©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Structure and Function of the Small Subunit of TFIIF (RAP30) from Drosophilamelanogaster(*)

(Received for publication, October 6, 1994; and in revised form, January 4, 1995)

Deborah J. Frank Curtis M. Tyree Catherine P. George James T. Kadonaga (§)

From the Department of Biology and Center for Molecular Genetics, University of California at San Diego, La Jolla, California 92093-0347

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

To study the mechanism of basal transcription by RNA polymerase II, a cDNA encoding the Drosophila homologue of the small subunit of TFIIF (also referred to as TFIIF30, RAP30, factor 5b, and ) was isolated. The Drosophila TFIIF30 gene is located at region 86C on the right arm of the third chromosome. The protein encoded by the cDNA, termed dTFIIF30, was synthesized in Escherichia coli and purified to greater than 95% homogeneity. In reconstituted transcription reactions with purified basal factors, the specific activity of dTFIIF30 was identical to that of its human homologue. Moreover, a carboxyl-terminal fragment, designated dF30(119-276), which contains the carboxyl-terminal 158 amino acid residues of dTFIIF30, was found to possess approximately 50% of the transcriptional activity as full-length dTFIIF30. The interaction of dTFIIF30 with the large subunit of TFIIF (also referred to as TFIIF74, RAP74, factor 5a, and beta) was investigated by glycerol gradient sedimentation analyses. In these experiments, dTFIIF30, but not dF30(119-276), assembled into a stable heteromeric complex with TFIIF74. These results, combined with those of previous work on TFIIF, support a model for TFIIF30 function in which the carboxyl-terminal region constitutes a functional domain that can interact with RNA polymerase II to mediate basal transcription, whereas the amino terminus comprises a domain that interacts with TFIIF74.


INTRODUCTION

Transcription by RNA polymerase II has a central role in a broad range of biological phenomena, and it is therefore useful to study the proteins that are involved in the basal transcription process (for recent reviews, see (1, 2, 3, 4, 5) ). RNA polymerase II, as conventionally defined, is capable of synthesis of RNA from a DNA template, but it additionally requires auxiliary proteins termed the ``basal'' or ``general'' factors for accurate and efficient transcription initiation and elongation. The basal factors include transcription factor (TF) (^1)IIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH, TFIIJ, TFIIS, and SIII. (^2)It appears that TFIIA, TFIIB, TFIID, TFIIE, TFIIF, and TFIIH are involved in the early steps of initiation and promoter clearance, while TFIIF, TFIIJ, TFIIS, and SIII facilitate elongation of the polymerase. It is postulated that the initial steps in the assembly of the transcription complex are the binding of TFIID, TFIIA, and TFIIB to the TATA box and initiation site sequences. Then, TFIIF and RNA polymerase II are incorporated to yield a minimal transcription complex(6, 7) , and, finally, the addition of TFIIE, TFIIH, and TFIIJ is thought to give the complete basal transcription complex. Although significant progress has been made in the identification, purification, and cloning of the basal transcription factors, the mechanistic analysis of basal transcription is still at a relatively early stage, and the current paradigms should be viewed as working models.

In this study, we will focus upon the small 30-kDa subunit of the Drosophila version of TFIIF, which we term dTFIIF30.^2 TFIIF was identified and purified by its ability to bind to immobilized RNA polymerase II and was named RAP30/74, for RNA polymerase II-associated proteins of 30 and 74 kDa apparent molecular mass(8, 9, 10, 11, 12, 13, 14, 15, 16) . In parallel studies, TFIIF was identified as a component of a fractionated HeLa cell transcription system and was found to be identical to RAP30/74(17, 18, 19, 20) . TFIIF has also been studied in rat, wherein it is designated beta(21, 22, 23, 24) ; in Drosophila, wherein it is designated factor 5(25, 26, 27, 28) ; and in Saccharomyces cerevisiae, wherein it is designated factor g(29) . The cDNA versions of the genes encoding the large subunit of TFIIF (RAP74) have been isolated from humans (30, 31) and Drosophila(27, 32) , while the cDNA versions of the genes encoding the small subunit of TFIIF (RAP30) have been isolated from humans(9, 33) , rat (24, 34) and Xenopus(35) .

A variety of experiments have led to significant insight into the function of TFIIF. In vitro transcription studies have suggested that TFIIF30 is required for transcription initiation and stimulation of elongation, whereas TFIIF74 stimulates transcriptional elongation and is required to varying degrees for transcription initiation(7, 16, 28, 36, 37) . The binding of TFIIF to RNA polymerase II is thought to occur through a region in TFIIF30 that appears to be conserved between TFIIF30 and bacterial (9, 12, 13, 20, 23) . The interaction of TFIIF30 with RNA polymerase II reduces the nonspecific binding of polymerase to DNA(14) , while both subunits of TFIIF will release polymerase from DNA to which it is nonspecifically bound(23) . In addition, competition experiments with excess wild-type TFIIF or a carboxyl-terminal deletion of TFIIF led to the suggestion that deletion of the carboxyl-terminal 54 amino acid residues of rTFIIF, which do not overlap with the homology region, does not affect the interaction of rTFIIF30 with hTFIIF74 but does impair the interaction of rTFIIF30 with polymerase(24) . Furthermore, experiments in which various fragments of hTFIIF30 and hTFIIF74 were co-expressed in vivo suggested that the amino-terminal 110 amino acid residues of hTFIIF30 are sufficient for interaction with hTFIIF74, whereas residues 62-171 of hTFIIF74 are sufficient for interaction with hTFIIF30(38) . These results collectively lead to a model wherein TFIIF30 comprises an amino-terminal domain that interacts with TFIIF74 and a carboxyl-terminal domain that interacts with RNA polymerase II and is required for basal transcription.

In this paper, we describe the isolation of a cDNA encoding Drosophila TFIIF30 and examine the biochemical function of the purified, recombinant protein. We have found that the Drosophila version of TFIIF30 is functionally homologous to the human factor and that a carboxyl-terminal fragment of the protein is defective in its interaction with TFIIF74 but remains functional for basal transcription. These studies provide direct biochemical evidence that the high affinity interaction of TFIIF30 with TFIIF74 is distinct from the ability of TFIIF30 to participate in basal transcription by RNA polymerase II.


EXPERIMENTAL PROCEDURES

Isolation of cDNA Encoding dTFIIF30

A short region (138 base pairs) of Drosophila genomic DNA was amplified by using PCR with degenerate oligonucleotide primers that corresponded to conserved regions of the mammalian (9, 24, 33, 34) and Xenopus(35) TFIIF30 cDNA sequences. DNA sequence analysis of the amplified genomic DNA revealed that the region between the two oligonucleotide primers contained an open reading frame with an amino acid sequence that was homologous to that of vertebrate TFIIF30. These intervening sequences were then used to prepare oligonucleotide probes for the isolation of a dTFIIF cDNA by the screening of a Drosophila embryo cDNA library. The dTFIIF30 cDNA sequence is available in GenBank (accession number U02461).

Expression and Purification of dTFIIF30

An NdeI site was introduced at the initiating methionine codon of the dTFIIF30 cDNA by PCR mutagenesis, and the resulting clone was resequenced to confirm that other mutations had not occurred during the PCR amplification. Then, an NdeI-BamHI fragment that contains the entire dTFIIF30 coding region was subcloned into pET-11a (Novagen). By using this construction, dTFIIF30 protein was synthesized in Escherichia coli strain BL21(DE3). The protein was partially purified by the denaturation and renaturation procedure that was previously employed by Finkelstein et al.(30) for the purification of hTFIIF30. dTFII30 was further purified to apparent homogeneity as follows. First, the protein was subjected to chromatography on a Mono S column (Pharmacia Biotech Inc.; column dimensions (diameter times height) = 0.5 cm times 5 cm; column volume = 1 ml; flow rate = 0.4 ml/min; fraction size = 0.2 ml; full scale absorbance range at 280 nm = 1.0) with HEMG buffer (25 mM Hepes-K, pH 7.6, containing 0.1 mM EDTA, 12.5 mM MgCl(2), 10% (v/v) glycerol, 1 mM dithiothreitol, 1 mM benzamidine-HCl, 0.1 mM phenylmethylsulfonyl fluoride, and the indicated concentrations of KCl). The protein was applied in HEMG buffer containing 0.1 M KCl, and the column was washed with three column volumes of the same buffer. Protein was eluted with a 5-ml linear gradient from 0.1 to 1.0 M KCl, and dTFIIF30 eluted at approximately 0.3 M KCl, as determined by the conductivity of the eluate. The peak fractions containing dTFIIF30 were adjusted to 1.0 M KCl (by the addition of 3.0 M KCl solution), and the resulting sample was subjected to chromatography on a Superdex 200 column (Pharmacia Biotech Inc.; column dimensions (diameter times height) = 1.6 cm times 60 cm; column volume = 120 ml; flow rate = 0.5 ml/min; fraction size = 1.0 ml; full scale absorbance range at 280 nm = 0.1) with HEMG buffer containing 1.0 M KCl. dTFIIF30 eluted from the Superdex 200 column with a K of approximately 0.375, and the protein was greater than 95% homogeneous, as determined by SDS-polyacrylamide gel electrophoresis and either silver staining or staining with Coomassie Brilliant Blue R-250. From a 2-liter culture of bacteria, 3.9 mg of dTFIIF30 was recovered at a concentration of 0.3 mg/ml. Protein concentration was determined by the Coomassie dye-binding assay (Pierce) by using bovine -globulin as a reference.

Expression and Purification of dF30(119-276)

The dTFIIF30 clone was mutagenized by PCR to yield a fragment encoding an initiating methionine codon (in an NdeI site) followed by amino acid residues 119-276 of dTFIIF30, and the resulting clone was resequenced to confirm that other mutations had not occurred during the PCR amplification. The NdeI-BamHI fragment was subcloned into pET-15b (Novagen). The resulting protein encoded by this clone, which is termed dF30(119-276), consists of amino acid residues 119-276 of dTFIIF30 (the carboxyl-terminal 158 amino acid residues of dTFIIF30) with a 21-amino acid residue amino-terminal extension that contains six consecutive histidine residues for Ni(II) affinity chromatography. The protein was purified to >95% homogeneity by chromatography on DEAE-Sepharose FF, SP-Sepharose FF, Mono S, and Superdex 200 resins (Pharmacia) followed by affinity chromatography with Ni(II)-nitriloacetic acid-agarose (Qiagen). A detailed account of the methodology is available upon request.

Purification of Basal Transcription Factors

The basal transcription factors, dTBP, dTFIIB, hTFIIE34, hTFIIE56, hTFIIF30, and hTFIIF74, were synthesized in E. coli and purified to greater than 90% homogeneity as described previously(7) . The recombinant hTFIIF74 that was used in the glycerol gradient sedimentation studies is termed RAP74-H(6)(39) . This version of hTFIIF74/RAP74 has a carboxyl-terminal extension that contains six consecutive histidine residues and was purified by Ni(II) affinity chromatography, as described by Wang et al.(39) . RNA polymerase IIA was purified to greater than 90% homogeneity from calf thymus, as described by George et al.(40) , and then subjected to further purification by glycerol gradient sedimentation. Purified TFIIH from HeLa cells (41) was the generous gift of Leigh Zawel and Danny Reinberg (University of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical School; Piscataway, NJ).

In Vitro Transcription

In vitro transcription and primer extension analyses from the Drosophila alcohol dehydrogenase proximal promoter and the adenovirus major late promoter were performed as described in Tyree et al.(7) .

Glycerol Gradient Sedimentation

Protein samples (the indicated combinations of hTFIIF74 (RAP74-H(6)) (10 µg), dTFIIF30 (5 µg), and dF30(119-276) (5 µg)) were incubated at 4 °C for 5 min in gradient buffer (25 mM Hepes-K, pH 7.6, containing 0.1 M KCl, 0.1 mM EDTA, 12.5 mM MgCl(2), 1 mM dithiothreitol, 0.01% (v/v) Nonidet P-40, and varying concentrations of glycerol, as indicated) containing 3% (v/v) glycerol and 6 M guanidine-HCl in a total volume of 120 µl. The resulting samples were dialyzed against the identical buffer lacking guanidine-HCl in a microdialyzer (Pierce) with 1 liter of circulating buffer for 6 h at 4 °C. The samples that were not subjected to denaturation with guanidine-HCl were incubated at 25 °C for 30 min in gradient buffer containing 3% (v/v) glycerol (120 µl total volume) prior to centrifugation. Each of the samples was then subjected to ultracentrifugation in a Beckman SW55 rotor at 50,000 rpm for 20 h at 4 °C in a 4.8-ml linear 7-22% (v/v) glycerol gradient with 300 µl of buffer containing 30% (v/v) glycerol at the bottom of the tube. Twelve fractions (425 µl) from each gradient were collected. The entire fractions were precipitated with 100% (w/v) trichloroacetic acid containing 0.4 mg/ml sodium deoxycholate (110 µl) and then analyzed by electrophoresis on a 12% poly-acrylamide-SDS gel and staining with Coomassie Brilliant Blue R-250.


RESULTS AND DISCUSSION

Isolation of a cDNA Encoding the Drosophila Homologue of TFIIF30

To provide an important component for the analysis of transcription in Drosophila, we have isolated a cDNA encoding the Drosophila version of the small subunit of TFIIF. By using PCR with oligonucleotides that correspond to regions of TFIIF30 that were conserved among vertebrate TFIIF30 proteins, we amplified a 138-base pair fragment of the Drosophila TFIIF30 gene. This segment was then used as a probe to isolate a Drosophila cDNA that contains the entire coding sequence (Fig. 1A). The Drosophila homologue of TFIIF30, as deduced from the cDNA sequence, is a protein of 276 amino acid residues and a calculated molecular mass of 32,544 Da. A comparison of the predicted amino acid sequences of the Drosophila, human, and Xenopus TFIIF30 proteins reveals 50% identity and 63% similarity between the human and Drosophila factors and 48% identity and 62% similarity between the Xenopus and Drosophila factors (Fig. 1B). Regions of similarity to bacterial factors, which have been previously identified in human, rat, and Xenopus TFIIF30(9, 15, 24, 33, 34, 35) , are also present in the Drosophila version of TFIIF30. Northern blot analysis with poly(A) RNA from Drosophila embryos (0-12 h after fertilization) revealed a single band of approximately 1.4 kilobase pairs (data not shown), which corresponds well with the length of the cDNA (1.1 kilobase pairs). Southern blot analysis indicated that dTFIIF30 is encoded by a single gene (data not shown). By in situ hybridization to polytene chromosomes, the Drosophila TFIIF30 locus was mapped to region 86C on the right arm of the third chromosome. (^3)


Figure 1: TFIIF30 from Drosophila. A, nucleotide sequence of a cDNA encoding dTFIIF30. The predicted amino acid sequence is given in the single-letter amino acid code. The dTFIIF30 cDNA sequence is in GenBank (accession number U02461). B, alignment of the amino acid sequences of TFIIF30 from Drosophila, Xenopus(35) , and human(9, 33) . The sequence homology was determined by using the PileUp program of the GCG sequence analysis package. The boldface lettering denotes amino acid residues that are identical in all three proteins.



Transcriptional Activity of Purified, E. coli-synthesized dTFIIF30

To characterize the biochemical properties of recombinant dTFIIF30, it was first necessary to synthesize and purify the protein and then to test the activity of the factor in a reconstituted in vitro transcription assay. We therefore modified the initiating methionine codon to create an NdeI site, which allowed subcloning of the dTFIIF30 cDNA into the E. coli expression vector, pET11a. The resulting E. colisynthesized dTFIIF30 was purified to greater than 95% homogeneity by conventional methods (Fig. 2, lane2).


Figure 2: Analysis of purified, E. coli-synthesized dTFIIF30 and dF30(119-276) by 12% SDS-polyacrylamide gel electrophoresis. The proteins were visualized by silver staining. Lane1, molecular mass standards (1 µg of total protein); lane2, purified dTFIIF30 (250 ng); lane3, purified dF30(119-276) (250 ng). The sizes of the molecular mass standards are given in kilodaltons.



We then tested the ability of the purified, recombinant dTFIIF30 to function in a reconstituted transcription system. These reactions were performed with purified, recombinant TFIIB, TBP, TFIIE, and TFIIF along with purified calf thymus RNA polymerase IIA and negatively supercoiled DNA templates under conditions that do not require TFIIH(6, 7, 36, 42) . The resulting transcription products were subjected to primer extension analysis, and, therefore, these assays detected the synthesis of relatively short transcripts (less than 100 nucleotides in length). In these basal transcription reactions, the specific activity of dTFIIF30 with the Drosophila alcohol dehydrogenase proximal promoter was similar to that of purified, bacterially-synthesized hTFIIF30 (Fig. 3A). In addition, the activity of dTFIIF30 was virtually identical to that of hTFIIF30 at different concentrations of factors as well as with the adenovirus major late promoter (data not shown). We therefore conclude that the dTFIIF30 is functionally homologous to its human counterpart for basal transcription.


Figure 3: Both dTFIIF30 and dF30(119-276) are active in a purified, reconstituted transcription system. Transcription reactions were carried out in the presence or absence of dTFIIF30 derivative with the following purified factors: dTBP (7.5 ng), dTFIIB (5 ng), hTFIIE34 (4 ng), hTFIIE56 (10 ng), hTFIIF74 (10 ng), and calf thymus RNA polymerase II (50 ng). The resulting transcripts were subjected to primer extension analysis, and the reverse transcription products are shown. A, comparison of dTFIIF30 versus hTFII30 with the Drosophila Adh proximal promoter (100 ng of template DNA). Lane1, no TFIIF30; lane2, hTFIIF30 (6 ng); lane3, dTFIIF30 (5 ng); lane4, dTFIIF30 (5 ng) in the presence of 4 µg/ml of alpha-amanitin. B, comparison of dTFIIF30 versus dF30(119-276) with the adenovirus major late promoter (100 ng of supercoiled template DNA). Transcription reactions were performed with varying amounts of either dTFIIF30 (0, 1, 5, 10, or 15 ng) or dF30(119-276) (0, 1, 5, 10, or 15 ng). C, dF30(119-276) supports TFIIH-dependent transcription from a linear DNA template. Transcription reactions were carried out with the adenovirus major late promoter (100 ng of linearized template DNA) and the purified factors indicated above in the presence or absence of HeLa TFIIH(41) . Where noted, either dTFIIF30 (5 ng) or dF30(119-276) (10 ng) was included in the reactions.



Transcriptional Activity of an Amino-terminal Deletion Mutant of dTFIIF30

Examination of the amino acid sequence of the Drosophila and vertebrate TFIIF30 proteins, as depicted in Fig. 1B, revealed that the most highly conserved segment of TFIIF30 was the carboxyl-terminal region that corresponds to residues 119-265 of dTFIIF30 and residues 102-249 of hTFIIF30. This portion of TFIIF30 comprises the regions of homology to bacterial factors, which appear to be important for interactions with RNA polymerase II(9, 12, 13, 15, 24, 33, 34, 35) . In contrast, the segment of TFIIF30 (amino acid residues 67-118 of dTFIIF30, and residues 59-101 of hTFIIF30) that is immediately amino-terminal to the conserved carboxyl terminus displays little similarity between Drosophila and vertebrate TFIIF30. It therefore seemed possible that the carboxyl-terminal 158 amino acid residues of dTFIIF30 might constitute a functional domain that is, in itself, able to mediate transcription initiation.

To test this hypothesis, we constructed a truncated version of the dTFIIF30, which we termed dF30(119-276), that comprises the carboxyl-terminal 158 amino acid residues (residues 119-276). This protein was synthesized in E. coli and purified to greater than 95% homogeneity (Fig. 2, lane3). We then determined the ability of purified dF30(119-276) to substitute for dTFIIF30 in reconstituted in vitro transcription assays with the adenovirus major late promoter (Fig. 3B). The addition of either dTFIIF30 or dF30(119-276) to the transcription reactions resulted in a significant increase in transcription. Quantitative analysis of the reverse transcription products with a PhosphorImager revealed that the specific activity of dF30(119-276) was roughly 50% of that of the full-length dTFIIF30 at the various concentrations of the factors. In addition, as shown in Fig. 3C, dF30(119-276) supports basal transcription from a linear DNA template under conditions where TFIIH is required(42) . These results thus indicate that the conserved carboxyl-terminal 158 amino acid residues of dTFIIF30 are sufficient for basal transcription by RNA polymerase II.

hTFIIF74 Forms a Stable Complex with Full-length dTFIIF30, but Not with dF30(119-276)

Further examination of the amino acid sequence of Drosophila and vertebrate TFIIF30 revealed that the amino-terminal region (amino acid residues 13-66 of dTFIIF30 and residues 6-58 of hTFIIF30) is also highly conserved. Based on the finding that the amino-terminal 110 amino acid residues of hTFIIF30 are required for binding to hTFIIF74 in vivo in a co-transfection assay(38) , it seemed possible that dF30(119-276), which is lacking the amino-terminal 118 amino acid residues of dTFIIF30 but is nevertheless functional for basal transcription (Fig. 3B), may be defective in its interaction with TFIIF74.

We therefore characterized the association of the TFIIF subunits by carrying out sedimentation analyses of dTFIIF30 and hTFIIF74 with linear 7-22% (v/v) glycerol gradients (Fig. 4). When the factors were combined in nondenaturing conditions (incubated in the buffer that was used in the transcription reactions for 30 min at 25 °C) and then subjected to glycerol gradient sedimentation, the majority of the dTFIIF30 did not co-sediment with hTFIIF74. We thus examined whether denaturation and renaturation of the proteins would result in the assembly of a stable complex. The factors, both alone and in combination, were incubated in a buffer containing 6 M guanidine-HCl, and then the denaturant was removed by dialysis to allow refolding of the proteins prior to glycerol gradient sedimentation. The denaturation and renaturation of the individual factors alone did not affect their sedimentation properties relative to proteins that were not treated with denaturant (data not shown). By comparison, denaturation and renaturation of dTFIIF30 with hTFIIF74 resulted in a significant increase in the sedimentation of virtually all of the dTFIIF30, as it co-sedimented with the faster migrating hTFIIF74 in the glycerol gradient. Hence, it appears that denaturation and renaturation of dTFIIF30 and hTFIIF74 is required for the efficient assembly of a stable heteromeric complex.


Figure 4: Glycerol gradient sedimentation analysis reveals interactions between dTFIIF30 and hTFIIF74, but not dF30(119-276) and hTFIIF74. The indicated samples were subjected in parallel to sedimentation in a 7-22% (v/v) glycerol gradient. Fractions from each of the gradients were analyzed by 12% polyacrylamide-SDS gel electrophoresis, and the proteins were visualized by staining with Coomassie Brilliant Blue R-250. Except where noted, the protein samples were subjected to a denaturation and renaturation treatment, in which guanidine-HCl was added to a final concentration of 6 M and then removed by dialysis. The sizes of the molecular mass standards (1.5 µg of total protein/lane) are given in kilodaltons. In the preparation of hTFIIF74, there is a contaminant that appears as a doublet with an apparent molecular mass of approximately 50 kDa.



We then examined the sedimentation of dF30(119-276) protein either alone or with hTFIIF74 (Fig. 4). These experiments revealed that the amino-terminally truncated dF30(119-276) did not co-sediment with hTFIIF74 when treated under conditions that were identical to those used for dTFIIF30. It therefore appears that dF30(119-276), which is functionally active for basal transcription, does not form a stable complex with hTFIIF74. These data, combined with that of previous work on TFIIF, provide evidence in support of a model for TFIIF30 function in which the carboxyl-terminal region constitutes a functional domain that can interact with RNA polymerase II to mediate basal transcription, whereas the amino terminus comprises a domain that interacts with TFIIF74.

It is pertinent to note that the sedimentation rate of hTFIIF74 was not significantly altered by the inclusion of dTFIIF30, even though it was presumably in a stable complex with dTFIIF30. There are many possible explanations for this effect, but it is perhaps most likely that the TFIIF complex displays atypical sedimentation properties, because the apparent native molecular mass of TFIIF as determined by glycerol gradient sedimentation (120 kDa for the Drosophila homologue) (26) is roughly one-half of the native mass as estimated by gel filtration (either 240 or 250 kDa for the human or rat homologues, respectively)(19, 21) . Notwithstanding, in our studies, the increase in the sedimentation rate of dTFIIF30 such that it co-sediments with hTFIIF74 upon denaturation and renaturation of dTFIIF30 with hTFIIF74 indicates the formation of a stable heteromeric complex between the factors.

The observation that treatment with a denaturant is necessary for the establishment of a high affinity interaction between dTFIIF30 and hTFIIF74 is consistent with the findings of Kephart et al.(28) , wherein it was noted that physical association of hTFIIF30 and dTFIIF74 required denaturation and renaturation of the proteins. Because TFIIF that has been isolated from Drosophila, rat, or human is a multimer with equimolar amounts of each subunit(19, 21, 26) , it is likely that renatured heteromer more closely resembles the native form of the factor than the individual polypeptides. Nevertheless, as observed by Kephart et al.(28) with the hTFIIF30-dTFIIF74 hybrid protein, we have found that the denatured and renatured dTFIIF30-hTFIIF74 hybrid protein exhibits the same transcriptional activity as the two polypeptides added separately under nondenaturing conditions (data not shown). There are a few possible explanations for these results. First, a physical association of TFIIF30 and TFIIF74 may not be necessary for transcription either in vivo or in vitro. Second, TFIIF30 and TFIIF74 may assemble into the heteromeric form in the course of the transcription reaction, and, third, the transcription assays employed in vitro may not reflect an in vivo requirement for a high affinity interaction between the subunits. In this context, it is worth noting that the truncated dF30(119-276) protein is active for basal transcription, but it is unable to form a stable complex with hTFIIF74. Hence, a physical association between TFIIF30 and TFIIF74 does not appear to be absolutely required for transcription in vitro.

Summary and Perspectives

These studies provide information on both the structure and the function of the Drosophila homologue of the small subunit of TFIIF. The cDNA clone encoding Drosophila TFIIF30 should be useful for genetic studies, which include the analysis of the expression of the protein throughout development. We have also generated polyclonal antibodies against dTFIIF30, which are available upon request. The biochemical studies of dTFIIF30 and dF30(119-276) suggest that the carboxyl terminus of the TFIIF30 is sufficient for basal transcription, while the amino terminus of the factor is required for association with TFIIF74.

From a broader perspective, this subunit of TFIIF represents a single polypeptide out of a basal transcription complex that may comprise greater than 50 polypeptides. TFIIF30 is believed to function at an early stage in the assembly of the transcription complex, and is a component of the central catalytic core of the minimal transcriptional machinery, which comprises TFIIB, TBP, TFIIF30, and the polymerase. Thus, in spite of the apparent complexity of the overall process, TFIIF has a unique and essential role in the transcription reaction. The further analysis of the relation between the two TFIIF subunits as well as the functional interactions between TFIIF and the remainder of the transcriptional machinery will provide important information regarding gene transcription and regulation.


FOOTNOTES

*
This work was supported in part by Grant GM 41249 (to J. T. K.) from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U02461[GenBank].

§
A Presidential Faculty Fellow. To whom correspondence should be addressed: Dept. of Biology, 0347, Pacific Hall, Rm. 2212B, University of California, San Diego, 9500 Gilman Dr., La Jolla, CA 92093-0347. Tel.: 619-534-4608; Fax: 619-534-0555.

(^1)
The abbreviations used are: TF, transcription factor; dTFIIF30, the 30-kDa subunit of RNA polymerase II transcription factor IIF, which is also known as RAP30 (RNA polymerase II-associated protein of 30 kDa apparent molecular mass); dF30(119-276), a fusion protein that consists of amino acid residues 119-276 of dTFIIF30 (the carboxyl-terminal 158 amino acid residues of dTFIIF30) with a 21-amino acid residue amino-terminal extension that contains six consecutive histidine residues; PCR, polymerase chain reaction.

(^2)
Transcription factors IIA through IIS are abbreviated as TFIIA, TFIIB, and so on. The TATA box-binding polypeptide of the TFIID complex is referred to as TBP. The purified basal factors used in this study were synthesized in E. coli. A lower case d, r, or h designates the original source of the recombinant factors as either Drosophila, rat, or human, respectively. The 34- and 56-kDa subunits of human TFIIE are referred to as hTFIIE34 and hTFIIE56. The 30- and 74-kDa subunits of human TFIIF are designated as hTFIIF30 and hTFIIF74. Alternate nomenclature for the basal transcription factors (for reviews, see (1, 2, 3, 4, 5) ) is as follows: TFIIB (alpha, FA, factor e); TFIID (, factor d, BTF-1); TFIIE (, factor a); TFIIF (RAP30/74, beta, factor 5, FC, BTF-4, factor g); TFIIH (BTF-2, factor b, ); TFIIS (S-II).

(^3)
T. Laverty and G. Rubin, unpublished results.


ACKNOWLEDGEMENTS

We thank Leigh Zawel and Danny Reinberg for the generous gift of purified HeLa TFIIH, Todd Laverty and Gerry Rubin for determination of the cytological location of the dTFIIF30 gene, Lucy DeVito for help with the structural analysis of the dTFIIF30 gene, Bo Qing Wang and Zach Burton for the gift of the RAP74-H(6) clone, Mike Pazin for assistance with PCR, Rohinton Kamakaka and Doug Smith for assistance with the computer sequence analysis, and Edd Lee for technical assistance. We also thank Mike Pazin, Lucy DeVito, and Tom Burke for critical reading of the manuscript.


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