INTRODUCTION

Top Abstract Introduction Materials and Methods Results Discussion References

In eukaryotes, transcription of protein-encoding genes by RNA polymerase II (Pol II) is the first step in expression of those genes (for reviews, see references 4, 35, 44, and 51). Two sequential stages are now recognized in the establishment of Pol II processivity: transcription initiation and the transition from initiation to elongation. At initiation, five general transcription factors (TFIIB, TFIID, TFIIE, TFIIF, and TFIIH) together with Pol II form the preinitiation complex (PIC) on the core promoter. Two models of PIC formation have been proposed on the basis of recent analyses. One model involves stepwise association of the general transcription factors and Pol II on promoter DNA, while the other model entails promoter sequences binding to a preassembled Pol II holoenzyme that contains most of the general transcription factors as well as SRB (suppressor of RNA polymerase B)- and Med-containing complex (reviewed in references 3 and 22). In vitro analyses of stepwise assembly of the PIC using purified factors have demonstrated that TFIIE joins the complex at a position near the transcription start site (between positions 14 and 2), after Pol II and TFIIF have joined the complex (25, 49). TFIIE then recruits TFIIH, and these two factors stabilize and activate the PIC, resulting in isomerization of double-stranded (ds) promoter DNA (promoter melting) upon transcription initiation. TFIIE and TFIIH are also involved in the transition from initiation to elongation, the stage during which they act to remove from the complex general transcription factors that have already completed their roles in the initiation step (promoter clearance) (reviewed in reference 35).

Human TFIIE (hTFIIE) consists of an ₂₂ heterotetramer of 57-kDa - and 34-kDa -subunits (41). hTFIIE is highly acidic (pI, 4.5) and possesses several putative structural motifs and characteristic sequences (40). The region essential for basal transcription is located within the N-terminal half of the molecule, in which all of the structural motifs reside (37, 38). The acidic region near the C terminus is the only region in the C-terminal half that has a stimulatory effect on basal transcription; this region binds directly to TFIIH. In contrast, hTFIIE is highly basic (pI, 9.5) and possesses several putative structural motifs and characteristic sequences different from those of hTFIIE (36, 48, 57). The internal region of hTFIIE is essential for basal transcription. It has been found that TFIIE binds to single-stranded (ss) DNA through the basic region near its C terminus; the other general transcription factors, TFIIB and TFIIF (RAP30), bind to this region as well (42). In addition, we have recently determined the three-dimensional structure of the central core region in TFIIE that binds to dsDNA (43).

Human TFIIH consists of nine subunits and has three ATP-dependent catalytic activities: kinase activity that phosphorylates the carboxy-terminal domain (CTD) of the largest subunit of Pol II, DNA-dependent ATPase activity, and DNA helicase activity (reviewed in reference 59). TFIIE regulates these TFIIH activities, stimulating the CTD kinase and ATPase activities and repressing the helicase activity (8, 31, 39). At transcription initiation, TFIIE binds to Pol II, TFIIB, and TFIIF, recruits TFIIH into the PIC to stabilize and activate the PIC, and binds to stabilize the ssDNA region in promoter melting. Recent studies have provided support for this model. (i) Photo-crosslinking studies demonstrated that TFIIE binds directly to the core promoter region (between positions 14 and 2), where the promoter melts upon transcription initiation (49). (ii) Two-dimensional crystallography of yeast TFIIE (yTFIIE) with Pol II demonstrated that yTFIIE binds to the active center of Pol II, which is located near the transcription initiation site on the promoter (25). (iii) Short mismatched heteroduplex DNA around the transcription initiation site in topologically relaxed linear templates was shown to alleviate the requirement for TFIIE, TFIIH, and ATP (10, 20, 45, 61).

The above description summarizes our current understanding of the roles of TFIIE and TFIIH before and during transcription initiation. In contrast to initiation, extensive studies of the later stages of transcription have yet to be carried out. Functional involvement of TFIIE and TFIIH in the transition from initiation to elongation has been suggested by studies using a negatively supercoiled immunoglobulin heavy chain (IgH) promoter and the short mismatched heteroduplex linear promoter described above (10, 13, 19, 20, 46, 62). It has been suggested that TFIIE and TFIIH may suppress abortive initiation, which produces short transcripts (around 2 to 15 nucleotides) by releasing the general factors TFIID and TFIIB from the PIC, and may convert Pol II to its elongation-competent form (10, 21, 24). It has been demonstrated that this elongation-competent Pol II is hyperphosphorylated (34, 70). Since TFIIH is the only kinase that can phosphorylate the CTD in the in vitro reconstituted active Pol II complex, TFIIH has been a primary candidate for the biologically relevant CTD kinase (31, 39).

The CTD contains multiple repeats of the heptapeptide sequence YSPTSPS, which occurs 52 times in the largest subunit of human Pol II (7, 71). Several lines of evidence indicate that the integrity of the CTD is essential for basal and activated transcription. The unphosphorylated form of Pol II (Pol IIa) is preferentially recruited into a PIC reconstituted with purified general transcription factors (30). CTD phosphorylation may initially occur between transcription initiation and the transition from initiation to elongation, converting Pol IIa to the phosphorylated form (Pol IIo) (34, 35, 59). Recently, it has been demonstrated that CTD phosphorylation is also important for recruiting the mRNA processing enzymes to the nascent transcript, presumably reflecting the fact that mRNA processing (splicing, capping, and polyadenylation) occurs during and/or after transcription (6, 33).

To further investigate the mechanisms of transcription initiation and the transition to elongation and the TFIIE functions during these steps, we isolated TFIIE cDNAs from the nematode Caenorhabditis elegans (ceTFIIE cDNAs) and expressed two subunits of ceTFIIE in bacteria, both together and independently, based on the idea that the basic transcriptional mechanisms might be conserved among eukaryotic species. We compared the ceTFIIE subunits with respect to their abilities to substitute for their human counterparts in a human in vitro transcription system, their specificities of binding to the general transcription factors, their effects on CTD phosphorylation by TFIIH and, finally, their abilities to convert Pol II to the elongation form. Importantly, we demonstrated for the first time that TFIIE is directly involved in the transition from transcription initiation to elongation and suggested, through the use of transcription transition assays together with analyses of the sites of phosphorylation in the CTD heptapeptide repeat sequence, that TFIIE-induced phosphorylation of serine at position 5 (Ser-5) in the CTD heptapeptide repeat might be essential for this transition.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results Discussion References

Cloning of C. elegans TFIIE cDNAs. The putative ceTFIIE coding sequence was identified using a TBLASTN homology search of the C. elegans translated expressed sequence tag (EST) databank (Sanger Centre, Cambridge, United Kingdom) to locate regions with significant homology to the hTFIIE amino acid sequence. Since there was an NdeI site approximately 25 bp upstream of the stop codon, four oligonucleotides were designed to perform two PCRs in order to amplify the N- and C-terminal halves independently. To amplify the N-terminal half of ceTFIIE, the oligonucleotide CEB1T (5'-CTGATCATATGGACCCGGAATTGTTAAGGC-3') was designed to create an NdeI site (underlined) at the first methionine codon and to disrupt a BamHI site by changing the third nucleotide of the second aspartate codon (T to C, bold and underlined) and was used in conjunction with the oligonucleotide CEB2B (5'-GTCATTGTAGAAGACGAC-3'). To obtain the C-terminal half, the oligonucleotide CEB1B (5'-CTTGAGGATCCAGAAGTGTGTAATTAAAATC-3') was designed to create a BamHI site (underlined) after the stop codon and was used in conjunction with the oligonucleotide CEB2T (5'-GTGGATTATATGAAGAAACG-3'). PCRs were performed using a C. elegans mixed-stage cDNA library (a kind gift from Yuji Kohara). After 50 cycles of PCR with an annealing temperature of 50°C, the PCR products (approximately 630 bp for the N-terminal portion and 640 bp for the C-terminal portion) were purified, blunt ended with the Klenow fragment of Escherichia coli DNA polymerase I, phosphorylated with T4 polynucleotide kinase, and subcloned into the SmaI site of pBluescript SK() (Stratagene). The nucleotide sequences of the cloned PCR products were confirmed using an ALFred DNA sequencer (Amersham Pharmacia Biotech).

Construction of ceTFIIE expression vectors. Plasmids containing either the N- or C-terminal portion of the ceTFIIE (ceTFIIE cDNA) open reading frame were digested with either NdeI and PstI (N-terminal clone; 0.54 kb) or PstI and BamHI (C-terminal clone; 0.33 kb). These fragments were subcloned into the pET3a and 6HisT-pET11d vectors to construct expression plasmids containing the entire coding region of ceTFIIE cDNA, expressing nontagged ceTFIIE (ceTFIIE) and six-histidine-tagged ceTFIIE (6H-ceTFIIE), respectively (17). Similarly, plasmids containing either the N- or C-terminal portion of the open reading frame of ceTFIIE (ceTFIIE cDNA) were digested with NdeI and SacI (N-terminal clone; 0.41 kb) or with SacI and BamHI (C-terminal clone; 0.89 kb). These fragments were subcloned into the pET3a and 6HisT-pET11d vectors to construct nontagged (ceTFIIE) and six-histidine-tagged (6H-ceTFIIE) ceTFIIE expression plasmids containing the full coding region of ceTFIIE cDNA (17).

Expression and purification of recombinant proteins. Recombinant proteins were expressed in E. coli BL21(DE3)pLysS by induction with isopropyl--D-thiogalactopyranoside (IPTG) (55). For general purification, soluble bacterial lysates were used. For miniscale preparations, lysates (1 ml) representing 50 to 100 ml of culture were mixed directly with 1 ml of buffer B (20 mM Tris-HCl [pH 7.9 at 4°C], 0.5 mM EDTA, 10% [vol/vol] glycerol, 1 mM phenylethylsulfonyl fluoride [PMSF], 2-µg/ml antipain, 2-µg/ml aprotinin, 1-µg/ml leupeptin, 0.8-µg/ml pepstatin, 10 mM 2-mercaptoethanol) containing 500 mM NaCl (BB500) and 100 µl of Ni-nitrilotriacetic acid (NTA) agarose resin (Qiagen) 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% [vol/vol] glycerol, 1 mM PMSF, 10 mM 2-mercaptoethanol) containing 500 mM KCl (BD500), and twice with 500 µl of BD500 containing 20 mM imidazole-HCl (pH 7.9). Bound proteins were eluted twice with 300 µl of BD500 containing 100 mM imidazole-HCl (pH 7.9). Typical preparations were >80% pure as judged by Coomassie blue staining of a sodium dodecyl sulfate (SDS)-polyacrylamide gel.

In vitro transcription assays. The general transcription factor TFIIH was purified either from HeLa nuclear extracts or from cytoplasmic S100 fractions as previously described (39). All other general transcription factors (TBP, TFIIB, TFIIF, and TFIIE) were purified essentially as follows: the recombinant proteins were expressed in E. coli, solubilized by sonication, and purified on a Ni-NTA agarose column. Pol II was purified to near-homogeneity from HeLa nuclear pellets by DE52, A25, P11, and high-performance liquid chromatography-DEAE 5PW columns. In vitro transcription was carried out as described previously (38). The plasmid pML(C₂AT)-50, which contains the adenovirus type 2 major late (AdML) promoter and gives a 390-nucleotide (nt) transcript, was used as a template for basal transcription assays (53). Autoradiography was performed at 80°C with Fuji RX-U X-ray film. The incorporation of [-³²P]CTP into transcripts was quantified using a Fuji BAS2500 Bio-Imaging analyzer.

Generation of antibodies against C. elegans TFIIE subunits. Both 6H-ceTFIIE subunits were expressed independently in E. coli, solubilized by sonication, and purified on a Ni-NTA agarose column. Since 6H-ceTFIIE was mostly soluble (>80% in soluble lysate) and 6H-ceTFIIE was mostly insoluble (>90% in pellet), 6H-ceTFIIE was purified from bacterial lysates and 6H-ceTFIIE was purified from bacterial pellets after solubilization with 4 M guanidine-HCl (pH 7.5). Two milligrams of each purified protein was subjected to SDS-polyacrylamide gel electrophoresis (PAGE), and the appropriate bands were excised from the gel after Coomassie blue staining.

Preparation of C. elegans embryonic nuclear extracts. The Bristol N2 wild-type strain of C. elegans was grown in liquid culture essentially as described previously (56). Liquid cultures were started by seeding two 9-cm plates of N2 into 1 liter of S medium (10 mM potassium citrate [pH 6], 50 mM potassium phosphate [pH 6], 50 µM EDTA, 5 µg of cholesterol/ml, 3 mM CaCl₂, 3 mM MgSO₄, 25 µM FeSO₄, 10 µM MnCl₂, 10 µM ZnSO₄, 1 µM CuSO₄) in a 2-liter flask with a culture paste of E. coli OP50 from a 2-liter culture. Worms were grown at 22°C with shaking at 350 rpm for 4 days, and growth was monitored until most worms were gravid hermaphrodites. Growth synchronization of C. elegans was then carried out essentially as described previously (28), except that the culture volume was 6 liters. Final recovery of embryos was 2.8 g. To prepare embryonic nuclear extracts, the embryos were harvested and homogenized as described previously (28).

Coimmunoprecipitation and depletion of C. elegans TFIIE from the nuclear extract. Rat polyclonal antisera against ceTFIIE (0.5 µl) and 6 µl (packed volume) of protein G-Sepharose 4FF (Amersham Pharmacia Biotech) were incubated in buffer C (20 mM Tris-HCl [pH 7.9 at 4°C], 0.5 mM EDTA, 20% [vol/vol] glycerol, 0.5 mM PMSF, 10 mM 2-mercaptoethanol, 0.002% [vol/vol] Nonidet P-40) containing 100 mM KCl (BC100) and 200 µg of bovine serum albumin (BSA)/ml for 2 h at 4°C with rotation. The protein G-Sepharose beads were precipitated and washed twice with 500 µl of buffer C containing 1 M KCl (BC1000) and twice with 500 µl of BC500. One hundred and fifty microliters of C. elegans nuclear extract (2.1 mg of protein/ml) pre-equilibrated with BC500 was then incubated with the prepared anti-ceTFIIE antibody-protein G beads in a 500-µl reaction volume for 4 h at 4°C with rotation. This step was repeated three times, and the resulting supernatant was used as a ceTFIIE-depleted nuclear extract. To check for complete depletion of ceTFIIE, the beads were washed twice with 500 µl of BC500 and twice with 500 µl of BC100 and boiled in SDS sample buffer, and the proteins released from the beads were analyzed by SDS-10% PAGE.

Primer extension reaction. In vitro transcription reactions were performed essentially as described previously (28) except that the reaction temperature was 24°C, 200 ng of the AdML promoter pMLH1 (14) was used as a supercoiled DNA template, and the reaction mixture volume was 50 µl. C. elegans embryonic nuclear extract (42 µg of total protein) or ceTFIIE-depleted nuclear extract was used for each reaction. Transcription was stopped by the addition of 75 µl of 450 mM sodium acetate (pH 5.3)-10 mM EDTA-0.5% SDS-yeast tRNA (50 µg/ml). Primer extension reactions were carried out as described previously (29). A synthetic oligonucleotide (5'-CTGACAATCTTAGCGCAGAAGTCATG-3') was 5' end labeled with [-³²P]ATP and T4 polynucleotide kinase and used as a primer. The products (92 nt) were analyzed on 12% denaturing polyacrylamide-urea gels. Autoradiography was performed at 80°C with Fuji RX-U X-ray film.

GST-pull down assay. GST fusion proteins were used for protein interaction assays. Each protein to be tested (200 ng) was incubated with lysates containing 400 ng of GST fusion proteins together with 5 µl (packed volume) of glutathione-Sepharose (Amersham Pharmacia Biotech) in a 500-µl reaction mixture in BC100 containing 200 µg of BSA/ml for 4 h at 4°C with rotation. The glutathione-Sepharose resin was then washed twice with 500 µl of buffer C containing 200 mM KCl (BC200) and once with 500 µl of BC100 and boiled in SDS sample buffer. The proteins released from the resin were analyzed by SDS-PAGE and Western blotting as described above.

Kinase assay. Kinase assays were carried out as described elsewhere (39) using the general transcription factors together with Pol II and a DNA fragment containing AdML promoter sequences from 39 to +29. Phosphorylation reactions were carried out at 30°C for 1 h and stopped by the addition of 75 µl of phosphorylation stop solution (10 mM EDTA, 0.1% Nonidet P-40, 0.05% SDS). Phosphorylated proteins were precipitated with trichloroacetic acid, analyzed by SDS-5% PAGE (5.5% acrylamide), and detected by autoradiography performed at 80°C with Fuji RX-U X-ray film. The extent of ³²P-phosphorylation of the CTD of the Pol II largest subunit was quantified using a Fuji BAS2500 Bio-Imaging analyzer.

Transcription transition assay. To measure the effects of various TFIIE constructs on the transition from initiation to elongation, the pML(C₂AT)100 insert was constructed by PCR using the 5' oligonucleotide ML100-1T (5'-GACTATCTAGAGTGTTCCTGAAGGGGG-3') to create an XbaI site (underlined) at the 5' end of the AdML core promoter and the 3' oligonucleotide ML100-1B (5'-CGATCTCCCGGGAAATATAGAAGAAGGAG-3') to create a SmaI site (underlined) at the 3' end of the short (97 bp) G-less cassette. The product of a PCR using these promers and pML(C₂AT)-50 as a template was subcloned into the SmaI site of pBluescript SK() (Stratagene) to yield the pML(C₂AT)100 transcription template, which gives a short 107-nt transcript. To provide a linear template, pML(C₂AT)100 was digested with SmaI. PICs for use in the transcription transition assays were performed in a 15-µl reaction mixture containing either no TFIIE or 15 ng of one of the four different TFIIE proteins, together with all other general transcription factors, Pol II, and 100 ng of the pML(C₂AT)100 template (linear or supercoiled). These mixtures were incubated for 45 min at 28°C under the in vitro transcription conditions, except that no nucleoside triphosphates were added. Transcription was then initiated by addition of 15 µl of reaction mixture containing 6 µCi of [-³²P]CTP (400 Ci/mmol; Amersham Pharmacia Biotech), 50 µM ATP, 50 µM UTP, 12.5 µM CTP, 40 µM 3'-O-methyl GTP, and 0.15 U of RNase T1 (Amersham Pharmacia Biotech). After transcription, reactions were stopped by heat treatment for 3 min at 68°C and treated with 4 U of calf intestine alkaline phosphatase for 20 min at 37°C to reduce the background signal due to nonincorporated [-³²P]CTP. The reactions were stopped, and transcripts were then ethanol precipitated and analyzed on 10% denaturing polyacrylamide-urea gels. Autoradiography was performed at 80°C with Fuji RX-U X-ray film. The incorporation of [-³²P]CTP into transcripts was quantified using a Fuji BAS2500 Bio-Imaging analyzer.

Transcription initiation reaction. Initiation reactions were performed essentially as described previously (13, 20). Each reaction (15 µl) contained 40 mM Hepes-KOH (pH 8.4), 12 mM Tris-HCl (pH 7.9 at 4°C), 60 mM KCl, 4 mM MgCl₂, 0.3 mM EDTA, 12% (vol/vol) glycerol, 0.3 mM PMSF, 6 mM 2-mercaptoethanol, and 150 µg of BSA/ml. By preincubation for 45 min at 28°C, the preinitiation complex was formed with 10 ng of the 158-bp AflIII-ScaI fragment of pMLH1 (containing the AdML promoter sequence from 111 to +47) (14) together with Pol II and all general transcription factors, except that TBP was used instead of TFIID and hTFIIE was replaced with various combinations of chimeric TFIIE. Transcription initiation was then carried out for 45 min at 28°C by addition of 5 µl of reaction mixture containing 60 µM ATP and 10 µCi [-³²P]CTP (800 Ci/mmol; NEN) in the same buffer composition. Reactions were stopped by heat treatment for 3 min at 68°C and treated with 10 U of calf intestine alkaline phosphatase for 20 min at 37°C. After inactivation for 10 min at 75°C, transcripts were analyzed on 20% denaturing polyacrylamide-urea gels. Autoradiography was performed at 80°C with Fuji RX-U X-ray film.

Nucleotide sequence accession numbers. The EMBL accession numbers for the ceTFIIE and ceTFIIE cDNA sequences are Y08816 and Y08815, respectively.

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

Isolation of C. elegans TFIIE cDNAs. In order to examine the relationship between TFIIE structure and its role in transcription, we have been isolating human TFIIE homologs from different species and studying their structural and functional similarities to their human counterparts. We isolated both subunits of a TFIIE homolog from Xenopus laevis (xTFIIE), and other groups isolated homologs from Drosophila melanogaster (dTFIIE) and yeast Saccharomyces cerevisiae (yTFIIE) (11, 36, 37, 68). When the functional exchangeabilities of these TFIIE homologs with human TFIIE were tested, xTFIIE and dTFIIE were functionally exchangeable in the human in vitro reconstituted transcription system but yTFIIE was not. In amino acid sequence comparison with their human counterparts, xTFIIE has 79% identity in xTFIIE and 84% in xTFIIE and dTFIIE has 46% identity in dTFIIE and 59% in dTFIIE, whereas yTFIIE has only 22% identity in yTFIIE and 23% in yTFIIE. Judging from these data, the border of a functional exchangeability may evolutionarily lie between Drosophila and yeast, indicating that an hTFIIE homolog from C. elegans will be of use in the examination of structure-function relationships in TFIIE.

View larger version (70K): [in this window] [in a new window]   FIG. 1.   Sequence analysis of C. elegans TFIIE. (A) Comparison of ceTFIIE (ceIIE) with hTFIIE (hIIE). The serine-rich sequence (Ser-rich), a region similar to the small subunit of TFIIF, TFIIF (IIF), a hydrophobic region (Hydrophobic), a BR-HLH and a BR-HL are indicated (42). In the middle panel, identity (above) and similarity (below) between ceIIE and hIIE over each structural motif and characteristic sequence region are indicated as percentages. The numbers presented above and below the diagram indicate the amino acid residues that delimit each structure. (B) Sequence alignment of TFIIE from five different species. Amino acid sequences of TFIIE from human (H), X. laevis (X), D. melanogaster (D), C. elegans (C), and yeast S. cerevisiae (Y) were aligned. Completely identical residues are shaded in black, and residues identical in four species are shaded in gray. Conserved similar residues are shown in bold type. Identical and similar amino acids were assigned as described previously (36). Putative structural motifs and characteristic sequences are shown as described for Fig. 1A and boxed (boxes a to f), except that a 3 homology region (box d, 3 homology) is additionally indicated. A hyphen indicates a gap.

View larger version (75K): [in this window] [in a new window]   FIG. 2.   Sequence analysis of C. elegans TFIIE. (A) Comparison of ceTFIIE (ceIIE) with hTFIIE (hIIE). The region with  subdomain homology and a leucine repeat (, LR), a zinc finger motif (ZF), a helix-turn-helix motif (HTH), a hydrophobic region (Hydrophobic), two regions rich in alanine and glycine residues (Ala, Gly-rich), and the first and second acidic regions (2nd acidic) are indicated (38). Identity (above) and similarity (below) between ceIIE and hIIE over each structural motif and characteristic sequence region are indicated as shown in Fig. 1A. (B) Sequence alignment of TFIIE from five different species. Amino acid sequences of TFIIE from four species were aligned as shown in Fig. 1B. Identical and similar amino acids were assigned as described previously (37). Putative structural motifs and characteristic sequences are shown as described for panel A and boxed (boxes a to j), except that two overlapping  subdomain homology regions (box a,  2.1-2.2 homology, and box b,  4.1-4.2 homology) and two alanine, glycine-rich regions (box g, 1st alanine, glycine-rich, and box h, 2nd alanine, glycine-rich) are additionally indicated. A hyphen indicates a gap.

Recombinant C. elegans TFIIE is identical to the natural form. In order to confirm that we had isolated bona fide ceTFIIE cDNAs, both ceTFIIE and ceTFIIE were expressed independently in bacteria with N-terminal six-histidine tags and purified on a Ni-NTA agarose column. Polyclonal antibodies were raised against recombinant ceTFIIE subunits by immunizing experimental animals with specific bands excised from SDS-PAGE gels. Recombinant and natural ceTFIIE subunits were compared with respect to migration on SDS-PAGE, antibody recognition, and function in primer extension assays (Fig. 3). Each subunit of natural ceTFIIE was immunoprecipitated from C. elegans embryonic nuclear extracts, and the precipitates were examined to determine whether the other subunit was coimmunoprecipitated. Rat anti-ceTFIIE antiserum was used to coimmunoprecipitate natural ceTFIIE, which produced a band of the same size as recombinant ceTFIIE on Western blots probed with rabbit anti-ceTFIIE antiserum (Fig. 3A, lane 4 versus lane 2). Natural ceTFIIE was similarly coimmunoprecipitated with rabbit anti-ceTFIIE antiserum and produced a band of the same size as recombinant ceTFIIE on Western blots probed with rat anti-ceTFIIE antiserum (Fig. 3B, lanes 3 and 4 versus lane 5).

View larger version (22K): [in this window] [in a new window]   FIG. 3.   Identification of natural C. elegans TFIIE. (A) Coimmunoprecipitation of natural ceTFIIE with ceTFIIE. To demonstrate association of natural ceTFIIE with ceTFIIE, 80 µg of C. elegans embryonic nuclear extract was incubated with anti-ceTFIIE antibody-protein G-Sepharose, and natural ceTFIIE was precipitated. After SDS-PAGE on a 10% polyacrylamide gel, coprecipitated ceTFIIE was detected with anti-ceTFIIE rabbit antibody after Western blotting. Lane 1, 5% input of embryonic nuclear extract (4 µg); lane 2, recombinant ceTFIIE (20 ng); lane 3, nuclear extract treated with preimmune serum (80 µg); lane 4, nuclear extract treated with anti-ceTFIIE serum (80 µg). An arrow indicates the position of ceTFIIE (ceIIE). (B) Coimmunoprecipitation of natural ceTFIIE with ceTFIIE. The same strategy as detailed for panel A was employed to study the association of natural ceTFIIE with ceTFIIE. Eighty micrograms of nuclear extract was incubated with anti-ceTFIIE antibody-protein G-Sepharose, and natural ceTFIIE was precipitated. Coprecipitated ceTFIIE was detected by anti-ceTFIIE rabbit antibody after Western blotting. Lane 1, 25% input of embryonic nuclear extract (20 µg); lane 2, nuclear extract treated with preimmune serum (80 µg); lanes 3 and 4, increasing amounts of nuclear extract treated with anti-ceTFIIE serum (40 and 80 µg, respectively); lane 5, recombinant ceTFIIE (200 ng). An arrow indicates the position of ceTFIIE (ceIIE). (C) Transcription complementation assay of ceTFIIE. ceTFIIE was depleted from nuclear extracts by treatment with anti-ceTFIIE antibody-protein G-Sepharose. In the same way, mock-depleted nuclear extracts were prepared by treatment with preimmune IgG-protein G-Sepharose. Complementation of natural ceTFIIE was studied by adding increasing amounts of purified recombinant ceTFIIE and carrying out primer extension reactions. Lane 1, ceTFIIE-depleted nuclear extracts (42 µg); lane 2, mock-depleted nuclear extracts (42 µg); lanes 3 to 5, ceTFIIE-depleted nuclear extracts (42 µg) with increasing amounts of recombinant ceTFIIE (5, 10, and 20 ng, respectively); lane 6, C. elegans nuclear extracts (42 µg). Arrows indicate the positions of the reverse transcript (92 nt) and the primer.

View larger version (19K): [in this window] [in a new window]   FIG. 4.   Characterization of recombinant C. elegans TFIIE. (A) SDS-PAGE analysis of four different recombinant TFIIE proteins, with subunits from either human or C. elegans. Recombinant TFIIE subunits were coexpressed in four different combinations from the coexpression plasmids described in Materials and Methods, purified, and analyzed by SDS-10% PAGE (lanes 1 to 4). The sizes of molecular mass markers are indicated on the left (in kilodaltons). The approximate positions of TFIIE (IIE) and TFIIE (IIE) are indicated by arrows on the right. Asterisks indicate degradation products derived from ceTFIIE and hTFIIE in lane 2. (B) Basal transcription activities of chimeric TFIIE. In vitro transcription assays were carried out with increasing amounts of chimeric TFIIE proteins. Lane 1, no TFIIE (); lanes 2 to 4, 10, 20, and 40 ng of TFIIE made up of 6H-ceTFIIE and nontagged ceTFIIE (ceEceE); lanes 5 to 7, 10, 20, and 40 ng of TFIIE made up of 6H-ceTFIIE and nontagged hTFIIE (ceEhE); lane 8, 12 ng of TFIIE made up of 6H-hTFIIE and nontagged ceTFIIE (hEceE); lane 9, 12 ng of TFIIE made up of 6H-hTFIIE and nontagged hTFIIE (hEhE); lanes 10 and 11, 3 ng of 6H-ceTFIIE (ceE) and 6H-hTFIIE (hE), respectively. The arrow indicates the position of the specific transcript (390 nt).

Transcriptional exchangeabilities of ceTFIIE subunits with their human counterparts. We then tested the abilities of ceTFIIE subunits to functionally replace their human counterparts using four different subunit combinations of TFIIE in a human in vitro transcription system with a supercoiled template (Fig. 4B). Although ceTFIIE, when complexed with hTFIIE, showed approximately 30% of wild-type hTFIIE activity (lanes 8 and 9), ceTFIIE showed less than 5% of the wild-type activity when complexed with hTFIIE (lanes 5 to 7). Moreover, wild-type ceTFIIE showed almost no activity regardless of the amount added (lanes 2 to 4). These results demonstrate that ceTFIIE can partially replace hTFIIE but ceTFIIE cannot replace hTFIIE in a human in vitro transcription system with a supercoiled template.

C. elegans and human TFIIE show similar binding specificities to human general transcription factors. In an attempt to determine why ceTFIIE was only partially able to replace its human counterpart, the binding specificities of ceTFIIE and hTFIIE for human general transcription factors were compared using GST-pull down assays (Fig. 5). Both C. elegans and human TFIIE bound strongly to TFIIB, TFIIE, and TFIIE (RAP30), weakly to TFIIF (RAP74) and TBP, and very weakly to TFIIA (Fig. 5A and B). The human TFIIH subunits were similarly tested (Fig. 5C and D); both C. elegans and human TFIIE bound to XPB, p62, and Cdk7, albeit relatively weakly (Fig. 5C and D, lanes 2, 4, and 8). The only difference detected between ceTFIIE and hTFIIE was that hTFIIE bound weakly to cyclin H (Fig. 5C, lane 9), whereas ceTFIIE did not bind to cyclin H but did bind weakly to p44 instead (Fig. 5D, lane 6). In addition, since we observed that human Pol II bound predominantly to TFIIB and hTFIIE, the binding of human Pol II to ceTFIIE was also tested; weaker but significant binding (about half-efficiency relative to that of hTFIIE) was observed (Y. Ohkuma, data not shown). Judging from these results, there was not much difference between ceTFIIE and hTFIIE in binding to the human general transcription factors. It therefore remains difficult to explain the partial exchangeability of ceTFIIE with hTFIIE simply from these binding results.

View larger version (21K): [in this window] [in a new window]   FIG. 5.   Binding assays using C. elegans TFIIE. (A) Binding of hTFIIE to the various general transcription factors. All of the human general transcription factors (400 ng each) except for hTFIIE, the TBP activation factors of TFIID, and the TFIIH subunits were fused to GST and expressed in bacteria. GST-pull down assays were carried out with 200 ng of 6H-hTFIIE. After SDS-PAGE on a 10% polyacrylamide gel and Western blotting, bound 6H-hTFIIE was detected with anti-hTFIIE antibody. Lane 1, control bacterial lysate (no GST protein) (); lane 2, GST alone (no fusion protein) (G); lane 3, GST-TFIIB (B); lane 4, GST-hTFIIE (E); lane 5, GST-TFIIF (F); lane 6, GST-TFIIF (F); lane 7, GST-TBP (T); lane 8, GST-TFIIA (A); lane 9, GST-TFIIA (A); lane 10, GST-TFIIA (A). An arrow indicates the position of 6H-hTFIIE (hIIE). (B) Binding of ceTFIIE to the various general transcription factors. GST-pull down assays were carried out as described for panel A, except that HA-ceTFIIE was used instead of 6H-hTFIIE. After Western blotting, bound HA-ceTFIIE was detected with anti-HA monoclonal antibody (12CA5). An arrow indicates the position of HA-ceTFIIE (ceIIE). (C) Binding of hTFIIE to the TFIIH subunits. The assay was done as described for panel A. Four hundred nanograms of each GST-fused TFIIH subunit, with GST alone (lane 1) as a control, were used to examine binding to hTFIIE. An arrow indicates the position of 6H-hTFIIE (hIIE). (D) Binding of ceTFIIE to the TFIIH subunits. GST-pull down assays were carried out as described for panel C, except that HA-ceTFIIE was used instead of 6H-hTFIIE. An arrow indicates the position of HA-ceTFIIE (ceIIE). Molecular mass markers are shown to the left.

C. elegans TFIIE does not bind efficiently to human TFIIE. In contrast to ceTFIIE, ceTFIIE was unable to replace hTFIIE in a human in vitro transcription system (Fig. 4B). To address this issue, the binding specificities of ceTFIIE and hTFIIE for various human general transcription factors were analyzed (Fig. 6). Of the general transcription factors, hTFIIE bound most strongly to hTFIIE (Fig. 6A, lane 3). However, ceTFIIE did not bind well to hTFIIE (about 10% of the efficiency of hTFIIE) (Fig. 6A and B, compare lanes 3). In addition, ceTFIIE bound more strongly to TFIIA than hTFIIE did (Fig. 6A and B, lanes 8). Binding to TFIIB, TFIIF (RAP30), and TBP was similar for C. elegans and human TFIIE (Fig. 6A and B, lanes 2, 5, and 6), and both also bound predominantly to p62 and weakly to p52 among the nine TFIIH subunits (Fig. 6C and D, lanes 4 and 5). These results indicate that a different binding affinity to hTFIIE might be a main reason for the inability of ceTFIIE to substitute functionally for hTFIIE.

View larger version (20K): [in this window] [in a new window]   FIG. 6.   Binding assays using C. elegans TFIIE. (A) Binding of hTFIIE to the various general transcription factors. All of the human general transcription factors (400 ng each) except for hTFIIE, the TBP activation factors of TFIID, and the TFIIH subunits were fused to GST and expressed in bacteria. GST-pull down assays were carried out as described for Fig. 5A using 200 ng of 6H-hTFIIE. After Western blotting, bound hTFIIE was detected with anti-hTFIIE antibody. Lane 1, GST alone (G); lane 2, GST-TFIIB (B); lane 3, GST-hTFIIE (E); lane 4, GST-TFIIF (F); lane 5, GST-TFIIF (F); lane 6, GST-TBP (T); lane 7, GST-TFIIA (A); lane 8, GST-TFIIA (A); lane 9, GST-TFIIA (A). Arrows indicate the position of 6H-hTFIIE (hIIE). (B) Binding of ceTFIIE to the various general transcription factors. GST-pull down assays were carried out as described for panel A using 200 ng of HA-ceTFIIE. After Western blotting, bound HA-ceTFIIE was detected with anti-HA monoclonal antibody (12CA5). An arrow indicates the position of HA-ceTFIIE (ceIIE). (C) Binding of hTFIIE to the TFIIH subunits. Assays were carried out as described for panel A. Four hundred nanograms of each GST-fused TFIIH subunit, with GST alone (lane 1) as a control, were used to examine binding to hTFIIE. An arrow indicates the position of 6H-hTFIIE (hIIE). (D) Binding of ceTFIIE to the TFIIH subunits. Assays were carried out as described for panel C, except that HA-ceTFIIE was used instead of 6H-hTFIIE. An arrow indicates the position of HA-ceTFIIE (ceIIE). Molecular mass markers are indicated to the left.

The intermediate shift in the Pol II phosphorylation state induced by C. elegans TFIIE may be caused by defective phosphorylation at serine-5 of the CTD heptapeptide sequence. In light of accumulated evidence suggesting a tight connection between CTD phosphorylation and transcription and our observation that ceTFIIE is partially able to replace its human counterpart in transcription, we investigated the effects of four different chimeric forms of TFIIE on CTD phosphorylation during PIC formation (Fig. 7A). Wild-type hTFIIE fully stimulated CTD phosphorylation, causing the largest subunit of Pol II to shift completely from the IIa to the IIo form (lanes 8 and 9). In contrast, chimeric TFIIE containing hTFIIE and ceTFIIE (hTFIIE-ceTFIIE) caused an intermediate shift of Pol II to a point between the IIa and IIo forms (lanes 6 and 7), while neither wild-type ceTFIIE nor TFIIE containing ceTFIIE and hTFIIE (ceTFIIE-hTFIIE) produced any significant Pol II shift upon phosphorylation reaction (lanes 2 to 5).

View larger version (29K): [in this window] [in a new window]   FIG. 7.   Effects of ceTFIIE subunits on CTD phosphorylation by TFIIH during preinitiation complex formation. Kinase assays (25 µl) were carried out as described in Materials and Methods. TFIIE proteins were prepared and purified as described for Fig. 4A and in Materials and Methods. (A) CTD phosphorylation of intact Pol II during PIC formation. Lane 1, kinase reaction without TFIIE (); lanes 2 and 3, ceTFIIE (ceIIEceIIE); lanes 4 and 5, chimeric TFIIE made up of ceTFIIE and hTFIIE (ceIIEhIIE); lanes 6 and 7, chimeric TFIIE made up of hTFIIE and ceTFIIE (hIIEceIIE); lanes 8 and 9, hTFIIE (hIIEhIIE). For lanes 2, 4, 6, and 8, 15 ng of each TFIIE was added. For lanes 3, 5, 7, and 9, 30 ng of each TFIIE was added. Phosphorylated proteins were analyzed on a 5.5% acrylamide-SDS gel and detected by autoradiography. Arrows indicate the positions of the phosphorylated (IIo) and unphosphorylated (IIa) forms of the largest subunit of Pol II. (B) Detection of the largest subunit of Pol II after treatment with kinases. The kinase reaction was carried out essentially as described for panel A, except that nonisotopic ATP was used instead of [-32P]ATP and a monoclonal antibody (8WG16) against the CTD heptapeptide was used to detect the largest subunit of Pol II. (C) Detection of phosphorylated Ser-2 in the CTD of the largest subunit of Pol II. Reactions were carried out as described for panel B, and phospho-Ser-2 in the CTD was detected using the monoclonal antibody H5. (D) Detection of phosphorylated Ser-5 in the CTD of the largest subunit of Pol II. Reactions were carried out as described for panel B, and phospho-Ser-5 in the CTD was detected using the monoclonal antibody H14.

C. elegans TFIIE shows a severe defect in its ability to support transcription at the transition from initiation to elongation on a linear DNA template. Since ceTFIIE showed a partial transcriptional exchangeability with hTFIIE on a supercoiled template and failed to induce Ser-5 phosphorylation well in the CTD of Pol II, we thought this defect of Ser-5 phosphorylation was a potential reason for the partial exchangeability. Thus, we characterized its function in transcription by focusing on the transition step to elongation. As shown in Fig. 8A, the PICs were preformed by incubation of Pol II with general transcription factors (with or without various forms of TFIIE) and either a linear or supercoiled AdML template [pML(C₂AT)100]. Transcription was started by addition of nucleoside triphosphates at a low concentration to limit Pol II processivity so as to allow study of the transition stage. Although transcription occurred at a very low level (0.8% of transcription in the presence of wild-type hTFIIE; Fig. 8B, lane 14 versus lane 18) even in the absence of TFIIE on the supercoiled template, hTFIIE was stringently required for efficient transcription on both linear and supercoiled templates (lanes 9, 10, 17, and 18). Chimeric TFIIE (hTFIIE-ceTFIIE) showed 26% of wild-type hTFIIE transcription activity on the supercoiled template (Fig. 8B, lane 16 versus lane 18), a level consistent with the results shown in Fig. 4B. However, hTFIIE-ceTFIIE showed only 5% of wild-type hTFIIE transcription on the linear template (Fig. 8B, lane 8 versus lane 10). The reason why we did not observe shorter (abortive) transcripts as well as unincorporated [-³²P]CTP so much may be that we treated samples with calf intestine alkaline phosphatase as described previously (21) and ethanol precipitated to reduce the background. The hTFIIE 278-291 mutant showed similarly reduced transcription (about 20% of the wild-type level) on both templates (Fig. 8B, lanes 11 and 12) (Ohkuma, data not shown; 42). This is clearly different from the transcription activity of hTFIIE-ceTFIIE. To examine whether this difference between linear and supercoiled templates reflects the difference at the transition stage to elongation, we carried out transcription initiation assays to see the first phosphodiester bond formation (Fig. 8C). The activity of hTFIIE