GTC
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE ADVANCED SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Genes to Cells (2009) 14, 395-405. doi:10.1111/j.1365-2443.2008.01278.x
© 2009 Blackwell Publishing or its licensors
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Tanaka, A.
Right arrow Articles by Ohkuma, Y.
Right arrow Search for Related Content
PubMed
Right arrow Articles by Tanaka, A.
Right arrow Articles by Ohkuma, Y.

Central forkhead domain of human TFIIEβ plays a primary role in binding double-stranded DNA at transcription initiation

 Aki Tanaka1,2, Tomomichi Watanabe2,a, Yukari Iida1, Fumio Hanaoka2,3,4 and Yoshiaki Ohkuma1,2,3,4,*

1 Laboratory of Gene Regulation, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama 930-0194, Japan
2 Graduate School of Frontier Biosciences, and
3 Graduate School of Pharmaceutical Sciences, Osaka University, Suita, Osaka 565-0871, Japan
4 Solution Oriented Research for Science and Technology, Japan Science and Technology Agency, Saitama 332-0012, Japan


   Abstract
Top
 Abstract
Introduction
 Results
 Discussion
 Experimental procedures
 References
 
The human general transcription factor, TFIIE, consists of two subunits, {alpha} and β. Structural analyses indicated the presence of a forkhead motif within the central region of TFIIEβ. This motif was essential for transcription and possessed a double-stranded DNA-binding activity. Protein-DNA photo-cross-linking studies indicated that TFIIEβ binds within the promoter region, adjacent to the transcription initiation site where promoter melting begins at transcription initiation. Furthermore, neither TFIIE nor the other general transcription factor TFIIH, were required for basal transcription of adenovirus major late promoter artificially pre-melted at the initiation site. These data suggest a model in which TFIIE binds to a position adjacent to the initiation site via the forkhead domain, enabling TFIIH to begin opening the promoter. Here, we used systematic point mutations to further investigate the functional roles of this domain. The mutant proteins were expressed in bacteria, purified and used to examine transcription of two different forms of template, phosphorylation of the C-terminal domain of RNA polymerase II, as well as dsDNA-binding. Taken together, our results strongly demonstrated that the primary function of the forkhead region is dsDNA-binding in transcription. In addition, we identified three positively charged lysine residues which play a key role in this function.


   Introduction
Top
 Abstract
 Introduction
Results
 Discussion
 Experimental procedures
 References
 
In eukaryotes, protein-coding genes are transcribed by RNA polymerase II (Pol II) and five auxiliary proteins called general transcription factors (TFIIB, TFIID, TFIIE, TFIIF and TFIIH) (reviewed in Roeder 1996; Orphanides et al. 1996). Before initiation of transcription, Pol II and the general transcription factors form a pre-initiation complex on the promoter. TFIIE is essential for two sequential steps, transcription initiation and the transition from initiation to elongation (reviewed in Ohkuma 1997; Hirose & Ohkuma 2007). It comprises of two subunits ({alpha} and β), both of which contain several structural motifs that are essential for either DNA binding or protein–protein interactions. Previously, we used NMR to identify a forkhead (or winged-helix) domain within the central core region of human TFIIEβ (hTFIIEβ; residues 66–146) and showed that this motif possessed a double-stranded DNA (dsDNA)-binding activity (Okuda et al. 2000). Using various deletion mutants, we found that this motif was located within a region essential for in vitro reconstituted transcription (Okamoto et al. 1998). When the promoter was artificially pre-melted around the transcription initiation site, hTFIIE and human TFIIH (hTFIIH) were no longer required for in vitro transcription (Holstege et al. 1995, 1996), suggesting that these two factors function in promoter melting. Before the binding of dsDNA and following promoter melting, hTFIIE recruits hTFIIH into the pre-initiation complex using the C-terminal acidic domain (AC-D) of hTFIIE{alpha}, which binds to the N-terminal Pleckstrin homology domain (PH-D) of the hTFIIH p62 subunit (Di Lello et al. 2008; Okuda et al. 2008). Recently, we used NMR to determine the structure of bound AC-D and PH-D (Okuda et al. 2008). Protein-DNA photo-cross-linking studies indicated that hTFIIEβ binds to the promoter region around –10, where hTFIIH-mediated melting starts transcription initiation (Douziech et al. 2000). In the light of these findings, we have suggested that this forkhead motif is essential for promoter melting during formation of the open pre-initiation complex immediately before transcription initiation.

The forkhead/winged-helix motif was first identified in Drosophila Forkhead (Fkh) and rat HNF-3{alpha} (FOXA1) proteins (Weigel & Jäckle 1990). These domains comprise approximately 100 amino acids and many of them possess DNA-binding activity. Proteins containing this motif belong to the large Fox (forkhead box) family and those in subfamily "O" (FOXO), are transcription factors that play roles in processes such as the cell cycle, apoptosis, DNA repair, glucose metabolism and longevity (Daitoku & Fukamizu 2007). The general transcription factors TFIIE and TFIIF are heterodimers of {alpha} and β subunits, both of which possess a single forkhead motif (Groft et al. 1998; Okuda et al. 2000; Kamada et al. 2001; Meinhart et al. 2003). It is noteworthy that in each factor, only the forkhead motifs of the smaller β subunits (TFIIEβ and TFIIFβ) exhibit DNA-binding activity and this activity is not observed in that motifs of the larger {alpha} subunits (TFIIE{alpha} and TFIIF{alpha}) (Kamada et al. 2001; Meinhart et al. 2003). Interestingly, the TFIIEβ forkhead domain use its opposite molecular surface for its DNA-binding activity to that employed by the canonical forkhead domains (Okuda et al. 2000). At present, the biological significance of the forkhead domains in TFIIE and TFIIF, remains poorly understood.

Here, we generated systematic point mutations within the forkhead domain of hTFIIEβ, in order to elucidate more precisely the functional roles played by this centrally located region. Following bacterial expression and affinity purification, we examined in vitro transcription, CTD kinase stimulation, and double-stranded DNA binding activities of these mutant proteins.


   Results
Top
 Abstract
 Introduction
 Results
Discussion
 Experimental procedures
 References
 
Forkhead/winged-helix domain of TFIIEβ is highly conserved among eukaryotes

A forkhead domain is centrally localized within hTFIIEβ (amino acids 66–146 residues in humans; Fig. 1A) (Okamoto et al. 1998) and this domain exhibits strong evolutionary conservation from yeast to humans (Fig. 1B). We generated several point mutations (single and double) at conserved residues (Fig. 1B) and following cloning the recombinant mutants were expressed as soluble His-tagged proteins in Escherichia coli BL21(DE3)pLysS. After purification with Ni-NTA agarose, the mutant proteins were subjected to SDS-polyacrylamide gel electrophoresis (Fig. 1C,D).


Figure 1
View larger version (36K):
[in this window]
[in a new window]

  		Figure 1  Purified hTFIIEβ proteins with point mutations in the forkhead domains. (A) Schematic diagram showing structural motifs and characteristic sequences of human TFIIEβ. Ser-rich: a serine-rich sequence (residues 26–71), TFIIFβ: a region similar to the Pol II binding region of TFIIFβ (residues 79–111), LR: a leucine repeat motif (residues 145–163), {sigma}3: a region similar to the bacterial {sigma} factor subdomain 3 (residues 163–193), bHLH: a basic region-helix-loop-helix motif (residues 197–238), bHL: a basic region-helix-loop sequence (residues 258–291). (B) Sequence alignment of forkhead domains from various species. Identical or similar residues are shadowed and conserved residues are shown in bold. A leucine residue, which is all identical throughout the species, is black shaded. The asterisks show the sites of single point mutations, and the lines show double point mutations. (C) SDS-PAGE analysis of purified hTFIIEβ proteins containing point mutations in the N-terminal half of the forkhead domain. Hexahistidine (6H)-tagged point mutants were expressed in E. coli BL21(DE3)pLysS, purified and then 400 ng of each protein was subjected to SDS-PAGE (12% acrylamide) and stained with Coomassie blue. Lane 1 shows wild-type 6H-hTFIIEβ, lanes 2-21 contain the mutant proteins. The positions of molecular weight markers are indicated on the right (in kDa). Mutated residues are indicated at the top of each lane. (D) SDS-PAGE analysis of the purified 6H-hTFIIEβ proteins containing point mutations in the C-terminal half of the forkhead domain. Expression, purification and SDS-PAGE were carried out as described in (C). Lane 1 shows wild-type 6H-hTFIIEβ, lanes 2–21 contain the mutant proteins.

 
Hydrophobic and basic residues in the hTFIIEβ forkhead domain are important for transcription

We used a supercoiled template containing the AdML promoter to assess how point mutations in the forkhead domain of hTFIIEβ might affect basal transcription (Fig. 2). A significant reduction in transcription activity (< 50% of wild-type activity) was observed for mutations at three hydrophobic residues and one lysine residue located within the N-terminal half of the domain (V77A L78A, V77D L78D and I102K; Fig. 2A–C) and at three lysine residues and one aromatic residue located within the C-terminal half (K129A, K129E, K129Q, K140A K142A, K140E K142E, K140Q K142Q and F139A; Fig. 2G,H). Increased transcription activity (> 140%) was observed for mutations at two residues in the middle of this domain (E105K, E105R and T106Y; Fig. 2D). In contrast, transcription was unaffected by any other mutations in the forkhead domain, including that of the remaining aromatic residue in the N-terminal half (Y84A; Fig. 2A–F).


Figure 2
View larger version (49K):
[in this window]
[in a new window]

  		Figure 2  Effects of forkhead domain point mutations on the basal transcription activity of hTFIIEβ using a supercoiled template. (A)–(H) In vitro transcription assays were carried out with a supercoiled template and increasing amounts (1, 4 and 16 ng) of wild-type 6H-hTFIIEβ (IIEβ wt) or its mutants. Following transcription, radio-labeled transcripts were subjected to urea-PAGE and detected by autoradiography (lower panels). Each transcript was quantified using a Fuji BAS2500 Bio-Imaging analyzer. Relative transcription activity (%; upper panels) was calculated using the activity of 16 ng of wild-type 6H-hTFIIEβ as 100%. Mutated residues are indicated below the panels. As a negative control, transcription was also carried out in the absence of hTFIIEβ protein (–β). The arrow on the right side of the lower panels indicates the position of 390 nucleotide transcripts.

 
Next, we examined the effects of these point mutations on basal transcription using the linearized template and found similar levels of transcription to those observed with the supercoiled template (Fig. 3A–C). As there were no additional defects in transcription with the linearized template, it is likely that the transcriptional defects shown here related primarily to transcription initiation and not to the transition from initiation to elongation (Yamamoto et al. 2001). However, it is important to note that some mutations (P96A, T106A and D110N) rather augmented transcription with linearized template (Fig. 3A,B). This suggests that these residues (Pro96, Thr106 and Asp110), which locate on the H2 helix and its surrounding region, might be located in the region essential to negatively regulate the transition to elongation.


Figure 3
View larger version (35K):
[in this window]
[in a new window]

  		Figure 3  Effects of forkhead domain point mutations on basal transcription activity using a linearized template. (A)–(C) In vitro transcription assays were carried out as described in Fig. 2, with the exception that linearized template was substituted for supercoiled and only 4 and 16 ng of wild-type 6H-hTFIIEβ (IIEβ wt) and its point mutants were tested. The radio-labeled transcripts (lower panels) and relative transcription activities (%; upper panels) are shown. Relative transcription activity was calculated using the activity of 16 ng of wild-type 6H-hTFIIEβ as 100%. Mutated residues are indicated below the panels. As a negative control, transcription was also carried out in the absence of hTFIIEβ protein (–β). The arrow on the right side of the lower panels indicates the position of 390 nucleotide transcripts.

 
Hydrophobic and basic residues are also important for CTD phosphorylation

It has been observed that there is a correlation between transcriptional activity and the extent of phosphorylation of the CTD of the largest subunit of Pol II (Ohkuma & Roeder 1994; Okamoto et al. 1998). To confirm and examine this result further, we tried to identify residues important for stimulation of hTFIIH-mediated CTD phosphorylation (Fig. 4). As expected, mutants with defects in transcription (V77A L78A, V77D L78D, I102K, I111K, K129E and K140Q K142Q) also showed defects in CTD phosphorylation (Fig. 4A, lanes 3, 4 and 16; Fig. 4B, lanes 8, 10 and 16). These findings indicate that transcription activity correlates well with CTD phosphorylation.


Figure 4
View larger version (40K):
[in this window]
[in a new window]

  		Figure 4  Effect of point mutations on TFIIH-mediated CTD phosphorylation. (A) Kinase assays for hTFIIEβ proteins containing point mutations in the N-terminal half of the forkhead domain. Kinase assays were carried out under the conditions of active initiation complex formation. Lane 1, no hTFIIEβ (–IIEβ); lane 2, wild-type 6H-hTFIIEβ (IIEβwt); lanes 3–16, 6H-hTFIIEβ point mutants. The mutated residues are indicated above each lane. Phosphorylation of the largest subunit of Pol II was analyzed on a 5.5% acrylamide-SDS gel and detected by autoradiography. Arrows indicate the phosphorylated (IIo) and unphosphorylated (IIa) forms of the largest subunit of Pol II. (B) Kinase assays for hTFIIEβ proteins containing point mutations in the C-terminal half of the forkhead domain. Kinase assays were carried out as described in Fig 4A. Lane 1, no hTFIIEβ (–IIEβ); lane 2, wild-type 6H-hTFIIEβ (IIEβwt); lanes 3–17, 6H-hTFIIEβ point mutants. Arrows indicate IIo and IIa.

 
hTFIIEβ forkhead domain is essential for binding to double-stranded DNA

Previously, chemical shift and biochemical binding studies were used to demonstrate that the hTFIIEβ forkhead domain binds dsDNA (Okuda et al. 2000). Here, we attempted to identify residues essential for its dsDNA-binding activity (Fig. 5). After binding to dsDNA agarose resin, all samples were subjected to SDS-PAGE and bound forkhead domain mutants were detected by Western blotting using anti-hTFIIEβ rabbit polyclonal antibody. Mutations in residues locate within the C-terminal half of this domain, that is, three lysine residues (Lys129, Lys140 and Lys142), one aromatic residue (Phe139), one acidic residue (Asp110) and one hydrophobic residue (Ile111) strongly affected dsDNA binding activity (Fig. 5A, lanes 13, 14, 16 and 17; Fig. 5B, lanes 8–10). In addition, some residues located within the N-terminal half of the forkhead domain, that is, a glutamine (Gln83), lysine (Lys86) and hydrophobic residue (Ile102), weakly affected binding of dsDNA (Fig. 5A, lanes 7 and 10; Fig. 5B, lane 5). There is a conspicuous overlap between residues essential for binding dsDNA and those essential for both transcription and stimulation of CTD phosphorylation. Thus, these results indicate that the dsDNA-binding activity of the forkhead domain is important for both transcription and CTD phosphorylation activities.


Figure 5
View larger version (31K):
[in this window]
[in a new window]

  		Figure 5  Ability of hTFIIEβ mutants to bind dsDNA. (A) Mutant hTFIIEβ proteins were mixed with dsDNA-cellulose beads. The mixtures were rotated for 4 h at 4 °C, washed and then subjected to SDS-PAGE. Bound mutants were detected by Western blotting using anti-hTFIIEβ antisera. Lane 1, no hTFIIEβ (–IIEβ); lane 2, wild-type 6H-hTFIIEβ (IIEβ wt); lanes 3–20, point mutant proteins. Mutated residues are indicated above each lane. (B) Mutant hTFIIEβ proteins were similarly treated as in (A). Lane 1, no hTFIIEβ (-IIEβ); lane 2, wild-type 6H-hTFIIEβ (IIEβ wt); lanes 3–11, point mutant proteins. Bound mutants were detected by Western blotting with anti-hTFIIEβ antisera.

 

   Discussion
Top
 Abstract
 Introduction
 Results
 Discussion
Experimental procedures
 References
 
Here, we analyzed the functional residues in the central forkhead domain of hTFIIEβ. We generated various point mutants of this domain and used them to identify residues essential for transcription initiation, stimulation of CTD phosphorylation, and binding of dsDNA (Figs 2–5). Our results are summarized in Fig. 6. As shown, transcription is severely affected by mutations of specific hydrophobic as well as basic residues, but it turned out from the present results that the primary function of the forkhead domain comes from the dsDNA-binding activity.


Figure 6
View larger version (25K):
[in this window]
[in a new window]

  		Figure 6  Effects of forkhead domain point mutations on hTFIIEβ function. Diagram summarizing the results from all of our functional studies. Asterisks indicate residues that form the hydrophobic core of the forkhead domain (residues 77–144). The secondary structure of this domain is indicated above the sequence. Mutated residues are shadowed. Essential residues are indicated by unbroken lines and conditional residues are indicated by dashed lines. In the column indicating relative transcription activities, circled residues represent transcription with a supercoiled template, whereas boxed residues represent transcription with a linearized template.

 
Mutations of hydrophobic residues cause structural distortions

Previously, we used NMR to determine the structure of the forkhead domain (PDB ID: 1D8K [PDB] ) and we used the data to map the hydrophobic residues essential for maintenance of the core spherical structure (Okuda et al. 2000). In addition, we used chemical shift analyses to determine the residues that bind dsDNA as well as point mutation analyses to identify four residues (Val77, Leu78, Ile102 and Phe139) that are of particular importance for structural maintenance (Okuda et al. 2000). From the structural analyses, we hypothesized that Val77 and Leu78 are essential for forming the H1 helix that Ile102 is essential for forming the H2 helix, and that Phe139 is essential for forming the S3 sheet.

Here in the present study, we observed that mutations at these residues affected transcription as well as CTD phosphorylation, suggesting that their replacement had resulted in a severely disrupted domain structure. It is clear from the structure presented in Fig. 7A, that residues facing inward play a significant role in maintaining structure. Specifically, Val77 does not extend its side chain directly inward, whereas Leu78 is actually facing toward three hydrophobic residues (Ile81, Leu119 and Leu124), and Ile102 is facing toward two hydrophobic residues (Ile81 and Leu124). Phe139 is farther away from Val77 and Leu78, and it is located on the S3 sheet facing Met85. As replacing these residues with hydrophilic amino acids abolishes hydrophobic interactions, it is likely that they are essential for maintenance of the forkhead structure.


Figure 7
View larger version (28K):
[in this window]
[in a new window]

  		Figure 7  Steric mapping of residues essential for TFIIE function. (A) Map of the essential hydrophobic residues in the forkhead domain of hTFIIEβ. The ribbon structure (PDB ID: 1D8J) was drawn using PyMOL (Okuda et al. 2000). {alpha}-helices (H1, H2, H3) are shown in red and β-sheets (S2 and S3) in cyan. Hydrophobic residues (V77, L78, I102, and F139) are indicated in yellow and their hydrophobic counterparts in blue. (B) Map of functionally important lysine residues (K86, K129, K140 and K142) in the forkhead domain of hTFIIEβ. The structure was drawn as in (A). Lysine residues are shown in yellow. The predicted dsDNA-binding surface is indicated by an ellipse using a purple line. (C) Residues at which point mutations cause transcriptional augmentation. These residues (Pro96, Glu101, Glu105, Thr106 and Asp110) are shown in yellow and their hydrophobic counterparts are indicated in blue. The view represents the opposite side of the domain to that shown in (A) and (B).

 
Essential dsDNA-binding lysines are present on one surface

Previously, we identified four lysines (Lys86, Lys129, Lys140 and Lys142) that were important for binding to dsDNA (Okuda et al. 2000). In this study, our mutational analyses confirmed these findings (Fig. 5A, lanes 13, 14, 17 and 20; Fig. 5B, lane 5). Mutation of these residues had a strong effect on basal transcription as well as a weak effect on CTD phosphorylation (Figs 2–4). As clearly demonstrated by the structure in Fig. 7B, these dsDNA-binding lysine residues (Lys86, Lys129, Lys140 and Lys142) are present on the same surface (drawn as an ellipse with a purple line). It is likely dsDNA-binding activity was affected by mutation of Ile102 and Phe139, as they are also located at this binding surface. Taken together, these results and those from previous structural and photo-cross-linking studies suggest that dsDNA-binding is the primary function of the hTFIIEβ forkhead domain (Douziech et al. 2000; Okuda et al. 2000). One mutant, K129A, bound to dsDNA but had reduced transcription activity (Figs 2G, 3C, 5A, lane 12). Although there is a discrepancy between those activities, it might be happened because dsDNA binding activity was studied by using only an hTFIIEβ mutant protein and dsDNA agarose, and, contrary to this, transcription was observed with many other general transcription factors together with Pol II. It is therefore possible that some unknown activities other than dsDNA binding activity might affect on transcription. These results also support our previous model, in which this domain uses the opposite surface from the DNA-binding surfaces of canonical forkhead/winged-helix domains such as HNF3-{gamma} and DP2 (Okuda et al. 2000). In addition, photo-cross-linking data suggest that hTFIIEβ must bind around the –10 position, upstream from the transcription initiation site, in order for promoter melting of the promoter to occur (Douziech et al. 2000).

Mutations of some residues cause transcriptional augmentation

In transcriptional studies with either supercoiled or linearized template, we observed enhanced transcription activity in mutants of residues on the H2 helix (Pro96, Glu101, Glu105, Thr106 and Asp110; Figs 2, 3). In particular, transcription increased by more than 30% for mutants in which these glutamates had been changed to basic residues, and transcription was raised by approximately 70% following substitution of a Tyr for Thr106. Figure 7C indicates that these residues are located on the opposite side of the DNA-binding surface shown in Fig. 7B and that they face to hydrophobic residues which might be essential for holding conformation (Ile81, Tyr84, Leu97, Ile102, Ile133 and Ala138). Taking the potential significance of these positions into consideration, it is possible that these residues might be involved in regulating the tightness of the structure itself and might cause stabilization of the structure of the DNA binding region. Thus, replacing glutamate with lysine or arginine, and threonine with tyrosine, might result in increased hydrophobicity of the side chains, causing further structural stabilization and thus enhanced transcription.

At present, the forkhead domain is known to function in both dsDNA-binding and protein–protein interactions, activities that are required for the wide variety of roles carried out by FOXO family proteins (Daitoku & Fukamizu 2007). In this paper, we used biochemical studies to further demonstrate that the forkhead domain plays both a primary and essential role in binding to promoter DNA, and we have suggested that it might effect efficient transcriptional initiation by mediating the transition of the pre-initiation complex from its inactive to active form. Contrary to the canonical forkhead domain, this domain binds DNA sequences nonspecifically, in addition to using the opposite surface for binding DNA (Okuda et al. 2000). It will be interesting to elucidate the mechanism by which this forkhead domain recognizes DNA within the pre-initiation complex and how it binds to the –10 region upstream of the transcription initiation site. These mechanisms will be explored in our future studies.


   Experimental procedures
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
References
 
Construction of human TFIIEβ point mutants

Subcloning of the full length wild-type hTFIIEβ cDNA into a bacterial expression vector was described previously (Watanabe et al. 2003). Various oligonucleotide-mediated point mutants were created using the site-directed mutagenesis kit Mutan-K (TaKaRa), and the hexa histidine (6H)-tagged hTFIIEβ pET21b expression plasmid as a template (Kunkel et al. 1987). In order to select mutants, restriction sites were placed in each oligonucleotide (Ohkuma et al. 1995; Okamoto et al. 1998) and potential clones were sequenced using an ABI PRISM 310 Genetic Analyzer (Applied Biosystems). Because of their large number, oligonucleotide sequences have not been included, but will be provided upon request.

Expression and purification of recombinant proteins

Following induction with isopropyl-β-D-thiogalactopyranoside (IPTG), recombinant point mutant 6H-hTFIIEβ proteins were expressed in E. coli BL21 (DE3) pLysS and then purified from bacterial lysates, as described previously (Watanabe et al. 2003).

In vitro transcription assays

Human TFIIH was purified from HeLa nuclear extracts or cytoplasmic S100 fractions, as described previously (Ohkuma & Roeder 1994). We also purified other recombinant general transcription factors, as well as native Pol II, in order to carry out the in vitro transcription reactions (Ohkuma et al. 1995). The plasmid pML(C2AT){Delta}-50 contains the adenovirus type 2 major late (AdML) promoter, which generates a 390-nucleotide (nt) transcript that was used as either a supercoiled or a linearized template for basal transcription assays (Yamamoto et al. 2001). To prepare linearized template, pML (C2AT){Delta}-50 was digested with SmaI. Autoradiography was carried out at –80 °C on Fuji RX-U X-ray film. Incorporation of [{alpha}-32P]-CTP into transcripts was quantified using a Fuji BAS2500 Bio-Imaging analyzer.

Kinase assays

As described previously, in vitro kinase assays were carried out using general transcription factors, Pol II and AdML promoter sequences from –39 to +29 (Ohkuma & Roeder 1994). Phosphorylation reactions were carried out at 30 °C for 1 h and then stopped with 75 µL of phosphorylation stop solution (10 mM EDTA, 0.1% NP40, 0.05% SDS). After addition of a carrier protein (2 µg BSA), phosphorylated proteins were precipitated with 10% TCA and analyzed by SDS-PAGE (5.5% acrylamide). Autoradiographs were carried out at –80 °C with RX-U X-ray film (Fuji Film). A Fuji BAS2500 Bio-Imaging analyzer was used to quantify the extent of 32P-phosphorylation of the CTD of the largest subunit of Pol II.

Double-stranded DNA-binding assays

Three hundred ng of 6H-hTFIIEβ (wild type or mutant) were mixed with 7 µL (packed volume) of dsDNA-cellulose (Sigma) in 500 µL reactions containing buffer C (20 mM Tris–HCl [pH 7.9 at 4 °C], 0.5 % EDTA, 20 % [vol/vol] glycerol, 0.5 mM PMSF, 10 mM 2-mercaptoethanol, 0.002% [vol/vol] Nonidet P-40, 100 mM KCl; BC100) and 200 µg/mL BSA. Reactions were incubated for 4 h at 4 °C with rotation, after which the resin was washed twice with 500 µL of buffer C containing 200 mM KCl (BC200) and once with 500 µL of BC100. Reactions were then boiled in SDS sample buffer, and analyzed by SDS-PAGE (15% acrylamide). Bound mutants were detected by Western blotting using anti-hTFIIEβ antisera (1 : 3000 dilution), as described above (Okamoto et al. 1998).


   Acknowledgements
 
We thank Dr Masahiko Okuda for discussions regarding the structure–function relationships, as well as Drs Yoko Arai and Yoshinori Watanabe for initial studies on the forkhead domain. We also thank the laboratory members at Osaka University and University of Toyama for discussions throughout this work. This work was supported in part by grants from the Protein 3000 project (to Y.O.), the Grants-in-Aid for Scientific Research from the Ministry of Educations, Culture, Sports, Science and Technology of Japan (to F.H. and Y.O.), and the Solution Oriented Research for Science and Technology, Japan Science and Technology Agency (to F.H. and Y.O.).


   Footnotes
 
Communicated by: Hiroshi Handa

aPresent address: Discovery Research Laboratories III, Pharmaceutical Research Division, Takeda Chemical Industries Ltd., 2-17-85 Juso-honmachi, Yodogawa-ku, Osaka 532-8686, Japan. Back

* Correspondence: ohkumay{at}pha.u-toyama.ac.jp


   References
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
 References
 
Daitoku, H. & Fukamizu, A. (2007) FOXO transcription factors in the regulatory networks of longevity. J. Biochem. 141, 769–774.[Abstract/Free Full Text]

Di Lello, P., Miller Jenkins, L.M., Mas, C., Langlois, C., Malitskaya, E., Fradet-Turcotte, A., Archambault, J., Legault, P. & Omichinski, J.G. (2008) p53 and TFIIE{alpha} share a common binding site on the Tfb1/p62 subunit of TFIIH. Proc. Natl Acad. Sci. USA 105, 106–111.[Abstract/Free Full Text]

Douziech, M., Coin, F., Chipoulet, J.M., Arai, Y., Ohkuma, Y., Egly, J.-M. & Coulombe, B. (2000) Mechanism of promoter melting by the xeroderma pigmentosum complementation group B helicase of transcription factor IIH revealed by protein-DNA photo-cross-linking. Mol. Cell. Biol. 20, 8168–8177.[Abstract/Free Full Text]

Groft, C.M., Uljon, S.N., Wang, R. & Werner, M.H. (1998) Structural homology between the Rap30 DNA-binding domain and linker histone H5: implications for preinitiation complex assembly. Proc. Natl Acad. Sci. USA 95, 9117–9122.[Abstract/Free Full Text]

Hirose, Y. & Ohkuma, Y. (2007) Phosphorylation of the C-terminal domain of RNA polymerase II plays central roles in the integrated events of eukaryotic gene expression. J. Biochem. 141, 601–608.[Abstract/Free Full Text]

Holstege, F.C.P., Tantin, D., Carey, M., van der Vliet, P.C. & Timmers, H.T.M. (1995) The requirement for the basal transcription factor IIE is determined by the helical stability of promoter DNA. EMBO J. 14, 810–819.[Medline]

Holstege, F.C.P., van der Vliet, P.C. & Timmers, H.T.M. (1996) Opening of an RNA polymerase II promoter occurs in two distinct steps and requires the basal transcription factors IIE and IIH. EMBO J. 15, 1666–1677.[Medline]

Kamada, K., De Angelis, J., Roeder, R.G. & Burley, S.K. (2001) Crystal structure of the C-terminal domain of the RAP74 subunit of human transcription factor IIF. Proc. Natl Acad. Sci. USA 98, 3115–3120.[CrossRef]

Kunkel, T.A., Roberts, J.D. & Zakour, R.A. (1987) Rapid and efficient site-specific mutagenesis without phenotypic selections. Methods Enzymol. 154, 367–382.[Medline]

Meinhart, A., Blobel, J. & Cramer, P. (2003) An extended winged helix domain in general transcription factor E/IIE{alpha}. J. Biol. Chem. 278, 48267–48274.[Abstract/Free Full Text]

Ohkuma, Y. (1997) Multiple functions of general transcription factors TFIIE and TFIIH in transcription: possible points of regulation by trans-acting factors. J. Biochem. 122, 481–489.[Abstract/Free Full Text]

Ohkuma, Y., Hashimoto, S., Wang, C.K., Horikoshi, M. & Roeder, R.G. (1995) Analysis of the role of TFIIE in basal transcription and TFIIH-mediated carboxy-terminal domain phosphorylation through structure–function studies of TFIIE-{alpha}. Mol. Cell. Biol. 15, 4856–4866.[Abstract/Free Full Text]

Ohkuma, Y. & Roeder, R.G. (1994) Regulation of TFIIH ATPase and kinase activities by TFIIE during active initiation complex formation. Nature 368, 160–163.[CrossRef][Medline]

Okamoto, T., Yamamoto, S., Watanabe, Y., Ohta, T., Hanaoka, F., Roeder, R.G. & Ohkuma, Y. (1998) Analysis of the role of TFIIE in transcriptional regulation through structure–function studies of the TFIIEβ subunit. J. Biol. Chem. 273, 19866–19876.[Abstract/Free Full Text]

Okuda, M., Tanaka, A., Satoh, M., Mizuta, S., Takazawa, M., Ohkuma, Y. & Nishimura, Y. (2008) Structural insight into the TFIIE–TFIIH interaction: TFIIE and p53 share the binding region on TFIIH. EMBO J. 27, 1161–1171.[CrossRef][Medline]

Okuda, M., Watanabe, Y., Okamura, H., Hanaoka, F., Ohkuma, Y. & Nishimura, Y. (2000) Structure of the central core domain of TFIIEβ with a novel double-stranded DNA-binding surface. EMBO J. 19, 1346–1356.[CrossRef][Medline]

Orphanides, G., Lagrange, T. & Reinberg, D. (1996) The general transcription factors of RNA polymerase II. Genes Dev. 10, 2657–2683.[Free Full Text]

Roeder, R.G. (1996) The role of general initiation factors in transcription by RNA polymerase II. Trends Biochem. Sci. 21, 327–335.[CrossRef][Medline]

Watanabe, T., Hayashi, K., Tanaka, A., Furumoto, T., Hanaoka, F. & Ohkuma, Y. (2003) The carboxy terminus of the small subunit of TFIIE regulates the transition from transcription initiation to elongation by RNA polymerase II. Mol. Cell. Biol. 23, 2914–2926.[Abstract/Free Full Text]

Weigel, D. & Jäckle, H. (1990) The fork head domain: a novel DNA binding motif of eukaryotic transcription factors? Cell 63, 455–456.[CrossRef][Medline]

Yamamoto, S., Watanabe, Y., van der Spek, P.J., Watanabe, T., Fujimoto, H., Hanaoka, F. & Ohkuma, Y. (2001) Studies of nematode TFIIE function reveal a link between Ser-5 phosphorylation of RNA polymerase II and the transition from transcription initiation to elongation. Mol. Cell. Biol. 21, 1–15.[Abstract/Free Full Text]

Received: 27 July 2008
Accepted: 3 December 2008





This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Tanaka, A.
Right arrow Articles by Ohkuma, Y.
Right arrow Search for Related Content
PubMed
Right arrow Articles by Tanaka, A.
Right arrow Articles by Ohkuma, Y.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE ADVANCED SEARCH TABLE OF CONTENTS