(Received for publication, April 27, 1995; and in revised form, August 1, 1995)
From the
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
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) ()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 promoter-specific. 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
(Hanna and Meares, 1983a, 1983b). In this report, recombinant CTD is
first labeled by phosphorylation with casein kinase II in the presence
of [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
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
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.
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 µg/ml phenylmethylsulfonyl fluoride,
and 1 µg/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
µg/ml phenylmethylsulfonyl fluoride, and 1 µg/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)
SO
fractionation. Approximately
90% of GST-CTD was precipitated in the presence of 30%
(NH
)
SO
. The 30%
(NH
)
SO
precipitate was dissolved in
and dialyzed against 50 mM NH
HCO
, 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
HCO
, 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
HCO
, 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 µg in a volume of 400
µl at a concentration of 8.7 µM as determined by amino
acid analysis.
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).
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).
Figure 1:
Inhibition of transcription by
recombinant CTD. P-Labeled RNA transcripts were analyzed
by electrophoresis on a 5% polyacrylamide, 8 M urea gel and
autoradiography. A 147-base pair
P-labeled DNA fragment
serves as an internal control for recovery of RNA transcripts. A, reactions contained 6 µl of DE0.25 extract and either
0.017 pmol of Ad2-ML template (lanes 1-5) or 0.19 pmol
of DHFR template (lanes 6-10). Reactions in lanes 1 and 6, lanes 2 and 7, lanes 3 and 8, and lanes 4 and 9 contained 0,
1, 10, and 42 pmol of GST-CTD, respectively. Reactions in lanes 5 and 10 contained 42 pmol of GST. Sp1 (30 ng) was included
in reactions containing the DHFR promoter. Reaction components were
preincubated prior to the addition of 0.073 pmol of RNAP IIA (8
milliunits). The positions of the 560- and 295-nucleotide transcripts
from the Ad2-ML and DHFR templates, respectively, are indicated on the left in addition to the position of the 147-base pair internal
standard. B, reactions in lanes 1-4 contained 6
µl of DE0.25 extract, 0.017 pmol of Ad2-ML template, and 0, 3.1,
6.2, and 18.6 pmol of rCTD, respectively. Reactions were preincubated
prior to the addition of 0.073 pmol of RNAP IIA. Reactions in lanes
5-8 contained 3 µl of DE0.25 extract, 0.19 pmol of DHFR
template, 30 ng of Sp1, and 0, 6.5, 13, and 26 pmol of rCTD,
respectively. Reactions were preincubated prior to the addition of
0.018 pmol of RNAP IIA. The positions of the run-off transcripts and
internal standard are indicated as in A.
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.
Figure 2:
Synthesis of azidophenacyl-modified SpCTD (N
R
SpCTD) and its use in
photolabeling experiments. A, the reactions involved in the
modification of rCTD to generate N
R
SpCTD are
shown. rCTD and [
S] ATP
S are incubated with
casein kinase II to produce
SpCTD, which is then reacted
with azidophenacyl bromide to produce the final product,
N
R
SpCTD. B, the reactions in which
proteins in the transcription extract can be photolabeled by virtue of
their interaction with N
R
SpCTD are shown.
Irradiation by ultraviolet light (300 nm) photoactivates the azide
moiety of N
R
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
S-P bond with phenylmercuric acetate results in the
transfer of the radioactive label from the rCTD to the protein that it
contacts.
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 is approximately
70,000, due to aberrant mobility on SDS-polyacrylamide gel
electrophoresis. A previously reported M
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
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
of
N
R
SpCTD is the same as that of rCTD (Fig. 3B, lane 1). Both the spectrum of
photocross-linked proteins following irradiation (Fig. 3B, lane 3) and the spectrum of
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
of 34,000, 70,000, and 74,000 (Fig. 3B, lane 4). The 70-kDa band is either
S-labeled CTD, which could result from inter- or
intramolecular cross-linking of N
R
SpCTD
itself, or N
R
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
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.
Figure 3:
Specificity of photolabeling by
NR
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 µl of
transcription extract, respectively. Lane 3 contains molecular
mass markers, with their sizes (
10
kDa)
indicated on the right. B, reactions in lanes
1-9 contained 0.34 pmol of N
R
SpCTD,
and reactions in lanes 10-12 contained 0.22 pmol of
SpCTD. All reactions with the exception of lanes 7 and 8 contained 11 µl of DE0.25 extract. Lanes with h
(+) at the top were irradiated
for 5 min at 300 nm, and lanes with PMA (+) were
incubated with phenylmercuric acetate overnight. Reactions in lanes
5 and 6 were incubated with 100 µg/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
R
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
R
SpCTD and
SpCTD are indicated
on the left and right, respectively. M1 and M2 are marker lanes and contain
P-labeled phosphorylase a and
P-labeled
autophosphorylated
subunit of casein kinase II, respectively. The
sizes (
10
kDa) of these markers are
indicated on the left.
The specificity of
photolabeling was also demonstrated by the following controls. Both the
formation of cross-linked and S-labeled proteins in the
transcription extract are photosensitive. When the reactions were not
irradiated, neither photocross-linked 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
R
SpCTD to photocross-link to proteins (Fig. 3B, lane 9). Furthermore, in the absence
of the azide moiety (N
R),
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
R
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
S-labeled CTD derived from intra- or
intermolecular cross-linking of N
R
SpCTD. Both
the absence of a photocross-linked band (CTD dimer, etc.) (Fig. 3B, lane 7) and the near absence of the
N
R
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
R
SpCTD can be used to identify proteins that
interact with the CTD.
Figure 4:
RNAP IIA, RNAP IIB, and rCTD as
competitors of photolabeling. All reactions contained 0.22 pmol of
NR
SpCTD and 11 µl of DE0.25 extract. Some
reactions contained 0.18 pmol of RNAP IIA and 0.19 pmol of DHFR
template (lanes 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 Fig. 3B.
Marker lanes M1 and M2 are also as described in the
legend to Fig. 3B.
Figure 5:
Comigration of photolabeled proteins with
recombinant transcription factors. A, reactions contained 0.36
pmol of NR
SpCTD and either 11 µl of DE0.25
extract (lanes 1 and 2), 8 pmol of rTFIIB (lanes
3 and 4), 8 pmol of rTFIIE (lanes 5 and 6), 8 pmol of rTFIIF (lanes 7 and 8), 8 pmol
each of rTFIIB, -IIE, and -IIF (lanes 9 and 10), or 8
pmol of rTBP (lanes 11 and 12). All reactions were
irradiated (lanes 1-12). Reactions in lanes 2, 4, 6, 8, 10, and 12 were
incubated with PMA. Reactions were analyzed as described in the legend
to Fig. 3B. Marker lanes M1 and M2 are also as described in the legend to Fig. 3B.
The autoradiogram is a 10-day exposure. B, autoradiogram of a
44-day exposure of the same gel shown in A. Lane designations are identical to those in A.
The 34- and 74-kDa proteins photolabeled prominently by
NR
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).
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 azido-modified SpCTD
(N
R
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
[
S]ATP
S and the subsequent alkylation of
the
S with the photoprobe. Cleavage of the cross-linked
proteins results in the transfer of
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
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 NR
SpCTD. The
two most intense photolabeled bands have a M
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
S to proteins, as indicated by proteinase K
sensitivity.
The photolabeling of the 34- and 74-kDa proteins by
NR
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 CTD-less 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
NR
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 NR
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
R
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
R
SpCTD, 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
R
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.