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(Received for publication, June 5, 1996, and in revised form, July 19, 1996)
From the Department of Biochemistry, University of Iowa,
Iowa City, Iowa 52242
ABSTRACT The entry of RNA polymerase II into a productive
mode of elongation is controlled, in part, by the postinitiation
activity of positive transcription elongation factor b (P-TEFb)
(Marshall, N. F., and Price, D. H. (1995) J. Biol. Chem.
270, 12335-12338). We report here that removal of the
carboxyl-terminal domain (CTD) of the large subunit of RNA polymerase
II abolishes productive elongation. Correspondingly, we found that
P-TEFb can phosphorylate the CTD of pure RNA polymerase II.
Furthermore, P-TEFb can phosphorylate the CTD of RNA polymerase II when
the polymerase is in an early elongation complex. Both the function and
kinase activity of P-TEFb are blocked by the drugs
5,6-dichloro-1- INTRODUCTION The expression of many genes is controlled in part at the level of
transcription elongation (reviewed in Refs. 1, 2, 3, 4). As is frequently
found in control processes, there is a negative control mechanism that
is manifest as a blockage during early elongation. Such blocks, usually
referred to as premature termination, have been observed during
transcription of a number of genes including c-myc (5, 6, 7),
c-fos (8), c-myb (9), c-fms (10),
adenosine deaminase (11, 12), A model for the control of elongation has been described that is based
on results obtained from a Drosophila in vitro transcription
system (21, 22) and is consistent with data obtained in
vitro and in vivo from many studies. Key features of
the model are that all RNA polymerase II molecules that initiate from a
promoter are destined to produce only short transcripts in a process
termed abortive elongation. Abortive elongation is distinct from
abortive initiation because the abortive transcripts are 10-20 times
longer during abortive elongation, and presumably the polymerase in the
abortive elongation complexes must relocate the promoter after
producing an abortive transcript to bring about reinitiation. Escape
from this negative control is accomplished through the action of
positive transcription elongation factor (P-TEF), which allows
productive elongation. Fractionation studies have recently identified
three components required to efficiently generate productive elongation
complexes, P-TEFa, P-TEFb, and factor 2 (23). P-TEFb was purified to
apparent homogeneity and was shown to act after initiation (23).
The carboxyl-terminal domain (CTD) of RNA polymerase II is
phosphorylated during the transcription cycle at a time coincident with
elongation regulation (reviewed in Refs. 24 and 25). The CTD can be
phosphorylated by the kinase associated with the general transcription
factor TFIIH (26, 27, 28) and a CTD kinase is known to be present in
preinitiation complexes at several promoters (29, 30). A kinase/cyclin
pair (SRB10/11) is part of the holoenzyme form of yeast RNA polymerase
II (31). A number of other kinases including casein kinase I and II
(32, 33), DNA-dependent protein kinase (34), and a murine
kinase related to cdc2 and Cdc28 (35), are capable of
phosphorylating the CTD. Also, the kinases CTD-K1 and CTD-K2 purified
from HeLa cells (36), CTK1 from yeast (37), and KI, KII, and KIII from
Aspergillus nidulans (38) can all phosphorylate the CTD. It
has been suggested that the stress-activated mitogen-activated protein
kinases are involved in phosphorylating RNA polymerase II during heat
shock (39). While all of the above are serine/threonine kinases, there
is one example of a tyrosine kinase, c-Abl, that can phosphorylate the
CTD (40). While phosphorylation of the CTD has been correlated with the
elongation phase of transcription, none of these kinases have been
shown to modify the functional properties of RNA polymerase II during
elongation.
Results presented here link the process of productive elongation and
phosphorylation of the CTD. We found that removal of the CTD by limited
proteolysis prohibits the transition into productive elongation. In
correlation with this, we found that a factor required for the
transition into productive elongation, P-TEFb, is a CTD kinase. P-TEFb
was shown to be distinct from Drosophila TFIIH by its
function in in vitro transcription, CTD kinase activity, and
polypeptide composition. A detailed investigation of the CTD kinase
function of P-TEFb and its inhibition by the drugs DRB and H-8 is also
presented.
EXPERIMENTAL PROCEDURES [ Partially purified RNA
polymerase II was treated with 0.27 µg/ml chymotrypsin
(Sigma) at 27 °C in HGED (25 mM HEPES,
pH 7.6, 15% glycerol, 0.1 mM EDTA, 1 mM
dithiothreitol) plus 110 mM KCl for times ranging from 0 to
20 min. Digestions were terminated by the addition of trypsin inhibitor
(Sigma) to 20 µg/ml. The extent of proteolysis of
the RNA polymerase II subunits was assayed by SDS-PAGE followed by
silver staining.
Preinitiation
complexes were formed on an immobilized actin template digested with
HpaII (780-nucleotide run-off) as described by Marshall and
Price (22). The preinitiation complexes were isolated, washed once with
55 mM HKB (20 mM HEPES, 55 mM KCl,
and 200 µg/ml bovine serum albumin), and resuspended into 55 mM HKB. Transcription was initiated by the addition of a
pulse solution, which contained 5 µCi of [ Recombinant GST-rpII1 fusion protein was produced in
Escherichia coli using a T7 polymerase-dependent
expression system. rpII1 is an amino-terminal portion
(Pro117 to Lys205) of Drosophila RNA
polymerase II large subunit. First, a GST coding sequence from pEG(KT)
was used to replace the NdeI/SalI fragment in
pET21a to construct a GST-expression plasmid (pET21a-GST). Second, The
rpII1 coding sequence was amplified by polymerase chain reaction using
an upstream primer (5 Transcription reactions containing
partially purified factors and the actin Act5C template (21) linearized
with HpaI were carried out as described (23). In addition to
RNA polymerase II, transcription reactions (12.5 µl) contained
P11-FT, factor 2, transcription factor IIE (factor 3), P11-0.4M step,
and P-TEFb, all derived from Drosophila Kc cell
nuclear extract (KcN) (41). Transcription reactions using
KcN (22) or Kc-FT (23) were carried out as
described previously. H-8 and DRB were incubated with KcN
for approximately 3 min at 22 °C prior to the addition of template
and NTPs.
P-TEFb used in Figs. 1 and 3 was
purified from KcN as described earlier (23) and summarized
in Fig. 3A. Phosphocellulose, phenyl-Sepharose, Mono Q, and
Mono S columns were used. A sample of the Mono S-purified material was
analyzed on a glycerol gradient as described (23).
The P-TEFb used in all other figures was purified from
Drosophila embryonic nuclear extract (23) by a protocol
similar to that diagrammed in Fig. 3A with one addition.
Material eluting from the phenyl-Sepharose column was loaded directly
onto a 10.0-ml ceramic hydroxylapatite column (Bio-Rad CHT10). The
column was then eluted with a linear gradient of potassium phosphate
from 10 mM to 750 mM in 25 mM
HEPES, pH 7.6, 15% glycerol. P-TEFb eluted between 400 and 500 mM phosphate. Pooled fractions containing P-TEFb were then
dialyzed and chromatographed on Mono S. P-TEFb eluting from Mono S
still had significant nucleic acid contamination, so the material was
subjected to chromatography on Mono Q followed by Mono S for
reconcentration. The peak fraction from the final Mono S column
contained about 0.5 mg/ml P-TEFb (see Fig. 7).
0.4 µl of purified RNA polymerase II and
various protein samples were mixed in 18 µl of 55 mM
HMKB. The reaction was then initiated by the addition of 2 µl of a
solution containing 2 µCi of [
RESULTS We examined the
involvement of the CTD in elongation control using a
Drosophila RNA polymerase II transcription system that
supports DRB-sensitive productive elongation (23).
Drosophila RNA polymerase II was treated with chymotrypsin
for increasing times to gradually remove the CTD (Fig.
1A). Trypsin inhibitor was added to aliquots
of the digestion reaction after 0, 2, 8, or 20 min. When trypsin
inhibitor was added to a similar reaction before the chymotrypsin, no
digestion took place during a subsequent 20-min incubation (Fig.
1A, lane 20*), indicating that the protease was
inactivated. Intact or truncated forms of the polymerase were then used
to drive transcription from the Act5C promoter using fractions derived
from Drosophila Kc cell nuclear extract
containing factors needed for initiation and productive elongation.
Although most of the RNA polymerase II was removed by the fractionation
procedure, some run-off transcripts were detected in the absence of
added RNA polymerase II (Fig. 1B). When intact RNA
polymerase II was added, the run-off signal increased dramatically and
was sensitive to DRB. The amount of DRB-sensitive, run-off transcript
decreased as the CTD was removed (Fig. 1B). The amount of
shorter, DRB-insensitive, abortive transcripts increased with added RNA
polymerase II but did not change in amount as the CTD was removed.
Quantitation of the autoradiograph and protein gel showed that the
level of run-off transcript was directly related to the amount of
polymerase containing the CTD, while the generation of abortive
transcripts was unaffected by loss of the CTD (Fig. 1C).
Earlier findings, using a minimal set of Drosophila
fractions required for initiation, indicated that there was no effect
when substituting CTD-less polymerase for intact polymerase (42). The
earlier results were obtained using fractions that did not contain
factors needed for the generation of DRB-sensitive productive
elongation complexes (23). Therefore, the negative effect of removal of
the CTD we observed here suggests that the CTD is involved in the
transition into productive elongation.
To address the formal possibility that truncation of the CTD in the
previous experiment had an effect on initiation that resulted in the
formation of exclusively DRB-insensitive complexes, we developed a
protocol for the truncation of the CTD after initiation (Fig.
2A). Early elongation complexes were formed
on an immobilized template and then washed with buffer containing 1 M KCl, which removes uninitiated RNA polymerase II (see
Fig. 6). The proteins found in early elongation complexes were stripped
from the beads with SDS and analyzed by SDS-PAGE followed by Western
blotting. Antibodies to a non-CTD-containing domain of the large
subunit of RNA polymerase II were generated (see ``Experimental
Procedures'') and used to probe the Western blot. Treatment with
increasing amounts of chymotrypsin resulted in removal of the CTD from
RNA polymerase II in early elongation complexes (Fig. 2B).
Essentially complete truncation occurred when 0.2 µg/ml or more of
chymotrypsin was used. The protease treatment did not negatively affect
the ability of the isolated early elongation complexes to continue
elongation during the subsequent chase (Fig. 2C,
none). Except for the increase in the longest transcripts at
the higher protease concentrations, the typical pattern of transcripts
seen during abortive elongation on the actin template (22, 43) was
detected (Fig. 2C). The increase in the longest transcripts
is due to the removal of the remaining trace amount of factor 2 (41),
which normally exerts a negative effect on elongation (43). To assess
the ability of the polymerase in early elongation complexes to enter
productive elongation, nuclear extract was added back with the chase.
In this experiment the extracts were complemented with a constant
amount of additional P-TEFb, which is normally limiting in the
extracts. DRB-sensitive run-off transcripts due to P-TEF action were
visible in the lanes using nonproteolyzed complexes (Fig.
2C, compare KcN and
KcN + DRB lanes). As
increasing amounts of chymotrypsin were used, the early elongation
complexes lost the ability to form long DRB-sensitive transcripts. The
long transcripts seen at high protease levels in the early elongation
complexes alone were not seen when KcN was added because of
the effect of endogenous factor 2 in the extract (43). The results
shown in Figs. 1 and 2 strongly suggest that the CTD is required for
the generation of long DRB-sensitive transcripts.
The requirement of the CTD for the
generation of DRB-sensitive long transcripts and the inhibition of the
process by the kinase inhibitor DRB (22, 23) prompted us to determine
if P-TEFb was a CTD kinase. Our first experiments (not shown) indicated
that incubation of P-TEFb with intact RNA polymerase II caused the
incorporation of phosphate into the large subunit and a shift to the
IIo form. Truncation of the CTD by chymotrypsin resulted in a loss of
the ability of P-TEFb to phosphorylate the polymerase (data not shown).
To correlate the CTD kinase activity with P-TEFb function in
transcription, fractions from a gradient elution of P-TEFb from a Mono
S column and a subsequent glycerol gradient (Fig.
3A) were tested in both assays. P-TEFb was
about 25% pure in the peak fraction (29) from Mono S (23). CTD kinase
activity coeluted with the transcription activity of P-TEFb on Mono S
(Fig. 3B and C). Fractions from the glycerol
gradient analysis of P-TEFb from Mono S fraction 30 were analyzed by
SDS-PAGE (Fig. 3D). Both activities again co-migrated with
each other (Fig. 3, E and F) and with the 124- and 43-kDa subunits of P-TEFb previously identified (23). P-TEFb
subunits, transcription function, and CTD kinase activity have
correlated across all columns assayed thus far.
We next wanted to
establish the basic parameters of the CTD kinase activity of P-TEFb.
P-TEFb was incubated for 5 min with RNA polymerase II and various cold
nucleotides. The products were subjected to SDS-PAGE and silver-stained
to visualize the RNA polymerase II (Fig. 4A).
The mobility shift from the unphosphorylated IIa form to the highly
phosphorylated IIo form of the large subunit was used to ascertain
kinase activity. Partial shifts indicated lower levels of incorporation
of phosphate (see Fig. 3). P-TEFb was capable of utilizing the purine
nucleotides in rough order of efficiency ATP > dATP = GTP > dGTP. P-TEFb was unable to utilize any of the pyrimidine
nucleotides at 100 µM, and other data (not shown)
indicates that these nucleotides are not substrates even at 600 µM.
The kinetics of phosphorylation of the CTD was examined with three
concentrations of P-TEFb and 10 µM ATP (Fig.
4B). At all concentrations of P-TEFb, intermediates between
IIa and IIo were seen at early time points, indicating a progressive
increase in phosphorylation. At the lowest concentration of P-TEFb
~50% of the large subunit molecules were progressively
phosphorylated, although the molar ratio of P-TEFb to RNA polymerase II
was about 1:50. This suggests that P-TEFb was not stably associated
with the polymerase during the addition of all phosphates and,
therefore, was not completely processive. As the concentration of
P-TEFb was increased, the fraction of the large subunit that was
shifted to IIo increased to about 90%. Comparison of the kinetics of
the appearance of the intermediate forms indicates that increasing the
P-TEFb concentration had a modest but nonlinear effect on the rate of
the phosphorylation. For example, comparing 1 min with 0.01 µl of
P-TEFb to 30 s with 0.05 µl indicates that although fewer large
subunit molecules were affected, an equivalent mobility shift was
obtained. At all P-TEFb concentrations tested, there were at least some
polymerase molecules that received no or very few phosphates, while
others were heavily phosphorylated to the IIo form. Our data are
consistent with a slow initial phosphorylation event(s) followed by
more rapid subsequent phosphate incorporation. The different rates
could be due to conformational changes in the polymerase or CTD
required for initial kinase action or could be due to increased
reactivity of partially phosphorylated CTD.
We next tested the sensitivity of the kinase to both DRB and H-8 under
various conditions (Fig. 4C). At 10 µM ATP
(the same concentration of ATP used in the labeling assay), P-TEFb was
strongly inhibited by DRB at 10 µM. Under the same
conditions, approximately 10 times as much H-8 was required to inhibit
the kinase to the same level (compare 3 µM DRB with 30 µM H-8 in Fig. 4C). This 10-fold difference
was seen consistently under all conditions tested. At 100 µM ATP, both drugs were less effective (Fig.
4C, right lanes). The inhibition by DRB and H-8
was directly related to the concentration of nucleotides used (compare
Fig. 4C, 10 µM ATP + 1 µM DRB
with 100 µM ATP + 10 µM DRB). Because the
inhibition by DRB and H-8 was affected by nucleotide levels, the
relative inhibition between DRB and H-8 may be a more useful way to
compare P-TEFb with other kinases.
Since the CTD kinase activity of P-TEFb was sensitive to H-8, we tested
the effect of the drug during transcription in nuclear extracts.
Increasing amounts of DRB or H-8 were included in a continuous labeling
assay (Fig. 5A). As seen earlier, run-off
transcription was very sensitive to DRB, with a 50% inhibition point
of 0.7 µM (Fig. 5B). Under identical
transcription conditions, H-8 had a 50% inhibition point of 7 µM (Fig. 5C). This 10-fold difference is the
same as that seen with the CTD kinase assay. As expected for an
inhibitor of productive elongation, initiation and abortive
transcription were unaffected by H-8 even at concentrations that
severely inhibited the appearance of run-off (data not shown). These
data support the hypothesis that P-TEFb is the target of these kinase
inhibitors during transcription.
We have provided evidence that an intact CTD
is required for productive elongation and that P-TEFb can phosphorylate
pure RNA polymerase II. Since P-TEFb acts during elongation (23), it
should be able to phosphorylate the CTD if the polymerase is in an
early elongation complex. To test this hypothesis we used Western blot
analysis to determine the phosphorylation state of the polymerase in
isolated transcription complexes. Preinitiation complexes were formed
on an immobilized actin template as described under ``Experimental
Procedures.'' When these complexes were washed with low salt buffer
they remained intact, and Western blot analysis indicated that the
large subunit of RNA polymerase II was hypophosphorylated (Fig.
6, lane 3). Incubation of the preinitiation
complexes with ATP caused a significant shift of the polymerase to the
IIo form (Fig. 6, lane 1). Note that the antibodies used to
detect the large subunit of RNA polymerase II reacted more strongly
with the IIa form of the large subunit than the phosphorylated IIo form
(Fig. 2B, markers). This phosphorylation was
unaffected by the presence of 20 µM DRB (Fig. 6,
lane 2). This result is consistent with our earlier data
that P-TEFb is not associated with preinitiation complexes (22, 23).
Essentially all of the RNA polymerase II was removed by washing with
buffer containing 1 M KCl (Fig. 6, lane 4). When
the preinitiation complexes were incubated under transcription
conditions for a short time, a portion of the polymerases initiated
and, therefore, remained associated with the template during the high
salt wash (Fig. 6, lane 5). The CTD kinase found in the
preinitiation complex was no longer associated with the polymerase
because incubation with ATP had no effect (Fig. 6, lane 6).
However, when increasing amounts of P-TEFb were incubated with the high
salt-washed early elongation complexes, the IIa form of the polymerase
decreased and the IIo form increased (Fig. 6, lanes 7 and
8). The ability of P-TEFb to phosphorylate RNA polymerase II
in an early elongation complex was inhibited by 20 µM DRB
(Fig. 6, lanes 9 and 10).
To
determine whether P-TEFb was related to the protein kinase associated
with TFIIH (44, 45, 46, 47, 48), we compared P-TEFb with Drosophila
TFIIH purified from Drosophila embryos by the method of
Austin and Biggin (49). The two proteins did not have any subunits in
common when analyzed by SDS-PAGE and silver staining (Fig.
7A). The large subunit of P-TEFb ran as a
doublet, which may be due to phosphorylation, since we have observed
autophosphorylation of both P-TEFb subunits (data not shown). To
compare the activities of the two factors, both were tested in the CTD
kinase assay under identical conditions (Fig. 7B). Using
equal concentrations of the two kinases, as determined by relative
protein staining, both were able to incorporate similar amounts of
32PO4 into the CTD of RNA polymerase II and
cause the shift to the IIo form. The CTD kinase activity of P-TEFb was
sensitive to 20 µM DRB, while that of TFIIH was
unaffected. Since DRB inhibition could be affected by enzyme levels, we
first performed titration experiments and determined that the levels of
P-TEFb and TFIIH were not saturating (data not shown). To determine if
TFIIH could functionally replace P-TEFb, similar amounts of both
proteins were tested in the P-TEFb-dependent
Kc-FT (23) transcription assay (Fig. 7C). As
expected, the addition of increasing amounts of P-TEFb led to an
increase in DRB-sensitive run-off transcripts (Fig. 7D). In
contrast, addition of even the highest levels of TFIIH had little
effect on the amount of run-off although the added TFIIH had high CTD
kinase activity when assayed using purified RNA polymerase II.
Therefore, it appears that TFIIH cannot substitute for P-TEFb during
the transition into productive elongation.
DISCUSSION The DRB-sensitive process whereby RNA polymerase II escapes
abortive elongation and enters a productive mode of elongation is
important in controlling transcription. We have shown that an intact
CTD is essential to achieve this transition into productive elongation
and that an indispensable elongation control factor, P-TEFb, is a CTD
kinase. The drugs DRB and H-8 inhibit the transition into productive
elongation as well as the CTD kinase activity of P-TEFb. In addition,
we have shown that pure P-TEFb can phosphorylate the CTD of RNA
polymerase II in early elongation complexes. We propose that the
postinitiation phosphorylation of the CTD by P-TEFb controls the
transition into productive elongation.
The CTD is
required for transcription in vivo (42, 50, 51, 52, 53), but
demonstrating a general requirement for the CTD in vitro
requires the use of crude extracts (54) or a more purified system that
includes specific elongation control factors (23). However, in more
purified systems lacking the elongation control factors, some promoters
still demonstrate a requirement for the CTD. The CTD is not required
for transcription from the Drosophila actin 5C promoter in a
Drosophila system (42) or from the Ad-2 major late promoter
in a HeLa system (55). The CTD is required for transcription from the
murine dihydrofolate reductase promoter in a HeLa system (30, 56, 57).
Akoulitchev et al. (57) showed that an early step in
initiation from the dihydrofolate reductase promoter (formation of the
first phosphodiester bond) required the CTD. Since our results indicate
a CTD requirement during elongation, the CTD may be involved in more
than one step in transcription.
Elongation control requires an activity, negative transcription
elongation factor (N-TEF), that suppresses the appearance of long
transcripts and causes abortive elongation or premature termination. We
found that the generation of abortive elongation products was
unaffected by CTD removal. This indicates that the CTD is not required
for the negative effects of N-TEF.
Our results
show that P-TEFb is distinct from a number of CTD kinases. Subunit
composition, DRB sensitivity, and functional properties distinguish
P-TEFb from TFIIH or the TFIIH-associated kinase. P-TEFb functions
during elongation and is not stably associated with the transcription
complex at any time (21, 22, 23). This further discriminates between P-TEFb
and TFIIH as well as the kinase associated with the SRB complex and DNA
PK, which are part of the initiation complex or otherwise bound to the
template. The subunit composition and chromatographic properties of
P-TEFb are not similar to any known kinase; however, the
Drosophila homologues of all kinases shown to phosphorylate
the CTD have not been purified. Anti-phosphotyrosine antibodies failed
to react with RNA polymerase II phosphorylated by P-TEFb (data not
shown); therefore, P-TEFb is probably a serine/threonine kinase.
No other published results demonstrate a functional role of a CTD
kinase during elongation, but several studies suggest a link between
CTD kinase activity and function. Yeast CTK1 (58) and KIN28 (59) have
been shown to be required for efficient phosphorylation of the CTD
in vivo. However, it is difficult to know if the reduction
of CTD phosphorylation observed with CTK1 or KIN28 mutants is direct or
the result of decreased transcription initiation. P-TEFb or a similar
protein may play a role in HIV-1 transcription, since productive
elongation is similar to the stimulation of highly processive
elongation complexes by Tat (20). Zhou and Sharp identified a factor,
Tat-SF, required for Tat activation of HIV transcription (60), but the
relation of Tat-SF to P-TEFb is unclear. Recently, a kinase that can
associate with the HIV-1 Tat protein was shown to have DRB-sensitive
CTD kinase activity, and it may represent the human equivalent of
P-TEFb (61).
We determined
the substrate specificity of P-TEFb and examined the kinetics of CTD
phosphorylation under a variety of conditions. The nucleotide
requirements of P-TEFb CTD kinase were similar to those reported for
HeLa TFIIH (26), CTD-K1 (36), and rat liver The studies presented indicate that caution should be taken when using
inhibitors to characterize CTD kinases. The concentration of ATP used
has a direct effect on the concentration of inhibitor required to
achieve 50% inhibition. This makes it difficult to compare the effects
of inhibitors in normal kinase assays using low ATP concentrations with
transcription assays, which have higher levels of all NTPs. In
addition, the concentrations of the polymerase and kinase, the time of
the reactions, and other assay conditions influence the effect of the
inhibitors. RNA polymerase II is the natural substrate, but the
phosphorylation of the CTD is complicated. Each phosphorylation site is
unique and may be influenced by phosphorylation of other sites. It is
possible to saturate the assay with kinase such that even in the
presence of inhibitors all large subunit molecules are completely
shifted to the IIO form (data not shown). In addition, the exact
relationship of the mobility of the large subunit to the number of
phosphates added is not known. It is also not clear what level of
phosphorylation is required for function during transcription or how
other factors might influence kinase action on an elongation complex.
We believe the best correlation between kinase activity and function
during transcription can be made by comparing the effects of two
inhibitors under the same set of conditions. The 10-fold difference
between levels of DRB and H-8 required to achieve equal inhibition of
CTD phosphorylation remained constant under all conditions tested.
Since we also observed a 10-fold difference in the levels of DRB and
H-8 required to inhibit transcription, we suggest that the effect of
the drugs in transcription is due to inhibition of the CTD kinase
activity of P-TEFb.
Yankulov et al. (62) proposed that the effect of DRB and H-8
on transcription was due to inhibition of the kinase activity of TFIIH
and suggested that the TFIIH-associated kinase controlled elongation by
RNA polymerase II. Although we do not want to rule out the possibility
of TFIIH playing a role in elongation control (see below), our results
indicate directly that P-TEFb functions to control elongation and that
TFIIH cannot substitute for P-TEFb. Their conclusions were based on the
observation that DRB or H-8 inhibited the TFIIH kinase and on the
appearance of run-off transcripts during transcription with similar
dose-response curves. Their kinase assays were performed at 7.5 µM ATP, and the transcription assays were performed at
500 µM NTPs. Because different triphosphate conditions
were used in the two assays, a valid comparison of inhibition curves
cannot be made. It is likely that the DRB sensitivity they detected
during transcription was due to inhibition of P-TEFb rather than
TFIIH.
Our hypothesis that the
transition into productive elongation is controlled by
P-TEFb-dependent phosphorylation of the CTD is supported by
several in vivo studies. A number of Drosophila
genes including HSP70 have early blocked polymerases in the
IIA form, while downstream elongating polymerases are highly
phosphorylated (63). In injected Xenopus oocytes, GAL4-based
activators stimulated elongation in a DRB-sensitive manner (64). It is
possible that transcriptional activators work with or through P-TEFb.
Dubois et al. (65) found that the addition of DRB or H-8 to
HeLa cells caused a rapid decrease in the amount of RNA polymerase IIO.
Egyhazi et al. (66) recently showed that DRB inhibited the
phosphorylation of RNA polymerase II to the IIO form in
Chironomus tentans. Their results indicated that addition of
DRB immediately stopped the incorporation of phosphate into the
polymerase but that polymerases in productive elongation complexes
maintained their hyperphosphorylated states until they completed their
current round of transcription.
Although TFIIH cannot replace P-TEFb in reconstruction of productive
elongation, our results do not rule out the involvement of TFIIH or
other kinases in elongation control. It is not clear if phosphorylation
of RNA polymerase II or another factor by TFIIH is required for P-TEFb
function, but it is clear that TFIIH is not sufficient for the
functional transition into productive elongation. Isolated early
elongation complexes did not contain detectable amounts of the
hyperphosphorylated RNA polymerase II. However, a low level of
prephosphorylation by TFIIH or other CTD kinases might stimulate the
activity of P-TEFb, which has a preference for a CTD containing some
phosphate. Recently, mitogen-activated protein kinases have been
implicated in controlling the phosphorylation state of RNA polymerase
II during heat shock (39). The control of transcription by protein
phosphorylation is complex (reviewed by Karin and Hunter (67)), and the
signal transduction pathways involved have a number of transcriptional
targets. The CTD is clearly one of these targets, but conclusive
evidence concerning which kinase(s) is directly responsible and which
kinases are upstream will require further study.
The mechanism by which CTD phosphorylation induces the transition into
productive elongation remains to be determined. Our original model
suggested that the action of P-TEF counteracted the activity of N-TEF
(22). The simplest model in which the interaction of N-TEF with the CTD
is eliminated by the action of P-TEFb CTD kinase has been ruled out by
our data that indicates that at least some component of N-TEF is still
associated with early elongation complexes lacking a CTD. N-TEF may
interact with another region of the elongation complex, and
phosphorylation of the CTD by P-TEFb may counteract a specific action
of N-TEF. The role in elongation control of proteins associated with
RNA polymerase II in a holoenzyme complex (68) remains to be
established. Rasmussen and Lis (69) discussed several models for the
involvement of CTD in elongation control. Tethering of the polymerase
to the promoter by the unphosphorylated CTD remains an attractive
possibility, although no direct evidence has been found. Further work
will need to be done to determine the fate of polymerases that stop
early but do not enter productive elongation. Our early data suggested
that termination of the polymerases allowed new polymerases to
occupy the early pause sites and that this termination had a half-life
of a few minutes (22). We have recently purified and characterized an
RNA polymerase II termination factor called factor 2 (43). In these
studies we found preliminary evidence that some early elongation
complexes were resistant to termination by factor 2. The role of
termination factors and potential antitermination factors in the
elongation control process remains to be clarified.
After the initial transition into productive elongation has been
passed, it may be necessary to maintain the highly phosphorylated state
of the polymerase. However, the results of Egyhazi et al.
(66) and Kephart et al. (21) suggest that if CTD kinases are
needed for maintenance of the IIO form during elongation then these
kinases are not sensitive to DRB. Therefore, P-TEFb is not likely
involved in maintenance. Maintenance could be accomplished by the
inhibition of CTD phosphatase (70) activity. It is not clear what
causes termination once the 3 FOOTNOTES Acknowledgments We thank Rick Austin and Mark Biggin (Yale)
for the very generous gift of Drosophila TFIIH. We thank
Will Zehring for providing us with a Drosophila embryonic
nuclear extract fraction that contained P-TEFb.
REFERENCES
Volume 271, Number 43,
Issue of October 25, 1996
pp. 27176-27183
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
Acknowledgments
REFERENCES
-D-ribofuranosylbenzimidazole (DRB) and
H-8. P-TEFb is distinct from transcription factor IIH (TFIIH) because
the two factors have no subunits in common, P-TEFb is more sensitive to
DRB than is TFIIH, and most importantly, TFIIH cannot substitute
functionally for P-TEFb. We propose that phosphorylation of the CTD by
P-TEFb controls the transition from abortive into productive elongation
mode.
-tubulin (13, 14), adenovirus (15),
SV40 (16), minute virus of mice (17), and human immunodeficiency virus
(HIV)1 (18). RNA polymerase II molecules
are found blocked, during elongation, near the promoter on many genes
in Drosophila melanogaster (19). Except for the involvement
of the viral Tat protein in HIV gene expression (20), little is known
about the molecular mechanisms involved in elimination of this
block.
-32P]CTP (3000 Ci/mmol) was
from ICN. Ribonucleoside triphosphates were from Pharmacia Biotech Inc.
DRB (Sigma) was dissolved in ethanol to 10 mM and stored at 
80 °C. H-8 was from Seikagaku America
and was dissolved in 20 mM HEPES, pH 7.6, to 20 mM and stored at 4 °C. The magnetic concentrator used
was the MPC-E from Dynal. All other chemicals were reagent grade.
-32P]CTP
and brought the reaction mixture to 600 µM in ATP, GTP,
UTP, and 2 mM MnCl2. We have found that
MnCl2 increases the rate of initiation of preinitiation
complexes, thereby increasing the number of polymerases in early
elongation complexes after a short pulse.2
After 15 s, the reaction was stopped by the addition of EDTA to 10 mM. These early elongation complexes were washed 3 times
with 1 M HMKB (20 mM HEPES, 5 mM
MgCl2, 1 M KCl, and 200 µg/ml bovine serum
albumin) and then once with 55 mM HMKB (20 mM
HEPES, 5 mM MgCl2, 55 mM KCl, and
200 µg/ml bovine serum albumin). The washed early elongation
complexes were resuspended in 55 mM HMKB and incubated with
the indicated amount of chymotrypsin for 10 min. Proteolysis was
terminated by adding trypsin inhibitor to 0.1 mg/ml. After
concentration, digested early elongation complexes were either
resuspended into 55 mM HMKB and chased with 600 µM of each NTP for 10 min in the presence or absence of
Kc nuclear extract and 0.1 µl P-TEFb, or they were
analyzed by SDS-PAGE followed by immunoblotting with affinity-purified
polymerase II antibody.
-ACGAATTCCACACAATCCAAAGATC-3) and a downstream
primer (5-CAGAATTCCTATTGCCGATCCCCAGA-3) and subcloned downstream of
GST in pET21a-GST. The fusion protein was expressed, purified using a
glutathione affinity column according to the Pharmacia protocol, and
then used to immunize rabbits (Pocono Rabbit Farm). Antibodies to RNA
polymerase II were purified by first passing the crude serum through a
GST column and then passing the flow-through over a GST-rpII1 column.
Antibodies were eluted from the affinity column with low pH buffer as
described by the manufacturer.
Fig. 1.
Requirement of the CTD for productive
elongation. A, silver-stained, 6-15% SDS-polyacrylamide
gel analysis of RNA polymerase II treated with chymotrypsin for the
indicated times. Lane 20*, 20-min digestion in the presence
of trypsin inhibitor. Lane M, 10-kDa ladder markers (Life
Technologies, Inc.) with sizes indicated. IIa and
IIb indicate positions of intact and CTD-less forms of the
largest polymerase subunit, respectively. IIc indicates the
position of the second largest polymerase subunit. B,
transcription analysis of polymerases after CTD truncation
(520-nucleotide run-off from actin promoter). DRB was added to 40 µM where indicated. C, quantitation of
transcription autoradiograph and protein gel. Autoradiographs were
scanned using a Bio-Rad model GS-670 imaging densitometer. Areas of the
silver-stained gel corresponding to subunit IIa were quantitated,
normalized to the 0 digestion time, and plotted as CTD remaining. The
portions of the autoradiograph indicated as run-off and abortive
transcripts were quantitated and plotted in parallel.
[View Larger Version of this Image (72K GIF file)]
Fig. 3.
Correlation of P-TEFb transcription and CTD
kinase functions. A, P-TEFb purification scheme. Elution
points are indicated. Angled lines indicate gradient
elution, and straight lines indicate step elution. B and
C, fractions from the Mono S column were analyzed in
transcription reactions (B) and for CTD kinase activity
(C). D, E, and F, fractions
from the glycerol gradient were analyzed by SDS-PAGE (D),
for transcription (E) and CTD kinase activities
(F). B and E, Kc-FT based
transcription assays detailed under ``Experimental Procedures''
(520-nucleotide run-off). D, silver-stained, 6-15%
SDS-polyacrylamide gel with indicated positions of 124- and 43-kDa
subunits of P-TEFb. C and F, CTD kinase assays
(10 µM ATP + [
-32P]ATP, 10-min
incubation).
[View Larger Version of this Image (57K GIF file)]
Fig. 7.
Comparison of the composition and properties
of P-TEFb and TFIIH. A, silver-stained, 6-15%
SDS-polyacrylamide gel with indicated positions (arrows) of
124- and 43-kDa subunits of P-TEFb. P-TEFb is Mono S step fraction 13 (1 × = 0.01 µl) from material purified from
Drosophila embryonic nuclear extract, and TFIIH is a
Superdex 200 fraction (49), the last step in purification.
B, CTD kinase assay with 10 µM ATP and 20-min
reactions. P-TEFb is same as in A. TFIIH is a Mono S
fraction (49) (1 × = 0.1 µl), which is slightly less pure but
approximately 10 times more concentrated than that used in
A. C, continuous labeling Kc-FT-based
transcription assays detailed under ``Experimental Procedures''
(520-nucleotide run-off). P-TEFb and TFIIH are same as in B.
D, quantitation of transcription gel in C. Dried
polyacrylamide gels were imaged using the Packard InstantImagerTM, and
the portions of the gel indicated as run-off were quantitated and
normalized to the no addition (none) lanes.
[View Larger Version of this Image (49K GIF file)]
-32P]ATP (ICN) and
unlabeled ATP at 10 µM (Figs. 3 and 4) or only unlabeled
ATP at concentrations of 1-100 µM (Fig. 4) or other NTPs
or dNTPs at concentrations of 1-100 µM (Fig. 4).
Reactions were incubated for the indicated times at 23 °C and then
terminated with SDS loading buffer. Samples were analyzed on a 6-15%
SDS-polyacrylamide gel, which was silver-stained, dried, and subjected
to autoradiography if the assay contained label.
Fig. 4.
Characterization of P-TEFb kinase
activity. CTD kinase assays were performed without label and then
analyzed on silver-stained, 6-15% SDS-polyacrylamide gels with
indicated positions of RNA polymerase II subunits. A,
nucleotide usage analysis. Reactions were performed with the indicated
nucleotide and 0.05 µl of P-TEFb (see Fig. 7A) for 5 min.
B, P-TEFb titration. Reactions were performed with 10 µM ATP for the indicated times. C, ATP effect
on DRB or H-8 inhibition. Reactions were performed with 0.05 µl of
P-TEFb for 5 min.
[View Larger Version of this Image (65K GIF file)]
Fig. 2.
Removal of the CTD during elongation.
Early elongation complexes were formed, isolated, treated with
chymotrypsin, and subjected to various elongation conditions as
outlined in A and described under ``Experimental
Procedures.'' B, Western blot of complexes before and after
chymotrypsin treatment. The mobilities of intact (IIa) and
proteolyzed (IIb) RNA polymerase II large subunit are
indicated. C, early elongation complexes (EECs)
in the first lane were treated with the indicated levels of
chymotrypsin and then chased for 10 min with no further addition, the
addition of KcN supplemented with P-TEFb, or the addition
of KcN supplemented with P-TEFb and 20 µM
DRB. Labeled transcripts were analyzed on a 6% gel, and transcript
sizes are indicated.
[View Larger Version of this Image (47K GIF file)]
Fig. 6.
Phosphorylation of CTD by P-TEFb in early
elongation complexes. Preinitiation complexes (PICs)
were formed, isolated, washed with 200 mM KCl, and then
incubated for 5 min in the presence of 600 µM ATP with or
without 20 µM DRB. Early elongation complexes were
formed, isolated, washed with 1 M KCl, and then incubated
for 5 min under the indicated conditions. The treated preinitiation
complexes and early elongation complexes were resolved by 6-15%
gradient gel followed by Western blot using the affinity-purified RNA
polymerase II antibodies (see ``Experimental Procedures'').
[View Larger Version of this Image (32K GIF file)]
Fig. 5.
Inhibition of productive elongation by DRB
and H-8. A, transcription in KcN extract
(520-nucleotide actin run-off). Transcription reactions were for 20 min
in the presence of the indicted concentrations of H-8 and DRB.
B and C, quantitation of levels of run-off
transcripts as determined using a Packard InstantImagerTM.
[View Larger Version of this Image (54K GIF file)]
(27) but were different
from those for yeast factor b (28) and CTD-K2 (36). The kinetics of
phosphorylation using different concentrations of kinase are not
consistent with processive P-TEFb action during the phosphorylation of
purified RNA polymerase II. P-TEFb does seem to prefer to phosphorylate
a CTD that has already been phosphorylated, which may have implications
for its function after initiation (see below).
-end of a gene has been transcribed, but
it is possible that a reduction in CTD phosphorylation might act as a
termination signal.
To whom correspondence should be addressed. Tel.: 319-335-7910;
Fax: 319-335-9570; E-mail: david-price{at}uiowa.edu.
1
The abbreviations used are: HIV, human
immunodeficiency virus; P-TEF, positive transcription elongation
factor; N-TEF, negative transcription elongation factor; TFIIH,
transcription factor IIH; CTD, carboxyl-terminal domain; PAGE,
polyacrylamide gel electrophoresis; KcN, KcN
cell nuclear extract.
2
D. Chafin, J. Peng, and D. Price, manuscript in
preparation.
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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G. Nogues, M. J. Munoz, and A. R. Kornblihtt Influence of Polymerase II Processivity on Alternative Splicing Depends on Splice Site Strength J. Biol. Chem., December 26, 2003; 278(52): 52166 - 52171. [Abstract] [Full Text] [PDF]
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A. K. Boehm, A. Saunders, J. Werner, and J. T. Lis Transcription Factor and Polymerase Recruitment, Modification, and Movement on dhsp70 In Vivo in the Minutes following Heat Shock Mol. Cell. Biol., November 1, 2003; 23(21): 7628 - 7637. [Abstract] [Full Text] [PDF]
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 S. C. Howard, A. Hester, and P. K. Herman The Ras/PKA Signaling Pathway May Control RNA Polymerase II Elongation via the Spt4p/Spt5p Complex in Saccharomyces cerevisiae Genetics, November 1, 2003; 165(3): 1059 - 1070. [Abstract] [Full Text] [PDF]
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B. E. Schwartz, S. Larochelle, B. Suter, and J. T. Lis Cdk7 Is Required for Full Activation of Drosophila Heat Shock Genes and RNA Polymerase II Phosphorylation In Vivo Mol. Cell. Biol., October 1, 2003; 23(19): 6876 - 6886. [Abstract] [Full Text] [PDF]
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M.-C. Keogh, V. Podolny, and S. Buratowski Bur1 Kinase Is Required for Efficient Transcription Elongation by RNA Polymerase II Mol. Cell. Biol., October 1, 2003; 23(19): 7005 - 7018. [Abstract] [Full Text] [PDF]
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K. D. Johnson, J. A. Grass, C. Park, H. Im, K. Choi, and E. H. Bresnick Highly Restricted Localization of RNA Polymerase II within a Locus Control Region of a Tissue-Specific Chromatin Domain Mol. Cell. Biol., September 15, 2003; 23(18): 6484 - 6493. [Abstract] [Full Text] [PDF]
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M. Gerber and A. Shilatifard Transcriptional Elongation by RNA Polymerase II and Histone Methylation J. Biol. Chem., July 11, 2003; 278(29): 26303 - 26306. [Abstract] [Full Text] [PDF]
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T. E. Adamson and D. H. Price Cotranscriptional Processing of Drosophila Histone mRNAs Mol. Cell. Biol., June 15, 2003; 23(12): 4046 - 4055. [Abstract] [Full Text] [PDF]
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B. D. Nguyen, K. L. Abbott, K. Potempa, M. S. Kobor, J. Archambault, J. Greenblatt, P. Legault, and J. G. Omichinski NMR structure of a complex containing the TFIIF subunit RAP74 and the RNA polymerase II carboxyl-terminal domain phosphatase FCP1 PNAS, May 13, 2003; 100(10): 5688 - 5693. [Abstract] [Full Text] [PDF]
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 F. Estruch and C. N. Cole An Early Function during Transcription for the Yeast mRNA Export Factor Dbp5p/Rat8p Suggested by Its Genetic and Physical Interactions with Transcription Factor IIH Components Mol. Biol. Cell, April 1, 2003; 14(4): 1664 - 1676. [Abstract] [Full Text] [PDF]
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F. Zhang, M. Barboric, T. K. Blackwell, and B. M. Peterlin A model of repression: CTD analogs and PIE-1 inhibit transcriptional elongation by P-TEFb Genes & Dev., March 15, 2003; 17(6): 748 - 758. [Abstract] [Full Text] [PDF]
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K. Fujinaga, D. Irwin, R. Taube, F. Zhang, M. Geyer, and B. M. Peterlin A Minimal Chimera of Human Cyclin T1 and Tat Binds TAR and Activates Human Immunodeficiency Virus Transcription in Murine Cells J. Virol., November 13, 2002; 76(24): 12934 - 12939. [Abstract] [Full Text] [PDF]
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S. S. Mandal, H. Cho, S. Kim, K. Cabane, and D. Reinberg FCP1, a Phosphatase Specific for the Heptapeptide Repeat of the Largest Subunit of RNA Polymerase II, Stimulates Transcription Elongation Mol. Cell. Biol., November 1, 2002; 22(21): 7543 - 7552. [Abstract] [Full Text] [PDF]
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 I. Gojo, B. Zhang, and R. G. Fenton The Cyclin-dependent Kinase Inhibitor Flavopiridol Induces Apoptosis in Multiple Myeloma Cells through Transcriptional Repression and Down-Regulation of Mcl-1 Clin. Cancer Res., November 1, 2002; 8(11): 3527 - 3538. [Abstract] [Full Text] [PDF]
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A. Michienzi, S. Li, J. A. Zaia, and J. J. Rossi A nucleolar TAR decoy inhibitor of HIV-1 replication PNAS, October 29, 2002; 99(22): 14047 - 14052. [Abstract] [Full Text] [PDF]
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S. R. Eberhardy and P. J. Farnham Myc Recruits P-TEFb to Mediate the Final Step in the Transcriptional Activation of the cad Promoter J. Biol. Chem., October 11, 2002; 277(42): 40156 - 40162. [Abstract] [Full Text] [PDF]
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K. Fujinaga, D. Irwin, M. Geyer, and B. M. Peterlin Optimized Chimeras between Kinase-Inactive Mutant Cdk9 and Truncated Cyclin T1 Proteins Efficiently Inhibit Tat Transactivation and Human Immunodeficiency Virus Gene Expression J. Virol., October 2, 2002; 76(21): 10873 - 10881. [Abstract] [Full Text] [PDF]
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X. Lin, R. Taube, K. Fujinaga, and B. M. Peterlin P-TEFb Containing Cyclin K and Cdk9 Can Activate Transcription via RNA J. Biol. Chem., May 3, 2002; 277(19): 16873 - 16878. [Abstract] [Full Text] [PDF]
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 G. Prelich RNA Polymerase II Carboxy-Terminal Domain Kinases: Emerging Clues to Their Function Eukaryot. Cell, April 1, 2002; 1(2): 153 - 162. [Full Text] [PDF]
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C. J. Bonangelino, J. J. Nau, J. E. Duex, M. Brinkman, A. E. Wurmser, J. D. Gary, S. D. Emr, and L. S. Weisman Osmotic stress-induced increase of phosphatidylinositol 3,5-bisphosphate requires Vac14p, an activator of the lipid kinase Fab1p J. Cell Biol., March 18, 2002; 156(6): 1015 - 1028. [Abstract] [Full Text] [PDF]
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B. Zhang, I. Gojo, and R. G. Fenton Myeloid cell factor-1 is a critical survival factor for multiple myeloma Blood, March 15, 2002; 99(6): 1885 - 1893. [Abstract] [Full Text] [PDF]
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M. Kimura, H. Suzuki, and A. Ishihama Formation of a Carboxy-Terminal Domain Phosphatase (Fcp1)/TFIIF/RNA Polymerase II (pol II) Complex in Schizosaccharomyces pombe Involves Direct Interaction between Fcp1 and the Rpb4 Subunit of pol II Mol. Cell. Biol., March 1, 2002; 22(5): 1577 - 1588. [Abstract] [Full Text] [PDF]
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M. Hoque, T. M. Young, C.-G. Lee, G. Serrero, M. B. Mathews, and T. Pe'ery The Growth Factor Granulin Interacts with Cyclin T1 and Modulates P-TEFb-Dependent Transcription Mol. Cell. Biol., March 1, 2002; 23(5): 1688 - 1702. [Abstract] [Full Text] [PDF]
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J. Martin-Serrano, K. Li, and P. D. Bieniasz Cyclin T1 Expression Is Mediated by a Complex and Constitutively Active Promoter and Does Not Limit Human Immunodeficiency Virus Type 1 Tat Function in Unstimulated Primary Lymphocytes J. Virol., January 1, 2002; 76(1): 208 - 219. [Abstract] [Full Text] [PDF]
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S. R. Eberhardy and P. J. Farnham c-Myc Mediates Activation of the cad Promoter via a Post-RNA Polymerase II Recruitment Mechanism J. Biol. Chem., December 14, 2001; 276(51): 48562 - 48571. [Abstract] [Full Text] [PDF]
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R. E. Kiernan, S. Emiliani, K. Nakayama, A. Castro, J. C. Labbe, T. Lorca, K.-i. Nakayama, and M. Benkirane Interaction between Cyclin T1 and SCFSKP2 Targets CDK9 for Ubiquitination and Degradation by the Proteasome Mol. Cell. Biol., December 1, 2001; 21(23): 7956 - 7970. [Abstract] [Full Text] [PDF]
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D. B. Renner, Y. Yamaguchi, T. Wada, H. Handa, and D. H. Price A Highly Purified RNA Polymerase II Elongation Control System J. Biol. Chem., November 2, 2001; 276(45): 42601 - 42609. [Abstract] [Full Text] [PDF]
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H. L. Jenkins and C. A. Spencer RNA Polymerase II Holoenzyme Modifications Accompany Transcription Reprogramming in Herpes Simplex Virus Type 1-Infected Cells J. Virol., October 15, 2001; 75(20): 9872 - 9884. [Abstract] [Full Text] [PDF]
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 M. Ljungman and M. T. Paulsen The Cyclin-Dependent Kinase Inhibitor Roscovitine Inhibits RNA Synthesis and Triggers Nuclear Accumulation of p53 That Is Unmodified at Ser15 and Lys382 Mol. Pharmacol., October 1, 2001; 60(4): 785 - 789. [Abstract] [Full Text] [PDF]
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D. L. Lindstrom and G. A. Hartzog Genetic Interactions of Spt4-Spt5 and TFIIS With the RNA Polymerase II CTD and CTD Modifying Enzymes in Saccharomyces cerevisiae Genetics, October 1, 2001; 159(2): 487 - 497. [Abstract] [Full Text] [PDF]
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P. Licciardo, L. Ruggiero, L. Lania, and B. Majello Transcription activation by targeted recruitment of the RNA polymerase II CTD phosphatase FCP1 Nucleic Acids Res., September 1, 2001; 29(17): 3539 - 3545. [Abstract] [Full Text] [PDF]
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S. Murray, R. Udupa, S. Yao, G. Hartzog, and G. Prelich Phosphorylation of the RNA Polymerase II Carboxy-Terminal Domain by the Bur1 Cyclin-Dependent Kinase Mol. Cell. Biol., July 1, 2001; 21(13): 4089 - 4096. [Abstract] [Full Text] [PDF]
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 A. De Luca, A. Tosolini, P. Russo, A. Severino, A. Baldi, L. De Luca, I. Cavallotti, F. Baldi, A. Giordano, J. R. Testa, et al. Cyclin T2A Gene Maps on Human Chromosome 2q21 J. Histochem. Cytochem., June 1, 2001; 49(6): 693 - 698. [Abstract] [Full Text]
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S.-H. Chao, A. L. Greenleaf, and D. H. Price Juglone, an inhibitor of the peptidyl-prolyl isomerase Pin1, also directly blocks transcription Nucleic Acids Res., February 1, 2001; 29(3): 767 - 773. [Abstract] [Full Text] [PDF]
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S. M. Foskett, R. Ghose, D. N. Tang, D. E. Lewis, and A. P. Rice Antiapoptotic Function of Cdk9 (TAK/P-TEFb) in U937 Promonocytic Cells J. Virol., February 1, 2001; 75(3): 1220 - 1228. [Abstract] [Full Text]
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 C. Herrmann and M. Mancini The Cdk9 and cyclin T subunits of TAK/P-TEFb localize to splicing factor-rich nuclear speckle regions J. Cell Sci., January 4, 2001; 114(8): 1491 - 1503. [Abstract] [PDF]
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M. E. Garber, T. P. Mayall, E. M. Suess, J. Meisenhelder, N. E. Thompson, and K. A. Jones CDK9 Autophosphorylation Regulates High-Affinity Binding of the Human Immunodeficiency Virus Type 1 Tat-P-TEFb Complex to TAR RNA Mol. Cell. Biol., September 15, 2000; 20(18): 6958 - 6969. [Abstract] [Full Text]
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Y. W. Fong and Q. Zhou Relief of Two Built-In Autoinhibitory Mechanisms in P-TEFb Is Required for Assembly of a Multicomponent Transcription Elongation Complex at the Human Immunodeficiency Virus Type 1 Promoter Mol. Cell. Biol., August 15, 2000; 20(16): 5897 - 5907. [Abstract] [Full Text]
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S. M. Carty, A. C. Goldstrohm, C. Suñé, M. A. Garcia-Blanco, and A. L. Greenleaf Protein-interaction modules that organize nuclear function: FF domains of CA150 bind the phosphoCTD of RNA polymerase II PNAS, July 19, 2000; (2000) 160266597. [Abstract] [Full Text]
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M. Zhou, M. A. Halanski, M. F. Radonovich, F. Kashanchi, J. Peng, D. H. Price, and J. N. Brady Tat Modifies the Activity of CDK9 To Phosphorylate Serine 5 of the RNA Polymerase II Carboxyl-Terminal Domain during Human Immunodeficiency Virus Type 1 Transcription Mol. Cell. Biol., July 15, 2000; 20(14): 5077 - 5086. [Abstract] [Full Text]
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J. W. Steinke, S. J. Kopytek, and D. O. Peterson Discrete promoter elements affect specific properties of RNA polymerase II transcription complexes Nucleic Acids Res., July 15, 2000; 28(14): 2726 - 2735. [Abstract] [Full Text] [PDF]
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M. N. Szentirmay and M. Sawadogo SURVEY AND SUMMARY: Spatial organization of RNA polymerase II transcription in the nucleus Nucleic Acids Res., May 15, 2000; 28(10): 2019 - 2025. [Abstract] [Full Text] [PDF]
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A. L. Lehman and M. E. Dahmus The Sensitivity of RNA Polymerase II in Elongation Complexes to C-terminal Domain Phosphatase J. Biol. Chem., May 12, 2000; 275(20): 14923 - 14932. [Abstract] [Full Text] [PDF]
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D. Ivanov, Y. T. Kwak, J. Guo, and R. B. Gaynor Domains in the SPT5 Protein That Modulate Its Transcriptional Regulatory Properties Mol. Cell. Biol., May 1, 2000; 20(9): 2970 - 2983. [Abstract] [Full Text]
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D. H. Price P-TEFb, a Cyclin-Dependent Kinase Controlling Elongation by RNA Polymerase II Mol. Cell. Biol., April 15, 2000; 20(8): 2629 - 2634. [Full Text]
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H. Tang, Y. Liu, L. Madabusi, and D. S. Gilmour Promoter-Proximal Pausing on the hsp70 Promoter in Drosophila melanogaster Depends on the Upstream Regulator Mol. Cell. Biol., April 1, 2000; 20(7): 2569 - 2580. [Abstract] [Full Text]
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J. T. Lis, P. Mason, J. Peng, D. H. Price, and J. Werner P-TEFb kinase recruitment and function at heat shock loci Genes & Dev., April 1, 2000; 14(7): 792 - 803. [Abstract] [Full Text]
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