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Analysis of the Role of TFIIE in Transcriptional Regulation through Structure-Function Studies of the TFIIEβ Subunit*

The general transcription factor TFIIE plays important roles at two distinct but sequential steps in transcription as follows: preinitiation complex formation and activation (open complex formation), and the transition from initiation to elongation. The large subunit of human TFIIE (TFIIEα) binds to and facilitates the enzymatic functions of TFIIH, but TFIIE also functions independently from TFIIH. To determine functional roles of the small subunit of human TFIIE (TFIIEβ), deletion mutations were systematically introduced into putative structural motifs and characteristic sequences. Here we show that all of these structures that lie within the central 227-amino acid region of TFIIEβ are necessary and sufficient for both basal and activated transcription. We further demonstrate that two C-terminal basic regions are essential for physical interaction with both TFIIEα and single-stranded DNA, as well as with other transcription factors including theDrosophila transcriptional regulator Krüppel. In addition, we analyzed the effects of the TFIIEβ deletion mutations on TFIIH-dependent phosphorylation of the C-terminal domain of RNA polymerase II and on wild type TFIIEβ-driven basal transcription. Both responsible regions also mapped within the essential 227-amino acid region. Our results suggest that TFIIE engages in communication with both transcription factors and promoter DNA via the TFIIEβ subunit.

In eukaryotes productive transcription initiation by RNA polymerase II (Pol II) 1 plays a key role in the regulation of gene expression in response to various developmental and environ-mental signals. Initiation by Pol II requires five general transcription factors (TFIIB, TFIID, TFIIE, TFIIF, and TFIIH) that act through core promoter elements and is regulated by tissueand/or gene-specific factors that act through distal control elements (for reviews, see Refs. [1][2][3][4][5]. Recent studies of preinitiation complex (PIC) formation have indicated the existence of the following alternative pathways: one involving the stepwise assembly of multiple general transcription factors, and one involving preassembly of a Pol II holoenzyme containing several general transcription factors and various coactivators (for reviews, see Refs. 6 -8). Analysis of the PIC assembly pathway using isolated factors has revealed that this process begins with the binding of TBP (the TATA-binding protein) component of TFIID to the TATA box. This is then followed by the sequential interactions of TFIIB, a complex containing Pol II and TFIIF, TFIIE, and TFIIH. TFIIH, which is recruited through direct interaction with TFIIE, may stabilize and activate the PIC through its enzymatic activities, resulting in open complex formation.
In addition to their key roles in PIC formation and initiation, TFIIE and TFIIH play important roles in the transition from initiation to elongation (promoter clearance) (for reviews, see Refs. 1, 2, and 5). Human TFIIE consists of 57-kDa (␣) and 34-kDa (␤) subunits and forms an ␣ 2 ␤ 2 heterotetramer with a molecular mass of 180 kDa (9,10). Human TFIIH consists of 9 subunits, and surprisingly, some of these subunits have been implicated in nucleotide excision repair and cell cycle regulation (for a review, see Ref. 11). This multisubunit general transcription factor is quite unique in that it contains the following three ATP-dependent catalytic activities: a kinase activity that phosphorylates the CTD (C-terminal domain) of the largest subunit of Pol II, a DNA-dependent ATPase, and a DNA helicase. Importantly, TFIIE plays essential roles in the regulation of these TFIIH activities; the CTD kinase and ATPase are positively regulated by TFIIE whereas the DNA helicase activity is negatively regulated (12)(13)(14)(15). This coordinated regulation, as well as multiple interactions of TFIIE and TFIIH with other transcription factors (reviewed in Ref. 5), probably provides a basis for the control of the two distinct steps of transcription.
Human TFIIE␣, which is a highly acidic (pI 4.5) protein of 439 amino acids, possesses several putative structural motifs and characteristic sequences (10,16). Previous studies demonstrated that the N-terminal half of TFIIE␣, which contains all the evolutionarily conserved structural motifs, is essential for basal transcription, whereas the C-terminal half is dispensable (15). A putative leucine zipper motif and a similar hydrophobic repeat domain, which are separated by a putative zinc finger motif, are important for heterodimerization with the TFIIE␤ subunit. Furthermore, TFIIH binds to the C-terminal acidic region of TFIIE␣. This interaction may be important for re-cruiting TFIIH into the PIC and for the modulation of the two functions of this factor in transcription initiation and in the transition from initiation to elongation.
Human TFIIE␤, which is a highly basic (pI 9.5) protein of 291 amino acids, also possesses several conserved structural motifs and characteristic sequences (10,17,18). Photocrosslinking studies revealed that TFIIE␤ binds to promoter DNA in the region between positions Ϫ14 and Ϫ2 from the transcription initiation site (ϩ1), a property that distinguishes it from TFIIE␣ which cannot be cross-linked with DNA (19). Twodimensional crystallography of the highly related yeast TFIIE with Pol II has confirmed these observations by showing that TFIIE interacts with the active center of Pol II relative to the transcription initiation site (20). Other recent studies have demonstrated that the introduction of short mismatched heteroduplex DNA regions around the initiation site (minimally from positions Ϫ4 to ϩ2) in topologically relaxed templates abolishes the requirement for TFIIE, TFIIH, and ␤-␥ ATP hydrolysis (21)(22)(23). Importantly, changes in the short mismatched region create differential requirements for TFIIE and TFIIH with, most notably, a continued requirement for TFIIE but lack of a requirement for the function of TFIIH being observed (23). These results indicate two possibilities as follows: first, that TFIIE and TFIIH play a role around the transcription initiation site during open complex formation, and second, that TFIIE has a unique function, possibly in promoter melting, distinct from its role with TFIIH.
To elucidate further mechanisms of TFIIE function, and especially that of TFIIE␤, we constructed a series of deletion mutants of TFIIE␤. We tested the ability of these mutants to support both basal and activated transcription and to associate with other general transcription factors and transcriptional regulatory factors, and we examined their effects on CTD phosphorylation by TFIIH as well as their dominant negative effects on basal transcription. In so doing, we have succeeded in identifying a central core that is important for the mediation of TFIIE function and which contains the C-terminal basic regions essential for binding both to general transcription factors and to single-stranded (ss) DNA. In addition, we provided a new clue to approach that TFIIE is not only recruiting TFIIH into the PIC and functioning as a bridge between Pol II and TFIIH for its CTD phosphorylation but also playing an unidentified important role during transcription initiation and in the transition from initiation to elongation.

EXPERIMENTAL PROCEDURES
DNA Templates-For basal transcription assays, the plasmid pML(C 2 AT)⌬-50 containing the adenovirus type 2 major late promoter was used as a template (24). To study transcriptional activation, the plasmid pG5HM(C 2 AT) was used as the test template (25), with the plasmid pML(C 2 AT)⌬-53Sh as the base-line control (26). pG5HM(C 2 AT) contains five GAL4-binding sites and the core promoter as described previously (15). The two templates pML(C 2 AT)⌬-50 and pG5HM(C 2 AT) give 390-nucleotide transcripts, and pML(C 2 AT)⌬-53Sh gives a 290nucleotide transcript.
Construction of Various Expression Vectors-The isolated plasmid p2EB contains the complete open reading frame of human TFIIE␤ (TFIIE␤ cDNA) cloned into pBluescript II SK(Ϫ) phagemid (Stratagene) (17). This was first digested with XbaI and ClaI, and the 1.7kilobase pair cDNA fragment was subcloned into the pGEM-7Zf(ϩ) vector (Promega) to create a BamHI site at the 3Ј-end of TFIIE␤ cDNA. The oligonucleotide 5Ј-CCCTTCTCACTCAGCCATATGGACCCAAGC-CTGTTG-3Ј was then used to create an NdeI site at the first methionine codon of TFIIE␤ cDNA and to disrupt a BamHI site located just after the first methionine by site-directed mutagenesis (27). Finally, the NdeI-BamHI fragment of this cDNA clone (p2EBT) was subcloned into the 6HisT-pET11d vector to construct the histidine (His)-tagged TFIIE␤ (6His-TFIIE␤) expression plasmid.
To set up the coexpression system of the two TFIIF subunits for the purpose of preparation of transcriptionally active recombinant TFIIF, the human TFIIF␤ (RAP30) expression plasmid (in pET11d) (29) was digested with XbaI and EcoRI, and both ends of this fragment containing TFIIF␤ cDNA were converted to blunt ends, whereas the expression plasmid of human TFIIF␣ (RAP74) with six His tags at the C terminus (in pET23d) (30) was digested with XbaI, and the XbaI sites were converted to blunt ends, and the resulting fragment was treated with calf intestine phosphatase. Then, the TFIIF␤ cDNA fragment was subcloned into the XbaI sites (blunt) of the TFIIF␣ expression plasmid to place both cDNAs tandemly and the 6His-TFIIF expression plasmid was constructed.
The plasmid pBSKR containing cDNA encoding the Drosophila developmental gene product Krü ppel (Kr) was kindly provided by F. Sauer and H. Jä ckle (31,32). To create an NdeI site at the first methionine codon, two oligonucleotides Kr1 (5Ј-CAGCCTCATATGTCCATAT-CAATGCTTC-3Ј) and Kr2 (5Ј-CATGGAACGGTCTAGATGAACGTC-3Ј) were used for polymerase chain reaction. This 82-base pair polymerase chain reaction product was digested with NdeI and XbaI, and the NdeI-XbaI fragment was then subcloned with the XbaI-EcoRI fragment of Kr cDNA into the NdeI and EcoRI sites of the HA (influenza hemagglutinin epitope sequence)-pET11d vector to construct the HA-tagged Kr (full length) expression plasmid (Fig. 7A). The HA-pET11d vector was constructed by inserting the NcoI-NdeI fragment of HA(S)-pGEM-7 2 into the NcoI and NdeI sites of 6HisT-pET11d.
Construction of TFIIE␤ Mutants-Deletion mutants of TFIIE␤ were constructed using plasmid p2EBT containing the wild type TFIIE␤ cDNA and the described procedure of oligonucleotide-mediated mutagenesis (27). A restriction site was designed in each oligonucleotide to select for properly mutated plasmids as described elsewhere (28), and the mutants were then checked by sequencing. The NdeI-BamHI fragments of all mutants were subcloned into 6HisT-pET11d to create a His tag at the N terminus. N-terminal and internal deletions were constructed by deleting the indicated amino acid residues and C-terminal deletions by creating termination codons at the residues shown in Fig.  1. (Because of the large number of oligonucleotides used, we have refrained from describing their exact sequences, but this information will be provided on request.) Expression and Purification of Recombinant Proteins-Recombinant proteins were expressed in E. coli BL21(DE3)pLysS by induction with isopropyl-␤-D-thiogalactopyranoside (34). For purification, soluble bacterial lysates were used (17). His-tagged proteins were purified on an Ni 2ϩ -nitrilotriacetic acid column (Qiagen) by eluting with 100 mM imidazole HCl (pH 7.9). The large scale preparation of TFIIE␤ was as described before (15) and resulted in Ͼ95% purity as judged by Coomassie Blue staining of an SDS-polyacrylamide gel. All the deletion mutants of TFIIE␤ were miniscale preparations. Lysates (1 ml) representing 50 -100 ml of culture were directly resuspended in Eppendorf tubes with 1 ml of buffer B (20 mM Tris-HCl (pH 7.9 at 4°C), 0.5 mM EGTA, 10% (v/v) glycerol, 1 mM phenylmethylsulfonyl fluoride, 2 g/ml antipain, 2 g/ml aprotinin, 1 g/ml leupeptin, 0.8 g/ml pepstatin, 10 mM 2-mercaptoethanol) containing 500 mM NaCl (BB500) and 100 l of Ni 2ϩ -nitrilotriacetic acid resin and incubated for 4 h at 4°C. The resin samples were washed twice with 1 ml of BB500, twice with 1 ml of buffer D (20 mM Tris-HCl (pH 7.9 at 4°C), 20% (v/v) glycerol, 1 mM phenylmethylsulfonyl fluoride, 10 mM 2-mercaptoethanol) containing 500 mM KCl (BD500), and twice with 500 l of BD500 containing 20 mM imidazole HCl (pH 7.9). Bound proteins were eluted twice with 300 l of BD500 containing 100 mM imidazole HCl (pH 7.9). Typical preparations were Ͼ75% pure.
Recombinant TFIIF was also expressed in E. coli BL21(DE3)pLysS. More than 90% of both TFIIF␣ (RAP74) and TFIIF␤ (RAP30) subunits became soluble in bacterial lysate when they were coexpressed, and 6His-TFIIF was purified through Ni 2ϩ -nitrilotriacetic acid column just as 6His-TFIIE␤. Purified TFIIF was Ͼ95% purity, contained both subunits stoichiometrically, and was fully active in transcription. 3 HA-tagged and GST fusion proteins were expressed in E. coli BL21(DE3)pLysS by isopropyl-␤-D-thiogalactopyranoside induction. Bacteria from 50 to 100 ml of culture was harvested, resuspended in 1 ml of BB500, and sonicated. Soluble lysates were separated from insoluble debris by ultracentrifugation and stored at Ϫ80°C until use.
Generation of Antibody against TFIIE␤-Two hundred micrograms (100 l) of purified 6His-TFIIE␤ (Ͼ99% pure) was mixed with the same volume (100 l) of complete Freund's adjuvant (Difco) and injected into each rabbit. Two weeks after the first injection, a second injection was carried out with 100 g (100 l) of purified TFIIE␤ in 100 l of incomplete Freund's adjuvant (Difco). A third injection was carried out 2 weeks later using the same procedures as described for the second injection. Blood was collected 8 days after the third injection. The generated antibody recognized all of the TFIIE␤ deletion mutants used in this study. 3 Coimmunoprecipitation of TFIIE␤ Mutants with TFIIE␣-Polyclonal antisera against TFIIE␣ (0.01 l) and 5 l (packed volume) of protein G-agarose (Pierce) were incubated in buffer C (20 mM Tris-HCl (pH 7.9 at 4°C), 0.5% EDTA, 20% (v/v) glycerol, 0.5 mM phenylmethylsulfonyl fluoride, 10 mM 2-mercaptoethanol, 0.002% (v/v) Nonidet P-40) containing 100 mM KCl (BC100) and 200 g/ml bovine serum albumin for 2 h at 4°C with rotation. The protein G-agarose beads were precipitated and washed with 500 l of washing buffer I (10 mM Tris-HCl (pH 7.9 at 4°C), 500 mM NaCl, 0.1% Tween 20), twice with 500 l of buffer C containing 1 M KCl (BC1000), and twice with 500 l of BC100. Various TFIIE␤ mutant proteins (200 ng) were incubated with TFIIE␣ (300 ng) for 1 h at room temperature, and bound proteins were coimmunoprecipitated after incubation with anti-TFIIE␣-protein G beads in a 500-l reaction volume for 4 h at 4°C with rotation. The beads were washed twice with 500 l of washing buffer I, once with 500 l of BC100, boiled in SDS sample buffer, and analyzed by SDS-PAGE (12% acrylamide).
Coimmunoprecipitated TFIIE␤ mutants were detected by Western blotting with anti-TFIIE␤ polyclonal antiserum (1:3000 dilution) after transfer to an Immobilon-P polyvinylidene difluoride membrane (Millipore) as described previously (15). Signals were detected using the enhanced chemiluminescence detection (ECL) system (Amersham Pharmacia Biotech) after incubation of the immunoblots with horseradish peroxidase-linked secondary antibodies. PBD-1 film (Kodak) or RX-U film (Fuji Film) was used to record the chemiluminescence.
GST Pull Down Assays-GST fusion proteins were used for protein interaction assays. Each tester protein (200 ng) was incubated with lysates containing 500 ng of GST proteins together with 5 l (packed volume) of glutathione-Sepharose (Amersham Pharmacia Biotech) in a 500-l reaction volume of BC100 with 200 g/ml bovine serum albumin for 4 h at 4°C with rotation. The glutathione-Sepharose resin was washed twice with 500 l of buffer C containing 200 mM KCl (BC200), once with 500 l of BC100, boiled in SDS sample buffer, and analyzed by SDS-PAGE. Pulled down tester proteins were detected by Western blotting as described in the coimmunoprecipitation assay.
Single-stranded DNA Binding Assay-Four hundred nanograms of His-tagged TFIIE␤ deletion mutants were incubated with 5 l (packed volume) of ssDNA-agarose (Life Technologies, Inc.) in a 500-l reaction volume of BC100 with 200 g/ml bovine serum albumin for 4 h at 4°C with rotation. The ssDNA-agarose resin was washed twice with 500 l of buffer C containing 250 mM KCl (BC250), once with 500 l of BC100, boiled in SDS sample buffer, and analyzed by SDS-PAGE (12% acrylamide). Bound mutants were detected by Western blotting with anti-TFIIE␤ antisera (1:3000 dilution) as described above.
Kinase Assay-Assays were carried out essentially as described (14,15) with all general transcription factors in the presence of Pol II and a DNA fragment containing the adenovirus type 2 major late promoter sequences from Ϫ39 to ϩ29, except that 16 ng of bacterially expressed recombinant TFIIF was used instead of 30 ng of high performance liquid chromatography heparin-purified TFIIF from HeLa nuclear extract. Phosphorylation reactions were done at 30°C for 1 h and stopped by addition of 75 l of phosphorylation stop solution (10 mM EDTA, 0.1% Nonidet P-40, and 0.05% SDS). Phosphorylated proteins were trichloroacetic acid-precipitated, analyzed by SDS-PAGE (5.5% acryl-amide), and detected by autoradiography performed at Ϫ80°C with Fuji RX-U x-ray film.

The Central Region of TFIIE␤ Is Important for Both Basal and Activator-dependent Transcription-Human
TFIIE␤ is the small subunit of TFIIE and consists of 291 amino acid residues (10,17). As judged from the predicted open reading frame of TFIIE␤ cDNA, it possesses several putative structural motifs and characteristic sequences: a serine-rich sequence (residues 26 -71), a region similar to the Pol II binding region of RAP30 (TFIIF␤) (residues 79 -111), a leucine repeat motif (residues 145-193), a region similar to the bacterial sigma factor subdomain 3 (residues 163-193), a basic region-helix-loop-helix motif (residues 197-238), and a region with basic region-helix-loop sequence (residues 258 -291). Except for the leucine repeat, these regions are different from the putative motifs and sequences observed in TFIIE␣ (15). Based on these sequences, a systematic series of N-terminal, C-terminal, and internal deletion mutants were constructed (shown in Fig. 1, A and B). Vectors encoding hexahistidine-tagged mutant and wild type TFIIE␤ proteins were expressed in bacteria, purified through nickel affinity chromatography, and analyzed by SDS-PAGE ( Fig. 2A and 3A). All mutants were highly soluble in the bacterial cell extracts and were easily purified, although some (⌬224 -291, ⌬96 -119, and ⌬117-153) were poorly expressed ( Fig The basal transcription activity of each deletion mutant was measured by complementation of a reconstituted transcription system containing the adenovirus type 2 major late promoter and all general transcription factors except for TFIIE␤ (15). As shown in Fig. 2B, the N-terminal deletion mutants ⌬8 -25 and ⌬8 -50 were almost as active as the wild type (lanes 4 and 5 versus lane 3). However, further deletions from the N terminus, including that extending to residue 75, abolished basal transcription activity (lanes 6 -9). In contrast, none of the C-terminal mutants (lanes 10 -14) other than mutant ⌬278 -291 supported basal transcription activity, and the activity of ⌬278 -291 was less than 20% that of the wild type TFIIE␤ (lane 14 versus lane 3). In a further investigation, the internal deletion mutants were analyzed (Fig. 3B). The internal mutant ⌬26 -47 was as active as the wild type (lane 5 versus lane 3), but the other internal mutants (lanes 6 -14) failed to identify another region dispensable for basal transcription. Therefore, the minimal functional region of TFIIE␤ was deduced to lie between residues 51 and 277. This means that the N-terminal half of the serine-rich sequence (residues 26 -50) and the C-terminal third of the helix region and all of the loop region of the basic region-helix-loop region (residues 278 -291) are non-essential, although loss of the latter C-terminal region (residues 278 -291) did reduce the basal transcription level at least 5-fold (Fig.  2B, lane 3 versus lane 14). Apart from these regions, all other putative motifs and characteristic sequences were found to be indispensable for TFIIE function.
Recent studies have revealed the existence of several targets for transcriptional regulatory factors among the general transcription factors (for a review, see Ref. 38), and these include TFIIE (for a review, see Ref. 5). This suggests that TFIIE functions not only as a signal transducer for transcriptional regulation as a result of its binding to other general transcription factors such as TFIID and TFIIB but that such signals are also generated through the direct binding of regulators. In an attempt to identify the region essential for transcriptional activation in TFIIE␤, the deletion mutants that supported basal transcription were tested with various activators. Fig. 4A shows the effects of the acidic activator GAL4-VP16 on these N-terminal, C-terminal, and internal deletion mutants. All four mutants that exhibited basal transcription activity (⌬8 -25, ⌬8 -50, ⌬278 -291, and ⌬26 -47), as well as wild type TFIIE␤, supported a 5-7-fold level of transcriptional activation. Similar results (7-8-fold activation) were observed even when the proline-rich activator GAL4-CTF1 was used (Fig. 4B). These results indicate that the 227-amino acid region (residues 51-277) supports both basal and activated transcription. Of course, it is still possible that this region might be actually important for activation, but this issue that can only be definitively resolved by the isolation and testing of point mutations.
The C-terminal Basic Regions of Human TFIIE␤ Bind to Several General Transcription Factors-TFIIE functions in two distinct but sequential steps, one during open complex formation and the other during the transition from transcription initiation to elongation. During both steps, the binding of TFIIE to various general transcription factors and to Pol II is expected to be quite important. In the present study, pull down assays (39) using GST fusion proteins for each subunit of the general transcription factors and recombinant TFIIE␤ showed that TFIIE␤ binds strongly to TFIIE␣, TFIIB, and TFIIF␤ (RAP30) and weakly to TFIIF␣ (RAP74) and TBP (Fig. 5A). Human TFIIE␤ also bound to Pol II and strongly to itself. 3 These results suggest that TFIIE␤ plays essential roles in both steps of transcription by binding to various general transcription factors. To better understand these roles, we first identified the binding regions of TFIIE␤ for three strongly associating factors TFIIE␣, TFIIB, and TFIIF␤.
The interactions of TFIIE␤ with TFIIB and TFIIF␤ (RAP30) were analyzed next (Fig. 5A). The TFIIE␤ mutants were mixed with GST-fused TFIIB (GST-TFIIB), pulled down with GST-TFIIB, and detected by anti-TFIIE␤ antibody in an immunoblot analysis (Fig. 6). Mutants lacking the basic and helix regions (residues 257-277) of the basic region-helix-loop sequence near the C terminus failed to bind to TFIIB (Fig. 6A, lanes 8 -11; Fig. 6B, lane 12). Almost equivalent results were obtained with GST-TFIIF␤ (RAP30). 3 Taken together, these results indicate that the two basic and adjacent C-terminal regions of TFIIE␤ may be targets of various general transcription factors.
Two C-terminal Basic Regions of TFIIE␤ Are Involved in Binding to the Drosophila Transcriptional Repressor Krü ppel-Recently, it has become apparent that vast numbers of transcriptional regulators target various transcription factors, as well as histones, during the multiple steps of gene transcription (for reviews, see Refs. 1 and 38). Since TFIIE plays key roles in two of those steps, transcription initiation and promoter clearance, it would be surprising if it were not a target for transcriptional regulatory factors (for a review, see Ref. 5). As analyzed both in tissue culture and in vitro, the Drosophila segmentation gene product Krü ppel (Kr), which is a zinc finger protein, functions as a transcriptional regulatory factor (31,32). It has been demonstrated that monomeric Kr acts as a transcriptional activator by binding to TFIIB and that the Kr dimer, on the other hand, acts as a transcriptional repressor by binding to TFIIE␤ (36,40). Since this result was the first report of the existence of transcriptional regulatory factor targeting general transcription factor TFIIE, we analyzed Kr binding to TFIIE␤.  (17,18). The numbers presented either above or below the diagram indicate the amino acid residue numbers that delimit each structure.
As shown in Fig. 7A, Kr binding to general transcription factors was studied using a GST pull down assay. Various GST general factor fusion proteins were tested for their ability to interact with HA-tagged Kr (HA-Kr). Kr binds tightly to TFIIB and TFIIE␤, as demonstrated by Sauer et al. (40), and weakly to TBP and TFIIF␤ (RAP30) (25-30% of TFIIB and TFIIE␤) (Fig. 7A, lanes 2 and 4 versus lanes 6 and 7). Kr also bound to TFIIA␤ but only weakly (less than 5% of TFIIB and TFIIE␤) (Fig. 7A, lane 10). These results are in good agreement with the observations described above. Therefore, the Kr binding regions within TFIIE␤ were identified by using a GST-Kr fusion protein in conjunction with TFIIE␤ deletion mutants (Fig. 7, B  and C). Kr bound to all the N-terminal deletion mutants, but binding was considerably reduced by deletion of the C-terminal basic region (mutant ⌬257-291) and completely abolished by further deletion of the other basic region (mutant ⌬176 -291) (Fig. 7B, lanes 3-8 versus lanes 10-12). Internal deletion mu-tants confirmed that deletion of either basic region had a severe effect upon Kr binding (Fig. 7C, lanes 11 and 13).
The C-terminal Basic Region of Basic Region-Helix-Loop Sequence Binds to Single-stranded DNA-As we reported previously (17), the C-terminal basic region of the basic region-helixloop (BR-HL) sequence in TFIIE␤ is similar to the basic regions of the basic region-helix-loop-helix (BR-HLH) domains of the Myc-related family of proteins (such as Myo-D1 and E12) (41)(42)(43). Thus, it was predicted that this basic region may bind directly to single-stranded (ss) and/or double-stranded (ds)DNA. Recent studies have lent further support to this idea as follows: (i) photocross-linking studies revealed that TFIIE␤ binds to a core promoter region (between Ϫ14 and Ϫ2) where dsDNA is melted by transcription initiation (19); (ii) two-dimensional crystallography of yeast TFIIE (yTFIIE) with Pol II revealed that yTFIIE actually interacts with the active center of Pol II, which is located near the transcription initiation site on DNA (20); (iii) short mismatched heteroduplex DNA around the initiation site in topologically relaxed templates abolishes the requirement for TFIIE, TFIIH, and ATP (22,23). Therefore, we tested whether TFIIE␤ can bind to DNA by using both the gel retardation assay and the pull down binding assay with ssDNA and dsDNA. While TFIIE␤ preferentially bound to ssDNA, TFIIE␣ alone bound only weakly to ssDNA (less than 5% of the level observed for wild type TFIIE␤), and neither subunit alone was able to bind to dsDNA. Surprisingly, how- ever, binding to dsDNA was observed when both subunits were mixed together to form active TFIIE. 3 Since TFIIE␤ binds strongly to ssDNA, ssDNA binding region in TFIIE␤ was identified by using ssDNA-agarose with the TFIIE␤ deletion mutants (Fig. 8). Deletions from the N terminus up to amino acid residue 152 did not abolish ssDNA binding (Fig. 8A, lanes 1-8). In contrast, the C-terminal deletion mutant ⌬257-291 failed to bind to ssDNA, although the mutant ⌬278 -291 could still bind (Fig. 8A, lane 12 versus lane 13). Analysis of the internal deletion mutants showed that all except ⌬257-291 could bind to DNA (Fig. 8B, lanes 1-12 versus  lane 13). These results clearly demonstrate that TFIIE␤ binds to ssDNA through its C-terminal basic region.

Effects of TFIIE␤ Mutations on TFIIH-dependent CTD Phosphorylation Correlate with Effects on Basal Transcription-
Previous studies demonstrated that TFIIE strongly stimulates CTD phosphorylation by TFIIH by itself and during formation of the active initiation complex (12,14) and that TFIIE␣ is essential for this stimulation which correlates well with the increase in basal transcription activity (15). It was considered that TFIIE might simply provide a bridge between TFIIH and Pol II to assist TFIIH-mediated phosphorylation of Pol II, as TFIIH by itself had been found not to interact well with Pol II.
However, as described above, there exists the possibility that TFIIE may have unique function(s) to stabilize promoter melting by binding to the ssDNA region of the promoter DNA (Fig.  8, Ref. 23). 3 To check this novel function of TFIIE, the effects of TFIIE␤ deletion mutations on CTD phosphorylation were analyzed (Fig. 9).
All four mutants of TFIIE␤ with basal transcription activity (⌬8 -25, ⌬8 -50, ⌬278 -291, and ⌬26 -47) stimulated CTD phosphorylation (completely shifted the largest subunit of Pol II from IIa to IIo form), whereas there was almost no stimulation observed in the absence of both TFIIE␣ and TFIIE␤ or even in the presence of TFIIE␣ (without TFIIE␤) (Fig. 9A, lanes 3-5  and 14 versus lanes 1 and 2, and Fig. 9B, lanes 3-5 versus lanes  1 and 2). It is important to note that most of transcriptiondefective TFIIE␤ mutants also stimulated CTD phosphorylation, but the extent of phosphorylation was less and different from each other. In addition, all those mutants made Pol II less shifted after phosphorylation (the largest subunit of Pol II could not be shifted to IIo form and stopped in between IIa and IIo). There are two interesting mutants found in this assay: one is the internal deletion mutant ⌬230 -255 that showed strong stimulation of CTD phosphorylation, but Pol II shift was incomplete (stopped in between IIa and IIo) (Fig. 9B, lane 13  versus lane 3), and the other is the N-terminal deletion mutant ⌬4 -75 that showed complete Pol II shift to IIo form, but the extent of stimulation was less (26% of wild type) (Fig. 9A, lane  6 versus lane 3). It should be noted that both mutants did not possess basal transcription activity (Fig. 2B, lane 6 and Fig. 3B,  lane 13).
At Least Two Regions of TFIIE␤ Are Essential for Productive Transcription Initiation-Structure-function analyses of TFIIE␤ revealed that except for the N-terminal serine-rich sequence, most of the putative structural motifs and characteristic sequences are functionally important for basal transcription (Figs. 2 and 3). Importantly, the conserved basic and helix regions near the C terminus are targets for the general transcription factors TFIIB and TFIIF␤ (RAP30), for the transcriptional regulator Kr (Figs. 6 and 7) 3 as well as for ssDNA (Fig.  8). To get an insight to proceed with the characterization of the regions in TFIIE␤ in addition to the above-described C-terminal regions by demonstrating the active effects of these regions on basal transcription, we employed the deletion mutants in a dominant negative competition assay (Fig. 10A). In this way, we found that the deletion mutant (⌬257-291) lacking the C-terminal basic region-helix-loop sequence actively suppressed basal transcription to 13% of the wild type level in a dose-dependent manner (Fig. 10A, lanes 12-16). Two other deletion mutants (⌬4 -125 and ⌬75-96), each of which lacks a region similar to the Pol II binding region of RAP30 (TFIIF␤) and the serine-rich sequence in the case of mutant ⌬4 -125, also reduced basal transcription to about 30% of the wild type level (Fig. 10A, lanes 7-11 and 17-21). On the other hand, the internal deletion mutant (⌬197-232) lacking the TFIIE␣ binding region had no effect on transcription even when added at a 64-fold excess over the amount of wild type TFIIE␤ (Fig. 10A,  lane 26 versus 22). The ⌬230 -255 mutant described above showed a modest negative effect on basal transcription (56% of wild type transcription at a 64-fold excess) although this mutant has so far failed to display an interaction with other transcription factor or with DNA (Fig. 10A, lane 31 versus 27). DISCUSSION Elucidation of the precise mechanisms involved in transcription initiation by Pol II has been a long-standing issue in molecular biology. During stepwise assembly of the preinitiation complex (PIC), TFIIE is essential for TFIIH recruitment and completion of PIC formation, as well as for stabilization and activation of the PIC. Recent studies have indicated that TFIIE, through interactions with other factors and possibly with DNA, is localized near the transcription initiation site (between positions Ϫ14 and Ϫ2) within the PIC and near the active center of Pol II (19 -21). It also appears that TFIIE has a novel but unclear function during promoter melting (23). Here, we have investigated the structure and function of the small subunit of human TFIIE (TFIIE␤). This included an examination of the effects of TFIIE␤ deletion mutations on both basal and activator-mediated transcription and identification and characterization of TFIIE␤ interactions with the general transcription factors TFIIE␣, TFIIB, and TFIIF␤ (RAP30), the Drosophila transcriptional repressor Kr, and ssDNA.
Structure-Function Relationships in TFIIE␤-Analysis of TFIIE␤ deletion mutants showed that a central 227-amino acid region (residues 51-277) was sufficient to mediate both basal and activated transcription. As summarized in Fig. 10B, this region contains all of the previously noted structural motifs and characteristic sequences with the exception of the N-terminal half of the serine-rich sequence and the loop region of the basic region-helix-loop sequence. All of the internal subdomains of the central 227 amino acid region were essential for transcription activity (Figs. 2B and 3B). Protein interaction studies indicated that two basic regions and associated sequences located near the C terminus (residues 197-210 and 258 -270) are important for direct TFIIE␤ interactions with various general transcription factors and transcriptional regu-latory factors as well as with ssDNA (Figs. 5-8). 3 . These basic regions have similar primary structures but may have different (context-dependent) binding preferences, as summarized in Fig. 10B. Thus the N-terminal half of the basic region-helixloop-helix (BR-HLH) motif is involved in TFIIE␣ interactions, whereas the second basic region and the following N-terminal half of the helix region may be part of a basic region-helix-loop (BR-HL) domain involved in interactions with TFIIB and TFIIF␤ (RAP30) as well as with ssDNA. In contrast, interaction of the Drosophila transcriptional repressor Kr with TFIIE␤ appears to involve both regions.
We also demonstrated that the TFIIE␤ subunit is essential for the stimulation of TFIIH-dependent CTD phosphorylation of Pol II by TFIIE as is TFIIE␣ (Fig. 9 and Ref. 15). The complete shift of the largest subunit of Pol II from IIa to IIo form upon CTD phosphorylation was dependent entirely on the basal transcription activity of TFIIE␤. An important difference with respect to TFIIE␣ was that some TFIIE␤ mutants supported neither basal transcription nor CTD phosphorylation even though they could bind to TFIIE␣ subunit, Pol II and ssDNA. This is in contrast to TFIIE␣ whose stimulation of CTD phosphorylation was dependent on its binding to TFIIE␤. Another difference was that the transcription-defective TFIIE␤ mutants gave various extents of Pol II shift upon phosphorylation, whereas all TFIIE␣ mutants showed a unique profile of Pol II shift (IIo form) except for mutants lacking the TFIIH binding region (Fig. 9 and Ref. 15). It is also interesting that TFIIE␣ together with Pol II and TFIIH stimulated CTD phosphorylation more than 10-fold in the absence of DNA (14) but that almost no stimulation was observed when Pol II and all the general transcription factors including TFIIE␣ were incubated with promoter DNA in the absence of TFIIE␤ (Fig. 9, A  and B, lane 2 versus lane 1). Under these latter conditions, stimulation could only be restored by adding TFIIE␤ along with the other general transcription factors (Fig. 9, A and B,  lane 3 versus lane 2). These results strongly suggest that TFIIE␤ plays important but different roles from TFIIE␣ for both transcription and CTD phosphorylation in the active initiation complex.
Novel Functional Role(s) of TFIIE Mediated by Binding to Single-stranded DNA-Intriguingly, as described above, TFIIE␤ was found to bind to ssDNA through its second basic region (Fig. 8). During the preparation for initiation, TFIIE joins the PIC after recruitment of TFIIF and Pol II and becomes located near the active center of Pol II through interactions with both Pol II and, most likely, with promoter DNA in the region between positions Ϫ14 and Ϫ2, resulting in the recruitment of TFIIH (for a review, see Refs. 1, 2, and 5). In the step where Pol II is phosphorylated by TFIIH, TFIIE activates TFIIH by stimulating its kinase and ATPase activities (12,14). However, the recent finding of differential requirements for TFIIE and TFIIH when short mismatched heteroduplex DNAs are created around the initiation site has raised the possibility that TFIIE has a TFIIH-independent function during promoter melting (23). Our results showing ssDNA binding activity by TFIIE suggest that TFIIE␤ may play an additional role by binding to the single-stranded region present within melted promoter DNA. Thus, although it is generally held that TFIIE and TFIIH work together during the promoter clearance step necessary for the transition from initiation to elongation (44), it also is possible that TFIIE works independently of TFIIH to remove certain general transcription factors from the initiation complex on the promoter.
Functionally Important Regions of TFIIE␤ in Transcription-In addition to identifying the regions important for basal transcription, the effects of adding excess amounts of TFIIE␤ mutant proteins on basal transcription assays containing wild type TFIIE␤ were studied (Fig. 10A). This approach is especially effective for both the identification of dominant negative mutants that lack the active center of TFIIE␤ but maintain functional interactions with other PIC components and, conversely, for the identification of dominant negative mutants that have lost their capacity to interact with other PIC components but still possess an active center. The deletion mutant ⌬257-291 of the C-terminal basic region of the basic regionhelix-loop sequence was the strongest dominant negative mutant (13% of wild type transcription level when added at a 64-fold excess) (Fig. 10A, lanes 12-16). Since this basic region was identified as a target for the general transcription factors TFIIB and TFIIF␤ (RAP30), as well as the Drosophila transcriptional repressor Kr and ssDNA, it appears to be quite important for TFIIE␤ function. Mutant ⌬257-291 may still possess Pol II and TFIIE␣ binding regions, resulting in the depletion of factors necessary for functional PIC formation. Two other deletion mutants (⌬4 -125 and ⌬75-96) have similar dominant negative effects (about 30% of wild type level when added at a 64-fold excess) (Fig. 10A, lanes 7-11 and lanes  17-21). The former mutant lacks the serine-rich sequence and the region similar to the Pol II binding region of RAP30 (TFIIF␤), whereas the latter lacks only the region similar to the Pol II binding region of RAP30 (TFIIF␤). However, because the first 50 amino acids of TFIIE␤, which contain the N-terminal half of the serine-rich sequence, are dispensable for transcription, the former mutant, ⌬4 -125, may be almost equivalent to the latter mutant, ⌬75-96. Importantly, we confirmed here that the region similar to the Pol II binding region of RAP30 (TFIIF␤) might also be essential for TFIIE␤ function. These mutants may not be recruited to their proper position in the PIC complexes because of a failure to bind to Pol II.
Functional Implications of TFIIE (and TFIIH) in Productive Transcription-In this study we have presented an initial characterization of TFIIE␤ structure and function, and we have proposed novel function(s) for TFIIE during both transcription initiation and in the transition from initiation to elongation. As described above, three recent studies dealing with the role of TFIIE in transcription initiation have suggested that TFIIE, as part of the PIC, binds both to the core promoter just upstream from the transcription initiation site (between positions Ϫ14 and Ϫ2) and to Pol II near its active center (for a review, see Refs. 1, 2, and 5). In this way, in conjunction with TFIIH, it would play an important role in promoter opening, a step which can be circumvented by premelting the promoter between positions Ϫ4 to ϩ2 (19 -23). Our results demonstrate that the C-terminal basic region of TFIIE␤ binds to both TFIIB and TFIIF␤ (RAP30). These observations agree with photocrosslinking results showing that TFIIB and TFIIF␤ (RAP30) bind to the promoter DNA just upstream of TFIIE␤ and stabilize the Pol II-TBP interaction at around position Ϫ19 (19). Since TFIIB and TFIIF␤ (RAP30) may bind to different surfaces on the promoter DNA (19) and since TFIIE␤ exists as a dimer in the PIC (9, 10), we can envisage a model in which TFIIB and TFIIF␤ (RAP30) bind to different TFIIE␤ subunits that are docked to the DNA in parallel with their C termini facing upstream. As mentioned above, TFIIE was able to bind to dsDNA, in contrast to the TFIIE␣ or TFIIE␤ subunit which could not bind to dsDNA when added individually. 3 On the other hand, TFIIE␤ was able to bind to ssDNA (Fig. 8). 3 One possibility is that TFIIE is recruited into the PIC and at that time TFIIE␣ assists TFIIE␤ to bind to the promoter region (between Ϫ14 and Ϫ2). This result fits well with the observation made by Coulombe and colleagues (19) that TFIIE␣ could not bind to dsDNA but that TFIIE␤ could bind to dsDNA with the assistance of TFIIE␣.
The promoter premelting study using short mismatched heteroduplex DNAs and the KMnO 4 sensitivity assays described by Timmers and co-workers (23) have demonstrated that TFIIE and TFIIH open DNA from positions Ϫ9 to ϩ1 in the presence of hydrolyzable ATP. Importantly, our finding of ssDNA binding by TFIIE␤ (Fig. 8) supports the possibility that this factor may contribute to the stabilization of single-stranded regions with the promoter DNA, thereby assisting the DNA unwinding activity of TFIIH which depends on energy supplied by ATP hydrolysis. At the same time, TFIIE may stimulate the CTD kinase activity of TFIIH and, ultimately, open the DNA between positions Ϫ9 to ϩ1. The stimulation of CTD phosphoryl-ation by TFIIE␤ in the PIC complex ( Fig. 9) also supports this model. The "open" region further extends downstream to ϩ8 after formation of the first phosphodiester bond (23). During the transition from initiation to elongation, "promoter clearance" occurs before the transcript attains the size of 10 nucleotides. It is noteworthy that TFIIE is released from the transcription complex before Pol II reaches position ϩ10 and that TFIIH, on the other hand, is released later between positions ϩ30 and ϩ68 (45). Within the extended DNA bubble found during promoter clearance, TFIIE might stabilize the singlestranded region and might work together with TFIIH to alter the conformation of the PIC, resulting in the disassembly and removal of the general transcription initiation factors (TFIID and TFIIB) from the initiation complex. In addition, TFIIE could help recruit transcription elongation factors. It is intriguing to consider the possibility that the general cofactor PC4, which is also an ssDNA-binding protein, may be involved in promoter clearance by displacing TFIIB from the initiation complex, as suggested by studies on the yeast PC4 homolog SUB1 (46 -49). Nonetheless, elucidation of the mechanisms of promoter clearance still awaits further structural and functional studies of TFIIE, TFIIH, and Pol II in conjunction with studies focused upon the elongation step that follows initiation.
An increasing number of transcriptional regulatory factors have been reported to target TFIIE and/or TFIIH in order to express their functions (for a review, see Ref. 5). Thus, transcription activation may, in part, affect promoter opening either by stabilizing single-stranded regions or by regulating the functions of TFIIE and/or TFIIH. The Drosophila developmental gene product and transcriptional regulatory factor, Kr, is one such factor that may target TFIIE (40). Our study clearly demonstrated that Kr predominantly binds to TFIIB and TFIIE␤ (Fig. 7A). By using TFIIE␤ deletion mutants, the Cterminal basic regions of both the basic region-helix-loop-helix motif and the basic region-helix-loop sequence were identified as Kr binding regions in TFIIE␤ (Fig. 7, B and C). Since these basic regions are targets for at least three general transcription factors and for ssDNA, Kr binding might inhibit their binding through competition and, thus, may cause transcriptional repression. Furthermore, it was recently found that the human TFIID subunit TAF II 80 (TBP Associated Factor for RNA polymerase II) binds to TFIIE␣ (50). Therefore, the possibility exists that TAF II s transduces signals not only from activators to the general transcription factors (like TFIID and TFIIB) involved in early PIC formation but also to the general transcription factors that affect open complex formation and promoter clearance (like TFIIE and TFIIH). Another recent finding of the histone-like structures in human TAF II 80, TAF II 60, and TAF II 20 and modification of these structures by the enzymes, histone acetyltransferases and histone deacetylases, has evoked renewed interest in the mechanisms of transcriptional regulation (3,8,33,51). This modification might be important for chromatin remodeling by modifying histones but also for transcriptional regulation. In addition, the recent finding that several histone acetyltransferases also acetylate TFIIE␤ (52) suggests TFIIE involvement in such acetylationmediated transcriptional regulation. In eukaryotic cells, DNAs are wrapped in histones, resulting in the formation of chromatin in the nucleus. Therefore, it is important to consider at the same time both the contributions of histone regulation and general transcription factors in transcription initiation. We are currently pursuing our investigation of the mechanisms of promoter opening and promoter clearance in an attempt to understand better how transcriptional regulatory factors contribute to transcription initiation and elongation through studies on TFIIE within this context.