(Received for publication, November 7, 1994; and in revised form, December 22, 1994)
From the
Transcription factor IIB (TFIIB) plays a central role in the assembly of the RNA polymerase II initiation complex. Monoclonal antibodies (mAbs) that react with human TFIIB were prepared and used as probes to identify portions of TFIIB that are accessible when the factor is in solution and when it is contained in a complex with DNA. Seven mAbs were examined and were mapped to three regions of the TFIIB molecule. Only the mAbs that mapped to residues 52-105 inhibited transcription, immunoprecipitated recombinant TFIIB and TFIIB from HeLa cell nuclear extract (NE), and supershifted a complex containing TFIIB, the TATA-binding protein, and DNA. The mAbs that mapped to residues 1-51 and the mAb that mapped to residues 106-316 did not show activity in the functional assays, with the exception of the far N-terminal mAbs (residues 1-51), which immunoprecipitated recombinant TFIIB, but not TFIIB from HeLa cell NE. These data indicate that the region containing residues 52-105 is exposed in solution and when TFIIB is part of the preinitiation complex and that some far N-terminal epitopes are accessible on the purified protein, but become blocked when TFIIB is in HeLa cell NE or in the preinitiation complex.
Initiation of transcription by RNA polymerase II requires a
variety of proteins to interact in the promoter region of the gene. The
proteins that must assemble on the DNA immediately upstream of the
initiation start site and/or interact with RNA polymerase II at the
start site are called general transcription factors (GTFs) ()and have been the subject of recent
reviews(1, 2, 3) . The GTFs purified from
human cells have been given the designation TFIIA, -B, -D, -E, -F, and
-H. The designations refer to the original chromatographic fractions
that contained transcriptional activity when combined and assayed in vitro using a linear template containing the adenovirus-2
major late promoter (MLP). In recent years, most of these GTFs have
been cloned, and the role of each factor in transcription initiation is
beginning to be defined. Homologous sets of factors have been obtained
from a variety of organisms; the most extensively studied are those
from yeast, rat, and Drosophila.
Most of the GTFs are
multimeric proteins, consisting of two or more subunits. Human TFIID is
a multimeric complex containing the TATA-binding protein (TBP) and at
least eight TBP-associated factors (4) . The binding of TFIID
(or TBP) to the TATA element at position -30 (with respect to the
transcriptional start site at position +1) nucleates the formation
of the transcription complex by recruiting TFIIA and TFIIB(5) .
TFIIA is nonessential and is thought to function by stabilizing the
TFIIDDNA complex and/or relieving the effect of inhibitory
proteins. TFIIB recruits the RNA polymerase II
TFIIF complex to
the preinitiation complex. TFIIE is then thought to bind to the
complex, recruiting TFIIH(6) . Recently, it has been
established that on certain supercoiled templates, accurate
transcription can be accomplished with just a subset of these
factors(7, 8) . This subset consists of TBP, TFIIB,
and RNA polymerase II, although the addition of the RAP30 component of
TFIIF seems to greatly stimulate the transcription reaction.
TFIIB
is unique among the GTFs in being a single protein, with the human
protein consisting of 316 amino acids. In addition to bridging the
TFIID (or TBP)DNA complex to the RNA polymerase II
TFIIF
complex, TFIIB binds to some activator
proteins(9, 10, 11, 12, 13) ,
which might help recruit TFIIB into the preinitiation complex. TFIIB
shares some general structural features with TBP, having an imperfect
direct repeat in the C-terminal region with a basic region between the
repeats(14, 15) . Recent reports from several
laboratories indicate that TFIIB has a bipartite structure. The
C-terminal domain forms a trypsin-resistant
structure(16, 17) . Truncated protein containing only
this C-terminal domain can bind to the TBP
DNA complex, but does
not support transcription(16, 17, 18) . The
N-terminal region is susceptible to proteolytic digestion, suggesting a
less compact structure. Protein lacking part or all of the N-terminal
region is deficient in recruiting the RNA polymerase II
TFIIF
complex to the assembling preinitiation
complex(16, 17, 18, 19) . In
addition, point mutations within a putative zinc finger structure
located at the far N terminus are also deficient in recruiting the RNA
polymerase II
TFIIF complex to the preinitiation
complex(20) . Deletion of almost any part of human TFIIB
results in loss of transcriptional activity(18, 19) .
In this study, we have used monoclonal antibodies (mAbs) as
structural probes to help dissect some of the regions of human TFIIB
that are accessible when the protein is in solution and when it is
complexed with other factors on promoter DNA. Surprisingly, although
the C-terminal domain has been shown to bind to the TBPDNA
complex, we found that some epitopes in the N-terminal region become
inaccessible when TFIIB is contained in the TFIIB
TBP
DNA
complex, while other epitopes within the N-terminal region remain
accessible.
For
production of mAbs, hybridoma cells were injected into Pristane-primed
Balb/c ByJ mice. Mouse IgG and IgG
mAbs were
purified from ascites fluid by chromatography on protein A-agarose
(Repligen, Cambridge, MA) as described(21) . Mouse IgG
mAbs were purified from ascites fluid by chromatography on
DEAE-cellulose as described(24) .
Figure 1: Epitope mapping of the TFIIB mAbs using protein fragments. A, Coomassie Blue-stained SDS-polyacrylamide gel electrophoresis of TFIIB treated with proteolytic enzymes. Lane1, prestained molecular weight markers; lane 2, 7.5 µg of untreated TFIIB; lanes 3 and 4, TFIIB treated with S. aureus V8 protease and trypsin, respectively. B, Western blot of the TFIIB treated with V8 protease and trypsin. Lanes1, 4, and 7, prestained molecular weight markers (these markers do not show up well on the photograph of the blot, but they served as markers to allow us to cut the blot accurately between sets of lanes); lanes 2, 5, and 8, TFIIB treated with V8 protease; lanes 3, 6, and 9, TFIIB treated with trypsin. The blot was reacted with mAbs IIB1 (lanes 2 and 3), IIB8 (lanes 5 and 6), and IIB5 (lanes 8 and 9). Because both digestions contained a considerable amount of undigested TFIIB, an untreated sample was not included on the blot. C, schematic representation of human TFIIB showing the protein fragments generated and the mAbs that mapped to each region.
Figure 2: Immunoprecipitation of purified recombinant TFIIB and TFIIB contained in HeLa cell NE. mAbs IIB1, IIB8, and IIB5 were adsorbed onto Formalin-treated S. aureus cells, and HeLa cell nuclear extract (upperpanel) or a solution of purified TFIIB (lowerpanel) was treated with the mAb-containing S. aureus cells as described under ``Materials and Methods.'' A Western blot of the TFIIB left in solution after treatment with each mAb was detected by reaction with mAb IIB1 and developed with the enhanced chemiluminescence reagents. Lane1, material treated with S. aureus cells; lanes 2-4, material treated with S. aureus cells adsorbed with mAbs IIB1, IIB8, and IIB5, respectively.
Figure 3:
Inhibition of transcription initiation
with the TFIIB mAbs. mAbs IIB1, IIB8, and IIB5 were added to HeLa cell
NE and used to transcribe from the MLP (A) or to purified
proteins and used to transcribe from the IgH promoter (B). A: lane 1, untreated NE; lane 2, NE treated
with 1 µg of -amanitin/ml; lanes 3-8, NE
preincubated with the designated amounts of TFIIB-specific mAb; lanes 9 and 10, NE preincubated with mAb 8WG16, a mAb
that reacts with RNA polymerase II and is known to inhibit
transcription initiation (22) and that was included as a
positive control. B: mAbs, as indicated, were added to the
TFIIB
TBP
DNA complex before the RNA polymerase II and NTPs
were added. mAb 3D3 (lanes 8 and 9), a mAb that
reacts with the
subunit of E. coli RNA
polymerase (24) , was included as a nonspecific antibody
control.
We also examined the ability of each mAb to inhibit
transcription when purified proteins are used in a minimal
transcription assay. Supercoiled DNA containing the IgH promoter cloned
in front of a G-less cassette was incubated with purified human TBP and
TFIIB for 10 min at room temperature. The complex was then incubated
with varying amounts of purified mAbs before the purified RNA
polymerase II was added. This reaction was incubated for 10 min at room
temperature before NTPs (containing
[-
P]UTP) were added, and transcription was
allowed to proceed for 50 min at 30 °C. Reactions were stopped, the
radiolabeled RNA was separated on a denaturing gel, and an
autoradiogram was prepared. A representative example of the inhibition
assay is shown in Fig. 3B, and the results for all of
the mAbs are summarized in Table 1. Only the mAbs that mapped to
residues 52-105 were able to inhibit transcription from the
supercoiled IgH promoter DNA template.
Because mAbs IIB1 and IIB10, which mapped to residues 1-51, were able to immunoprecipitate purified TFIIB, we preincubated the purified TFIIB with mAb IIB1 before the TFIIB was added to the minimal transcription system. This mAb was not able to inhibit transcription even when preincubated with TFIIB, although mAb IIB8 was still able to inhibit (data not shown). However, incubation of the minimal transcription reaction for varying periods of time indicated that multiple rounds of transcription were occurring in this assay (data not shown).
Figure 4:
Reactivity of the TFIIB mAbs with the
TFIIBTBP
DNA complex. mAbs were added to the
TFIIB
TBP
DNA complex, and a gel shift experiment was
performed. A: lane 1, TBP
DNA complex; lane
2, TFIIB
TBP
DNA complex; lanes 3-6,
TFIIB
TBP
DNA complex incubated with 40 ng of mAbs IIB1,
IIB8, IIB5, and 3D3, respectively. mAb 3D3 was included as a
nonspecific antibody control. B: lanes 1 and 2, TBP
DNA and TFIIB
TBP
DNA complexes,
respectively; lane 3, TFIIB
TBP
DNA complex
incubated with 40 ng of mAb IIB8; lane 4, TFIIB incubated with
40 ng of mAb IIB8 before it was added to the TBP
DNA complex; lane 5, TFIIB
TBP
DNA complex incubated with 40 ng
of mAb IIB1; lane 6, TFIIB incubated with 40 ng of mAb IIB1
before it was added to the TBP
DNA
complex.
Because mAbs IIB1 and IIB8 were
able to immunoprecipitate purified TFIIB (Fig. 2), we examined
the ability of these mAbs to inhibit the binding of TFIIB to the
TBPDNA complex by incubating the TFIIB with the mAb before
addition to the TBP
DNA. mAb IIB8 did not inhibit the binding of
TFIIB to the complex and was able to supershift the complex even when
preincubated with TFIIB (Fig. 4B, lane4). mAb IIB1 did not inhibit the binding of TFIIB to the
complex (Fig. 4B, lane6) even when
the mAb concentration was raise to 120 ng (data not shown).
In recent years, considerable information about the proteins involved in transcription initiation from RNA polymerase II-transcribed promoters has become available. However, information about how the structural features of these proteins interact with DNA and with other proteins to accomplish accurate transcription initiation is still lacking. The most powerful tool for detecting molecular interactions is x-ray crystallography. The crystal structure of the DNA-binding domain of TBP has been determined by itself (29) and as a co-crystal with DNA (30, 31) , and a low resolution, three-dimensional structure of yeast RNA polymerase II has been derived from two-dimensional crystals formed on a lipid bilayer(32) . However, until the crystal and co-crystal structures of all of the proteins involved in the initiation complex have been determined, we will need to rely on other investigative methods to examine the molecular interactions that must occur to accomplish transcription initiation. In this study, we have used a panel of mAbs as probes to help define regions of the human TFIIB protein that are on the surface of the molecule when the protein is in solution and when the protein becomes part of the nascent preinitiation complex. All of the mAbs also react with mouse TFIIB; therefore, the information obtained with the mAbs regarding the human protein might be applicable to many mammalian species.
The mAbs mapped to three distinct regions of the TFIIB molecule (Fig. 1C). Clearly, the mAbs within a group behave similarly in the functional assays (Table 1). However, we have not been able to determine if mAb IIB1 is different from IIB10 or if mAbs IIB7, IIB8, IIB12, and IIB20 are different from each other.
mAb IIB5, which maps to the C-terminal domain (residues 106-316), does not react with native TFIIB, but does react with TFIIB immobilized on polystyrene (enzyme-linked immunosorbent assay) and with denatured TFIIB immobilized on nitrocellulose (Western blot assay). Thus, the epitope for this mAb is probably buried within the compact structure of the C-terminal domain and is not accessible unless the molecule is distorted in some fashion. Consistent with this interpretation is the fact that this mAb does not inhibit transcription and does not supershift a preinitiation complex.
The mAbs that
mapped to residues 52-105 in the N terminus of the molecule
showed the greatest activity in the functional assays (Table 1).
These mAbs immunoprecipitated purified TFIIB and TFIIB contained in
HeLa cell NE (Fig. 2); they inhibited transcription in both HeLa
cell NE and in the IgH minimal promoter system (Fig. 3); and
they supershifted a complex containing TFIIB, TBP, and DNA (Fig. 4). These data indicate that this region of the molecule
is accessible to the mAbs when the protein is in solution and when it
is contained in the TFIIBTBP
DNA complex. These results are
in agreement with other studies (16, 17, 18, 20) showing that the C
terminus is involved in the binding to the TBP
DNA complex and
that the N-terminal region is important in the binding to the RNA
polymerase II
TFIIF complex. However, the exact sites of
interaction on TFIIB with RNA polymerase II and/or the RAP30 protein of
TFIIF are not clear. Ha et al.(18) , using protein
binding studies with TFIIB containing deletions, showed that the N
terminus was responsible for the binding of RAP30, while the major
interactions with RNA polymerase II could be localized to a large
region of the C-terminal domain. Malik et al.(17) showed that a mutant lacking amino acids 3-55
did not bind to RNA polymerase II; however, the RNA polymerase II in
this study did contain residual amounts of RAP30. Thus, these
investigators might have been actually measuring the binding to the
RAP30
RNA polymerase II complex. Clearly, mAbs that map to region
52-105 inhibited transcription in the IgH minimal promoter system (Fig. 3B). RAP30 was not included in this assay, and we
were not able to detect any residual RAP30 in our
immunoaffinity-purified calf thymus RNA polymerase II when we probed it
with a mAb (prepared against human RAP30) that cross-reacts with bovine
RAP30 (data not shown). However, we cannot discount the possibility
that the binding of the mAb to region 52-105 simply results in a
steric hindrance that precludes the RNA polymerase II from binding to
the sites in the C-terminal domain defined by Ha et
al.(18) .
Curiously, when Hisatake et al.(19) tested their deletion mutants for transcriptional
activity, two mutants within region 52-105 (67-80 and
83-103) were able to support a very low level of
transcriptional activity. All of the other deletion mutants that they
tested showed no transcriptional activity. Although we do not know the
exact amino acids within region 52-105 involved in the epitopes
for mAbs IIB7, IIB8, IIB12, and IIB20, these mAbs were very effective
in inhibiting transcription both in HeLa cell NE and in the IgH minimal
promoter system (Fig. 3). Thus, the binding of a mAb to this
region disrupts the ability of TFIIB to support transcription. We have
not yet been able to determine if the binding of a mAb inhibits the
binding of the RNA polymerase II molecule to the complex or inhibits
some undefined activity of the TFIIB molecule.
mAbs that reacted
with the far N terminus (residues 1-51) were able to
immunoprecipitate the purified recombinant protein, but were not able
to immunoprecipitate the native TFIIB from HeLa cell NE effectively (Fig. 2). This indicates that the far N terminus is not
accessible to the mAb in HeLa cell NE, possibly due to interaction with
another protein. It is possible that TFIIB is interacting with the RNA
polymerase IITFIIF complex in HeLa cell NE. However, in a
previous study(21) , we showed that HeLa cell NE that was
immunodepleted of TFIIB by mAb IIB8 lost transcriptional activity and
that this activity could be completely restored by the addition of
recombinant immunoaffinity-purified TFIIB. Thus, we were not removing
any other factor necessary for transcription from the MLP. We cannot
discount the possibility that reaction of mAb IIB8 with TFIIB (in
region 52-105) might disrupt the interaction of TFIIB with the
RNA polymerase II
TFIIF complex, but the reaction of mAb IIB1 (in
region 1-51) could not disrupt this interaction. Other proteins
in the NE might be interacting with the far N-terminal region,
precluding the interaction of TFIIB with mAbs IIB1 and IIB10. For
example, Baniahmad et al.(33) recently showed that
the human thyroid hormone
-receptor can bind to TFIIB, resulting
in a down-regulation of transcriptional activity (silencing). These
investigators showed that the ligand-binding domain of the human
thyroid hormone
receptor interacted with the far N terminus of
TFIIB and that it was this interaction that resulted in the silencing
function of the hormone receptor. We showed in a previous study (21) that the addition of excess TFIIB to HeLa cell NE that had
been immunodepleted of TFIIB increased the transcriptional activity
over the untreated NE. Although our interpretation of this observation
is that TFIIB is limiting in the NE, another interpretation is that
immunodepletion removes TFIIB that is complexed with another protein
that down-regulates the activity of TFIIB in HeLa cell NE.
Finally,
although the mAbs that reacted with residues 1-51 were able to
immunoprecipitate the purified TFIIB from solution, they did not
inhibit transcription when the purified TFIIB was used in the IgH
minimal promoter system (Fig. 3) and did not supershift a
TFIIBTBP
DNA complex (Fig. 4A). Thus,
although the C-terminal domain has been shown to bind to the
TBP
DNA complex (16, 17, 18, 20) , some epitopes
within the far N-terminal region also become inaccessible when the
TFIIB enters the preinitiation complex. However, although the mAbs
react with the purified protein, preincubating purified TFIIB with mAb
IIB1 did not inhibit the binding of TFIIB in the gel shift assay (Fig. 4B) and did not inhibit transcription from the
IgH minimal promoter (data not shown). These results might be explained
by a conformational change in TFIIB when it binds to the TBP
DNA
complex, resulting in a loss of ability of the mAb to bind to the
protein. An alternative, but less likely, explanation is that the
affinity of the TFIIB for the TBP
DNA complex is greater than the
affinity for the mAb, resulting in the displacement of the mAb from the
complex and masking of the epitope from reaction with the mAb.