PURIFICATION AND FUNCTIONAL ANALYSIS OF GENERAL TRANSCRIPTION FACTOR IIE*

Mammalian RNA polymerase I1 transcription factor IIE (TFIIE) was purified to apparent homogeneity. The activity copurified with polypeptides of 34 and 56 kDa. The 56-kDa subunit was sufficient for low levels of transcription activity in a transcription system reconstituted in vitro with highly purified general transcription factors and RNA polymerase 11. The 34-kDa polypeptide was found to be stimulatory. The native molecular mass of TFIIE, as determined by gel filtration, was estimated to be approximately 200 kDa, suggesting that TFIIE exists in solution as a tetramer composed of two 56-kDa and two 34-kDa polypeptides. Consistent with previous studies demonstrating an interaction of TFIIE with RNA polymerase 11, we found that the entry of TFIIE into the transcription cycle was subsequent to the entry of RNA polymerase 11.

Mammalian RNA polymerase I1 transcription factor IIE (TFIIE) was purified to apparent homogeneity. The activity copurified with polypeptides of 34 and 56 kDa. The 56-kDa subunit was sufficient for low levels of transcription activity in a transcription system reconstituted in vitro with highly purified general transcription factors and RNA polymerase 11. The 34-kDa polypeptide was found to be stimulatory. The native molecular mass of TFIIE, as determined by gel filtration, was estimated to be approximately 200 kDa, suggesting that TFIIE exists in solution as a tetramer composed of two 56-kDa and two 34-kDa polypeptides. Consistent with previous studies demonstrating an interaction of TFIIE with RNA polymerase 11, we found that the entry of TFIIE into the transcription cycle was subsequent to the entry of RNA polymerase 11. Transcription of protein-coding genes in higher eukaryotes is carried out by a multienzymatic complex that includes RNA polymerase I1 and several accessory transcription factors. One class of factors consists of DNA-binding proteins that recognize specific promoter or enhancer elements, through which they regulate gene-or tissue-specific transcription by RNA polymerase I1 (reviewed in Mitchell and Tjian, 1989;Johnson and McKnight, 1989). Another group termed general transcription factors act through the core promoter elements (TATA box and initiator motif) and are required for basal levels of specific transcription initiation at all class two promoters (for review, see Saltzman and Weinmann, 1989;Mermelstein et al., 1989). The mechanisms by which the general transcription factors determine the position and directionality of transcription initiation and the biochemical events by which they trigger the formation of the first phosphodiester bond by RNA polymerase I1 are not yet understood.
In order to answer these questions and to learn about the participation of the general transcription factors in the regulation of transcription initiation, our approach has been to isolate each factor in a pure form and to elucidate their individual activities. We have identified and extensively purified six human general transcription factors (TFIIA, -IID, * This work was supported by National Institutes of Health Grant GM 37110 and National Science Foundation Grant DMB 88-19342. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. -IIb, -IIE, -IIF, and -1IH)'~' that, together with RNA polymerase 11, are sufficient for specific transcription initiation from DNA templates containing minimal promoter elements (TATA and initiator motifs). Binding of TFIID to the TATA motif appears to be the first step in the formation of a transcription-competent complex, providing a recognition site for the association of the other general transcription factors and RNA polymerase I1 (Buratowski et Maldonado et al., 1990). Kinetic analyses and complex formation studies have suggested that TFIIA acts at an early step of complex formation Fire et al., 1984;Davison et al., 1983), presumably by facilitating binding of TFIID to the TATA box, thereby generating the DA complex (Buratowski et al., 1989;Maldonado et al., 1990). The gene encoding human TFIID has been isolated (Peterson et al., 1990;Kao et al., 1990;Hoffmann et al., 1990), and TFIIA has been purified to apparent homogeneity (Samuels and Sharp, 1986).3 TFIIB has been purified to apparent homogeneity, and a cDNA clone encoding this activity has been i~olated.~ TFIIF has also been purified to apparent homogeneity (Flores et al., 1990), and a cDNA clone encoding the small subunit of TFIIF (RAPSO; Flores et al., 1988) was isolated (Sopta et al., 1989). The roles of TFIIB and TFIIF in complex formation have also been analyzed .5s6 TFIIB associates with the DA complex, resulting in the formation of the DAB complex . It was shown by glycerol gradient sedimentation  and affinity chromatography (Sopta et al., 1985) that TFIIF binds to RNA polymerase I1 and, more recently, that this interaction accounts for the specific association of RNA polymerase I1 with the preinitiation complex (Flores et al.)? The formation of a transcription-competent complex requires, in addition to the stable association of RNA polymerase I1 and TFIIF with the DAB complex (DABPolF complex),6 the participation of at least two other activities (TFIIE and TFIIH). TFIIH is a newly identified general transcription factor that has been partially purified.' Previous studies using a partially purified TFIIE fraction have indicated that IIE is a general transcription factor that interacts with RNA polymerase I1 (Flores et ' The abbreviations used are: TF, transcription factor; Hepes, 4-(2-hydroxyethy1)-1-piperazineethanesulfonic acid; Ad-MLP, adenovirus major late promoter; HPLC, high pressure liquid chromatography; FPLC, fast protein liquid chromatography; SDS, sodium dodecyl sulfate.

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al., 1989). A better understanding of the functional properties of TFIIE has been hampered by the slow progress in its purification. In the work presented here, we report the purification to apparent homogeneity of transcription factor IIE. In agreement with previous studies indicating an interaction between TFIIE and RNA polymerase I1 Flores et al., 1989), we found that the entry of TFIIE into the transcription cycle is dependent on RNA polymerase I1 and results in the formation of a stable "putative intermediate" during the production of a transcriptioncompetent complex.
Purification of TFIZE-TFIIE was purified from HeLa cell nuclear extracts (Dignam et al., 1983). Twelve different preparations of nuclear extract (from approximately 2.5 X 10" cells, 9.8 g of protein, 1000 ml) were pooled and used as starting material for a representative purification. The first three fractionation steps (chromatography on phosphocellulose, DE-52, and HPLC DEAE-5PW columns) were as described previously by Flores et al. (1989). TFIIE activity derived from the DEAE-5PW chromatographic step (80 ml, 44 mg) was concentrated to 4 ml of using a Mono-S column (HR10/10, Pharmacia LKB Biotechnology Inc.) by step elution with buffer C (20 mM Tris; HCI buffer, pH 7.9, 0.1 mM EDTA, 20% glycerol, 10 mM 0-mercaptoethanol, 0.2 mM phenylmethylsulfonyl fluoride) containing 0.8 M KC1 and 0.01% Nonidet P-40. The material was then loaded onto a Superdex S200 (HR16/30, Pharmacia) gel filtration column equilibrated in buffer C with 0.8 M KCl. Column fractions were dialyzed against buffer C with 0.1 M KC1 and assayed for TFIIE activity as described above. Fractions containing TFIIE activity were pooled (10 ml, 2.4 mg), dialyzed, and loaded onto a Mono-S column (HR5/5, Pharmacia) equilibrated with buffer C with 0.1 M KC1. The hound material was eluted with a 25-ml linear gradient of 0.1-0.5 M KC1 in buffer C. TFIIE activity eluted between 0.25 and 0.3 M KC1. Active fractions were pooled ( 3 ml, 300 pg) and dialyzed against buffer C with 1.4 M ammonium sulfate for 2 h. TFIIE was then loaded onto a phenyl-Superose column (HR5/5, Pharmacia) equilibrated with buffer C with 1.4 M ammonium sulfate. The bound material was eluted with a linear gradient (25 ml) of 1.4-0 M ammonium sulfate in buffer C. The fractions containing TFIIE activity (50 pg, eluting between 0.4 and 0.2 M ) were pooled, dialyzed against buffer C with 1.4 M ammonium sulfate, and loaded onto a phenyl-Superose microcolumn (PC 1.6/5) using the Smart system (Pharmacia). The bound protein was first step-eluted with buffer C with 0.6 M ammonium sulfate, followed by a 2.5-ml linear gradient from 0.6 to 0.2 M ammonium sulfate in buffer C. TFIIE activity eluted a t approximately 0.3 M ammonium sulfate. Transcriptionally active fractions were stored at -80 "C.
Separation of the Subunits of TFIZE by Chromatography on a Reverse Phase Column-TFIIE (phenyl-Superose microcolumn fraction, 5 pg) was fractionated by reverse phase chromatography on an RPC C2/C18 microcolumn (PC 3.2/3, Pharmacia) using the Smart system (Pharmacia). Proteins were eluted with a linear gradient (12 ml) of 0-60% acetonitrile in 0.1% trifluoroacetic acid. Fractions of 200 pl were collected, and aliquots of 2 pl were analyzed by SDSpolyacrylamide gel electrophoresis followed by silver staining. The 34-and 56-kDa polypeptides eluted at 37 and 47% acetonitrile, respectively.
Renaturation of TFZZE Actiuity-The 34-and 56-kDa polypeptides that copurified with TFIIE activity were separated by reverse phase chromatography as described above. Fractions containing the 34-or 56-kDa polypeptides were pooled, dried in a speed vacuum concentrator, and resuspended in 40 pl of buffer C containing 6 M guanidinium HCl,O.l% Nonidet P-40, and 100 pg/ml bovine serum albumin. This suspension was incubated a t room temperature for 30 min. The guanidinium HC1 was removed by chromatography on a fast desalting column (PC 32/10, Pharmacia) equilibrated with buffer C with 0.1 M KC1. In one case, both polypeptides were mixed before removal of the guanidinium HCl. Recovery of TFIIE activity was measured using the specific transcription assay.
Estimation of the Native Molecular Mass of TFZZE-The native molecular mass of TFIIE was estimated by size exclusion chromatography of the IIE phenyl-Superose microcolumn fraction (FPLC-Superdex S200 column, HR16/30, Pharmacia). The elution volume (V,) of TFIIE was determined using a specific transcription assay for TFIIE activity and by silver staining. The native molecular mass of TFIIE was estimated by interpolation from a curve constructed using five different protein standards (apoferritin, 440 kDa; /3-amylase, 200 kDa; alcohol dehydrogenase, 150 kDa; bovine serum albumin, 68 kDa; and cytochrome c, 14 kDa). DNA Binding Assays-The protein components, as indicated in the figure legends, were incubated with a DNA fragment (approximately 0.5 ng, 5000 cpm) containing sequences from the adenovirus major late promoter (Ad-MLP) and extending from residue -40 to +20. Incubation was a t 30 "C for 30 min. Reaction mixtures (25 p l ) contained 10 mM Hepes-KOH, pH 7.9,4 mM MgClz, 5 mM (NHd)SOd, 8% (v/v) glycerol, 2% (w/v) polyethylene glycol 8000,50-60 mM KC1, 5 mM P-mercaptoethanol, 0.2 mM EDTA, and 25 pg/ml of poly(dG-dC). (dG-dC). Protein-DNA complexes were separated by electrophoresis through a 4% polyacrylamide gel containing 3% (v/v) glycerol using Tris borate/EDTA buffer, p H 8.2 (40 mM Tris, 40 mM boric acid, 1 mM EDTA) as the running buffer. Electrophoresis was performed a t 100 V until the bromphenol blue dye reached the bottom of the gel.
Other Methods-Protein kinase assay conditions were as described by Cisek and Corden (1989) and contained as substrates either histone H1, casein, a peptide containing four copies of the heptapeptide repeat present at the C terminus of the largest subunit of RNA polymerase I1 (Allison et al., 1988), the phosphorylated (IIo), or the dephosphorylated (IIa) forms of RNA polymerase 11. DNA helicase and topoisomerase assays were performed essentially as described by Sopta et al. (1989) and Darby and Vosberg (1985), respectively.

RESULTS
Purification of TFZZE-TFIIE activity was purified using a transcription assay reconstituted with the general transcription factors, RNA polymerase 11, and Ad-MLP. The first three chromatographic steps in the purification of TFIIE (phosphocellulose, DEAE-cellulose, HPLC-DEAE-5PW) were essentially as described previously by Flores et al. (1989). The purification of TFIIE was accomplished by chromatography of the DEAE-5PW protein pool on Superdex S200, Mono-S, and phenyl-Superose columns, as described under "Materials and Methods" and summarized in Table I. Purification of TFIIE to apparent homogeneity was accomplished by chromatography of the phenyl-Superose protein pool on a phenyl-Superose microcolumn as described under "Materials and Methods." This procedure resulted in TFIIE activity coeluting with polypeptides of 34 and 56 kDa (data not shown, see Fig. 2). The polypeptide compositions of the different chromatographic steps of the purification of TFIIE are shown in Fig. 1.
This highly purified preparation of TFIIE was transcriptionally active and devoid of DNA-dependent and -independent ATPase, topoisomerase, and DNA helicase activities (data not shown). The TFIIE preparation was also devoid of detectable protein kinase activity, as measured using different substrates as described under "Materials and Methods" (data not shown).
The Renatured 56-kDa S u b u n i t of TFIZE Is Sufficient for "One unit is defined as the amount of protein that catalyzed the incorporation of 1 pmol of nucleotide into a specific transcript in 60 min under the conditions specified under "Materials and Methods." The high recovery of activity on the Mono-S chromatographic step is due to the removal of an inhibitor of transcription. This procedure has been repeated four times with similar results. ND, not determined. Basal Leuels of Actiuity-The results presented above suggested that TFIIE activity resided in a heterodimer composed of 34-and 56-kDa subunits. In order to assess the native molecular mass of TFIIE, the highly purified preparation was subjected to gel filtration chromatography on a FPLC-Superdex S200 column. TFIIE activity eluted from the column with an apparent molecular mass of approximately 200 kDa (Fig.  2C). Both the 34-and 56-kDa polypeptides coeluted with TFIIE activity (Fig. 2, compare panels A and B ) and appeared to be present in stochiometric amounts, as determined by densitometric scanning of a protein gel stained with Coomassie Blue (data not shown). Together these results strongly suggest that in solution TFIIE is a tetramer composed of two 56-and two 34-kDa polypeptides.
In order to analyze further whether the 34-and 56-kDa polypeptides copurifying with TFIIE activity were both necessary for transcriptional activity, the polypeptides were separated by chromatography on a reverse phase column (Fig.  3A). The 34-and/or 56-kDa polypeptides were denatured, followed by renaturation as described under "Materials and Methods," and then assayed independently or in combination for their ability to support transcription from the Ad-MLP in a system reconstituted with the other general transcription factors and RNA polymerase 11. The 34-kDa polypeptide was unable to support transcription (Fig. 3B, lane 3 ) ; however, the 56-kDa polypeptide was capable of low levels of activity (lane 4 ) . The level of activity observed with the 56-kDa polypeptide was approximately 11% of that observed with native TFIIE (compare lane 4 with 9, respectively). The addition of renatured 34-kDa polypeptide to reaction containing the 56-kDa renatured protein resulted in an approximately 2-fold increase of transcriptional activity (compare lane 4 with lanes 5-7). A different situation was observed when the isolated 34-and 56-kDa polypeptides were renatured together. Under this condition, the level of TFIIE activity recovered was higher than that observed with the independently renatured polypeptides (Fig. 3B, compare lanes l and 2 with lanes 5-7). The recovered TFIIE activity approached approximately 40% of the native TFIIE (compare lane 2 with 9, data not shown). These results may indicate that the association of the two subunits is optimal or that the renaturation of the 34-and/or 56-kDa subunit is better when both polypeptides are renatured together. Similar results were observed when the transcription assay was reconstituted using a partially purified human TFIID protein or with recombinant human or yeast TFIID proteins isolated from overproducing Escherichia coli cells. The results presented above indicate that the 56-kDa subunit of TFIIE is sufficient for basal levels of activity and that the 34-kDa subunit is stimulatory.
The Entry of TFIIE into the Transcription Cycle Is Dependent on RNA Polymerase 11-Previous studies analyzing a partially purified preparation of TFIIE indicated that TFIIE interacted with RNA polymerase I1  and was required prior to the initiation of transcription Flores et al., 1989). We have analyzed, using a gel mobility shift assay and highly purified TFIIE, whether a specific DNA-protein complex could be isolated that resulted from the entry of TFIIE into the transcription cycle.
We have demonstrated previously that the entry of RNA polymerase I1 into the transcription cycle was dependent on a DNA-protein complex formed at the TATA motif and surrounding sequences of the Ad-MLP and included transcription factors IIA, IID, and IIB (DAB complex). In addition, we also showed that the binding of RNA polymerase I1 to the DAB complex was dependent on TFIIF. 6 We have observed that a highly purified preparation of TFIIE (renatured polypeptides) could bind to the DABPolF complex (Fig.  4B, lane 1 ). The association of TFIIE with the transcription complex resulted in the production of a DNA-protein complex (DABPolFE) migrating more slowly than the DABPolF complex on a native polyacrylamide gel (Fig. 4A, compare lanes  9-12; Fig. 4B, compare lanes 1 and 2). In agreement with previous results demonstrating an interaction of TFIIE with RNA polymerase 11, the entry of TFIIE into the transcription cycle was dependent on RNA polymerase 11. The omission of RNA polymerase I1 from a DNA-binding assay containing the Ad-MLP and factors IIA, IID, IIB, IIF, and IIE resulted in the formation of the DAB complex (Fig. 4B, lane 3 ) . The migration of the DAB complex was not affected by the presence of TFIIE (Fig. 4A, lanes 5 and 6). Also, the omission of TFIIB from the DNA-binding assay resulted in the formation of the DA complex. (Fig. 4B, lane 4 ) ; the migration of this complex was also not affected by the presence of TFIIE (Fig.  4A, compare lanes 3 and 4 ) . The formation of the DABPolFE complex was, as expected, also dependent on TFIID and TFIIA (Fig. 4B, lanes 5 and 6, respectively).
Interestingly, while we were able to demonstrate that the 56-kDa component of TFIIE was sufficient for basal levels of transcriptional activity, we found that both the 56-and 34-kDa polypeptides copurifying with TFIIE activity were required to form the DABPolFE complex (Fig. 4B, lane 1 ) . Independently, neither the 34-nor the 56-kDa polypeptides altered the mobility of the DABPolF complex in a mobility shift assay (data not shown). It is possible that the 56-kDa subunit binds weakly to the complex and requires the 34-kDa subunit for stable isolation using the mobility shift assay. On the other hand, it is also possible that the 34-kDa component of TFIIE is necessary for transcription and binding of TFIIE to the DABPolF complex and is present as a contaminant in the TFIIH protein preparation, the only factor not present in the DNA-binding assay but present in the transcription assay (see "Discussion"). Another less likely possibility is that the 56-kDa polypeptide, in the absence of the 34-kDa component of TFIIE, can bind stably to the complex; however, any change (in conformation, charge, or mass) induced to the DABPolF complex is such that it cannot be resolved using the mobility shift assay.
The results presented above demonstrate that TFIIE enters the transcription cycle after RNA polymerase 11, in agreement with studies demonstrating an interaction between TFIIE and RNA polymerase 11.

DISCUSSION
In this study, we report the purification to apparent homogeneity of TFIIE. Transcription factor IIE copurified with polypeptides of 34 and 56 kDa and eluted from gel filtration columns performed in high salt (0.8 M KC1) with an apparent mass of approximately 200 kDa. These results suggest that TFIIE exists in solution as a tetramer composed of two 56and two 34-kDa polypeptides. However, we observed that the 56-kDa polypeptide was sufficient for low levels of activity and that addition of the 34-kDa polypeptide resulted in stimulation of transcription. Studies analyzing TFIID, the TATAbinding protein, have indicated that recombinant yeast and human TFIID proteins (isolated from overproducing E. coli cells) were capable of directing basal levels of transcription, but not capable of mediating the response to activators (Lewin, 1990). The existence of molecules or coactivators capable of transmitting activation through TFIID and the other general transcription factors has been postulated to explain this finding (Pugh and Tjian, 1990;Lewin, 1990, and references therein). We have found that both the 56-and 34-kDa subunits of TFIIE were capable of mediating basal levels of transcription independently of the source of TFIID (see Fig. 4B). The role of the 34-kDa polypeptide in response to upstream activators has not yet been analyzed. However, the renaturation data (Fig. 4) strongly suggests that the 34-kDa subunit of TFIIE is primarily involved in basal transcription, probably by mediating a correct interaction between the 56-kDa subunit of TFIIE and the other general transcription factors.
It is interesting to note that a factor termed FE, with properties similar to TFIIE, was recently described by Kawaguchi et al. (1990). They observed that FE was capable of binding to RNA polymerase I1 and copurified with polypeptides of 33 and 60 kDa. However, they indicated, using renaturated polypeptides isolated by chromatography on a denaturing gel filtration column, that the 33-kDa polypeptide was sufficient to reconstitute FE activity in a system reconstituted with the different general transcription factors and RNA polymerase 11. It is possible that TFIIE and FE are two different factors; however, the similarity of the polypeptides copurifying with the activities (TFIIE and FE) and the observation that both factors are capable of interacting with RNA polymerase I1 strongly suggests that they are the same. It is possible that the observed differences may be explained by the purity of the other factors and/or RNA polymerase I1 used in the transcription assay. Even though our transcription assay is reconstituted with highly purified general transcription factors and RNA polymerase 11, it is currently difficult to assess whether the 34-kDa component of TFIIE is present as a contaminant in the other factor preparations. We have found that a component of TFIIA is a 34-kDa p~lypeptide,~ that TFIIH activity copurifies with polypeptides of 35 and 90 kDa,' and that TFIIB activity is contained in a 33-kDa polypeptide .4 Different general transcription factors have been isolated from rat liver extracts (Conaway and Conaway, 1989;Conaway et al., 1990); it is currently unknown which of those factors corresponds to TFIIE. The molecular cloning of the different general transcription factors will certainly answer most of these questions. The purification of TFIIE to apparent homogeneity and isolation of the polypeptides composing TFIIE activity is a step toward this goal.