(Received for publication, October 6, 1994; and in revised form, January 4, 1995)
From the
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
) 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.
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) ()IIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH, TFIIJ, TFIIS, and
SIII. (
)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. 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
(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.
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.
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 -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.
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.
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.
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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U02461[GenBank].