The photoactivated cross-linking of recombinant C-terminal domain to proteins in a HeLa cell transcription extract that comigrate with transcription factors IIE and IIF.

The C-terminal domain (CTD) of RNA polymerase II (RNAP II) is essential for the assembly of RNAP II into preinitiation complexes on some promoters such as the dihydrofolate reductase (DHFR) promoter. In addition, during the transition from a preinitiation complex to a stable elongation complex, the CTD becomes heavily phosphorylated. In this report, interactions involving the CTD have been examined by protein-protein cross-linking. As a prelude to the study of CTD interactions, the effect of recombinant CTD on in vitro transcription was examined. The presence of recombinant CTD inhibits in vitro transcription from both the DHFR and adenovirus 2 major late promoters, suggesting that the CTD is involved in essential interactions with a general transcription factor(s). Factors in the transcription extract that interact with the CTD were identified by protein-protein cross-linking. Recombinant CTD was phosphorylated at its casein kinase II site, at the C terminus of the CTD, in the presence of [S]adenosine 5′-O-(thiotriphosphate) and alkylated with azidophenacyl bromide. Incubation of azido-modified S-labeled CTD with a HeLa transcription extract followed by ultraviolet irradiation results in the covalent cross-linking of the CTD to proteins in contact with the CTD at the time of irradiation. Subsequent incubation with phenylmercuric acetate results in the transfer of S from the CTD to the protein to which it was cross-linked. The two major photolabeled bands have a M of 34,000 and 74,000. The specificity of CTD interactions was demonstrated by a reduction in photolabeling in the presence of unmodified CTD or RNAP II containing an intact CTD (RNAP IIA) but not in the presence of a CTD-less RNAP II (RNAP IIB). The S-labeled 34- and 74-kDa proteins comigrate on SDS-polyacrylamide gel electrophoresis with the β subunit of transcription factor IIE and the 74-kDa subunit of transcription factor IIF, respectively. Moreover, some of the minor S-labeled bands comigrate with other subunits of the general transcription factors.

An intriguing aspect of class II transcription is that RNA polymerase II (RNAP II) 1 contains an unusual structure known as the CTD (C-terminal domain). This highly repetitive domain located at the end of the largest RNAP II subunit consists of the consensus sequence Tyr-Ser-Pro-Thr-Ser-Pro-Ser repeated 52 times in mammalian cells. Genetic studies have established that the CTD is essential for cell viability (Nonet et al., 1987;Allison et al., 1988;Bartolomei et al., 1988;Zehring et al., 1988). The conspicuous presence of the CTD in eukaryotes in addition to its unique nature has generated considerable attention in recent years.
Basal levels of transcription catalyzed by RNAP II require the concerted action of the general transcription factors (TF) IIA, IIB, IIE, IIF, and IIH and TATA-binding protein (TBP) (for a review, see Zawel and Reinberg (1993)). Activated transcription occurs in the presence of the multisubunit TFIID complex (TBP and TBP-associated factors) (Dynlacht et al., 1991;Tanese et al., 1991) and sequence-specific TFs whose binding sites may be located proximal or distal to the promoter (Mitchell and Tjian, 1989).
In vivo, the CTD is either unphosphorylated (RNAP IIA) or heavily phosphorylated (RNAP IIO). A CTD-less enzyme (RNAP IIB) is found only in purified preparations of RNAP II as a result of facile proteolytic cleavage of the CTD. Transcription studies have shown that although RNAP IIA assembles into the preinitiation complex, transcript elongation is catalyzed by RNAP IIO (for a review, see Dahmus (1994)). However, the role or even the significance of CTD phosphorylation during the transition from initiation to elongation has yet to be established. The CTD's function also appears to be promoterspecific. During transcription from the dihydrofolate reductase (DHFR) promoter, the CTD is necessary for the assembly of the preinitiation complex (Kang and Dahmus, 1993). Transcription from the viral adenovirus 2 major late promoter (Ad2-MLP), however, proceeds efficiently in the absence of the CTD (Kim and Dahmus, 1989;Kang and Dahmus, 1993). Other studies have demonstrated that the CTD is involved in the transcriptional activation of some but not all genes (Scafe et al., 1990;Liao et al., 1991;Gerber et al., 1995).
One approach to the analysis of CTD function is to define the interactions in which the CTD participates during the course of transcription. Both genetic and biochemical studies have implicated a functional interaction between the CTD and the multisubunit TFIID (Koleske et al., 1992;Conaway et al., 1992). Indeed, a direct interaction between the CTD of RNAP IIA and TBP has been demonstrated (Usheva et al., 1992). TFIIE, via its 56-kDa subunit, has also been shown to selectively interact with RNAP IIA and thus to be sensitive to the phosphorylation state of the CTD. However, a direct and stable interaction between the CTD and TFIIE could not be rigorously established (Maxon et al., 1994). Genetic evidence suggests a functional interaction between the CTD and SIN1, a negative regulator of transcription (Peterson et al., 1991). Genetic evidence also suggests a functional interaction between the CTD and SRBs (Thompson et al., 1993). Finally, recent studies have established that yeast RNAP II holoenzyme is comprised of core RNAP II and a large multiprotein complex consisting of these SRBs as well as some of the general TFs (Thompson et al., 1993;Kim et al., 1994;Koleske and Young, 1994). The CTD may be directly involved in the interaction of RNAP II with this macromolecular complex termed the mediator (Kim et al., 1994).
A novel approach to defining CTD interactions during transcription is to photolabel CTD-interacting proteins with a cleavable, radioactive azide-based photoprobe attached to the CTD itself. These studies were patterned after previous studies that utilized azide photochemistry to cross-link Escherichia coli RNA polymerase to nascent RNA transcripts Meares, 1983a, 1983b). In this report, recombinant CTD is first labeled by phosphorylation with casein kinase II in the presence of [ 35 S]ATP␥S. Only the most C-terminal serine is flanked by acidic residues and is phosphorylated by CKII. The photoprobe is then coupled to the 35 S-thiophosphate. This procedure results in the positioning of the photoprobe at a unique site at the very C terminus of the CTD. The probe consists of an aryl azide, which can be photoinduced to covalently cross-link to nearby proteins in the presence of ultraviolet light. Following cleavage by phenylmercuric acetate, the 35 S is transferred from the CTD to the protein to which it was cross-linked. Therefore, the term "photolabel" is used here to indicate the transfer of radioactive label from the CTD to CTD-interacting proteins and is distinct from the term "photocross-link." The studies presented here demonstrate that the CTD interacts with a limited number of proteins in a HeLa cell transcription extract and that some of these proteins comigrate on SDS-polyacrylamide gel electrophoresis with a subset of the general TFs.

EXPERIMENTAL PROCEDURES
Materials-Glutathione-agarose, human thrombin, benzamidine-Sepharose, azidophenacyl bromide, phenylmercuric acetate, phosphorylase b, and phosphorylase kinase were purchased from Sigma. [ 35 S]ATP␥S, [␥-32 P]ATP, and En 3 Hance were purchased from DuPont NEN. [␣-32 P]CTP (800 Ci/mmol) was purchased from Amersham Corp. Proteinase K was purchased from Boehringer Mannheim. Ultrapure ribonucleotides and protein molecular weight markers were purchased from Pharmacia Biotech Inc. Diagnostic film was purchased from Kodak. Diethylaminoethyl cellulose (DE52) was obtained from Whatman, and Bio-Gel P-10 was obtained from Bio-Rad. Expression plasmid pGCTD was kindly provided by W. Dynan (University of Colorado, Boulder). Glutathione S-transferase (GST) was kindly provided by V. Lippuner and C. Gasser (University of California, Davis). Amino acid analysis was done by the Protein Structure Laboratory of University of California, Davis.
Purification of Mouse Recombinant CTD-The fusion protein glutathione S-transferase-CTD (GST-CTD) was purified by a modification of the method described in Peterson et al. (1992). Competent E. coli cells were transformed with pGCTD plasmid, which contains sequences encoding the GST-CTD fusion protein. Exponentially growing cultures (A 600 ϭ 0.6) were induced with 0.5 mM isopropyl-1-thio-␤-D-galactopyranoside at 30°C for 7-9 h. Cells were centrifuged at 3800 ϫ g for 10 min (4°C) and resuspended at 20 ml/liter of culture volume in Buffer A. The cells were frozen in liquid nitrogen and quickly thawed in a 24°C water bath. Lysozyme was added to a final concentration of 100 g/ml, and the mixture was incubated on ice for 20 min. Triton X-100 was then added to a final concentration of 1%, and the mixture was incubated on ice for an additional 15 min. The viscous mixture was sonicated on ice until the viscosity decreased significantly and then centrifuged at 6800 ϫ g for 20 min (4°C). The supernatant was brought to a final volume of 500 ml/12 liters of culture volume with Buffer A containing 1% Triton X-100.
Glutathione-agarose, equilibrated with Buffer A containing 1% Triton X-100, was added to the supernatant mixture at a ratio of 1.25 ml of resin/liter of culture volume, and the mixture was incubated for 2 h at 4°C with constant rotation. At 4°C, the resin was first washed with 20 volumes of buffer containing 50 mM Tris-HCl, pH 7.9, 1 M NaCl, 15 mM ␤-mercaptoethanol, 20 g/ml phenylmethylsulfonyl fluoride, and 1 g/ml each pepstatin A, leupeptin, and soybean trypsin inhibitor and then washed with 20 volumes of phosphate-buffered saline containing 1% Triton X-100, 15 mM ␤-mercaptoethanol, 20 g/ml phenylmethylsulfonyl fluoride, and 1 g/ml pepstatin A, leupeptin, and soybean trypsin inhibitor. The washed resin was then poured into a column, and the GST-CTD fusion protein was eluted with 3 column volumes of Buffer A containing 15 mM glutathione and 20% glycerol. Column fractions were analyzed by 10% SDS-polyacrylamide gel electrophoresis and silver staining.
The peak fractions containing GST-CTD were pooled and subjected to (NH 4 ) 2 SO 4 fractionation. Approximately 90% of GST-CTD was precipitated in the presence of 30% (NH 4 ) 2 SO 4 . The 30% (NH 4 ) 2 SO 4 precipitate was dissolved in and dialyzed against 50 mM NH 4 HCO 3 , pH 8.0. The GST-CTD protein concentration was estimated by SDS-polyacrylamide gel electrophoresis, silver staining, and comparison to markers. Human thrombin was added to the GST-CTD fraction at a concentration of 10 units/mg of GST-CTD, and digestion was allowed to take place for 1 h at 24°C. To remove thrombin from the digested sample, benzamidine-Sepharose equilibrated with 50 mM NH 4 HCO 3 , pH 8.0, was added at a 2000 -4000-fold molar excess of thrombin, and the mixture was rotated for 1-2 h at 4°C. The mixture was pipetted into a microcolumn (Isolab), and the eluate was collected first by gravity and then by centrifugation. To remove GST from the digested sample, glutathione-agarose equilibrated with 50 mM NH 4 HCO 3 , pH 8.0, was added at an approximately 400-fold molar excess of GST, and the mixture was rotated for 1 h at 4°C. The eluate containing recombinant CTD (rCTD) was collected as described above. A representative preparation of rCTD from a 6-liter culture yielded approximately 140 g in a volume of 400 l at a concentration of 8.7 M as determined by amino acid analysis.
In Vitro Transcription Reactions-The DE0.25 transcription extract, DNA templates containing either the DHFR or Ad2-ML promoter, Sp1, and RNAPs IIA and IIB were prepared as described previously (Kang and Dahmus, 1993).
Transcription reactions were carried out as described previously (Kang and Dahmus, 1993) with the following modification. Increasing amounts of either GST-CTD or rCTD (as indicated in the figure legends) were preincubated with the DE0.25 extract and DNA template(s) (as indicated) in the absence of RNAP IIA for 15 min. RNAP IIA was then added, and the reaction was incubated for 45 min to allow preinitiation complex assembly. Reactions were stopped after an additional incubation for 15 min in the presence of ribonucleotides. Quantitation of specific run-off transcripts was carried out by phosphor-imaging analysis (Fuji BAS1000).
Preparation of 35 SpCTD-Purified rCTD (195 pmol) was incubated with 2 M [ 35 S]ATP␥S (Ͼ1300 Ci/mmol) in the presence of 45 units of casein kinase II in a total reaction volume of 210 l containing 25 mM Hepes-KOH, pH 6.8, 27 mM KCl, 9 mM MgCl 2 , 0.05 mM EDTA, 16% glycerol, and 0.025% Tween 80 for 30 min at 37°C. Casein kinase II was purified from calf thymus as described previously (Dahmus, 1981). The reaction was loaded onto a DE52 column previously equilibrated with Buffer B. The major fraction of 35 SpCTD came out in the flow-through and in the first few fractions of the wash, while casein kinase II and [ 35 S]ATP␥S were retained on the column. The specific activity of 35 SpCTD of an average preparation was approximately 4 ϫ 10 Ϫ2 Ci/pmol. Preparation of N 3 R 35 SpCTD-All manipulations involving azido compounds were carried out under reduced lighting using a 6-watt incandescent bulb. A typical reaction contained 0.39 M 35 SpCTD, 3.9 mM NaHCO 3 , 3.9 mM azidophenacyl bromide, and 30% methanol. Azidophenacyl bromide, which is very insoluble in water, was initially dissolved into methanol and was the last reagent to be added to the reaction. After the reaction was incubated for 45 min at 24°C, N 3 R 35 SpCTD was purified by gel filtration chromatography using P-10 resin previously equilibrated with buffer containing 20 mM Hepes-KOH, pH 7.9, 100 mM KCl, 0.2 mM EDTA, 20% glycerol, and 0.025% Tween 80.
Photolabeling of Proteins in a Transcription Extract with N 3 R 35 SpCTD-Reactions (18 l) containing 11 l of DE0.25 extract or 8 pmol of each rTF (as indicated in the figure legends) along with 0.2-0.4 pmol of N 3 R 35 SpCTD (as indicated) in final buffer conditions of 30 mM Hepes-KOH, pH 7.9, 80 mM KCl, 7 mM MgCl 2 , 0.15 mM EDTA, 15.5% glycerol, and 0.015% Tween 80 were incubated in the dark for 60 min at 24°C. The reactions were transferred to individual wells in a microtiter plate, and the plate was placed in a glass Pyrex dish containing a thin layer of water. The dish containing the microtiter plate was then placed on a Fotodyne transilluminator ( max ϭ 300 nm) and irradiated for 5 min. Cleavage of photocross-linked proteins was achieved by the addition of 20 l of phenylmercuric acetate (PMA) (saturated solution in 0.1% SDS) and incubation overnight at room temperature in the dark. Reactions containing cross-linked proteins without cleavage were also incubated overnight after the addition of 20 l of 0.1% SDS. Dithiothreitol was then added to a final concentration of 10 mM to reduce any azide remaining in the reaction. Reactions were analyzed by electrophoresis on a 5-17% gradient SDS-polyacrylamide gel. Gels were soaked in En 3 Hance according to the manufacturer's instructions before they were dried and exposed to diagnostic x-ray film.

Recombinant CTD Inhibits in Vitro
Transcription-To determine whether the CTD of RNAP IIA is involved in important and perhaps critical interactions during transcription, increasing amounts of rCTD were added to transcription reactions containing either the Ad2-ML or DHFR promoter. Run-off transcription produces 560-nucleotide and 295-nucleotide transcripts from the purified templates containing the Ad2-ML and DHFR promoters, respectively. The DE0.25 transcription extract used for these transcription reactions requires the addition of exogenous RNAP II for transcriptional activity (Kang and Dahmus, 1993). Recombinant CTD was expressed as a fusion protein with GST in E. coli cells. With the addition of increasing amounts of GST-CTD, transcription diminished from each promoter (Fig. 1A, lanes 1-4 and 6 -9). At the highest concentration of GST-CTD added, inhibition was 67% from the Ad2-MLP (lane 4) and 77% from the DHFR promoter (lane 9). The fact that an equimolar amount of GST at the highest concentration did not inhibit transcription from either promoter suggests that the inhibition is specifically caused by the CTD (Fig. 1A, lanes 5 and 10).
To confirm that the CTD moiety is responsible for the inhibition of transcription, purified rCTD was added to transcription reactions containing either the Ad2-ML or DHFR promoter. Because the GST-CTD fusion protein contains a genetically engineered recognition site for thrombin between the two proteins, the CTD moiety was released by digestion of the fusion protein with thrombin and purified as described under "Experimental Procedures." The presence of increasing amounts of purified rCTD, up to 6.2 pmol, results in a 74% inhibition of transcription from the Ad2-MLP (Fig. 1B, lanes  1-3). Curiously, the addition of 18.6 pmol of rCTD or a 3-fold higher amount caused only a 30% inhibition (Fig. 1B, lane 4). Although the reason for the biphasic response is not known, this effect was reproducible. The presence of increasing amounts of rCTD, up to 26.0 pmol, results in a 58% inhibition of DHFR transcription (Fig. 1B, lanes 5-8). Thus, the presence of excess rCTD leads to an inhibition of transcription, suggesting that an essential TF(s) is being sequestered by the rCTD.
N 3 R 35 SpCTD Photolabels a Limited Number of Proteins in a Transcription Extract-In an effort to identify the contacts made by the CTD during the initial stages of transcription, a derivatized rCTD containing an azide photoprobe was used to cross-link proteins in a transcription extract. The placement of the photoprobe within the CTD was accomplished by phosphorylation of rCTD with casein kinase II in the presence of [ 35 S]ATP␥S and subsequent alkylation of 35 SpCTD with azidophenacyl bromide to generate N 3 R 35 SpCTD ( Fig. 2A). Photolabeling of CTD-interacting proteins was accomplished by incubation of N 3 R 35 SpCTD with a transcription extract followed by irradiation and then cleavage of cross-linked proteins by PMA (Fig. 2B). Because PMA specifically cleaves at the 35 S-P bond, the radioactive label of N 3 R 35 SpCTD is thus transferred to the protein(s) in contact with the CTD at the time of irradiation.
The DE0.25 transcription extract, though depleted of RNAP II and less crude than a whole cell or nuclear extract, still contains a multitude of proteins of various sizes as shown in a silver-stained gel (Fig. 3A, lanes 1 and 2). This is the same extract used in the transcription experiments shown in Fig. 1. Although the calculated molecular weight of rCTD is approximately 40,000, the M r is approximately 70,000, due to aberrant mobility on SDS-polyacrylamide gel electrophoresis. A previously reported M r of 65,000 was obtained for a CTD derived by CNBr cleavage of calf thymus RNAP subunit IIa (Cadena and Dahmus, 1987). The difference in M r may be due to the presence of additional amino acids on rCTD and/or the variation in the polyacrylamide gel electrophoresis system used. The M r of N 3 R 35 SpCTD is the same as that of rCTD (Fig. 3B, lane 1). Both the spectrum of photocross-linked proteins following irradia- SpCTD, which is then reacted with azidophenacyl bromide to produce the final product, N 3 R 35 SpCTD. B, the reactions in which proteins in the transcription extract can be photolabeled by virtue of their interaction with N 3 R 35 SpCTD are shown. Irradiation by ultraviolet light (300 nm) photoactivates the azide moiety of N 3 R 35 SpCTD into a highly reactive nitrene, which covalently cross-links the CTD to proteins in contact with the CTD at the time of irradiation. Cleavage of the 35 S-P bond with phenylmercuric acetate results in the transfer of the radioactive label from the rCTD to the protein that it contacts. tion (Fig. 3B, lane 3) and the spectrum of 35 S-labeled proteins (photolabeled) following cleavage of photocross-linked proteins (Fig. 3B, lane 4) consist of only a limited number of labeled bands. This indicates that the CTD interacts with a specific subset of proteins present in the transcription extract. The three most intense photolabeled bands have a M r of 34,000, 70,000, and 74,000 (Fig. 3B, lane 4). The 70-kDa band is either 35 S-labeled CTD, which could result from inter-or intramolecular cross-linking of N 3 R 35 SpCTD itself, or N 3 R 35 SpCTD, which was not cleaved by PMA (see below). The latter is unlikely, given the high efficiency of cleavage ( Fig. 3B; compare  lanes 1 and 2). Proteins corresponding to a M r of 76,000 and 102,000 were also specifically labeled in the transcription extract (see Fig. 3B, lane 4). The degree of specificity displayed in this photolabeling approach is remarkable in light of the complexity of the DE0.25 extract.
The specificity of photolabeling was also demonstrated by the following controls. Both the formation of cross-linked and 35 Slabeled proteins in the transcription extract are photosensitive. When the reactions were not irradiated, neither photocrosslinked nor photolabeled bands were observed (Fig. 3B, lanes 1  and 2, respectively). Cross-linking is also dependent on a reactive azide moiety. The addition of dithiothreitol, which reduces the azide to an unreactive amine, abolishes the ability of N 3 R 35 SpCTD to photocross-link to proteins (Fig. 3B, lane 9). Furthermore, in the absence of the azide moiety (N 3 R), 35 SpCTD does not photocross-link or photolabel proteins in the extract (Fig. 3B, lanes 10 -12). The labeled bands are proteins and not nucleic acids, as demonstrated by the sensitivity of the photolabeled bands to proteinase K (Fig. 3B, compare lane 5 with buffer control in lane 6). Finally, the presence of photolabeled proteins is dependent on the presence of the HeLa cell transcription extract. No photolabeled bands except for the 70-kDa band were observed when N 3 R 35 SpCTD was incubated in the absence of the DE0.25 transcription extract (Fig. 3B,  lanes 7 and 8). As previously mentioned, the 70-kDa band likely corresponds to 35 S-labeled CTD derived from intra-or intermolecular cross-linking of N 3 R 35 SpCTD. Both the absence of a photocross-linked band (CTD dimer, etc.) (Fig. 3B, lane 7) and the near absence of the N 3 R 35 SpCTD band in nonirradiated, cleaved reactions (Fig. 3B, lane 2 and Fig. 4, lane 2) suggest that the 70-kDa band is a result of intramolecular cross-linking. These experiments establish the specificity of the cross-linking reaction and indicate that N 3 R 35 SpCTD can be used to identify proteins that interact with the CTD.
Photolabeling of the 34-and 74-kDa Proteins by N 3 R 35 Sp-CTD Is Effectively Competed by RNAP IIA and rCTD-The photolabeling results in Fig. 3B indicate that rCTD interacts primarily with two proteins in the DE0.25 transcription extract with M r of 34,000 and 74,000. To determine whether the 34and 74-kDa proteins were photolabeled specifically as a result of their interaction with N 3 R 35 SpCTD, RNAP IIA, RNAP IIB, and rCTD were added individually to the reactions as potential competitors of these interactions. Reactions contained RNAP II (0.18 pmol or 20 milliunits) and the DHFR template in order to allow preinitiation complex formation. RNAP IIA but not RNAP IIB can form preinitiation complexes on the DHFR promoter (Kang and Dahmus, 1993). In the presence of approximately the same molar amount of N 3 R 35 SpCTD (0.22 pmol), RNAP IIA efficiently reduced the photolabeling of the 34-and 74-kDa proteins (Fig. 4, compare lanes 3 and 4 with lanes 5 and  6). However, the presence of RNAP IIB did not diminish the photolabeling of these proteins (Fig. 4, compare lanes 3 and 4  with lanes 7 and 8). The photolabeling of the 34-and 74-kDa proteins was effectively abolished by the presence of a large molar excess of unmodified rCTD (39 pmol) (Fig. 4, lanes 9 and  10). The fact that the 70-kDa band was not affected by either RNAP IIA or rCTD also suggests that it results from intramolecular cross-linking of N 3 R 35 SpCTD (compare the 70-kDa band in lanes 4, 6, and 10). Therefore, the 34-and 74-kDa photolabeled bands result from specific interactions with the CTD.
The 34-and 74-kDa Photolabeled Bands Comigrate with TFIIE-␤ and RAP74, Respectively-The observation that rCTD FIG. 3. Specificity of photolabeling by N 3 R 35 SpCTD. A, proteins present in the DE0.25 transcription extract were analyzed by electrophoresis on a 5-17% gradient SDS-polyacrylamide gel and silver staining. Lanes 1 and 2 contain 0.5 and 1.0 l of transcription extract, respectively. Lane 3 contains molecular mass markers, with their sizes (ϫ 10 Ϫ3 kDa) indicated on the right. B, reactions in lanes 1-9 contained 0.34 pmol of N 3 R 35 SpCTD, and reactions in lanes 10 -12 contained 0.22 pmol of 35 SpCTD. All reactions with the exception of lanes 7 and 8 contained 11 l of DE0.25 extract. Lanes with h (ϩ) at the top were irradiated for 5 min at 300 nm, and lanes with PMA (ϩ) were incubated with phenylmercuric acetate overnight. Reactions in lanes 5 and 6 were incubated with 100 g/ml proteinase K or proteinase K buffer (10 mM Tris-HCl, pH 7.9, 2.5 mM calcium acetate), respectively, for 85 min at 37°C after PMA incubation. In the reaction in lane 9, N 3 R 35 SpCTD was incubated with 33.3 mM dithiothreitol for 15 min prior to addition of DE0.25 extract. All of the reactions were loaded on a 5-17% gradient polyacrylamide-SDS gel which was enhanced, dried, and exposed to diagnostic x-ray film for several weeks. The positions of N 3 R 35 SpCTD and 35 SpCTD are indicated on the left and right, respectively. M1 and M2 are marker lanes and contain 32 P-labeled phosphorylase a and 32 P-labeled autophosphorylated ␤ subunit of casein kinase II, respectively. The sizes (ϫ 10 Ϫ3 kDa) of these markers are indicated on the left. inhibits transcription from class II promoters suggests that the CTD interacts with an essential TF and that the 34-and 74-kDa photolabeled proteins may correspond to subunits of specific TFs. With this view in mind, photolabeling reactions were carried out with equimolar amounts of each of the recombinant TFs (rTFs) incubated with N 3 R 35 SpCTD in the absence of transcription extract. Surprisingly, rTFIIB (33 kDa), ␣ (56 kDa) and ␤ (34 kDa) subunits of rTFIIE and RAP74 (74 kDa) and RAP30 (30 kDa) subunits of rTFIIF were photolabeled when incubated with N 3 R 35 SpCTD alone (Fig. 5A, lanes 3-8).
Especially noteworthy was the intense photolabeling of RAP74 as compared with RAP 30 (Fig. 5A, lane 8). When equimolar amounts of rTFIIB, -IIE, and -IIF were incubated together with N 3 R 35 SpCTD, subunits of each of the rTFs were again photolabeled, with RAP74 being photolabeled the most intensely (Fig. 5A, lane 10). Contrary to results from previous studies (Usheva et al., 1992), rTBP (38 kDa) was not efficiently photolabeled under these conditions relative to the other rTFs (Fig.  5A, lanes 11 and 12). An autoradiogram of a longer exposure, however, revealed that rTBP was in fact photolabeled weakly (Fig. 5B, lane 12). In the interpretation of these results it is important to remember that the photoprobe is at the very C terminus of the CTD and therefore may not contact proteins that bind to internal or N-terminal sites on the CTD.
The 34-and 74-kDa proteins photolabeled prominently by N 3 R 35 SpCTD in the DE0.25 extract comigrate with the ␤ subunit of TFIIE and the RAP74 subunit of TFIIF, respectively (Fig. 5A, compare lane 2 with lanes 6, 8, and 10). rTFIIB migrates faster than the 34-kDa photolabeled band and produced a doublet of photolabeled bands (as in Fig. 5A, lane 10) when reactions containing the DE0.25 extract were supplemented with rTFIIB (data not shown). In addition, a more careful analysis of a longer exposure of the same gel (Fig. 5B) reveals that some of the weaker photolabeled bands comigrate with RAP30, the ␣ subunit of TFIIE, and TBP (Fig. 5, A and B, lanes 2).

DISCUSSION
The CTD is an essential but enigmatic structure found in the largest subunit of RNAP II. The objective of the studies presented here is to determine the interactions between the CTD and proteins present in a transcription extract using a cleavable, radioactive azide-based photoprobe. The demonstration that rCTD inhibits in vitro transcription from either the Ad2-ML or DHFR promoter indicates that the CTD plays an important if not essential role in the transcription of class II genes. The simplest interpretation of these results is that rCTD is interacting with a general TF(s) that the CTD of RNAP IIA would normally contact during the course of transcription.
Notably, the addition of rCTD inhibits transcription not only from the DHFR promoter but also from the Ad2-MLP. Previous studies have shown that in vitro transcription from the Ad2-MLP does not require the CTD and that the CTD-less RNAP IIB readily assembles into preinitiation complexes on the Ad2-MLP (Kang and Dahmus, 1993). On the other hand, these same studies have shown that in vitro transcription from the DHFR promoter requires the CTD, and that RNAP IIB is unable to assemble into preinitiation complexes on the DHFR promoter. Therefore, the fact that rCTD is able to inhibit transcription from the Ad2-MLP with RNAP IIA indicates that rCTD is interacting with an essential TF(s) required in the transcription from both the DHFR and Ad2-ML promoters.
In an attempt to identify factors that bind the CTD, an  5 and 6), 0.18 pmol of RNAP IIB and 0.19 pmol of DHFR template (lanes 7 and 8), or 39 pmol of rCTD (lanes 9 and 10). The reactions were treated as follows: not irradiated (lanes 1 and 2); irradiated (lanes 3-10); and incubated with PMA overnight (lanes 2, 4, 6, 8, and 10). The analyses of photocross-linked and photolabeled proteins are as described in the legend to azido-modified 35 SpCTD (N 3 R 35 SpCTD) was added to a transcription extract and irradiated with ultraviolet light to covalently cross-link the derivatized CTD with proteins that contact the CTD. The novelty of this cross-linking approach is that the photoprobe is positioned within the CTD by phosphorylation with protein kinase in the presence of [ 35 S]ATP␥S and the subsequent alkylation of the 35 S with the photoprobe. Cleavage of the cross-linked proteins results in the transfer of 35 S from the CTD to the protein in contact with the CTD at the time of irradiation. Accordingly, the specificity of the cross-linking reaction is determined by the specificity of the protein kinase that positions the 35 S within the CTD. Since casein kinase II phosphorylates the most C-terminal serine, these studies specifically identify proteins that contact the C terminus of the CTD.
Despite the myriad of proteins present in the transcription extract, only a few proteins were prominently photolabeled by N 3 R 35 SpCTD. The two most intense photolabeled bands have a M r of 34,000 and 74,000. Photoaffinity labeling was specific in that cross-linking was dependent on (a) ultraviolet irradiation, (b) a reactive aryl azide moiety, and (c) the transcription extract. Furthermore, PMA cleavage resulted in the transfer of 35 S to proteins, as indicated by proteinase K sensitivity. The photolabeling of the 34-and 74-kDa proteins by N 3 R 35 SpCTD was a direct result of their interactions with CTD as indicated by the observation that RNAP IIA and the unmodified rCTD reduced and abolished, respectively, the photolabeling of these proteins. In contrast, photolabeling of the 34-and 74-kDa proteins was unaffected by the presence of the CTDless RNAP IIB. This suggests that the 34-and 74-kDa proteins in the transcription extract may be the TFs sequestered by excess rCTD leading to the inhibition of transcription. In support of this hypothesis, the 34-and 74-kDa proteins comigrate with TFIIE-␤ and RAP74 (TFIIF), respectively. Surprisingly, TFIIB, the 56-kDa subunit of TFIIE, the 30-kDa subunit of TFIIF (RAP30), and TBP were also photolabeled with differing efficiencies when the rTF was incubated alone with N 3 R 35 SpCTD. TBP was the most weakly photolabeled of all of the factors. Upon closer examination of the photolabeled bands derived from the transcription extract, it became obvious that in addition to labeled proteins of 34, 74, 76, and 102 kDa, there were less intense bands that comigrated with TFIIE-␣, RAP30, and TBP. A total of about 25 photolabeled proteins are apparent on a long exposure of the gel. The remarkable conclusion from these observations is that in a transcription extract that contains a multiplicity of proteins, the CTD contacts only a small subset of these proteins and that within this subset many proteins correspond in mobility to subunits of the general TFs. One interpretation of this result is that the CTD interacts with a macromolecular complex in the transcription extract that contains many of the general TFs. Additional studies, however, are necessary to establish whether or not these photolabeled proteins indeed correspond to general TFs and to establish the nature of the other proteins that contact the CTD.
The observation that incubation of rTFIIB with N 3 R 35 -SpCTD results in efficient photolabeling appears to contradict the apparent absence of a photolabeled TFIIB in the transcription extract. Furthermore, there is a striking difference in the relative labeling of the putative ␣ and ␤ subunits of TFIIE in the extract compared with the labeling of rTFIIE (Fig. 5, compare lanes 2 and 6). When N 3 R 35 SpCTD is incubated with rTFIIE, the photoprobe contacts the ␣ and ␤ subunits to comparable extents, whereas in the transcription extract the photoprobe contacts primarily the ␤ subunit. The question then is why the interactions between the CTD and TFIIB/TFIIE within the context of the transcription extract differ from the interactions that occur between the CTD and rTFs alone. The differential labeling may be a consequence of the positioning of the photoprobe at the C terminus of the CTD and the precise organization of proteins within a putative macromolecular complex that interacts with the CTD. According to this interpretation, many of the general TFs have an affinity for the CTD and can contact the CTD at the location of the photoprobe when incubated alone. In the transcription extract, however, the TFs may assemble into a specific complex, in which case only TFIIE-␤ and RAP74 are accessible to the photoprobe. This interpretation is supported by the observation that when TFIIB, -IIE, and -IIF were incubated together with N 3 R 35 Sp-CTD, the photolabeling of rTFIIB diminished significantly as compared with when it was present alone (Fig. 5A, compare  lanes 4 and 10). Another determinant that has not been addressed is the relative amounts of each of the general TFs present in the DE0.25 extract. When the rTF was incubated alone with N 3 R 35 SpCTD, 8 pmol of each rTF was added to the reaction. Conceivably, the relative molar ratios of each of the general TFs may influence the pattern of photolabeling. However, since the extract is optimal for transcription from class II promoters, the relative amount of each factor present reflects what is necessary for in vitro transcription and should also reflect its relative concentration in vivo.
The results presented in this report are consistent with the recent finding that yeast RNAP II holoenzyme is comprised of core RNAP II in addition to several of the general TFs, the SRBs, and other proteins (Koleske and Young, 1994;Kim et al., 1994). The formation of the holoenzyme appears to involve a direct interaction of the RNAP II core with a mediator complex comprised of some 20 polypeptides (Kim et al., 1994). The CTD may play a direct role in the mediator-core interaction. One attractive interpretation is that mammalian cells also contain a mediator-like complex that interacts directly with the CTD and that the different intensities of photolabeling of the general TFs in the transcription extract reflect the various positioning of these factors around the CTD as well as around each other. In other words, TFIIE-␤ and RAP74 may be positioned near the end of the CTD molecule, where the photoprobe has facile access to them. The other factors may be present in a more interior location of the CTD, which makes their accessibility to the photoprobe less likely. TBP appears to be the least accessible to the photoprobe. Thus, positioning the photoprobe in a different location within the CTD may yield a different pattern of photolabeling intensities.
An alternative interpretation to the variable photolabeling intensities of the general TFs in the extract is that the intensities truly reflect the frequency with which the CTD interacts with each factor. This would mean that TFIIE-␤ and RAP74 interact with the CTD to a greater extent than any other TF, whereas TBP interacts very weakly with CTD relative to the other TFs. As previously noted, a strong interaction between TBP and the CTD was demonstrated by protein affinity studies in which a column containing one heptamer repeat sufficiently depleted a transcription extract of TBP activity such that transcription was compromised (Usheva et al., 1992).
The placement of the azido-based photoprobe on rCTD is the first step in the determination of CTD interactions. Advantages to this photoaffinity labeling method are (a) the probe can be positioned at known sites within the CTD, (b) the aryl azide is highly reactive upon irradiation, and (c) the cross-linked proteins can be identified following efficient cleavage and transfer of the radioactive label. The long term goal is to place the photoprobe on the CTD of RNAP IIA itself so that the contacts made by the CTD can be examined as RNAP II progresses through transcription.