Factors Involved in Specific Transcription by Mammalian RNA Polymerase I1 RNA POLYMERASE 11-ASSOCIATING PROTEIN 30 IS AN ESSENTIAL COMPONENT OF TRANSCRIPTION FACTOR IIF*

Transcription from class I1 promoters requires five general factors, IIA, IIB, and IIF, in addition to RNA polymerase I1 for basal levels of transcription Molecular Biology of RNA: Perspective8 A protein fraction containing transcription factors (TF) IIE and IIF was able to reconstitute transcription from the adenovirus major late promoter when added to extracts depleted of the RNA polymerase 11-associating J. Biol. Chem. 260, 10353-10360). Studies with monoaffinity-purified antibodies directed against RAP 30 demonstrated, by Western blot analysis, that RAP 30 copurifies on five columns with transcription factor IIF. That RAP 30 is a functional component of TFIIF was also demonstrated; preincubation of anti- RAP 30 antibodies with purified TFIIF inhibited transcription. Inhibition of transcription was overcome by the addition of purified TFIIF. RAP 30 is an integral part of a preinitiation complex; the incubation of all the general transcription factors with a promoter-con-taining DNA, prior to the addition of the anti-RAP

Studies with monoaffinity-purified antibodies directed against RAP 30 demonstrated, by Western blot analysis, that RAP 30 copurifies on five columns with transcription factor IIF. That RAP 30 is a functional component of TFIIF was also demonstrated; preincubation of anti-RAP 30 antibodies with purified TFIIF inhibited transcription. Inhibition of transcription was overcome by the addition of purified TFIIF. RAP 30 is an integral part of a preinitiation complex; the incubation of all the general transcription factors with a promoter-containing DNA, prior to the addition of the anti-RAP 30 antibodies, resulted in the formation of a DNA-protein complex that was not inhibited by the antibodies. Incubation of the transcription factors in the absence of a promoter-containing DNA resulted in a complex that was partially resistant to the antibodies.
Several soluble cell-free systems that allow accurate initiation of transcription by RNA polymerase I1 on exogenously added DNA templates have been developed (1-3). The fractionation of these extracts has led to the identification of several factors required for transcription of class I1 promoters * This work was supported by National Institutes of Health Grant GM 37120, New Jersey Commission on Cancer Research Grant 687-035, by a grant from the Foundation of the University of Medicine and Dentistry of New Jersey (to D. R.), and by grants from the Medical Research Council of Canada (to. J. G.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
$ (4-9). We have identified five general factors, TFIIA,' -IIB, -IID, -IIE, and -IIF, that operate via the TATA sequence and were required, in addition to RNA polymerase 11, for basal levels of transcription (10). TFIID is a specific DNA binding protein that binds to the TATA box element (11). TFIIA and TFIID are required for the formation of the committed complex at the promoter (9). The transcriptional activity of factors IIB, IIE, and IIF appears to be mediated via proteinprotein interactions. Reinberg and Roeder (8) have shown that a preparation containing TFIIE and TFIIF could be made to co-sediment with RNA polymerase I1 and with TFIIB.
Other groups have also reported the isolation of factors required for transcription of class I1 promoters. Zheng et al. (12) have isolated a 27,000-dalton protein (BTF3) from HeLa cells and demonstrated that purified BTF3 co-sedimented with RNA polymerase 11. Sopta et al. (13), utilizing RNA polymerase I1 affinity columns, have isolated three RNA polymerase 11-associating proteins, RAP 30, RAP 38, and RAP 74. Burton et al. (14) showed that at least one of these RAPS was required for accurate initiation of transcription in vitro. RAP 38 was shown not to be required for transcription initiation but seemed to be an elongation factor (14,18) similar to 1 1 s (15) and SI1 (16,17). Furthermore, Burton et al. (18,19) showed that monoaffinity-purified antibodies against RAP 30 inhibited transcription when added to HeLa cell nuclear extracts and that RAP 30 is a generally required initiation factor. It was also demonstrated that a complex of RAP 30 and RAP 74 was required to restore accurate transcriptional activity to an extract depleted of these factors by immunoprecipitation with anti-RAP 30 antibodies (19).
In this article we demonstrated that TFIIF, a newly identified facto3 purified from the previously described preparation of TFIIE (8), contains RAP 30 as an essential component. The prior incubation of purified TFIIF with monoaffinitypurified anti-RAP 30 antibodies before addition to a reaction mixture containing purified components abolished transcription from the adenovirus major late promoter (Ad-MLP). Incubation of TFIIF with the other general transcription factors, RNA polymerase I1 and a promoter-containing DNA, led to the formation of a transcription-competent DNA-protein complex that was not inhibited by anti-RAP antibodies.

MATERIALS AND METHODS
The purification of the transcription factors was as previously described (8). RAP 30-74-depleted extract was prepared by immunoprecipitation of RAP 30 and RAP 74 from an extract derived from HeLa cell nuclei with affinity-purified anti-RAP 30 antibodies and protein A-Sepharose beads, as previously described (19). The affinity purification of the anti-RAP 30 antibodies and the Western blots were as previously described (19).
Transcription reactions (40 pl) were performed in a buffer containing 40 mM Hepes (pH 7.9), 12% glycerol, 55 mM KCI, 10 mM ammonium sulfate, 2% polyethylene glycol 8000,5 mM dithiothreitol, 0.6 mM ATP and CTP, 0.025 mM [32P]UTP, and template DNA as indicated in the figure legends. The DNA was pML(CAT) (20). The adenovirus major late promoter directed transcription from a 380base pair synthetic polydeoxynucleotide chain which lacks CMP residues in the transcribed DNA strand.

RESULTS AND DISCUSSION
Five General Factors Are Required for Transcription from the MLP-We and others have documented that transcription from the adenovirus major late promoter required at least four protein fractions in addition to RNA polymerase I1  (Fig. 1, lane 7). Furthermore, the addition of TFIIF did not obviate the requirements for the general factors or the upstream specific factor MLTF (11,22) ( Fig. 1).
Extracts Depleted of RAP 30-74 Can Be Complemented for Transcription from the Ad-MLP by Transcription Factors IIE and IIF-Previous studies have demonstrated that the fractionation of transcriptionally active extracts by chromatography on RNA polymerase I1 affinity columns resulted in the binding of three major polypeptides (RAP 30, 38, and 74) to the column (13) and the production of transcriptionally inactive extracts (break-through fractions) (14). However, when the flow-through fractions were supplemented with the RAP mixture they regained transcriptional activity (14). Removal of RAP 30 and RAP 74 from an extract as a RAP 30-74 complex with antibody to RAP 30 also inhibited the extract  (8)), major late transcription factor (MLTF) (5 X upstream promoter element (UPE) fraction (22), 2 p l ) , and a plasmid DNA (0.1 pg) containing the major late promoter (20). Reaction mixtures (40 p l ) were incubated for 60 min at 30°C. Products of reactions were separated by electrophoresis on 6% polyacrylamide-urea gels. The amount of radiolabeled nucleotide incorporated into the transcript was determined by excising the bands of a dry gel and counting in a scintillation counter. (19). In this case the depleted extract could be fully reconstituted with electrophoretically purified RAP 30 and RAP 74 (19). Reinberg and Roeder (8) demonstrated that the previously described preparation of TFIIE contained at least one transcription factor that bound to purified RNA polymerase I1 and TFIIB. On the basis of these results, we investigated whether the previously described TFIIB and/or TFIIE protein fractions were able to complement an RAP 30-74-depleted extract for transcription from the Ad-MLP. The addition of a DNA template containing the Ad-MLP to RAP 30-74depleted extract did not result in the generation of detectable levels of transcription (Fig. 2, lane I). However, addition of a preparation of TFIIE (containing TFIIF, see below) and a promoter containing DNA resulted in low but specific levels of transcription (Fig. 2, lane 3). The TFIIB protein fraction was unable to complement the RAP-depleted extract ( l a n e 2). The addition of the phosphocellulose 0.5 M fraction (containing TFIIB, TFIIE, and TFIIF, see Fig. 3A) resulted in transcription; however, the levels of activity were similar to those obtained when only the preparation of TFIIE was added to the depleted extract. These results suggested that the depleted extract contained TFIIB but was deficient in TFIIE and/or TFIIF. The low levels of transcriptional activity observed with the depleted extract, in comparison to those obtained with a system reconstituted with partially purified general transcription factors (compare lanes 3 and 5), could result from limiting IIA and/or IID, possible as a result of its partial removal by co-precipitation by the anti-RAP 30 antibodies (see below).
Inactivation of TFIZFActivity by Antibodies Directed against RAP30"The preparation of TFIIE containing TFIIF activity was able to partially complement the transcriptional activity of an RAP 30-74-depleted extract. Furthermore, addition of TFIIB had no effect on the transcriptional activity of the depleted extract. These results are in agreement with those of Reinberg and Roeder (8), which demonstrated that a partially purified preparation of TFIIE interacted with RNA polymerase I1 and that no stable complex was formed between TFIIB and the polymerase. However, we (8) and others (12,19) have suggested that RAP 30 might be equivalent to TFIIB. This was suggested by the observation that upon purification of TFIIB to near homogeneity (8)

RAP 30 Is an Essential Component of Transcription Factor IIF
glycerol gradient sedimentation and gel filtration, with a polypeptide of 30,000 daltons. Therefore, TFIIB has a molecular weight similar to that of RAP 30. In order to further investigate whether RAP 30 corresponded to TFIIB, TFIIE, or TFIIF, different fractions from the purification of these factors (see. Fig. 3A) were analyzed by Western blots using antibodies against RAP 30 (19). The anti-RAP 30 was monospecific because it was affinity-purified on a column containing an immobilized RAP 30-~-galactosidase fusion protein produced in Escherichia coli (19). These analyses indicated that the nuclear extract (Fig. 3R, lane 1) (lanes 7-9). A similar analysis utilizing the IIE fractions indicated that through the Affi-Gel blue chromatographic step (see. Fig. 3A) all contained equal amounts of RAP 30-reacting material (lanes [12][13][14].

30-reacting material was observed
Additional purification of the IIE-Affi-Gel blue protein fraction by chromatography on a high pressure liquid chromatography DEAE-5PW column resulted in the separation of two factors, TFIIE and TFIIF, both required to reconstitute transcription (see Fig. l).' Analysis of the Western blot indicated that the TFIIF, but not the TFIIE fraction, reacted with the anti -RAP 30 antibodies (lanes 11 and 10, respectively). These suggested that TFIIF corresponded to RAP 30. The presence of variable amounts of RAP 30-reacting material in the IIB-DEAE-cellulose protein fraction is in agreement with previous observations that indicated a copurification of variable amounts of TFIIE with TFIIB (8) and with the demonstration that a partially purified TFIIE protein fraction (containing TFIIF) interacted with TFIIB (8).
It has previously been shown that RAP 30 is required for accurate initiation in a crude transcription system (14, 19).
To test whether RAP 30 is also required for initiation in a transcription system reconstituted with purified factors, increasing amounts of the monoaffinity-purified anti-RAP 30 antibodies were incubated with a constant amount of TFIIF prior to the addition of TFIIF to mixtures containing the DNA template and the other purified factors. This resulted in an inhibition of transcription (Fig. 4A, lanes 1 and 2). The observed inhibition was specific for the anti-RAP 30 antibodies; preincubation of TFIIF with antibodies directed against @galactosidase had no effect on transcription (lanes 3 and 4).
In order to directly show that RAP 30 is a functional component of TFIIF activity, the experiment outline in the lower part of Fig. 4B was performed. A fraction containing TFIIE and TFIIF was incubated with antibodies (directed against RAP 30 or &galactosidase) for 20 min. Protein A-Sepharose saturated with bovine serum albumin was then added and the antibodies bound to protein A removed by centrifugation. Aliquots of the aqueous phase were added in place of IIE and IIF to transcription reaction mixtures. The result of this experiment (Fig. 423) indicated that incubation of TFIIE and TFIIF with anti-RAP 30 antibodies prevented complementation (lanes 3 and 4). This inhibition was specific for the anti-RAP 30 antibodies; the incubation of the TFIIE and TFIIF with antibodies directed against B-galactosidase had no effect on transcription (lanes 5 and 6). The observed inhibition by the anti-RAP 30 antibodies was overcome by the addition of purified TFIIF (devoid of TFIIE) (lanes 7 and  8); however, the addition of purified TFIIE (devoid of TFIIF) did not overcome this inhibition (lanes 9 and 10). Furthermore, the incubation of TFIIB with anti-RAP 30 antibodies had no effect on transcription (data not shown). These results demonstrated that anti-RAP 30 antibodies inhibited transcription by specifically interacting with TFIIF.  Fig. 1. However, TFIIF (0.4 pg) was first incubated with antibodies at 4°C for 20 min prior to the addition into the transcription reaction mixtures. B, reaction mixtures were as described in the legend to Fig. 1. However, a fraction containing TFIIE and TFIIF (Sephacryl AcA 44,17 pg) was first incubated with antibodies (anti-RAP 30 or @-galactosidase), as described in the panel at the bottom of the figure. Protein A bound to Sepharose (20 pg) was then added, and antibody-protein complexes were removed by centrifugation on a tabletop centrifuge for 5 min at 10,000 rpm. Aliquots of the aqueous phase were added as a source of TFIIE and TFIIF (lanes 3-12). In addition purified TFIIF (DEAE-5PW fraction, 0.35 pg, lanes I , 7, 8, 11.12) or purified TFIIE (DEAE-5PW fraction, 0.2 pg, lanes 1, [9][10][11][12] was also added. Reaction products were analyzed as described in the legend to Fig. 1. bodies or that TFIIF activity, after a preincubation with the factors, was no longer required. The formation of this complex was dependent on incubation of the transcription factors and DNA. When the antibodies directed against RAP 30 were added together with the transcription factors and the MLP to the reaction mixtures, transcription was significantly reduced ( compare lanes 4 and 5 ) . The preincubation of purified TFIIF with a DNA containing the Ad-MLP also did not overcome the observed inhibition by the anti-RAP 30 antibodies (lanes 2 and 3). In order to further study if TFIIF was able to associate with the other factors so that it became resistant (or partially resistant) to inactivation by anti-RAP 30 antibodies, different combinations of factors were incubated in the absence of DNA prior to the addition of the antibodies. Incubation of TFIIF with TFIIE did not affect the inhibition of transcription by the anti-RAP 30 antibodies (Fig. 5R, lane 3). The incubation of TFIIF with RNA polymerase I1 and TFIIE (lanes 4 and 5 ) , or in addition with TFIIB (lanes 6 and 7) yielded low amounts of transcription resistant to the antibodies. This was increased approximately %fold when all the transcription factors were preincubated ( l a n e 9); however, they were 2-3-fold lower than the control reaction  (hnes 2, 4, 6, and 8)). The most consistent interpretation of these results is that TFIIF can associate with the other transcription factors in a protein complex that is partially resistant to inactivation by the antibody. This partial resistance could be because of an instability of the complex. This protein complex became more stable if it was associated with a DNA molecule that contained the MLP.

CONCLUSIONS
The studies presented here indicate that RAP 30, a protein isolated using affinity chromatography on RNA polymerase I1 columns (13), is a functional component of TFIIF, a newly discovered general transcription factor? This conclusion was based on the following observations: (i) the TFI1F:RAP 30 ratio was constant during purification through five chromatographic steps (Fig. 3B); (ii) the incubation of TFIIF with monoaffinity-purified anti-RAP 30 antibodies inhibited transcription; (iii) a preparation containing TFIIF and TFIIE complemented the transcriptional activity of RAP 30-74depleted extracts (9). It has previously been demonstrated that preparations of TFIIE (8) (containing TFIIF), but not TFIIB, were able to interact with purified RNA polymerase 11. The studies presented here support this observation, namely that the preparation of TFIIE (containing TFIIF) was able to complement the transcriptional activity of an extract depleted of RAP 30-74 (13). Further investigations will determine whether purified TFIIE and/or purified TFIIF interacts with RNA polymerase 11. The studies of Zheng et al. (12) have demonstrated that a 27,000-dalton protein isolated from HeLa cells co-sedimented with RNA polymerase 11. Furthermore, Burton et al. (19) have shown that RAP 30 interacted with RAP 74 and that both RAP 30 and RAP 74 were required for transcription from the major late promoter. Thus, it is possible that TFIIF contains both RAP 30 and RAP 74 or that TFIIF interacts with the polymerase and that TFIIE is the equivalent to RAP 74 and interacts with TFIIF. This remains to be further investigated using purified fractions.