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Originally published In Press as doi:10.1074/jbc.M404722200 on September 22, 2004

J. Biol. Chem., Vol. 279, Issue 49, 51395-51403, December 3, 2004
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A Novel Zinc Finger Structure in the Large Subunit of Human General Transcription Factor TFIIE*{boxs}

Masahiko Okuda{ddagger}§, Aki Tanaka¶||, Yoko Arai||**, Manami Satoh{ddagger}{ddagger}{ddagger}, Hideyasu Okamura{ddagger}§, Aritaka Nagadoi{ddagger}, Fumio Hanaoka¶§§, Yoshiaki Ohkuma¶, and Yoshifumi Nishimura{ddagger}¶¶

From the {ddagger}Graduate School of Integrated Science, Yokohama City University, 1-7-29, Suehiro-cho, Tsurumi-ku, Yokohama 230-0045, the §Kihara Memorial Yokohama Foundation for the Advancement of Life Sciences, 1-7-29, Suehiro-cho, Tsurumi-ku, Yokohama 230-0045, the Graduate School of Frontier Biosciences, Osaka University, 1-3 Yamada-oka, Suita, Osaka 565-0871, the ||Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamada-oka, Suita, Osaka 565-0871, and the §§Cellular Physiology Laboratory, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan

Received for publication, April 28, 2004 , and in revised form, September 13, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The zinc finger domain in the large subunit of TFIIE (TFIIE{alpha}) is phylogenetically conserved and is essential for transcription. Here, we determined the solution structure of this domain by using NMR. It consisted of one {alpha}-helix and five {beta}-strands, showing novel features distinct from previously determined zinc-binding structures. We created point mutants of TFIIE{alpha} in this domain and examined their binding abilities to other general transcription factors as well as their transcription activities. Four Zn2+-ligand mutants, in which each of cysteine residues at positions 129, 132, 154, and 157 was replaced by alanine, possessed no transcription activities on a linearized template, whereas, on a supercoiled template, interesting functional asymmetry was observed: although the C-terminal two mutants abolished transcription activity (<5%), the N-terminal two mutants retained about 20% activities. The N-terminal two mutants bound stronger to the small subunit of TFIIF than the wild type and the C-terminal two mutants were impaired in their binding abilities to the XPB subunits of TFIIH. These suggest that the structural integrity of the zinc finger domain is essential for the TFIIE function, particularly in the transition from the transcription initiation to elongation and the conformational tuning of this domain for appropriate positioning of TFIIF, TFIIH, and polymerase II would be needed depending on the situation and timing.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
In eukaryotes, transcription initiation of protein-coding genes requires RNA polymerase II (pol II)1 and its auxiliary five general transcription factors, TFIIB, TFIID, TFIIE, TFIIF, and TFIIH (1, 2). Pol II and general transcription factors assemble together on a promoter DNA to form a preinitiation complex (PIC). TATA box-binding protein, a DNA binding subunit of TFIID, binds first to the TATA box. TFIIB then binds and recruits pol II to the complex (3). Pol II comes with TFIIF, which is important for accurate location of pol II in the complex around the transcription initiation site (4). Finally, TFIIE and TFIIH are incorporated to complete PIC formation (3).

During PIC formation, TFIIE interacts with various general transcription factors, pol II, and promoter DNA, recruits TFIIH into the PIC, and regulates the enzymatic activities of TFIIH; serine kinase of the C-terminal domain of the largest subunit of pol II, DNA-dependent ATPase, and DNA helicase activities (5–7). At transcription initiation, TFIIE binds both to pol II near its active site and to promoter DNA ~10 bp upstream (-10) from the transcription initiation site (+1), where the promoter melting starts (8). There TFIIE conducts pol II to sit at the initiation site, makes pol II processive upon C-terminal domain phosphorylation by stimulating C-terminal domain kinase activity of TFIIH, and simultaneously assists a helicase subunit XPB of TFIIH to start melting at the -10 position. At the transition from initiation to elongation, TFIIE also plays a direct role in promoter clearance by regulating kinase and DNA helicase activities of TFIIH (6, 9).

Human TFIIE (hTFIIE) is a heterotetramer consisting of two {alpha} (hTFIIE{alpha}; 57 kDa, 439 aa) and two {beta} (hTFIIE{beta}; 34 kDa, 291 amino acids) subunits (10–13). Both subunits possess several structural motifs and characteristic sequences. Although hTFIIE has been functionally characterized, little is known about its structure, especially on hTFIIE{alpha} (14–16). Main reason is its low solubility. Although high concentration of protein sample should be prepared for the structural studies, each hTFIIE subunit aggregates and precipitates at the concentration higher than 10 mg/ml. Thus we have been focusing on dissecting functional domains in each subunit. To identify core regions in hTFIIE{alpha}, limited proteolyses were performed and a highly conserved region (residues 113–174) containing a zinc finger motif, which is essential for transcription activity of TFIIE (14, 17) was isolated (Fig. 1). In this study, we have determined the solution structure of this core domain (hTFIIE{alpha}c) of hTFIIE{alpha} by NMR and provided insight into its role for the TFIIE function.



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  		FIG. 1.
Sequence alignment of the TFIIE{alpha}c. Conserved amino acids are shown in bold.Zn2+-coordinating cysteines are boxed in red. Residues that contribute to the formation of the hydrophobic core are boxed in yellow. Mutated residues in the mutation analyses are boxed and numbered. The secondary structure of the hTFIIE{alpha}c is shown above the aligned sequence. Arrows and a cylinder indicate {beta}-strands and an {alpha}-helix, respectively. Xenop, Xenopus; Droso, Drosophila; C.ele, Caenorhabditis elegans; S.pom, Schizosaccharomyces pombe; S.cer, Saccharomyces cerevisiae; A.per, Aeropyrum pernix; M.jan, Methanococcus jannaschii; M.the, Methanobacterium thermoautotrophicum; S.sol, Sulfolobus solfataricus; (GenBankTM accession numbers: human, CAA45068 [GenBank] mouse, BAB27686 [GenBank] Xenopus, CAA78505 [GenBank] Drosophila, AAD00187 [GenBank] C. elegans, CAA70050 [GenBank] S. pombe, CAB66310 [GenBank] S. cerevisiae, AAA62665 [GenBank] A. pernix, BAA81014 [GenBank] M. jannaschii, AAB98767 [GenBank] M. thermoautotrophicum, AAB86141 [GenBank] and S. solfataricus, AAK40605 [GenBank] .

 

   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Construction of hTFIIE{alpha}c Expression Plasmid—For subcloning the nucleotide sequence of the hTFIIE{alpha}c corresponding to the amino acid residues 113–174 into a bacterial expression vector, the oligonucleotide ES1T (5'-GTGTACCATATGAGAATTGAGACCGATGAGAGAG-3') was designed to create an NdeI site (underlined) and to add the methionine codon before the R113 residue and the nucleotide ES1B (5'-GAACGGGATCCTCAGCGTGCATCTTTTTTGGGCATTGC-3') was designed to create a BamHI site (underlined) right after the stop codon (bold letters). This nucleotide fragment was amplified by PCR using two oligonucleotides and the p2EA plasmid containing the complete open reading frame of human TFIIE{alpha} (hTFIIE{alpha} cDNA) as a template (11), digested with NdeI and BamHI, and subcloned into the NdeI and BamHI restriction sites of the pBluescript SK(-). The nucleotide sequences of the cloned PCR products were confirmed using an ABI PRISM 310 Genetic Analyzer (Applied Biosystems). The NdeI-BamHI fragment of hTFIIE{alpha}c cDNA was then subcloned into the pET11d vector (Novagen) to construct the N-terminal six histidine (His6)-tagged hTFIIE{alpha}c (6His-hTFIIE{alpha}c) expression plasmid.

Purification of hTFIIE{alpha}c—The hTFIIE{alpha}c plasmid was transformed into Escherichia coli BL21(DE3)pLysS (Novagen). The cells were grown at 37 °C in LB or in M9 minimal media containing [15N]ammonium chloride with or without [13C]glucose. 6His-hTFIIE{alpha}c was then induced by addition of 1 mM isopropyl-{beta}-D-thiogalactopyranoside. After 4–7 h growth the cells were harvested. The cell pellet was resuspended in buffer A (20 mM Tris-HCl (pH 7.0), 10% (v/v) glycerol, 30 µM ZnCl2, 1 mM phenylmethylsulfonyl fluoride, 1 mM benzamidine) containing 100 mM NaCl (BA100). The cells were lysed by sonication and centrifuged, and the supernatant was loaded onto the nickel-nitrilotriacetic acid (NTA)-agarose (Qiagen) column, equilibrated with BA100 containing 20 mM imidazole-HCl. The sample was eluted by linear gradient from 20 to 350 mM imidazole-HCl. The peak fractions were pooled, and the buffer was changed to 50 mM Tris-HCl (pH 8.0), 30 µM ZnCl2, 2.5 mM CaCl2 containing 150 mM NaCl. The sample was digested with thrombin for 16 h at 25 °C to remove the His6 tag, then, loaded onto the Ni-NTA-agarose column equilibrated with buffer B (20 mM potassium phosphate (pH 7.0), 30 µM ZnCl2) containing 200 mM NaCl. hTFIIE{alpha}c was passed over the column, concentrated using Centriprep (Amicon) and applied onto Superdex30 (Amersham Biosciences) equilibrated with the buffer (20 mM potassium phosphate (pH 7.0), 30 µM ZnCl2, 5 mM 1,4-dithiothreitol) containing 50 mM NaCl. The final fractions were used for analyses.

NMR Spectroscopy—Protein concentration for NMR experiments is about 3–4 mM in the buffer (20 mM potassium phosphate (pH 6.0), 30 µM ZnCl2, 5 mM deuterated 1,4-dithiothreitol, 50 mM NaCl) dissolved in either 90% H2O/10% D2O or 99.9% D2O. All NMR experiments were carried out at 32 °C on either Bruker DRX-500, DRX-600, AVANCE-500, or AVANCE-600 spectrometer. Backbone and side-chain resonances were assigned using the following experiments: CBCA(CO)NH, CBCANH, HN(CA)CO, HNCO, DQF-COSY, TOCSY, HBHA(CO)NH, 15N-edited TOCSY-HSQC, HCCH-COSY, HCCH-TOCSY (18), (HB)CB-(CGCD)HD, (HB)CB(CGCDCE)HE (19), CG(CB)H, CG(CD)H, and CG(CDCE)H (20). Stereospecific assignments were obtained from a combination of HNHB, HN(CO)HB, HNCG, and HN(CO)CG (21) and DQF-COSY and 15N-edited NOESY-HSQC (18). Distance information was obtained from NOESY, 15N-edited NOESY-HSQC, 13C-edited NOESY-HSQC, three-dimensional 15N,15N-edited HSQC-NOESY-HSQC, four-dimensional 15N,15N-edited HSQC-NOESY-HSQC, four-dimensional 13C,15N-edited HSQC-NOESY-HSQC and four-dimensional 13C,13C-edited HSQC-NOESY-HSQC (18). Backbone torsion angles {varphi} were obtained from HNHA and HNCA-J (18). Side-chain torsion angles {chi}1 and {chi}2 were obtained from a combination of HNHB, HN(CO)HB, HNCG, and HN(CO)CG (21) and 15N-edited NOESY-HSQC and 13C-edited NOESY-HSQC (18). Hydrogen bond restraints were obtained by backbone amide H/D-exchange experiment and CPD-H(N)CO (22) to detect trans-hydrogen bond 3hJNC' couplings. Spectra were processed using NMRPipe (23) and analyzed using the programs PIPP, CAPP, and STAPP (24) and NMRView (25). NMR relaxation rates for amide nitrogens of hTFIIE{alpha}c were measured according to the procedure described previously (26). The 15N T1 data were collected using 15N delays of 24, 64, 128, 256, 384, 512, 1024, 1536, and 2048 ms. The 15N T2 data were collected using 15N delays of 16.2, 32.3, 48.5, 64.6, 129.3, 193.9, 258.6, 387.8, and 517.1 ms. The steady-state {1H}-15N NOEs data were measured by taking the ratio of the peak intensities from experiments performed with and without application of 1H saturation.

Structure Calculation—Interproton distance restraints derived from NOE intensities were grouped into three distance ranges, 1.8–2.7 Å (1.8–2.9 Å for NOEs involving NH protons), 1.8–3.3 Å (1.8–3.5 Å for NOEs involving NH protons), and 1.8–5.0 Å, corresponding to strong, medium, and weak NOEs, respectively. The upper limit was corrected for constraints involving methyl groups, aromatic ring protons, and nonstereospecifically assigned methylene protons. Dihedral angle restraints for {varphi} were set to -90° < {varphi} < -40° for 3JHN{alpha} < 6.0 Hz and -160° < {varphi} < -80° for 3JHN{alpha} > 8.0 Hz, based on the short range NOEs and backbone chemical shifts. {chi}1 angles were restrained ±30° for three side-chain rotamers. The zinc was constrained to be tetrahedrally coordinated by four cysteines as follows. Zn–S{gamma} bond length sets to 2.3 Å, Zn-S{gamma}-C{beta} and S{gamma}-Zn-S{gamma} angles set to 108° and 109°. Hydrogen bond restraints in areas of regular secondary structure were introduced at the final stages of refinement. Structure calculations were performed by the distance geometry and simulated annealing using program X-PLOR (27). A total of 100 structures were calculated, and 69 structures had no NOE violations larger than 0.2 Å and no dihedral angle violations greater than 1°. Structural statistics for the 20 best structures are summarized in Table I. Structures were analyzed and displayed using PROCHECK-NMR (28), GRASP (29), MOLMOL (30), and SYBYL (Tripos Inc., St. Louis, MO).


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  		TABLE I
Structural statistics for the 20 best structures

 
Construction of hTFIIE{alpha} Point Mutants—By using the site-directed mutagenesis kit Mutan-K (TaKaRa) with the wild type hTFIIE{alpha} cDNA plasmid as a template, various oligonucleotide-mediated point mutants were created (31). A restriction enzyme site was placed in an oligonucleotide to select for properly mutated plasmids as described elsewhere, and the mutants were then checked by sequencing using an ABI Prism 310 Genetic Analyzer (Applied Biosystems). The oligonucleotides used for mutation were listed in Table II. Created enzyme sites were underlined, and the enzyme names were written in parentheses. Mutated sequences are written in bold, and each mutated codon is boxed. For three oligonucleotides creating C157A, D164A, and D164K, restriction enzyme sites were not created and mutated sequences were directly checked by sequencing. The NdeI-BamHI fragment of mutated hTFIIE{alpha} cDNA was then subcloned into the pET11d vector (Novagen) to construct the N-terminal His6-tagged hTFIIE{alpha} (6 His-hTFIIE{alpha}) expression plasmid.


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  		TABLE II
Oligonucleotide sequences used for point mutations

 
Purification of Recombinant Proteins—Recombinant point mutant hTFIIE{alpha} proteins were expressed in E. coli Rosetta(DE3)pLysS (Novagen) by induction with isopropyl-{beta}-D-thiogalactopyranoside. For general purification, soluble bacterial lysates containing with buffer B (20 mM Tris-HCl (pH 7.9 at 4 °C), 0.5 mM EDTA, 10% (v/v) glycerol, 1 mM phenylmethylsulfonyl fluoride, 2 µg/ml antipain, 2 µg/ml aprotinin, 1 µg/ml leupeptin, 0.8 µg/ml pepstatin, 10 mM 2-mercaptoethanol, 500 mM NaCl) were used. For miniscale preparations, lysates (1 ml) representing 25 ml of culture were mixed 100 µl of Ni-NTA-agarose resin (Qiagen), and incubated for 4 h at 4 °C. The resin samples were washed three times with 1 ml of buffer D500 (20 mM Tris-HCl (pH 7.9 at 4 °C), 20% (v/v) glycerol, 1 mM phenylmethylsulfonyl fluoride, 10 mM 2-mercaptoethanol, 500 mM KCl) containing 10 mM imidazole-HCl (pH 7.9), and washed twice with 1 ml of BD500 containing 40 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 judging by Coomassie Blue staining of an SDS-polyacrylamide gel.

In Vitro Transcription Assays—Recombinant general transcription factors as well as native pol II and TFIIH were purified as described previously (31). In vitro transcription with either a supercoiled or a linearized template was carried out with increasing amounts (12 and 24 ng) of wild type or mutant hTFIIE{alpha} proteins. The plasmid pML(C2AT){Delta}-50, which contains the adenovirus 2 major late promoter and gives a 390-nucleotide transcript, was used as a template (31). To prepare the linearized template, pML(C2AT){Delta}-50 was digested with SmaI. After transcription, radiolabeled transcripts were subjected to urea-PAGE and detected by autoradiography. The transcripts were quantified by a Fuji BAS2500 Bio-Imaging analyzer. Relative transcription activities of the mutant hTFIIE{alpha} proteins were calculated by defining the transcription activity of 24 ng of wild type hTFIIE{alpha} as 100%.

GST-Pull-down Assay—GST fusion proteins were performed as previously described (31). The bound proteins were detected by Western blotting with anti-hTFIIE{alpha} mouse monoclonal antibody (1:8,000 dilution) (Medical Biology Laboratory). Signals were detected using the enhanced chemiluminescence (ECL) detection system (Amersham Biosciences) using RX-U film (Fuji Film).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Identification of the Core Domain of hTFIIE{alpha}—TFIIE{alpha} is highly conserved among eukaryotes. In addition, TFE from archaea has recently been found to be a homolog of the N-terminal half of hTFIIE{alpha} (32–34). To identify the structural domain, the recombinant hTFIIE{alpha} was digested by limited proteolysis. Three structural domains consisting of the amino acid residues 113–174, 276–323, and 283–323 and two structural domains consisting of the amino acid residues 106–188 and 302–342 were identified by trypsin and chymotrypsin digestions, respectively, followed by mass spectrometries. Finally, a central core domain (hTFIIE{alpha}c) was identified to consist of the amino acid residues 113–174 containing a putative zinc finger motif (Fig. 1) (11). 6His-hTFIIE{alpha}c was expressed in E. coli BL21(DE3)pLysS, purified, and subjected to conventional multidimensional NMR measurements.

Overview of the Core Domain Structure—Almost all signals for each amino acid of hTFIIE{alpha}c could be assigned except for the {beta} protons of the N-terminal Arg-113. The hTFIIE{alpha}c has the compact structure consisting of one short {alpha}-helix and five {beta}-strands (Fig. 2, A and B); S1 strand (residues 126–129), t1 turn (residues 130–133), S2 strand (residues 134–136), {alpha}-helix (residues 138–144), S3 strand (residues 145–146), loop (residues 147–150), S4 strand (residues 151–154), t2 turn (residues 155–158), and S5 strand (residues 159–164). The three {beta}-strands (S1, S2, and C-terminal half of S5) and the other three {beta}-strands (S3, S4, and N-terminal half of S5) form antiparallel {beta}-sheets. Fig. 2C shows that two {beta}-sheets separated by the {alpha}-helix are symmetrical, including the positions of cysteine residues in the topology diagram. Zn2+ coordinates to Cys-129 and Cys-132 in the t1 turn and Cys-154 and Cys-157 in the t2 turn. The H-D exchange rates of amide protons of Val-131, Cys-132, Phe-156, and Cys-157 were found to be relatively slow. Judging from the structure, hydrogen bonds could be formed in Cys-129:S{gamma}-Val-131:HN, -Cys-132:HN and Cys-154: S{gamma}-Phe-156:HN, -Cys-157:HN, respectively (Fig. 2D). These bifurcating hydrogen bonds are consistent with the hydrogen bond pattern between S{gamma} of i residue and HN of i+2, i+3 residues observed in many zing finger domains (35–38). In the H-D exchange experiment almost all of amide protons were exchanged with deuterium within 40 min, so that little hydrogen bond information was obtained. We could directly observe more hydrogen bonds in the {beta}-sheets by measuring transhydrogen bond 3hJNC' couplings (39) (Supplemental Fig. S4).



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  		FIG. 2.
The hTFIIE{alpha}c structure. A, the superposition of a family of the final 20 NMR structures. Structures are superposed by minimizing the r.m.s.d. of backbone atoms of residues 126–164. B, ribbon representation of the mean structure. Five {beta}-strands, one {alpha}-helix, and two turns are indicated by S1–S5, H1, t1, and t2, respectively. Zn2+ are shown as pink spheres. C, topology diagram. First and second {beta}-sheets are colored yellow and green, an {alpha}-helix is colored light blue. Four Cs on the turns indicate Zn2+-conjugating cysteines. D, zinc-binding site. A pink sphere indicates Zn2+. Residues involved in Zn2+ coordination (indicated by dotted red lines) and bifurcated hydrogen bonds (indicated by dotted blue lines) are shown. E, hydrophobic core. Residues involved in the hydrophobic core and the zinc-binding site are shown with the side chains. Zn2+-coordinating cysteines are shown in green. Five phenylalanines are shown in red. The others are shown in blue. F, electrostatic potential on the molecular surface. Positive potential is colored blue, and negative potential is colored red. Conserved negative potential cluster are indicated by arrows.

 
The coordination of Zn2+ to four cysteine residues and the auxiliary two bifurcated hydrogen bonds fix the t1 and t2 turns rigidly, forming two anti-parallel {beta}-sheets approach around one side of the {alpha}-helix. Fig. 2E shows that a hydrophobic core is formed by hydrophobic amino acids: Ala-141, Leu-144, and Val-161 as well as five phenylalanine residues that are piled up each other and arranged like a capital character, L. This aromatic-aromatic interaction network seems to contribute significantly to the stability of the structure. These residues are well conserved among species (Fig. 1). Electrostatic potential on the molecular surface shows that the hTFIIE{alpha}c has a negative potential cluster, which is formed by conserved Asp-138, Glu-160, Glu-162, Glu-163, Asp-164, and Glu-165, and is extensively located on a concave surface opposite to the zinc-binding site (Fig. 2F).

N-terminal and C-terminal regions were disordered due to poor distance restraints. Nevertheless only about 40 amino acids can architect the rigid structure. Actually, 15N-T1,T2, and 15N–{1H}heteronuclear NOE values revealed flexibility of backbone in the N-terminal 13 and C-terminal 11 residues (Fig. 3).



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  		FIG. 3.
NMR relaxation analysis of the hTFIIE{alpha}c. 15N-T1, T2, and heteronuclear 1H-{15N} NOE values are plotted for each residue in top, middle, and bottom panels, respectively. All spectra were recorded on a Bruker AVANCE 600 spectrometer at 32 °C under the same solution conditions as for the structure determination. The absence of a bar is due to the overlapping of amide peaks. Asterisk indicates a position of Pro residue.

 
Novel Structural Features of the Zinc Finger Motif—Once hTFIIE{alpha}c was predicted to form a zinc-ribbon structure as observed in the C-terminal domain of transcription elongation factor TFIIS (TFIISc) (35), the N-terminal domain of TFIIB (TFIIBn) (36), and the C-terminal domain of RPB9 subunit of pol II (RPB9c) (37). However, hTFIIE{alpha}c holds an entirely different structure from so-called zinc-ribbon structures (Fig. 4A) (35–38, 40–42). The topology of TFIISc, TFIIBn, and RPB9c are {beta}{beta}{beta}, {beta}{beta}{beta}, and {beta}{beta}{beta}{beta} ({beta}:{beta}-strand), respectively, which form anti-parallel {beta}-sheets, whereas the topology of hTFIIE{alpha}c is more complicated as {beta}{beta}{alpha}{beta}{beta}{beta} ({alpha}:{alpha}-helix), forming two antiparallel {beta}-sheets separated by one {alpha}-helix. However, architectures of the zinc-binding sites of these proteins are very similar in each other (Fig. 4B). Comparing the backbone structures of two turns (CXXCXX and CXXCXX, 12 amino acid residues), root mean square deviations of hTFIIE{alpha}c to TFIISc, TFIIBn, and RPB9c are 0.78, 0.97, and 0.74 Å, respectively. Although we searched similar structures of hTFIIE{alpha}c by using the DALI server (43), nothing has been identified. So, we considered that hTFIIE{alpha}c holds an entirely novel type of zinc finger structures.



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  		FIG.4.
Structural comparison of the hTFIIE{alpha}c with the zinc-ribbon domains. A, ribbon structures of the zinc-coordinating domains. hTFIIE{alpha}c; the core domain of the human TFIIE{alpha}, hTFIISc; the C-terminal domain of the transcription elongation factor TFIIS from human, aTFIIBn; the N-terminal domain of the general transcription factor TFIIB from archaea, yRPB9c; the C-terminal domain of the RPB9 subunit of pol II from S. cerevisiae.Zn2+ are indicated as pink spheres. B, superposition of the zinc-binding sites. Two turns are shown by superposing with the minimum root mean square deviation. Red, hTFIIE{alpha}c; cyan, hTFIISc; green, aTFIIBn; and orange, yRPB9c.

 
Functional Roles of hTFIIE{alpha}c in Transcription—For functional analyses of hTFIIE{alpha}, we substituted each of the four Zn2+-binding cysteine residues at 129, 132, 154, and 157 with alanine, C129A, C132A, C154A, and C157A, as well as two highly conserved acidic residues, Glu-140 and Asp-164 on the negatively charged surface, with alanine or lysine, E140A, E140K, D164A, and D164K. All these mutants of hTFIIE{alpha} with His6 at the N terminus were expressed in E. coli, purified through Ni-NTA-agarose column, and subjected to SDS-PAGE (Fig. 5A). The effects of these point mutants on basal transcription by using a supercoiled adenovirus major late pML{Delta}-50(C2AT) template were analyzed (Fig. 5, B and C). Although C154A and C157A abolished transcription activity (<5%), C129A and C132A retained 12 and 25% activities, respectively (Fig. 5B). On the other hand, the acidic residue mutants were active in transcription; E140A possessed almost the same activity as the wild type, E140K was also active but with the reduced activity of 50%, and D164A and D164K mutants possessed higher activities at ~170–180% than the wild type (Fig. 5C).



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  		FIG. 5.
Effects of the point mutants of the hTFIIE{alpha} zinc finger region on transcription. A, SDS-PAGE of the purified point mutants of the hTFIIE{alpha} zinc finger region. Mutated residues are indicated on the top of the panel. The molecular masses and positions of the standard markers were indicated on the left (in kilodaltons). B and C, effect of zinc finger point mutations on the basal transcription activity of hTFIIE{alpha} with a supercoiled template. In vitro transcription assays with a supercoiled template were carried out with increasing amounts (6, 12, and 18 ng) of wild type hTFIIE{alpha} (IIE{alpha} wt) or hTFIIE{alpha} proteins with point mutations in the zinc finger region. After the transcription reaction, radio-labeled transcripts were subjected to urea-PAGE and detected by autoradiography (lower panels). Relative transcription activities of the mutant hTFIIE{alpha} proteins (%) were calculated by defining the transcription activity of 18 ng of wild type hTFIIE{alpha} as 100% and are shown as bars (upper panels). Mutated residues are indicated on the bottom. As a control, transcription was carried out without hTFIIE{alpha} protein (-{alpha}). Arrows on the right side indicate the position of the 390-nulceotide transcripts. D and E, effects of zinc finger point mutations on the basal transcription activity of hTFIIE{alpha} with a linearized template. In vitro transcription assays with a linearized template were carried out with increasing amounts (12 and 18 ng) of wild type hTFIIE{alpha} (IIE{alpha} wt) or hTFIIE{alpha} proteins with point mutations in the zinc finger region. Mutated residues are indicated on the bottom. As a control, transcription was carried out without hTFIIE{alpha} protein (-{alpha}). Arrows on the right side indicate the position of the 390-nulceotide transcripts.

 
To dissect the effects of these mutants on the transcription step more precisely and to see the transition activity from transcription initiation to elongation, a linearized template was used (44). Most of the transcription activities of the mutants on a linearized template were similar to the activities on a supercoiled template except that the activities of C129A and C132A were completely abolished (Fig. 5D), and the mutant D164A showed further augmented activity of 370% (Fig. 5E). These suggest that hTFIIE{alpha}c can be functionally dissected into two; the C-terminal half involves in transcription initiation alone, but the N-terminal half involves not only initiation but also the transition from initiation to elongation. Although Asp-164 is conserved in eukaryotes as well as even in archaea (Fig. 1), substitutions to alanine and lysine residues, D164A and D164K, rather stimulated transcription both on supercoiled and linear templates (Fig. 5, C and E).

Effects of Mutations on Binding to the General Transcription Factors—We next investigated the binding abilities of these mutants to the general transcription factors by GST-pull-down assays (Fig. 6, A and B). All mutants as well as the wild type gave the similar binding patterns except that C129A and C132A bound to TFIIF{beta} about 3-fold stronger than the wild type (Fig. 6A, lane 6) and that C154A completely lost and C157A reduced their binding abilities to the XPB subunit of TFIIH (Fig. 6B, lane 3). The bindings of the mutants to intact pol II were also tested but all, including the wild type failed to bind (data not shown). The stronger binding to TFIIF{beta} may contribute to the transcription initiation activities of C129A and C132A on a supercoiled template retained ~12–25%. TFIIF is important for transcription initiation and thus will be able to substitute for a part of TFIIE{alpha} function. The reduced binding of C154A and C157A to XPB of TFIIH may cause abolished transcription on a supercoiled template; C154A and C157A could not recruit XPB at the region on the promoter where promoter melting starts, and thus these mutants are defective in transcription initiation, because XPB functions for promoter opening utilizing its DNA helicase activity.



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  		FIG. 6.
Effects of point mutations at the hTFIIE{alpha} zinc finger region on binding to the subunits of general transcription factors. A, GST-pull-down assays were performed with GST-tagged subunits of all the general transcription factors except for TFIIH and the TAF subunits of TFIID. Bound mutants were detected by Western blotting with anti-hTFIIE{alpha} antisera. Each mutant name is indicated on the left side of each panel. An arrow on the right side of each panel indicates each hTFIIE{alpha} protein. The N-terminal zinc finger mutants C129A and C132A showed augmented binding to TFIIF{beta} (lane 6), and, therefore, the subunit name is boxed for TFIIF{beta}. B, GST-pull down assays were performed with GST-tagged TFIIH subunits. The assay was performed as described in A. The C-terminal zinc finger mutants C154A and C157A showed severely decreased binding to XPB (lane 3), and, therefore, the subunit name is boxed for XPB.

 
The Asp-164 mutants of hTFIIE{alpha}c, D164A and D164K, did not show so much difference from the wild type in binding to general transcription factors except that both bound more strongly to the XPB and XPD subunits of TFIIH. In addition, D164A bound to the p44 and Cdk7 subunits of TFIIH (Fig. 6, A and B). It is intriguing to consider that three TFIIH subunits (XPD, p44, and Cdk7) have been reported to be involved in the transition stage from initiation to elongation. This fits to the present experiment that D164A was more active in transcription, especially, on a linearized template (3.7-fold of the wild type activity) (Fig. 5E) and indicates that TFIIH may bind more rigidly to D164A than the wild type hTFIIE{alpha} and contributes to the transition stage to elongation.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
To elucidate the functions of hTFIIE{alpha}c, we investigated the effects of several point mutants on transcription and on the binding abilities to the general transcription factors (Figs. 5 and 6). For transcription activity assay we used the supercoiled and linearized templates of adenovirus major late promoter. Requirement of TFIIE and TFIIH for the promoter melting depends on promoter DNA (45–48). On the supercoiled template, TFIIE can melt the promoter independently of TFIIH. However, on the linearized template both TFIIE and TFIIH are necessary for the transition activity from the initiation to elongation. In mutants of Zn2+ ligand cysteine residues at 129, 132, 154, and 157, the interesting finding was the functional asymmetry in hTFIIE{alpha}c. The N-terminal two mutants C129A and C132A retained 12 and 25% activities in transcription with the supercoiled template, whereas the C-terminal two mutants C154A and C157A almost completely abolished the transcription activities (Fig. 5B). On the other hand, all of the cysteine mutants showed no transcription activities with the linearized template (Fig. 5D). It is suggested that a defect in transcription initiation should cause no transcription activity with a supercoiled template, whereas a defect in the transition from initiation to elongation could cause some transcription activities with a supercoiled template, but not at all with a linearized template. So C154A and C157A would be deficient in transcription initiation, whereas C129A and C132A would be somewhat deficient in initiation but significantly deficient in the transition stage. The N-terminal zinc finger mutants C129A and C132A showed stronger binding to TFIIF{beta} and the C-terminal mutants C154A and C157A, on the other hand, showed diminished binding to XPB (Fig. 6A, lane 6, and Fig. 6B, lane 3). It is noteworthy that the TFIIE deletion mutants of the zinc finger domain still possessed the stimulation activity of TFIIH-mediated C-terminal domain phosphorylation of pol II in the presence of template DNA and other general transcription factors but not in the absence (14). Thus, the structural integrity of hTFIIE{alpha}c might be essential for the TFIIE function, the conformational tuning of hTFIIE{alpha}c for appropriate positioning of TFIIF, TFIIH, and pol II would be needed depending on the situation and timing.

The functional meaning of Asp-164 is still under consideration, because it is still difficult to explain the reasons why the D164A and D164K mutants showed stronger transcription than the wild type. Because Asp-164 is well conserved among species, this acidic character may be essential for association with some ubiquitous transcription factors, for example, TRAP/Mediator, cofactors, and transcription elongation factors (49). One candidate is p100, a coactivator interacting with EBNA2 and STAT6, because this protein was reported to bind to both subunits of TFIIE (50, 51). p100 possesses the Tudor domain, which is supposed to bind to the methylated peptides (52). Recently, transcriptionally active pol II with Ser-5 phosphorylation of the heptapeptide repeat of the largest subunit was found to recruit histone H3 Lys-4-specific methyltransferase Set1 (reviewed in Ref. 53). Thus, it is intriguing to speculate that p100 and TFIIE work for the bridges to connect between methylated histones and pol II. Further studies are necessary to confirm those possibilities for the functional roles of hTFIIE{alpha}c.


   FOOTNOTES
 
The atomic coordinates and structure factors (code 1VD4 [PDB] ) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

* This work was supported by a Collaborative of Regional Entities for the Advancement of Technological Excellence (CREATE) from Japan Science and Technology agency, by a Project of Protein 3000, Transcription and Translation, and by Grants in Aid for Scientific Research from Ministry of Education, Culture, Sports, Science and Technology. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{boxs} The on-line version of this article (available at http://www.jbc.org) contains Table S1: Coupling constants for 3JHNH{alpha}, Fig. S1: Sequential and medium range NOEs, Fig. S2: Chemical shift indices, Fig. S3: Beta sheet diagram with long range (d{alpha}{alpha}, d{alpha}N and dNN) NOEs, Fig. S4: 2D CPD-H(N)CO spectra for the detection of trans-hydrogen bond 3hJNC' couplings, and Fig. S5: Ramachandran diagram. Back

** Present address: Graduate School of Medicine, Tohoku University, 2-1 Seiryo-machi, Aoba-ku, Sendai 980-8575, Japan. Back

{ddagger}{ddagger} Present address: RIKEN, Yokohama Institute, 1-7-22, Suehiro-cho, Tsurumi-ku, Yokohama, 230-0045, Japan. Back

¶¶ To whom correspondence should be addressed. Tel.: 81-45-508-7216; Fax: 81-45-508-7362; E-mail: nisimura{at}tsurumi.yokohama-cu.ac.jp.

1 The abbreviations used are: pol II, RNA polymerase II; PIC, preinitiation complex; hTFIIE{alpha}c, core domain of human TFIIE{alpha}; HSQC, heteronuclear single quantum correlation; NOE, nuclear Overhauser effect; GST, glutathione S-transferase. Back



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