Functional Correlation among Gal11, Transcription Factor (TF) IIE, and TFIIH in Saccharomyces cerevisiae
Gal11 AND TFIIE COOPERATIVELY ENHANCE TFIIH-MEDIATED PHOSPHORYLATION OF RNA POLYMERASE II CARBOXYL-TERMINAL DOMAIN SEQUENCES*

Hiroshi SakuraiDagger § and Toshio Fukasawa

From the Dagger  School of Health Sciences, Faculty of Medicine, Kanazawa University, 5-11-80 Kodatsuno, Kanazawa, Ishikawa 920-0942, Japan and the  Kazusa DNA Research Institute, 1532-3 Yana, Kisarazu, Chiba 292-0812, Japan

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

Saccharomyces cerevisiae Gal11, a component of the holoenzyme of RNA polymerase II, interacts through its functional domains A and B with the small (Tfa2) and large (Tfa1) subunits of the general transcription factor (TF) IIE, respectively. We have recently suggested that Gal11 functions through a common pathway with TFIIE in transcriptional regulation (Sakurai, H., and Fukasawa, T. (1997) J. Biol. Chem. 272, 32663-32669). Here, we report that the activity of the TFIIH-associated kinase, responsible for phosphorylation of the largest subunit of RNA polymerase II at the carboxyl-terminal domain (CTD), is enhanced cooperatively by Gal11 and TFIIE. The enhancement of CTD phosphorylation was observed in the holoenzyme of RNA polymerase II, but not in its core enzyme. The stimulatory effect was completely abolished in the absence of either domain B of Gal11 or the Tfa1 subunit of TFIIE, suggesting that the domain B-Tfa1 interaction is necessary, if not sufficient, for an extensive phosphorylation of the CTD by TFIIH. Stimulation of basal transcription by Gal11 was coupled with enhancement of TFIIH-catalyzed CTD phosphorylation in a cell-free transcription system, suggesting that Gal11 activates transcription by stimulating the CTD phosphorylation in the cell.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

In eukaryotes, RNA polymerase II (RNAPII)1 and a set of general transcription factors (TFs) including TATA-binding protein (TBP), TFIIB, TFIIE, TFIIF, and TFIIH assemble to form the preinitiation complex on the core promoter to initiate transcription from an accurate start site (1-4). The largest subunit (Rpb1) of RNAPII contains a repeated heptapeptide sequence at the carboxyl terminus, which is referred to as the carboxyl-terminal domain (CTD) (for review, see Ref. 5). The CTD is required for transcription of most, if not all, of the genes in vivo (5) as well as for mRNA processing (6). During the transcription cycle, the CTD is phosphorylated by a kinase present within TFIIH (4, 7, 8). Formation of the preinitiation complex requires RNAPII with unphosphorylated CTD, whereas elongation of transcripts is accomplished by RNAPII with phosphorylated CTD (4). These observations suggest that phosphorylation of the CTD by the TFIIH-associated kinase is an obligatory step in the transcription process from initiation to elongation.

In the yeast Saccharomyces cerevisiae, partial truncation of the CTD causes defects in expression of various genes (5, 9, 10). Genetic screening for suppressors of truncation mutations of the CTD by Nonet and Young (10) led to identification of a class of genes called SRB. Subsequent biochemical analyses suggested that nine Srb proteins (Srb2 and Srb4-11) form a complex, which is tightly associated with RNAPII at the CTD (11-14). The Srb-RNAPII complex also contains the general transcription factors TFIIB, TFIIF, and TFIIH and the global transcription regulators Gal11 and Swi/Snf. The whole complex has been termed RNAPII holoenzyme since it has been implicated to be a preformed initiation subcomplex (12-17). Another form of RNAPII holoenzyme was isolated by Kornberg and co-workers (18) as a "mediator"-RNAPII complex. The mediator was fractionated from whole cell extracts for the capacity that confers core RNAPII responsiveness to DNA sequence-specific activators in the presence of the general transcription factors in vitro (18). The mediator fraction, also associated with RNAPII at the CTD, contains TFIIF and global transcription regulators including Srb2, Srb4, Srb5, Srb6, Gal11, Sin4, Rgr1, Rox3, and Med6 (18-21). Thus, TFIIF, some of the Srb proteins, and Gal11 are common components in both forms of the holoenzyme, whereas the presence of the other components is still controversial. The holoenzyme possesses properties distinct from those of core RNAPII: stimulation of basal as well as activator-induced transcription, interaction with activators, and enhancement of CTD phosphorylation by TFIIH in vitro (12, 13, 18, 21).

Loss-of-function mutations of GAL11 result in a wide variety of mutant phenotypes, including inefficient utilization of galactose or nonfermentable carbon sources and temperature-sensitive growth on rich media (22). Purified or recombinant Gal11 stimulates basal transcription not only in cell-free systems consisting of nuclear or whole cell extracts (23), but also in a reconstituted transcription system (22, 24). Gal11 contains two domains (designated A and B) that are essential for Gal11 function in the cell. Domain A, comprising amino acid residues 866-929, is involved in binding to the small subunit of TFIIE, whereas domain B sequences (from 116 to 255) bind to the large subunit of TFIIE (24). Recently, we constructed a mutant form of TFIIE (TFIIE-Delta C) that fails to interact with Gal11 and found that the TFIIE-Delta C mutant shows phenotypes quite similar to those of gal11 null mutations (22). Furthermore, combination of TFIIE-Delta C with a gal11 null mutation did not result in an enhanced effect compared with the respective single mutations. Based on these findings, we have suggested that TFIIE and Gal11 function in a common regulatory pathway of transcription (22).

In this work, we addressed the question of how Gal11 and TFIIE regulate transcription in the light of recent findings concerning functional interactions between TFIIE and TFIIH in yeast (25, 26) as well as in mammalian cells (4, 8). Mammalian TFIIE has been shown to regulate the enzymatic activities of TFIIH such as CTD kinase, DNA helicase, and ATPase (27-31). Here, we show that Gal11 stimulates TFIIH-catalyzed phosphorylation of the CTD in the presence of TFIIE only when holo-RNAPII is used as substrate. We further demonstrate that the enhanced phosphorylation of the CTD by Gal11 is associated with stimulation of transcription in a cell-free transcription reaction. In light of these results, the role of Gal11 in the transcription process is discussed.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Plasmids

Plasmid pSK720 contains polyhistidine-tagged full-length GAL11 in the pQE32 vector (QIAGEN Inc.) (22). Expression constructs of Gal11-Delta A (pSK723) or Gal11-Delta B (pSK724) were created by removal of domain A (amino acids 866-929) or domain B (amino acids 48-326) of Gal11 (24) from pSK720, respectively.

Protein Purification

TFIIH purified from yeast (Mono Q fraction) was a gift from Drs. Jesper Svejstrup and Roger Kornberg (32). Holo-RNAPII prepared from a GAL11 wild-type or a gal11 null yeast, core RNAPII, recombinant TBP, and recombinant TFIIB were gifts from Drs. Young-Joon Kim and Roger Kornberg (18). Recombinant proteins (full-length Gal11, Tfa1, and Tfa2) were expressed in Escherichia coli and purified as described (22, 33).

Both Gal11-Delta A and Gal11-Delta B were expressed in E. coli JM109 cells harboring pSK723 and pSK724, respectively. The respective extract was adsorbed on Ni2+-nitrilotriacetic acid-agarose (QIAGEN Inc.) as described (22), and the slurry was washed with buffer F (0.1 M Hepes-KOH, pH 7.6, 10% glycerol, 0.1 M potassium acetate, and 0.1% Nonidet P-40) containing 20 mM imidazole.

Gal11-Delta A Purification-- After washing the resin with buffer F containing 100 mM imidazole, Gal11-Delta A was eluted with buffer F containing 200 mM imidazole and 0.5 M potassium acetate. The pooled fraction was loaded onto a Sephadex G-25 column (Amersham Pharmacia Biotech) equilibrated with buffer B (22) containing 0.1 M potassium acetate (buffer B-0.1) and eluted with the same buffer. Proteins were then loaded onto an S-Sepharose column (Amersham Pharmacia Biotech) equilibrated with the same buffer. After washing with buffer B-0.2, the proteins were eluted with buffer B-0.35. The pooled fraction was diluted with buffer B to give a potassium acetate concentration of 0.1 M and fractionated on a Q-Sepharose column (Amersham Pharmacia Biotech). Gal11-Delta A was eluted with buffer B-0.35 after washing with buffer B-0.2.

Gal11-Delta B Purification-- Gal11-Delta B was eluted from Ni2+-nitrilotriacetic acid-agarose with buffer F containing 100 mM imidazole. The pooled fraction was loaded onto a Q-Sepharose column equilibrated with buffer B-0.1. The flow-through fraction was applied to a HiTrap heparin column (Amersham Pharmacia Biotech), and Gal11-Delta B was eluted with buffer B-1.0. The yield of Gal11-Delta A and Gal11-Delta B each was ~0.2 mg/liter of starting culture.

CTD Kinase Assay

The reaction mixture (10 µl) contained TFIIH (30 ng) and either holo-RNAPII (100 ng) or its core polymerase (50 ng) in a buffer containing 10 mM Hepes-KOH, pH 7.6, 0.1 M potassium acetate, 5 mM MgSO4, 2 mM dithiothreitol, 0.02% Nonidet P-40, 5% glycerol, 50 µg/ml bovine serum albumin, 10 µM ATP, and 1 µCi of [gamma -32P]ATP. The reaction was carried out at 24 °C for 40 min and terminated by the addition of SDS-containing loading buffer. After heating at 94 °C for 7 min, the sample was loaded on an SDS-polyacrylamide gel. Labeled proteins were visualized by autoradiography and quantified by a BAS-1000 imaging analyzer (Fuji Film). All experiments were repeated at least three times, and similar results were obtained.

In Vitro Transcription Assay

Yeast nuclear extract was prepared from a gal11 null strain (23). A transcription assay (20 µl) was carried out using the GAL7 gene (pSK164, 40 ng) as template as described (23), except that concentrations of nucleoside triphosphates were 0.1 mM each CTP, GTP, and UTP; 20 µM ATP; and 10 µCi of [gamma -32P]ATP. After incubation at 24 °C for 1 h, the mixture was divided into two portions. One was subjected to primer extension to analyze transcripts (23), whereas the other was used for analysis of CTD phosphorylation. The latter sample was incubated at 45 °C for 10 min and then mixed with 200 ng of an anti-CTD antibody (8WG16) and 10 µl of protein A-Sepharose (Amersham Pharmacia Biotech) in 100 µl of a buffer containing 20 mM Tris-Cl, pH 7.6, 150 mM NaCl, and 0.1% Nonidet P-40. After incubation at 4 °C for 3 h on a rotating wheel, the resin was washed three times with the same buffer. Bound proteins were extracted with SDS loading buffer and fractionated on an SDS-polyacrylamide gel, and 32P-labeled proteins were visualized by autoradiography.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

Enhancement of TFIIH-catalyzed Phosphorylation of the CTD in the Presence of Both Gal11 and TFIIE-- First, the effect of TFIIE on the TFIIH-associated CTD kinase activity was determined by using holo-RNAPII purified by the method of Kornberg and co-workers (18) as substrate, which contains TFIIF and transcription regulators including Gal11. A holo-RNAPII preparation from a GAL11 wild-type yeast was incubated with TFIIH in the presence of [gamma -32P]ATP, and the phosphorylated proteins were fractionated on an SDS-polyacrylamide gel and visualized by autoradiography. As shown in Fig. 1A, a band with an approximate molecular mass of 205 kDa, corresponding to that of Rpb1 (see Refs. 5 and 7), was phosphorylated by TFIIH (lanes 1 and 2). When TFIIE was added to the reaction mixture, the phosphorylation of Rpb1 was enhanced by a factor of 5.4 ± 0.6 (compare lanes 2 and 3). Further addition of Gal11 did not significantly affect Rpb1 phosphorylation (lanes 4 and 5). By contrast, when holo-RNAPII from a gal11 null yeast was used as substrate (lanes 6-9), TFIIE alone could not stimulate Rpb1 phosphorylation (compare lanes 6 and 8), and a high level of the phosphorylation (11.3 ± 2.1-fold stimulation) was attained only in the presence of both Gal11 and TFIIE (lane 9). The successful stimulation of Rpb1 phosphorylation by TFIIE alone observed in holo-RNAPII from the wild-type yeast (lane 3) was therefore attributed to endogenous Gal11 in the holoenzyme preparation. From these results, we concluded that Gal11 and TFIIE cooperatively enhanced Rpb1 phosphorylation by TFIIH.


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Fig. 1.   Regulation of TFIIH-catalyzed CTD phosphorylation by Gal11 and TFIIE. A, holo-RNAPII prepared either from a GAL11 wild-type strain (100 ng; lanes 1-5) or from a gal11 null strain (100 ng; lanes 6-9) and core RNAPII (50 ng; lanes 10-13) were used as substrate. Kinase assay mixtures contained TFIIH (30 ng), TFIIE (20 ng), and Gal11 (5 ng) (indicated by +). After incubation at 24 °C for 40 min, samples were subjected to SDS-6% polyacrylamide gel electrophoresis followed by autoradiography. The position of Rpb1 is indicated by the arrowhead. Molecular masses are given in kilodaltons. Lanes 10-13 were subjected to autoradiography 10 times longer than lanes 1-9. B, reaction mixtures contained holo-RNAPII from a gal11 null yeast (100 ng), TFIIH (30 ng), TFIIE (20 ng), and Gal11 (5 ng). After the kinase reaction, mixtures were heated at 45 °C for 10 min to inactivate the TFIIH-associated kinase (7). 100 ng of either anti-CTD antibody 8WG16 (alpha -CTD; lane 3) or control antibody (alpha -con; lane 2) was added to the mixtures, and the samples were incubated at 24 °C for 1 h. After treatment with calf intestinal alkaline phosphatase (5 units) at 37 °C for 10 min, proteins were precipitated by the addition of 10% trichloroacetic acid and electrophoresed on an SDS-polyacrylamide gel. The arrowhead indicates the position of Rpb1.

Although TFIIH-associated protein kinase is capable of phosphorylating the CTD (7), it has not been determined if the phosphorylation of Rpb1 is restricted within the CTD or not. One might therefore argue that Gal11/TFIIE-enhanced activity could phosphorylate domains other than the CTD. To address this argument, products of the kinase reaction were incubated with a control or an anti-CTD antibody and then treated with phosphatase (Fig. 1B). The samples were electrophoresed on an SDS-polyacrylamide gel, and the labeled proteins were visualized by autoradiography. When the product of the kinase reaction was pretreated with the control antibody, phosphatase treatment resulted in the disappearance of the Rpb1 band on an autoradiogram (compare lanes 1 and 2). By contrast, preincubation with the anti-CTD antibody apparently caused no loss of the Rpb1 band, presumably because the antibody protected phosphoryl groups on the CTD from phosphatase activity (compare lanes 1 and 3). This result indicated that the Gal11/TFIIE-dependent enhancement of Rpb1 phosphorylation occurred at the CTD.

We then studied the effect of Gal11 and TFIIE on TFIIH-catalyzed CTD phosphorylation using core RNAPII as substrate. In accordance with previous reports (18, 21), the efficiency of CTD phosphorylation in core RNAPII was 20-40 times lower than that in holo-RNAPII (data not shown). As shown in Fig. 1A, neither Gal11 nor TFIIE alone affected CTD phosphorylation in core RNAPII (lanes 10-12). The addition of both proteins caused an increase in the efficiency of CTD phosphorylation, but only by a factor of 1.5 ± 0.2 (lane 13). These results indicated that the cooperative effect of Gal11 and TFIIE was much more pronounced in holo-RNAPII than in core RNAPII.

Tfa1 Subunit of TFIIE Is Required for Enhancement of CTD Phosphorylation-- To determine which subunit of yeast TFIIE, Tfa1 or Tfa2 (33), was responsible for the enhancement of CTD phosphorylation by TFIIH, the respective subunit was added to a reaction mixture containing holo-RNAPII prepared from a gal11 null yeast. As shown in Fig. 2A, neither Tfa1 nor Tfa2 affected CTD phosphorylation if Gal11 was not added to the reaction (lanes 3 and 5). The addition of Gal11 enhanced phosphorylation of the CTD when Tfa1 was incorporated into the reaction (12.0 ± 2.5-fold stimulation), whereas no enhancement of phosphorylation was seen when Tfa2 was added (lanes 4 and 6). The presence of both Tfa1 and Tfa2 also caused a significant stimulation of CTD phosphorylation if Gal11 was also added to the reaction (lanes 7 and 8). We therefore concluded that Tfa1 of the TFIIE subunits was responsible for the stimulation of TFIIH-catalyzed CTD phosphorylation in cooperation with Gal11; the conclusion is comparable to the previous finding in mammalian systems that the large subunit of TFIIE is sufficient to activate phosphorylation of the CTD by TFIIH (29-31).


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Fig. 2.   Effect of TFIIE subunits or mutant Gal11 proteins on TFIIH-catalyzed CTD phosphorylation in holo-RNAPII. A, the kinase reaction was carried out using Gal11-lacking holo-RNAPII as described under "Experimental Procedures." Tfa1 (10 ng), Tfa2 (10 ng), and Gal11 (5 ng) were added to the reaction mixtures (indicated by +). B, reaction mixtures contained Gal11-lacking holo-RNAPII, TFIIH, and TFIIE (20 ng). Kinase reactions were carried out in the absence (lane 1) or presence of 5 ng of full-length Gal11 (wild-type (wt); lane 2), Gal11-Delta A (lane 3), or Gal11-Delta B (lane 4). Stimulation of CTD phosphorylation by the addition of Gal11 is shown as -fold stimulation based on the value obtained with the BAS-1000 imaging analyzer (mean ± S.E. of three separate experiments).

Effect of Gal11 Mutants on Enhancement of CTD Phosphorylation-- To further assess the cooperative function of Gal11 and TFIIE, Gal11 derivatives with deletions of domain A or B, known to be responsible for the interaction with Tfa2 or Tfa1 of TFIIE, respectively (22, 24), were employed (Fig. 2B). In the presence of TFIIE, a Gal11 derivative lacking domain A (Gal11-Delta A) stimulated CTD phosphorylation, but only 3.0 ± 0.6-fold over the control without Gal11, whereas full-length Gal11 did so more than 10-fold (compare lanes 1-3). Deletion of domain B (Gal11-Delta B) resulted in total loss of the enhancement of CTD phosphorylation (lane 4). These results suggested that the interaction between domain B and Tfa1 was essential in the cooperative function of Gal11 and TFIIE, but might not be sufficient (see "Discussion").

CTD Phosphorylation in the Preinitiation Complex-- To study CTD phosphorylation in holo-RNAPII as incorporated into the preinitiation complex, the kinase reaction mixture contained TBP, TFIIB, TFIIE, TFIIH, holo-RNAPII (which contains TFIIF (18)), and DNA encompassing the promoter region of GAL7 from positions -93 to +43 (34). The latter provided the site of assembly for the preinitiation complex. Aliquots were withdrawn from the reactions at the indicated times for analysis of phosphorylated proteins (Fig. 3A). At all the time points, Rpb1 was two times more efficiently phosphorylated in the mixture in which the preinitiation complex was supposed to be formed than in the mixture in which the complex was not formed. Moreover, phosphorylated Rpb1 migrated slightly more slowly when the preinitiation complex was formed than when it was not formed, presumably due to an extensive phosphorylation of the CTD at multiple sites in the former (compare odd- and even- numbered lanes). An electrophoretic mobility shift of Rpb1 due to an extensive phosphorylation of the CTD at multiple sites by TFIIH was previously observed using core RNAPII as substrate (7). However, in our assay using holo-RNAPII, a mobility shift of Rpb1 was induced when it was integrated in the preinitiation complex. Phosphorylation of the CTD accompanying the mobility shift of the Rpb1 band was observed even in Gal11-lacking holo-RNAPII (Fig. 3B, compare lanes 1-3), suggesting that Gal11 is not absolutely required for the extensive phosphorylation of the CTD in the preinitiation complex. However, the addition of Gal11 resulted in a further increase in CTD phosphorylation by a factor of ~4.1 ± 0.3 (compare lanes 3 and 4), indicating that Gal11 induced further phosphorylation of the CTD. In agreement with the results shown in Fig. 2B, Gal11-Delta A stimulated CTD phosphorylation, but only 2.0 ± 0.2-fold over the background level (Fig. 3B, compare lanes 3 and 5), whereas Gal11-Delta B showed no obvious effect (compare lanes 3 and 6).


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Fig. 3.   CTD phosphorylation in the preinitiation complex. A, kinase reactions were performed using holo-RNAPII prepared from a GAL11 wild-type strain and were terminated at the times indicated. Reaction mixtures contained holo-RNAPII, TFIIH, and TFIIE (20 ng; lanes 1, 3, and 5). TBP (12 ng), TFIIB (6 ng), and the DNA fragment (10 ng) containing the promoter region of GAL7 were also added to assemble the preinitiation complex (lanes 2, 4, and 6). Faster and slower migrating bands are indicated on the left and right, respectively, by arrowheads. The relative levels of phosphorylation normalized to the value of lane 1 as 1.0 were 1.7 ± 0.2 (lane 2), 1.8 ± 0.1 (lane 3), 3.3 ± 0.3 (lane 4), 3.0 ± .0.2 (lane 5), and 5.4 ± 0.4 (lane 6). B, kinase reactions were carried out as described above in the absence (lanes 1 and 2) or presence of TBP, TFIIB, and the DNA fragment (lanes 3-6) using Gal11-lacking holo-RNAPII. The mixtures contained 5 ng of full-length Gal11 (wild-type (wt); lanes 2 and 4), Gal11-Delta A (lane 5), or Gal11-Delta B (lane 6). Faster and slower migrating bands are indicated on the left and right, respectively, by arrowheads.

Phosphorylation of the CTD during Transcription-- To elucidate the relationship of the Gal11/TFIIE-enhanced CTD phosphorylation to transcription, transcripts were analyzed in parallel with phosphorylated Rpb1 during the course of transcription in vitro. Li and Kornberg (35) reported that a close correlation of CTD phosphorylation with transcription was observed in a crude nuclear extract system, but not in the basal system comprising purified components. Thus, dependence of transcription on CTD phosphorylation was observed only in the nuclear extract system. We therefore prepared a nuclear extract from a gal11 null strain in which transcription occurred on a template DNA containing the promoter region of GAL7 in the presence of [gamma -32P]ATP. After the reaction was completed, transcripts were analyzed by primer extension, while Rpb1 precipitated with the anti-CTD antibody was subjected to SDS-polyacrylamide gel electrophoresis followed by autoradiography (Fig. 4). In accordance with Li and Kornberg (35), the addition of the protein kinase inhibitor H-8 to the reaction resulted in a complete arrest of both transcription of GAL7 and CTD phosphorylation of Rpb1 (compare lanes 1 and 2), suggesting that phosphorylation of the CTD is necessary for transcription in the nuclear extract. The addition of Gal11 enhanced transcription as well as CTD phosphorylation by factors of 3.6 ± 0.4 and 2.0 ± 0.2 over the background levels, respectively (compare lanes and 3). The stimulatory effect of Gal11 on CTD phosphorylation appeared rather small compared with that on transcription. This may well be due to the presence of nonspecific protein kinases other than TFIIH-associated kinase and free core RNAPII in the nuclear extract, both of which would lower the apparent effect of Gal11 on the CTD phosphorylation. Neither Gal11-Delta A nor Gal11-Delta B exerted appreciable effects on both transcription and CTD phosphorylation (lanes 4 and 5).


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Fig. 4.   Effect of Gal11 on transcription and CTD phosphorylation in the nuclear extract. Transcription and CTD phosphorylation assays were carried out as described under "Experimental Procedures." The protein kinase inhibitor H-8 was added to give a final concentration of 3 mM (lane 2). Reaction mixtures contained 10 ng of full-length Gal11 (wild-type (wt); lane 3), Gal11-Delta A (lane 4), or Gal11-Delta B (lane 5). Upper and lower arrowheads indicate the GAL7 transcripts and Rpb1, respectively. The molecular mass is given in kilodaltons. Relative levels of transcription and CTD phosphorylation are shown as -fold stimulation (mean ± S.E. of three separate experiments).

    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

Using yeast RNAPII holoenzyme as substrate, we have demonstrated that CTD phosphorylation catalyzed by TFIIH is significantly enhanced by a cooperative function of Gal11 and TFIIE. The observed stimulatory effect depends on both domain B of Gal11 and the subunit Tfa1 of TFIIE. Since Tfa1 binds both domain B (22, 24) and TFIIH (26), the domain B-Tfa1-TFIIH interaction may be essential for phosphorylation of the CTD in holo-RNAPII. A Gal11 derivative lacking domain A (Gal11-Delta A) stimulated CTD phosphorylation, but less efficiently than wild-type Gal11. Neither Gal11 nor TFIIE led to a significant effect on TFIIH-mediated CTD phosphorylation with core RNAPII as substrate. Since domain A of Gal11 is known to encompass the region required for interaction with holo-RNAPII as well (15), it is reasonable to suggest that the lowered stimulatory ability of Gal11-Delta A is due to its weak association with holo-RNAPII and that a tight association of Gal11 with holo-RNAPII is a prerequisite for efficient stimulation of CTD phosphorylation by TFIIH. Recent studies by Svejstrup et al. (36) have suggested that phosphorylation of the CTD causes dissociation of holo-RNAPII into the mediator and core RNAPII and that the dissociated core polymerase travels along the template to elongate the transcript. Taken all together, we suggest that the Gal11-TFIIE-TFIIH interaction is involved in regulation of the transition of holo-RNAPII to an elongation-competent complex in yeast.

The present experiments have demonstrated that neither of the mutant Gal11 proteins (Gal11-Delta A and Gal11-Delta B) is capable of enhancing transcription in a nuclear extract. These results are consistent with those of in vivo analyses showing that neither mutant is able to induce the expression of GAL7-lacZ (24). Since phosphorylation of the CTD is a key step in the transcription reaction (4), we assume that the observed enhancement of CTD phosphorylation would account for Gal11-dependent stimulation of transcription in the cell. Previous studies have strongly suggested that Gal11 reinforces interactions between TFIIE and holo-RNAPII (24). We therefore speculate that Gal11 in holo-RNAPII has at least two roles in transcription stimulation: recruitment of TFIIE to holo-RNAPII, which contributes to the formation of the preinitiation complex, and enhancement of CTD phosphorylation in cooperation with TFIIE, which triggers formation of the elongation complex.

We previously demonstrated that Gal11 stimulates basal transcription in a system reconstituted with core RNAPII and the general transcription factors (24). However, we failed to show a significant effect of Gal11 on CTD phosphorylation in core RNAPII (Fig. 1A). On the other hand, Li and Kornberg (35) have shown that CTD phosphorylation is dispensable for transcription initiation in the reconstituted system. These observations led us to speculate that Gal11 also regulates other enzymatic activities such as ATPase and DNA helicase of TFIIH besides its CTD kinase activity through TFIIE function since mammalian TFIIE has been shown to regulate TFIIH ATPase and DNA helicase activities (28-30). Although many more experiments are required to clarify the exact role of Gal11 in transcription, this work has clearly documented functional interactions among Gal11, TFIIE, and TFIIH and consequently further supports a model where Gal11 is involved in the transition from initiation to elongation in the transcription process.

    ACKNOWLEDGEMENTS

We thank Drs. Roger D. Kornberg, William J. Feaver, Young-Joon Kim, and Jesper Q. Svejstrup for providing plasmids and yeast general transcription factors.

    FOOTNOTES

* This work was supported by grants-in-aid for scientific research (to H. S.) from the Ministry of Education, Science, Sports, and Culture of Japan.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ To whom correspondence should be addressed: Tel.: 81-76-265-2588; Fax: 81-76-234-4360; E-mail: sakurai{at}kenroku.ipc.kanazawa-u.ac.jp.

1 The abbreviations used are: RNAPII, RNA polymerase II; TF, transcription factor; TBP, TATA-binding protein; CTD, carboxyl-terminal domain.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

  1. Sayre, M. H., Tschochner, H., and Kornberg, R. D. (1992) J. Biol. Chem. 267, 23376-23382[Abstract/Free Full Text]
  2. Conaway, R. C., and Conaway, J. W. (1993) Annu. Rev. Biochem. 62, 161-190[CrossRef][Medline] [Order article via Infotrieve]
  3. Roeder, R. G. (1996) Trends Biochem. Sci. 21, 327-335[CrossRef][Medline] [Order article via Infotrieve]
  4. Zawel, L., and Reinberg, D. (1995) Annu. Rev. Biochem. 64, 533-561[CrossRef][Medline] [Order article via Infotrieve]
  5. Young, R. A. (1991) Annu. Rev. Biochem. 60, 689-715[CrossRef][Medline] [Order article via Infotrieve]
  6. McCracken, S., Fong, N., Yankulov, K., Ballantyne, S., Pan, G., Greenblatt, J., Patterson, S. D., Wickens, M., and Bentley, D. (1997) Nature 385, 357-361[CrossRef][Medline] [Order article via Infotrieve]
  7. Feaver, W. J., Gileadi, O., Li, Y., and Kornberg, R. D. (1991) Cell 67, 1223-1230[Medline] [Order article via Infotrieve]
  8. Svejstrup, J. Q., Vichi, P., and Egly, J.-M. (1996) Trends Biochem. Sci. 21, 346-350[CrossRef][Medline] [Order article via Infotrieve]
  9. Nonet, M., Sweetser, D., and Young, R. A. (1987) Cell 50, 909-915[Medline] [Order article via Infotrieve]
  10. Nonet, M. L., and Young, R. A. (1989) Genetics 123, 715-724[Abstract/Free Full Text]
  11. Thompson, C. M., Koleske, A. J., Chao, D. M., and Young, R. A. (1993) Cell 73, 1361-1375[Medline] [Order article via Infotrieve]
  12. Koleske, A. J., and Young, R. A. (1994) Nature 368, 466-469[CrossRef][Medline] [Order article via Infotrieve]
  13. Hengartner, C. J., Thompson, C. M., Zhang, J., Chao, D. M., Liao, S.-M., Koleske, A. J., Okamura, S., and Young, R. A. (1995) Genes Dev. 9, 897-910[Abstract]
  14. Liao, S.-M., Zhang, J., Jeffery, D. A., Koleske, A. J., Thompson, C. M., Chao, D. M., Viljoen, M., van Vuuren, H. J. J., and Young, R. A. (1995) Nature 374, 193-196[CrossRef][Medline] [Order article via Infotrieve]
  15. Barberis, A., Pearlberg, J., Simkovich, N., Farrell, S., Reinagel, P., Bamdad, C., Sigal, G., and Ptashne, M. (1995) Cell 81, 359-368[Medline] [Order article via Infotrieve]
  16. Wilson, C. J., Chao, D. M., Imbalzano, A. N., Schnitzler, G. R., Kingston, R. E., and Young, R. A. (1996) Cell 84, 235-244[Medline] [Order article via Infotrieve]
  17. Thompson, C. M., and Young, R. A. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 4587-4590[Abstract]
  18. Kim, Y.-J., Bjorklund, S., Li, Y., Sayre, M. H., and Kornberg, R. D. (1994) Cell 77, 599-608[Medline] [Order article via Infotrieve]
  19. Li, Y., Bjorklund, S., Jiang, Y. W., Kim, Y.-J., Lane, W. S., Stillman, D. J., and Kornberg, R. D. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 10864-10868[Abstract]
  20. Gustafsson, C. M., Myers, L. C., Li, Y., Redd, M. J., Lui, M., Erdjument-Bromage, H., Tempst, P., and Kornberg, R. D. (1997) J. Biol. Chem. 272, 48-50[Abstract/Free Full Text]
  21. Lee, Y. C., Min, S., Gim, B. S., and Kim, Y.-J. (1997) Mol. Cell. Biol. 17, 4622-4632[Abstract]
  22. Sakurai, H., and Fukasawa, T. (1997) J. Biol. Chem. 272, 32663-32669[Abstract/Free Full Text], and references therein
  23. Sakurai, H., Ohishi, T., Amakasu, H., and Fukasawa, T. (1994) FEBS Lett. 351, 176-180[CrossRef][Medline] [Order article via Infotrieve]
  24. Sakurai, H., Kim, Y.-J., Ohishi, T., Kornberg, R. D., and Fukasawa, T. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 9488-9492[Abstract/Free Full Text]
  25. Li, Y., Flanagan, P. M., Tschochner, H., and Kornberg, R. D. (1994) Science 263, 805-807[Medline] [Order article via Infotrieve]
  26. Bushnell, D. A., Bamdad, C., and Kornberg, R. D. (1996) J. Biol. Chem. 271, 20170-20174[Abstract/Free Full Text]
  27. Lu, H., Zawel, L., Fisher, L., Egly, J.-M., and Reinberg, D. (1992) Nature 358, 641-645[CrossRef][Medline] [Order article via Infotrieve]
  28. Drapkin, R., Reardon, J. T., Ansari, A., Huang, J.-C., Zawel, L., Ahn, K., Sancar, A., and Reinberg, D. (1994) Nature 368, 769-772[CrossRef][Medline] [Order article via Infotrieve]
  29. Serizawa, H., Conaway, J. W., and Conaway, R. C. (1994) J. Biol. Chem. 269, 20750-20756[Abstract/Free Full Text]
  30. Ohkuma, Y., and Roeder, R. G. (1994) Nature 368, 160-163[CrossRef][Medline] [Order article via Infotrieve]
  31. Ohkuma, Y., Hashimoto, S., Wang, C. K., Horikoshi, M., and Roeder, R. G. (1995) Mol. Cell. Biol. 15, 4856-4866[Abstract]
  32. Svejstrup, J. Q., Feaver, W. J., LaPointe, J., and Kornberg, R. D. (1994) J. Biol. Chem. 269, 28044-28048[Abstract/Free Full Text]
  33. Feaver, W. J., Henry, N. L., Bushnell, D. A., Sayre, M. H., Brickner, J. H., Gileadi, O., and Kornberg, R. D. (1994) J. Biol. Chem. 269, 27549-27553[Abstract/Free Full Text]
  34. Tajima, M., Nogi, Y., and Fukasawa, T. (1986) Mol. Cell. Biol. 6, 246-256[Medline] [Order article via Infotrieve]
  35. Li, Y., and Kornberg, R. D. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 2362-2366[Abstract]
  36. Svejstrup, J. Q., Li, Y., Fellows, J., Gnatt, A., Bjorklund, S., and Kornberg, R. D. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 6075-6078[Abstract/Free Full Text]


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