge, and a C-terminal basic region. The N-terminal
region binds to RAP30(35, 44) . The central region
appears to be a largely unstructured hinge that controls accessibility
of the C-terminal region. The C-terminal region of RAP74 binds directly
to TFIIB (Fig. 3D) and to RNA polymerase II (35) . Polymerase-RAP74 contact stimulates a CTD phosphatase
that must bind to the ``body'' of polymerase to access the
CTD ``tail''(34) . Deletion from the N terminus of
RAP74 initially increases masking of TFIIB binding (Fig. 3D), RNA polymerase II binding(35) , and
stimulation of CTD phosphatase activity(34) . Further deletion
from the N terminus and within the central region unmasks TFIIB
binding, polymerase binding, and stimulation of CTD phosphatase
activity. The central region is also the site of phosphorylation by
casein kinase II, and phosphorylation by this and/or other kinases
appears to stimulate polymerase binding by TFIIF(48) . Although TFIIB can bind directly to RAP30, RAP74 blocks TFIIB-RAP30
binding through two mechanisms. By binding the N-terminal region of
RAP30, the N-terminal region of RAP74 blocks TFIIB-RAP30 binding (Fig. 4D, right panel). Also, by binding
TFIIB, the C-terminal region of RAP74 blocks TFIIB-RAP30 binding (Fig. 4D, left panel). The structure of the
TATA box-TBP-TFIIB complex strongly indicates that interaction between
TFIIB and TFIIF might be through the N-terminal region of
TFIIB(17) . TFIIB-TFIIF contact, therefore, is most likely
maintained through the N-terminal region of TFIIB and the C-terminal
region of RAP74. The strongest contacts between TFIIB and RAP74 map
between RAP74 amino acids 358-517 (Fig. 3D).
Although RAP74 blocks TFIIB-RAP30 binding, RAP74 can bind to RAP30 and
TFIIB simultaneously (Fig. 4A). This result is
consistent with the mapping of RAP74 functional domains, since the
N-terminal region binds RAP30 and the C-terminal region binds TFIIB.
Although RNA polymerase II and TFIIB bind to overlapping regions within
the C-terminal domain of RAP74, RNA polymerase II does not appear to
strongly compete TFIIB-RAP74 binding (Fig. 4E). That
TFIIB and RAP74 might interact in complexes containing RNA polymerase
II is also indicated by the genetic interaction between yeast SUA7 and SSU71(30) and by TFIIB-mediated suppression
of RAP74 stimulation of CTD phosphatase activity(34) .
Interaction between RAP74 and TFIIB, therefore, may be maintained in
complexes with RNA polymerase II. The SUA7 gene in yeast
encodes TFIIB, and the RAP74 homologue in yeast is encoded by SSU71/TFG1. sua7 mutants that confer cold sensitivity and
altered transcriptional start site selection, and are suppressed by ssu71 mutants, map to a particular region of TFIIB near the N
terminus(11) . From our work, we would have predicted that the ssu71 mutations that suppress sua7 might map within
the C-terminal region of SSU71 where human TFIIB and RAP74
physically interact. The ssu71 G363D and G363R alleles, ( Although the experiments shown here demonstrate
specific protein-protein contacts between general transcription factors
TFIIB and TFIIF, the importance of RAP74 blocking TFIIB-RAP30
interaction is not clear. TFIIB is expected to interact with TFIIF
through the RAP74 subunit, although in the absence of RAP74, a
TFIIB-RAP30 complex could be maintained. Such alternate structures are
most easily understood if TFIIF does not invariably function as an
intact unit, but rather, under some circumstances, as two independent
factors, RAP30 and RAP74. Chang et al.(23) demonstrated that accurate initiation could be
reconstituted in a TFIIF-depleted extract by addition of RAP30 alone.
In those experiments, elongation to the run-off position required
further addition of RAP74. Thus, RAP30 and RAP74 functions appear to be
partially separable in initiation and elongation, at least in an
extract transcription system. TFIIB-RAP30 interactions, therefore,
might be maintained within some promoter-bound complexes to support
initiation but not elongation. Addition of RAP74 would displace the
TFIIB-RAP30 interaction, and elongation would begin. Regulated
interaction between TFIIB, RAP30, and RAP74, therefore, might allow for
distinct pathways of pre-initiation complex assembly on different
promoters and for separate timing of RAP30 and RAP74 function in
initiation and elongation.
Volume 271,
Number 20,
Issue of May 17, 1996 pp. 11703-11709
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
)(TATA-binding
protein) first binds to this recognition sequence. TFIIA facilitates
this interaction. TFIIB can enter the complex either by binding to
these template-associated factors or by binding to RNA polymerase II.
For a tight association with the complex, polymerase must first
associate with TFIIF, so TFIIB and TFIIF cooperate to bring polymerase
into the pre-initiation complex (4, 5, 6, 7) . TFIIE and TFIIH are
additionally required for accurate initiation from linear DNA
templates.
-binding sequence that is important for
pre-initiation complex assembly(10) . Immediately adjacent to
the Zn-binding motif is a highly conserved sequence
that is important for selection of transcriptional start sites (11) . This N-terminal region of TFIIB binds the RAP30 subunit
of TFIIF and also RNA polymerase II(12, 13) , and
mutants in this region inhibit assembly of transcription intermediates,
probably because they fail to interact with either TFIIF or
polymerase(10, 12, 13, 14) . The
C-terminal region of TFIIB contains two imperfect direct repeats. Near
the end of the first repeat is a very basic sequence, and the region
surrounding this structure includes separate binding sites for TBP and
the acidic activation domain of herpes simplex virus
VP16(15, 16) . In the absence of other factors, the N-
and C-terminal regions of TFIIB fold on one another, masking binding
sites for other transcription factors. Binding to VP16 opens up TFIIB
to expose the C-terminal region, which binds TBP, and the N-terminal
region, which binds RNA polymerase II and TFIIF (16) . Thus,
TFIIB forms a bridge between TBP and RNA polymerase II-TFIIF, in
assembly of the pre-initiation complex, and the assembly steps in which
TFIIB is involved are influenced by regulators. Recent reports of an
x-ray crystal structure of TATA box DNA with C-terminal fragments of
TBP and TFIIB (17) and a nuclear magnetic resonance structure
of a C-terminal fragment of TFIIB (18) support this model for
TFIIB function in assembly. The C-terminal region of TFIIB binds TBP,
activators, and template, and the N-terminal region is presented as a
scaffold for assembly of RNA polymerase II-TFIIF into the
complex(17, 18) .
Protein Reagents
Fragments of a human RAP30 cDNA (36) were subcloned into pET23d (Novagen) and
transformed into Escherichia coli BL21(DE3) for
expression(37) . To construct RAP30-(1-227), an NcoI to BamHI (blunt: end-filled with Klenow DNA
polymerase I and deoxynucleoside triphosphates) fragment was subcloned
between the NcoI and HincII sites of the vector. To
construct RAP30-(1-176) an NcoI to XhoI (blunt)
fragment was cloned between the NcoI and HincII sites
of the vector. These mutants have a DKLAAALEHHHHHH C-terminal
extension. To construct RAP30-(1-118), an NcoI to PvuII fragment was subcloned between the NcoI and XhoI (blunt) sites of the vector. This mutant has an HHHHHH
C-terminal extension. To construct RAP30-(1-98) an NcoI
to SstI (blunt) fragment was subcloned between the NcoI and HincII sites of the vector. This mutant has
a DKLAAALEHHHHHH C-terminal extension. RAP30-(1-249),
RAP30-(27-249), RAP30-(51-249), RAP30-(110-249),
RAP(160-249), RAP30-(1-152), and RAP30-(27-152) were
constructed by polymerase chain reaction amplification using an
upstream primer containing an engineered NcoI site and a
downstream primer containing an engineered NotI site. The NcoI to NotI fragments were subcloned between the
same sites of the vector. These mutants have a AALEHHHHHH C-terminal
extension.-affinity chromatography (Qiagen).
RAP30-(110-249) was further purified by Mono S (Pharmacia Biotech
Inc.) chromatography. Protein concentrations were determined using
molar extinction coefficients calculated on the basis of aromatic amino
acid composition(38) .In Vitro Transcription
The transcriptional
activity of RAP30 mutants was tested as described
previously(37) . A TFIIF-depleted extract was prepared by
immunodepletion using anti-RAP30 antibodies. This extract was combined
with adenovirus major late promoter template DNA (pML digested with SmaI, 50 µg/ml), RAP74, and RAP30 wild type or mutants and
preincubated for 1 h in buffer containing 12 mM Hepes, pH 7.9,
12% glycerol, 60 mM KCl, 12 mM MgCl
, 0.2
mM EDTA, 3.2 mM EGTA, and 1.2 mM dithiothreitol. 600 µM ATP, CTP, UTP, and 25
µM [
P]GTP were added to the
reaction, and transcription was allowed to proceed for 30 min.
Transcripts were isolated and analyzed by polyacrylamide gel
electrophoresis in 50% (w/v) urea and 1 
TBE. The accurately
initiated transcript (217 nucleotides) was quantitated using a
Molecular Dynamics phosphorimager.Protein-Protein Interaction Assays, TFIIB Binding to
RAP30 and RAP74 Mutants
TFIIB was immobilized on Affi-Gel 10
(Bio-Rad) at a density of about 1 mg of TFIIB per ml of resin. 20
µl of TFIIB beads and 300 pmol of RAP30 or RAP74 mutant were
incubated at 4 °C for 1 h in 0.5-ml storage buffer containing 0.1 M KCl (SB 0.1) and 0.2% BSA. SB contains 20 mM Hepes
pH 7.9, 20% glycerol (w/v), 1 mM EDTA, 1 mM EGTA, and
variable KCl concentration (SB 0.1 contains 0.1 M KCl). Beads
were washed with 1 ml of SB 0.25, and bound proteins were eluted in 50
µl of SB 0.5. 30 µl of this eluate was analyzed on a 15%
SDS-PAGE gel developed with silver nitrate. 20-30% of input RAP30
and RAP74 were retained on TFIIB affinity beads in these experiments.
Affi-Gel beads without bound protein ligand were used as a negative
control.O, and 0.4 mg/ml o-phenylenediamine.
The development was stopped by addition of 100 µl of 4 N HSO
. The reaction was measured for
absorbance at 490 nm using a plate reader (Bio Tek Instruments; EL310).
All determinations were done in duplicate and reported as average
values.
-affinity bead procedure (data not shown).
TFIIB binding to RAP30 and RAP74 was determined by affinity bead and
ELISA procedures ( Fig. 3and Fig. 4). +, high
activity; ±, low activity; ?, barely detectable
activity; and -, no detectable activity; n.d., no
determination was made for a particular mutant; *, data are not shown
in this report.
, 1-249; 
, 1-227; , 1-176; 
,
27-249.
differs between the two experiments shown
in the left and right panels because of the time of
color development. RAP30 wild type has no histidine tag.
RAP30-(1-249) has a C-terminal histidine tag. D, RAP74
has a masked binding site for TFIIB located within a C-terminal region
between amino acids 358 and 517. RAP74 and RAP74 mutants were bound to
TFIIB affinity beads. Bound proteins were analyzed in a polyacrylamide
gel developed with silver nitrate. None of the RAP74 mutants bound to
negative control beads with no bound protein ligand (data not
shown).
-). TFIIB or
RAP74 (500 ng) was immobilized in the wells of a microtiter plate. A
mixture of a mobile protein (3.5 pmol) and increasing quantities of a
potential binding competitor (or facilitator) were added. Detection of
bound protein was with anti-RAP30 or anti-RAP74 antiserum. B,
RAP74 blocks formation of the TFIIB-RAP30 complex. A
Ni-affinity bead procedure was used. Histidine-tagged
RAP30 or RAP30-(1-176) (300 pmol) was bound to TFIIB (600 pmol)
in the presence or absence of RAP74 (600 pmol). In the presence of
RAP74, TFIIB did not bind to the Ni beads. A Western
blot is shown developed with anti-TFIIB antiserum. C, TFIIB
does not block formation of a TFIIF complex. Histidine-tagged RAP30 or
RAP30-(1-176) retained RAP74 on Ni beads in
both the presence and absence of TFIIB. A Western blot is shown
developed with anti-RAP74 antiserum. D, RAP74 blocks formation
of the TFIIB-RAP30 complex both by binding to TFIIB (left
panel) and by binding to RAP30 (right panel). RAP74 or
RAP74 mutants (competitor) were tested in the ELISA competition assay
as in A. The region of RAP74 that binds TFIIB maps between
amino acids 358 and 517 (Fig. 3D) and the region of
RAP74 that binds RAP30 maps between amino acids 1 and
172(35, 44) . E, calf thymus RNA polymerase
II (RNAP II) does not appear to block TFIIB-RAP74
interactions. The ELISA competition assay was done as indicated in the key.
Interactions between TFIIF subunits and TFIIB
Because of
genetic and physical interactions between TFIIF and
TFIIB(12, 30) , RAP30 and RAP74 were tested for
binding to TFIIB immobilized on agarose beads (Fig. 3). In Fig. 3A, TFIIB is shown to bind to both RAP30 (lane
2) and RAP74 (lane 4). This result confirms the previous
report of a direct interaction between TFIIB and RAP30 (12) and
demonstrates for the first time that TFIIB interacts directly with
RAP74.
137-356) bound with somewhat lower affinity (lanes
1, 2, and 6). RAP74-(207-517) and
-(407-517) bound much more weakly. RAP74 mutants from which the
C-terminal region was deleted were not observed to bind TFIIB (data not
shown). The TFIIB binding site on RAP74, therefore, appears to be
located within the C-terminal region between amino acids 358 and 517. 137-356), the C-terminal domain also appears to be masked
for TFIIB binding. RAP74-(207-517) shows the most dramatic
masking effect for polymerase binding (35) and phosphatase
stimulation (34) just as it does for TFIIB binding (Fig. 3D, lane 3). In summary, the central
region of RAP74 (amino acids 207-356) appears to mask the
C-terminal region, and the N-terminal region (amino acids 1-205)
makes the C-terminal region more accessible for protein-protein
interactions.RAP74 Blocks TFIIB-RAP30 Binding
Since both TFIIB
and RAP74 interact independently with overlapping regions of RAP30,
RAP74 might stimulate or antagonize TFIIB-RAP30 binding. In Fig. 4A, an ELISA test for interactions between TFIIB,
RAP30, and RAP74 was done in which one protein was immobilized in the
well of the microtiter plate, another was added to the well as a
binding partner, to be detected with antibody, and the third was added
as a potential competitor or facilitator of the binding interaction.
Using this protocol, RAP74 was shown to inhibit TFIIB binding to RAP30 (filled circles). This same conclusion was obtained using a
Ni-affinity bead procedure in which histidine-tagged
RAP30 was used to retain TFIIB in the presence or absence of RAP74 (Fig. 4B). So RAP74 can block the TFIIB-RAP30
interaction, but in a similar test, RAP30 was not observed to block the
interaction between TFIIB and RAP74 (Fig. 4A;
(-)). This was perhaps the expected result because
the N-terminal region of RAP74 binds to
RAP30(35, 44) , and the C-terminal region of RAP74
binds to TFIIB (Fig. 3D), so both contacts should be
maintained simultaneously. TFIIF subunit-subunit interactions are also
stable in the presence of TFIIB (Fig. 4, A (open
circles) and C). In the presence of RAP74, therefore,
TFIIB does not bind RAP30, but contact between RAP74 and RAP30 is
maintained. TFIIB-TFIIF interactions, therefore, are most likely
maintained through contact between TFIIB and the C-terminal region of
RAP74. For the TFIIB-RAP30 interaction to be maintained within a
transcription complex, TFIIF would have to dissociate into RAP30 and
RAP74 subunits.-) or RAP74 (filled squares). Of course
TFIIB and RAP74 could bind independently to polymerase without
maintaining the TFIIB-RAP74 interaction. Additional experiments will be
required to demonstrate TFIIB-RAP74 contacts within the pre-initiation
complex.
)and the C-terminal region binds
DNA(41, 42) . In the current work, we have mapped the
functional domains of human RAP30 that are required for accurate
transcription, that bind RAP74, and that bind TFIIB. Tight TFIIB-RAP30
binding requires primary sequence distributed over an extensive region
of RAP30 between amino acids 1 and 176 (Fig. 3, B and C).
)however, correspond to the Lys-111 position of human
RAP74(30, 35) , within the RAP30 binding region. So
both TFIIF subunits may cooperate with TFIIB in the process of start
site selection.
)))
We thank M. Hampsey for helpful comments on this
manuscript and for permission to mention ssu71 G363D and G363R
alleles. We thank D. Reinberg for a TFIIB production clone and for
instructions for TFIIB purification.
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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