©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Accessibility of Epitopes on Human Transcription Factor IIB in the Native Protein and in a Complex with DNA (*)

(Received for publication, November 7, 1994; and in revised form, December 22, 1994)

Nancy E. Thompson (§) Lee A. Strasheim Katherine M. Nolan Richard R. Burgess

From the McArdle Laboratory for Cancer Research, University of Wisconsin, Madison, Wisconsin 53706

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

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) (^1)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 TFIIDbulletDNA complex and/or relieving the effect of inhibitory proteins. TFIIB recruits the RNA polymerase IIbulletTFIIF 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)bulletDNA complex to the RNA polymerase IIbulletTFIIF 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 TBPbulletDNA 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 IIbulletTFIIF 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 IIbulletTFIIF 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 TBPbulletDNA complex, we found that some epitopes in the N-terminal region become inaccessible when TFIIB is contained in the TFIIBbulletTBPbulletDNA complex, while other epitopes within the N-terminal region remain accessible.


MATERIALS AND METHODS

Buffers and Reagents

All reagents were obtained from Sigma unless otherwise specified. TE buffer contained 50 mM Tris-HCl, pH 7.9, and 0.1 mM EDTA. Buffer D contained 20 mM HEPES, pH 7.9, 0.2 mM EDTA, 100 mM KCl, 0.5 mM dithiothreitol, and 20% glycerol. Phosphate-buffered saline contained 2 mM KH(2)PO(4), 10 mM Na(2)HPO(4), 3 mM KCl, and 150 mM NaCl, pH 7.2. All pH values were determined at 23 °C.

Proteins and Plasmids

Purified recombinant TBP and partially purified TFIIB for the preparation of mAbs were kind gifts from Promega (Madison, WI). TFIIB for biochemical and transcription assays was purified by immunoaffinity chromatography as described(21) . Calf thymus RNA polymerase II was purified by a modification of the immunoaffinity procedure previously described(22) . The mouse IgH minimal promoter (positions -47 to +1) cloned in front of a G-less cassette (23) was a kind gift from Jeffrey Parvin (Massachusetts Institute of Technology, Cambridge, MA). The phIIB plasmid, containing the human TFIIB gene on the pET11a vector(14) , was a kind gift from Danny Reinberg (Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, Piscataway, NJ). Mouse NE, prepared from NIH 3T3 cells, was a kind gift from Jill Slansky (McArdle Laboratory for Cancer Research).

Hybridomas and mAbs

Hybridomas producing TFIIB-specific mAbs were prepared as described(21) . Isotyping was performed using an enzyme-linked immunosorbent assay-based kit (Boehringer Mannheim). mAbs 8WG16 and 3D3 (both IgG molecules) were used as control mAbs in functional assays. mAb 8WG16 reacts with the heptapeptide repeat in the C-terminal domain of the largest subunit of RNA polymerase II(22) . mAb 3D3 reacts with a region in the C terminus of the subunit of Escherichia coli RNA polymerase(24) .

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(1) mAbs were purified from ascites fluid by chromatography on DEAE-cellulose as described(24) .

Electrophoresis and Western Blots

Proteins were separated by electrophoresis by the method of Laemmli (25) using 15% polyacrylamide and 0.1% SDS in the running gel. Western blots were prepared as described previously(26) , except for the immunoprecipitation experiments. Because the immunoprecipitation determinations required more sensitivity, we used anti-mouse IgG prepared in goats, conjugated to horseradish peroxidase (Hyclone Laboratories, Logan, UT), as the secondary antibody, and the substrate was the enhanced chemiluminescence system (ECL, Amersham Corp.). Prestained molecular weight markers (SDS-7B, Sigma) were included in all gels; these markers were visible when transferred to the nitrocellulose.

Transcription Reactions

Promoter-directed transcription assays using HeLa cell nuclear extracts and the MLP were performed as described previously(22) . Promoter-directed transcription reactions using the IgH minimal promoter were performed by a slight modification (21) of the procedure described by Parvin and Sharp(8) .

Gel Shift Assays

A 25-base pair oligonucleotide containing a consensus TATA element was purchased from Promega. Assays were performed essentially as described by Wiley et al.(27) , with a few modifications. The reaction volumes were 20 µl and contained 8 ng of end-labeled P-oligonucleotide, 10 ng of human TBP (Promega), and 14 ng of immunoaffinity-purified TFIIB. The reactions were incubated at 23 °C for 15 min. For the mAb supershift assays, 10-120 ng of mAb were added, and the reactions were allowed to incubate at 23 °C for an additional 15 min. The reactions were subjected to electrophoresis on a nondenaturing 4-15% gradient polyacrylamide gel on a PhastSystem (Pharmacia Biotech Inc.).

Immunoprecipitation Assays

Each mAb was adsorbed onto the surface of Formalin-treated Staphylococcus aureus cells (Sigma), and these immunosorbents were used to immunodeplete TFIIB from HeLa cell NE and from a solution containing bacterially expressed, purified TFIIB (10 ng/µl). Formalin-treated S. aureus cells (100 µl of a 10% solution) were washed twice with phosphate-buffered saline containing 0.5% bovine serum albumin. The S. aureus cells were then incubated with diluted ascites fluid (50 µl of ascites fluid diluted into 250 µl of phosphate-buffered saline containing 0.5% bovine serum albumin) for 30 min at 23 °C. The S. aureus cells were washed three times with 1 ml of phosphate-buffered saline containing bovine serum albumin and once with 1 ml of Buffer D. The mAb-containing S. aureus cells were resuspended in 25 µl of Buffer D and 80 µl of HeLa cell NE or 80 µl of purified TFIIB contained in Buffer D. After incubation at 23 °C for 20 min, the S. aureus cells were removed by centrifugation, and the supernatant fluids were assayed by Western blotting for the detection of TFIIB remaining in solution.

Preparation of Protein Fragments

Fragments of human TFIIB were generated by digestion of the purified protein with trypsin and S. aureus V8 protease. Trypsin (L-1-tosylamido-2-phenylethyl chloromethyl ketone-treated; Worthington) was used at a TFIIB:trypsin ratio of 200:1 (w/w). V8 protease (Sigma) was used at a TFIIB:V8 protease ratio of 20:1. Digestions were performed in Buffer D and incubated at 37 °C for varying times. The optimal time of digestion was 25 min for trypsin and 15 min for V8 protease, although digestion was not complete with either enzyme. A peptide containing amino acids 1-123 was prepared by digesting the phIIB plasmid with NcoI at nucleotide 370 in the TFIIB coding region and with HindIII in the vector. The ends were filled in with the Klenow fragment and ligated with T4 ligase; the plasmid was transformed into BL21(DE3)/pLysS for expression with isopropyl-1-thio-beta-D-galactopyranoside induction(28) .


RESULTS

Properties of TFIIB mAbs

The properties of the mAbs that react with human TFIIB are listed in Table 1. Some of these mAbs have been used in a previous study(21) . Because the TFIIB used as the immunogen and for screening for antibody production by enzyme-linked immunosorbent assay was only partially purified (50%), all of the enzyme-linked immunosorbent assay-reactive mAbs had to be rescreened by Western blot assays to identify specifically the TFIIB-reactive mAbs. This screening strategy eliminated all TFIIB-reactive mAbs that did not react with protein in the Western blot. The most sensitive mAb in the Western blot was IIB1. By using TFIIB that was purified by immunoaffinity chromatography, we determined that mAb IIB1 could detect 5 ng of human TFIIB in the Western blot assay (data not shown). All of the mAbs detected a protein of about the same size as human TFIIB in mouse nuclear extracts.



Epitope Mapping

Proteolytic fragments of human TFIIB were used to determine the relative position of each epitope. Trypsin digestion yields a protease-resistant fragment containing amino acid residues 106-316(16, 17) . V8 protease digestion yields a protease-resistant fragment containing amino acids 52-316(17) . Digestions of TFIIB with trypsin and V8 protease were assayed by SDS-polyacrylamide gel electrophoresis (Fig. 1A). The resultant peptide patterns were essentially as described by Malik et al.(17) . The reactivity of the mAbs with the protease-resistant fragments was determined by Western blot analysis. mAbs whose epitopes are contained, at least partially, within amino acids 1-51 react only with the undigested TFIIB. mAbs whose epitopes are contained, at least partially, within amino acids 52-105 react with the undigested TFIIB and the V8 protease-resistant fragment, but not with the trypsin-resistant fragment. mAbs whose epitopes are contained within amino acids 106-316 react with the undigested TFIIB, the V8 protease-resistant fragment, and the trypsin-resistant fragment. A representative Western blot showing the reactions of mAbs IIB1, IIB8, and IIB5 with the proteolytic fragments is in Fig. 1B. Because a considerable amount of TFIIB remained undigested in these samples, an undigested sample was not included in the blot. The reactivity of the TFIIB mAbs with the proteolytic fragments is summarized in Fig. 1C. To confirm the mAb reactivities, a peptide containing amino acids 1-123 was prepared by recombinant DNA methods as described under ``Materials and Methods'' using the NcoI restriction site shown in Fig. 1C. The truncated protein was expressed in E. coli, and Western blotting was performed with all of the mAbs. The truncated protein reacted with all of the mAbs except IIB5 (data not shown), indicating that the proteolytic fragments were identified correctly.


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.



Epitope Accessibility When TFIIB Is in Solution

The mAbs were tested for the ability to react with TFIIB in solution by immunodepletion assays using both native TFIIB contained in HeLa cell NE and bacterially expressed, purified TFIIB. After treatment of HeLa cell NE or purified TFIIB as described under ``Materials and Methods,'' the supernatant was tested for depletion of TFIIB by Western blot assays using mAb IIB1 as a probe for TFIIB remaining in solution. A representative Western blot using mAbs IIB1, IIB8, and IIB5 as the immunoprecipitating mAbs is shown in Fig. 2, and the results for all of the mAbs are summarized in Table 1. Only mAbs that mapped to residues 52-105 were able to immunoprecipitate the TFIIB from HeLa cell NE. mAbs IIB7, IIB8, IIB12, and IIB20 all immunodepleted the TFIIB from the NE. These mAbs were also able to remove purified TFIIB from solution ( Fig. 2and Table 1). Although mAbs that mapped to the far N terminus were unable to remove TFIIB from HeLa cell NE, they were able to remove purified TFIIB from solution ( Fig. 2and Table 1). HeLa cell NE that had been immunodepleted with mAbs lost transcriptional activity when tested on the MLP, and transcriptional activity could be restored to the immunodepleted extracts by the addition of immunoaffinity-purified TFIIB(21) .


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.



Effect of TFIIB mAbs on Transcription in HeLa Cell NE and in a Minimal Promoter System

The ability of each mAb to inhibit promoter-directed transcription in HeLa cell NE was determined by using a linear DNA template containing the MLP. Purified mAb was preincubated with HeLa cell NE using varying concentrations of mAb at 30 °C for 20 min. The DNA template was added, and the reaction was incubated at 30 °C for 15 min to allow the initiation complex to form. Then NTPs (containing [alpha-P]GTP) were added, and transcription was allowed to proceed for 30 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. 3A, and the results for all of the mAbs are summarized in Table 1. Only the mAbs that mapped to residues 52-105 inhibited transcription in the NE. These mAbs all inhibited transcription at 20-50 ng/µl. By quantitative Western blot assays (using purified TFIIB and mAb IIB1), we determined that there is 10-20 ng/µl TFIIB in the NE, which is 5-10 ng/µl TFIIB in the final transcription assay(21) . Therefore, inhibition occurred at a molar ratio of mAb to TFIIB of 1. mAbs IIB1 and IIB10 did not inhibit transcription at any concentration. mAb IIB5 also did not inhibit efficiently, although a slight inhibition was noted when 48 ng/µl mAb IIB5 was used in the assay (Fig. 3A, lane8). However, this reduction in transcript is probably due to a small amount of RNase in the antibody preparation because incubation of the transcript with purified mAb IIB5 gives a similar slight reduction in signal (data not shown), and we have observed a small amount of RNase in other mAbs purified on DEAE-cellulose. mAb 8WG16 was included as a control in this experiment; this mAb inhibits transcription initiation in HeLa cell NE(22) .


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 alpha-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 TFIIBbulletTBPbulletDNA 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 [alpha-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).

Epitope Accessibility When TFIIB Is Complexed with TBP and Linear DNA

The mAbs were tested for the ability to react with human TFIIB when it is contained in a preinitiation complex by examining the ability of the mAbs to supershift a TFIIBbulletTBPbulletDNA complex in the gel shift assay. Each purified mAb was added to the TFIIBbulletTBPbulletDNA complex that was formed in gel shift buffer at room temperature. After incubation for 15 min, the complexes were separated by electrophoresis, and an autoradiogram was prepared. In preliminary antibody titration experiments, we found that 40 ng, but not 20 ng, of mAb IIB8 could result in a supershift of the complex (data not shown). A representative gel shift assay using mAbs IIB1, IIB8, and IIB5 and a control mAb is shown in Fig. 4A, and the results for all of the mAbs are summarized in Table 1. Only mAbs that mapped to residues 52-105 supershifted the complex. mAbs IIB1, IIB5, and IIB10 did not supershift the complex even when the antibody concentration was raised to 100 ng (data not shown).


Figure 4: Reactivity of the TFIIB mAbs with the TFIIBbulletTBPbulletDNA complex. mAbs were added to the TFIIBbulletTBPbulletDNA complex, and a gel shift experiment was performed. A: lane 1, TBPbulletDNA complex; lane 2, TFIIBbulletTBPbullet DNA complex; lanes 3-6, TFIIBbulletTBPbullet 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, TBPbulletDNA and TFIIBbulletTBPbulletDNA complexes, respectively; lane 3, TFIIBbulletTBPbullet 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 TBPbulletDNA complex; lane 5, TFIIBbulletTBPbulletDNA complex incubated with 40 ng of mAb IIB1; lane 6, TFIIB incubated with 40 ng of mAb IIB1 before it was added to the TBPbulletDNA 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 TBPbulletDNA complex by incubating the TFIIB with the mAb before addition to the TBPbulletDNA. 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).


DISCUSSION

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 TFIIBbulletTBPbulletDNA 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 TBPbulletDNA complex and that the N-terminal region is important in the binding to the RNA polymerase IIbulletTFIIF 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 RAP30bulletRNA 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 (Delta67-80 and Delta83-103) 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 IIbulletTFIIF 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 IIbulletTFIIF 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 beta-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 betareceptor 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 TFIIBbulletTBPbulletDNA complex (Fig. 4A). Thus, although the C-terminal domain has been shown to bind to the TBPbulletDNA 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 TBPbulletDNA 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 TBPbulletDNA 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.


FOOTNOTES

*
This work was supported by Grants CA07175, CA23076, and CA60896 from NCI, Grant GM28575 from the National Institutes of Health, and a grant from Promega. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: McArdle Laboratory for Cancer Research, 1400 University Ave., Madison, WI 53706. Tel.: 608-263-3375; Fax: 608-262-2824.

(^1)
The abbreviations used are: GTFs, general transcription factors; TFII, transcription factor II; MLP, adenovirus-2 major late promoter; TBP, TATA-binding protein; mAb, monoclonal antibody; IgH, immunoglobulin µ-heavy chain; NE, nuclear extract.


ACKNOWLEDGEMENTS

We thank Danny Reinberg for the phIIB plasmid, Mark Knuth and Elaine Schenborn (Promega) for the partially purified human TFIIB that was used as the immunogen, Jeffrey Parvin for the IgH promoter, and Jill Slansky for the mouse nuclear extract.


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