From the
Each cycle of transcription appears to be associated with the
reversible phosphorylation of the repetitive COOH-terminal domain (CTD)
of the largest RNA polymerase (RNAP) II subunit. The dephosphorylation
of RNAP II by CTD phosphatase, therefore, plays an important role in
the transcription cycle. The following studies characterize the
activity of HeLa cell CTD phosphatase with a special emphasis on the
regulation of CTD phosphatase activity. Results presented here suggest
that RNAP II contains a docking site for CTD phosphatase that is
essential in the dephosphorylation reaction and is distinct from the
CTD. This is supported by the observations that (a)
phosphorylated recombinant CTD is not a substrate for CTD phosphatase, (b) RNAP IIB, which lacks the CTD, and RNAP IIA are
competitive inhibitors of CTD phosphatase and (c) CTD
phosphatase can form a stable complex with RNAP II. To test the
possibility that the general transcription factors may be involved in
the regulation of CTD phosphatase, CTD phosphatase activity was
examined in the presence of recombinant or highly purified general
transcription factors. TFIIF stimulates CTD phosphatase activity
5-fold. The RAP74 subunit of TFIIF alone contained the stimulatory
activity and the minimal region sufficient for stimulation corresponds
to COOH-terminal residues 358-517. TFIIB inhibits the stimulatory
activity of TFIIF but has no effect on CTD phosphatase activity in the
absence of TFIIF. The potential importance of the docking site on RNAP
II and the effect of TFIIF and TFIIB in regulating the
dephosphorylation of RNAP II at specific times in the transcription
cycle are discussed.
RNA polymerase (RNAP)
The CTD of
mammalian RNAP II contains more than 250 serine/threonine/tyrosine
residues and is subject to extensive phosphorylation. RNAP II with a
phosphorylated CTD is referred to as RNAP IIO whereas the
unphosphorylated form is referred to as RNAP IIA. Phosphorylation of
the CTD results in a decrease in the electrophoretic mobility of the
largest subunit in SDS-PAGE. The phosphorylated subunit, designated
IIo, has an apparent M
A multiplicity of protein kinases
capable of phosphorylating the CTD in vitro have been
characterized. It is not clear if this multiplicity is a reflection of
multiple CTD kinases functioning at the same step in the transcription
cycle, discrete CTD kinases functioning at different steps in the
transcription cycle, and/or the adventitious use of the CTD as an in vitro substrate by protein kinases that do not
phosphorylate the CTD in vivo. Of special interest is the CTD
kinase associated with TFIIH(2, 3, 4, 5, 6, 7) and yeast CTK1
(8). The association of CTD kinase with an essential transcription
factor suggests that this kinase functions in CTD phosphorylation in vivo. The observation that disruption of the gene encoding
the largest subunit of CTK1 results in a change in RNAP II
phosphorylation suggests that CTK1 is also one of several protein
kinases that influence phosphorylation of the CTD in vivo.
Much less is known about CTD phosphatases. A phosphatase that can
dephosphorylate RNAP IIO has been purified from a HeLa cell extract
(9). This enzyme is a type 2C phosphatase, requires Mg
Results from a variety
of studies indicate that RNAPs IIA and IIO have distinct functions in
transcription and that each round of transcription is associated with
the reversible phosphorylation of the CTD. Promoter-dependent
transcription in vitro requires a set of general transcription
factors including TFIIA, -B, -D, -E, -F, -H, and -J (for a recent
review see Ref. 10. TFIIA, -B, and -D assemble onto the promoter
followed by RNAP II which is recruited as a complex with TFIIF. Finally
TFIIE, -H, and -J assemble into the preinitiation complex. The state of
phosphorylation of the CTD appears to play a direct role in the
recruitment of RNAP as indicated by the observation that RNAP IIA is
preferentially recruited relative to RNAP IIO to both the adenovirus-2
major late (Ad2-ML) and dihydrofolate reductase
promoters(11, 12, 13) . Presumably the CTD
kinase associated with TFIIH phosphorylates the CTD immediately
preceding or concomitant with the initiation of transcription. The
functional consequences of phosphorylation of the CTD are not known but
likely alter interactions mediated by the unphosphorylated CTD in the
preinitiation complex. Phosphorylation of the CTD appears dispensable
for transcription in vitro from the Ad2-MLP(14) .
Transcript elongation is catalyzed by RNAP IIO. Recently, it has
been shown that RNAP II paused early in elongation on some Drosophila genes is unphosphorylated(15) . The
observation that activation of transcription correlates with
phosphorylation of the CTD suggests that CTD phosphorylation may be
essential to generate an elongation competent form of RNAP II.
Accordingly, the CTD may play a positive role in transcript elongation.
The CTD is presumably dephosphorylated by CTD phosphatase after
completion of the transcript.
Since RNAPs IIA and IIO have distinct
roles in transcription, CTD kinases and CTD phosphatase(s) that
catalyze the interconversion of these two forms must be carefully
regulated. Both CTD kinases and CTD phosphatases have the potential to
be positive or negative effectors of transcription depending on where
they act in the transcription cycle. For example, CTD phosphatase that
dephosphorylates free RNAP IIO may function as a positive effector of
transcription by increasing the amount of RNAP IIA available for
recruitment to the preinitiation complex. Conversely, CTD phosphatase
that dephosphorylates RNAP IIO in an elongation complex could in
principle act as a negative effector. This is based on the assumption
that the phosphorylated CTD plays a positive role in transcript
elongation. Although this assumption remains to be established, the
observation that transcript elongation in HeLa cells is catalyzed
exclusively by RNAP IIO suggests either that the CTD of elongating RNAP
II is not a substrate for CTD phosphatase or that dephosphorylation of
the CTD causes transcript elongation to cease. The objective of these
studies is to gain insights into the regulation of CTD phosphatase
activity. Studies presented here indicate that a site on RNAP II that
is distinct from the CTD acts as a docking site for CTD phosphatase.
Furthermore, TFIIF and TFIIB appear to be involved in the regulation of
CTD phosphatase activity.
The observation that CTD phosphatase inhibits
the assembly of RNAP II into a preinitiation complex suggests that it
may differentially influence transcription if added prior to or after
complex formation. Results presented in Fig. 4A show
that CTD phosphatase inhibits transcription if added before
preinitiation complex assembly but had a greatly reduced effect if
added 1 min after the initiation of transcription (compare lanes 2 and 3 with lanes 5 and 6). Since CTD
phosphatase inhibits transcription at a stage before CTD
phosphorylation normally occurs, it is unlikely that the inhibitory
effect of CTD phosphatase is due to dephosphorylation of the CTD.
Furthermore, magnesium is required for CTD phosphatase activity but was
not present during the preincubation reaction. Finally, the observation
that CTD phosphatase inhibits transcription from both RNAP IIA and IIB
establishes that the inhibitory effect is not mediated by the CTD (Fig. 4B).
The following results support the idea that there is a
docking site on RNAP II to which CTD phosphatase binds and thereby
gains access to the CTD. First, RNAPs IIA and IIB are competitive
inhibitors of CTD phosphatase activity. The docking site must be
outside the CTD since RNAP IIB, which lacks the CTD, is as effective as
RNAP IIA in inhibiting CTD phosphatase activity. Second, RNAP II can
form a stable complex with CTD phosphatase as indicated by the
observations that CTD phosphatase (a) binds to a RNAP II
affinity column and (b) prevents the assembly of RNAP IIA into
the preinitiation complex. Third, rCTDo cannot be dephosphorylated by
CTD phosphatase. An alternative interpretation of the finding that
rCTDo is not a substrate is that the conformation of the phosphorylated
CTD in RNAP IIO is different from that of the recombinant CTD and that
CTD phosphatase recognizes only the native conformation. This is
unlikely given the apparent independence of the CTD from the core of
RNAP II and that CTD kinases in general phosphorylate both rCTD and
RNAP IIA. Furthermore, this interpretation does not account for the
fact that RNAPs IIA and IIB are competitive inhibitors of the
phosphatase reaction.
The indication that CTD phosphatase must first
interact with a site on RNAP II distinct from the CTD, before the CTD
can be dephosphorylated, has important implications for the regulation
of CTD phosphatase activity. The accessibility of the docking site
could control the entry of CTD phosphatase and hence dephosphorylation
of the CTD. The accessibility of the docking site may be influenced by
the association of transcription factors and/or the conformation of
RNAP II in the initiation, elongation, and termination complex. This is
in contrast to a model in which CTD phosphatase has ready access to the
extended CTD.
Many protein phosphatases contain targeting subunits
that bind at specific sites to localize the phosphatase to its site of
action(35) . The domain in CTD phosphatase that binds to the
docking site on RNAP II may be analogous to a targeting subunit and
would explain the specificity of CTD phosphatase for RNAP II. The
process of docking could account for the processive behavior of CTD
phosphatase and may also be required to generate an active conformation
of CTD phosphatase (Fig. 9). Alternatively, the docking process
may ``load'' CTD phosphatase onto the CTD which could then
disengage from the docking site and proceed along the CTD to
dephosphorylate it.
The stimulatory activity of TFIIF
appears to reside in the RAP74 subunit. The addition of RAP30, either
alone or in combination with RAP74, has no effect on CTD phosphatase
activity. However, since it is unlikely that RAP30 and RAP74 assemble
into intact TFIIF under these conditions, the possibility that RAP30
participates directly in TFIIF mediated stimulation cannot be excluded.
Interestingly, TFIIF and RAP74 differ in their level of stimulation, 5-
and 20-fold, respectively, and the amount required for stimulation. The
ability of TFIIF to stimulate CTD phosphatase at lower concentrations
than RAP74 suggests that TFIIF has a higher affinity for RNAP II than
the RAP74 subunit.
The minimal region of RAP74 sufficient for
stimulation is the COOH-terminal region spanning residues
358-517. This COOH-terminal region includes an RNAP II binding
site and accessibility of this site is affected by
NH
TFIIB can bind to both RNAP II and TFIIF and was
found to inhibit TFIIF-stimulated CTD phosphatase activity. It is
important to note that TFIIB does not influence the basal level of CTD
phosphatase activity. TFIIB functions early in initiation and
facilitates the recruitment of RNAP II/TFIIF. The mechanism by which
TFIIB facilitates the recruitment of RNAP II is not clear but likely
involves a direct interaction of TFIIB with RNAP II, RAP30, and/or
RAP74(37, 39) .
The finding that CTD phosphatase
gains access to the CTD by interaction with a docking site on RNAP II
distinct from the CTD and that both TFIIF and TFIIB influence CTD
phosphatase activity suggests that the regulation of CTD
dephosphorylation may be complex. One interpretation of these results
is that the association of TFIIF (or RAP74) with RNAP II facilitates
the interaction of CTD phosphatase with RNAP II either by a direct
interaction between TFIIF and CTD phosphatase or by inducing a
conformational change in RNAP II that makes the docking site more
accessible. CTD phosphatase activity would then be stimulated at
specific steps in the transcription cycle by the interaction of TFIIF
with RNAP II.
TFIIF is a component of the preinitiation complex and
could in principle promote dephosphorylation of the CTD during
initiation. However, phosphorylation of RNAP II occurs during the
transition to a stable elongation complex, and although its role in
this transition remains to be defined, the stimulation of
dephosphorylation of RNAP II at this stage appears antagonistic to the
transcription process. TFIIB may function at this step to inhibit the
normal stimulation of CTD phosphatase activity by TFIIF. In addition,
the binding site for CTD phosphatase may be occluded in the
preinitiation complex. To test these ideas, it will be of considerable
interest to examine the ability of CTD phosphatase to dephosphorylate
RNAP II at various stages in the transcription cycle.
We gratefully acknowledge Jon Chesnut for preparation
of the general transcription factors, Grace Dahmus for preparation of
RNAP I, RNAP IIA, and CKII and technical assistance, and Mona Kang for
preparation of RNAP IIB and recombinant CTD. We also thank Mona Kang,
Sang Soo Lee, and Alan Lehman for their helpful comments during the
course of these studies and for their review of this manuscript.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(
)II is a
multisubunit enzyme responsible for the transcription of protein coding
genes in eukaryotes. The largest subunit contains at its COOH terminus
a highly conserved domain not found in other RNAPs (for a recent review
see Ref. 1). This COOH-terminal domain (CTD) is composed of tandem
repeats of the consensus sequence YSPTSPS. RNAP II from plasmodium
contains 19 repeats, mammals contain 52 repeats, and other eukaryotes
contain an intermediate number of repeats. Genetic studies show that
the CTD is essential in vivo although it is dispensable for
transcription from some promoters in vitro.
of 240,000 in mammalian
cells whereas the unphosphorylated subunit, designated IIa, has an
apparent M
of 214,000. Interconversion of the two
forms and separation on SDS-PAGE form a simple assay for CTD kinases
and phosphatases. A third form, RNAP IIB, which lacks the CTD as a
consequence of limited proteolysis during the purification process, is
not found in vivo.
for activity, and has a molecular weight of about 200,000. The
CTD phosphatase is specific for the dephosphorylation of serine and
threonine in the consensus CTD repeat in that it does not
dephosphorylate RNAP II phosphorylated by casein kinase II on the most
COOH-terminal serine adjacent to the consensus repeat. Furthermore, CTD
phosphatase does not dephosphorylate RNAP IIO prepared in vitro by phosphorylation of RNAP IIA with c-Abl tyrosine
kinase.(
)
CTD phosphatase can dephosphorylate
RNAP IIO purified from calf thymus or RNAP IIO prepared in vitro by the phosphorylation of RNAP IIA with two different HeLa cell
CTD kinases. Interestingly, CTD phosphatase and several CTD kinases
appear to act processively resulting in the complete conversion of RNAP
IIO to IIA and RNAP IIA to IIO, respectively.
Materials
Radiolabeled ribonucleotides
[-
P]ATP (3000 Ci/mmol) and
[
-
P]CTP (800 Ci/mmol) were obtained from
DuPont NEN. The Resource Q column was obtained from Pharmacia Biotech
Inc. Recombinant mouse CTD was expressed in and purified from Escherichia coli and was kindly provided by Mona Kang
(University of California, Davis). The original clone was provided by
Bill Dynan (University of Colorado, Boulder). The RAP30 and RAP74
antisera was prepared as described in Wang et al.(16) .
Purification of Proteins
CTD phosphatase was
purified from HeLa cells as described by Chambers and
Dahmus(9) . RNAPs IIA and IIB were purified from calf thymus by
the method of Hodo and Blatti (17) with the modifications
described by Kang and Dahmus(13) . RNAP I was purified from calf
thymus as described by Dahmus(18) . TFIIB(19) , TBP (20),
and TFIIE (21) were expressed in E. coli. TFIIF was
expressed in baculovirus and purified as described
previously(22) . TFIIH (23) and TFIIJ (24) were
purified from HeLa cells. RAP30, RAP74, and RAP74 deletion proteins,
each modified with a COOH-terminal His tag, were expressed in E.
coli and purified using Ni affinity
chromatography.
(
)Although TFIIF, RAP30, and
RAP74 form multimeric complexes in solution, for simplicity, the molar
amounts are calculated assuming a heterodimeric structure for TFIIF and
monomeric structures for RAP30 and RAP74. The DE0.25 transcription
extract was purified from HeLa cells as described
previously(13) . CTD kinase was purified from the DE0.25
transcription extract. The DE0.25 fraction was loaded on a Resource Q
column and developed with a 15-ml linear gradient of 0.1-0.6 M KCl. The peak of CTD kinase activity eluted at 0.23 M and contained CTD kinase 1(25) . Casein kinase II was
purified as described previously(26) .
RNAP II Affinity Chromatography
A HeLa cell
nuclear extract was chromatographed on either a RNAP II or control
column as described previously(27) .
Preparation of
RNAP IIA was labeled with casein kinase II and
[P-Labeled
Substrates
-
P]ATP as described by Chesnut et
al.(12) .
P-Labeled RNAP IIA was incubated
with excess cold ATP (2 mM) and CTD kinase to generate RNAP
IIO.
P-Labeled RNAP IIO was purified by DE52
chromatography as described previously(12) .
P-Labeled recombinant CTDo (rCTDo) was prepared by the
same procedure using 20 fmol of recombinant CTD except it was not
further purified on DE52.
P-Labeled RNAP IIO used as a
control in experiments with
P-labeled CTDo was prepared in
the same manner using 20 fmol of RNAP IIA.
P-Labeled
subunit IIo was isolated from
P-labeled RNAP IIO by
electrophoresis on a SDS 5% polyacrylamide gel. The subunit IIo band
was located by autoradiography and excised, the protein electroeluted
from the gel, the SDS extracted as described previously(28) ,
and the dried pellet dissolved in and dialyzed against buffer A (50
mM Tris, pH 7.9, 20% glycerol, 0.5 mM dithiothreitol,
0.1 mM EDTA, 0.025% Tween 80, 10 mM KCl, and 10
mM MgCl
).
CTD Phosphatase Assay
CTD phosphatase assays were
performed in buffer A as described previously(9) . SDS-PAGE
analysis of the reactions was carried out according to the method of
Laemmli(29) . The resolving gel was either 5% polyacrylamide or
a linear gradient of 5-17.5% polyacrylamide. CTD phosphatase
activity was quantitated on a BAS1000 PhosphorImager (Fuji) and is
given in photo-stimulated luminescence values. One unit of CTD
phosphatase converts 1 pmol of RNAP IIO to RNAP IIA in 1 min.
Transcription Reactions
The 822-base pair DNA
template containing the adenovirus-2 major late promoter (Ad2-MLP) was
prepared essentially as described by Payne et al.(30) .
Transcription reactions were performed as described previously (31) with the following modifications. Preinitiation complexes
were assembled with 5 µl of DE0.25 (replacing the DE0.15 and DE0.25
fractions), 5-7 10
units of RNAP IIA,
35 ng of the DNA template in a final volume of 31 µl. Reactions
were incubated at 30 °C for 30 min. A
P-labeled
147-base pair DNA fragment was added to the stop solution to serve as
an internal control for recovery of the RNA during the extraction
procedure. Transcription reactions with the purified transcription
factors were performed as described above except that reactions
contained 1.4 pmol of TFIIB, 0.5 pmol of TFIIE, 1.4 pmol of TFIIF, 0.5
µg of TFIIH, 1 µg of TFIIJ, and 1.6 pmol of TBP in place of the
DE0.25 fraction.
Fractionation of Preinitiation Complexes with Sepharose
CL-4B
Preinitiation complexes were formed in the presence of the
DE0.25 extract and P-labeled RNAP IIA in 3.5
transcription reactions as described above. Complexes were fractionated
on Sepharose CL-4B as described previously(13) .
CTD Phosphatase Interacts with a Site on RNAP II
Distinct from the CTD
Previous studies indicate that CTD
phosphatase specifically dephosphorylates phosphoserine and
phosphothreonine within the consensus CTD repeat and that it
dephosphorylates the CTD in a processive manner(9) . In an
effort to further characterize the specificity of CTD phosphatase, its
ability to utilize recombinant CTD phosphorylated with CTD kinase was
examined. The phosphorylated recombinant CTD is referred to as rCTDo
and undergoes the mobility shift in SDS-PAGE characteristic of RNAP
subunit IIo (Fig. 1A, compare lanes 1 and 2). Surprisingly, CTD phosphatase did not dephosphorylate
rCTDo (Fig. 1A) but did dephosphorylate an equimolar
sample of RNAP IIO prepared in the same manner (Fig. 1B). The addition of RNAP IIA did not stimulate
dephosphorylation of rCTDo (data not shown). Furthermore, the largest
subunit of RNAP IIO, purified by SDS-PAGE, was also not
dephosphorylated by CTD phosphatase (Fig. 1C). One
interpretation of these results is that a site on native RNAP II
outside the CTD must be recognized by CTD phosphatase before it can
proceed to dephosphorylate the CTD.
Figure 1:
Recombinant CTD and purified subunit
IIo are not substrates for CTD phosphatase. The ability of CTD
phosphatase to dephosphorylate CTDo, RNAP IIO, and purified subunit IIo
was determined as described under ``Experimental
Procedures.'' Panel A, aliquots of P-labeled
CTDo (0.5 fmol) assayed in the presence of varying amounts of CTD
phosphatase and analyzed on a 5-17.5% polyacrylamide-SDS gel. Lane 1, CTDo; lane 2, CTDa; lanes 3-6,
0, 0.1, 0.2, and 0.4 milliunits of CTD phosphatase, respectively,
incubated with CTDo. Panel B, aliquots of
P-labeled RNAP IIO (0.5 fmol) assayed with CTD phosphatase
and analyzed on a 5% polyacrylamide-SDS gel. Lane 1, RNAP IIO; lane 2, RNAP IIO with 0.1 milliunits of CTD phosphatase. Panel C, CTD phosphatase assay with gel-purified
P-labeled subunit IIo analyzed on a 5% polyacrylamide-SDS
gel. Lane 1, purified subunit IIo; lane 2, subunit
IIo incubated with CTD phosphatase; lane 3, mixture of subunit
IIo and RNAP IIO incubated with CTD
phosphatase.
If indeed, CTD phosphatase
interacts with a site on RNAP II prior to dephosphorylation of the CTD,
RNAP IIA may act as a competitive inhibitor of CTD phosphatase. To test
this idea, CTD phosphatase activity was determined in the presence of
increasing amounts of unlabeled RNAP IIA (Fig. 2). The addition
of 100 fmol of RNAP IIA to an assay containing 3 fmol of
[P]RNAP IIO strongly inhibited CTD phosphatase
activity. The addition of comparable amounts of RNAP I or rCTD had no
effect on CTD phosphatase activity. However, RNAP IIB, which lacks the
CTD, was as effective as RNAP IIA in inhibiting CTD phosphatase
activity providing further support for the idea that the recognition
site for CTD phosphatase is distinct from the CTD.
Figure 2:
RNAP IIA and IIB inhibit CTD phosphatase
activity. CTD phosphatase was assayed in the presence of varying
amounts of RNAP I, recombinant CTD, RNAP IIA, or RNAP IIB as described
under ``Experimental Procedures.'' The CTD phosphatase
activity was quantitated on a PhosphorImager and the percent activity
remaining plotted versus picomoles of
competitor.
CTD Phosphatase Can Inhibit Preinitiation Complex
Assembly
If CTD phosphatase stably interacts with a site on RNAP
II, high concentrations of phosphatase may interfere with transcription
either by preventing the recruitment of RNAP II to the preinitiation
complex or by interfering with phosphorylation of the CTD that
accompanies transcript initiation. Given that RNAP IIA is in excess in
most in vitro transcription reactions, it is unlikely that CTD
phosphatase would stimulate the rate of transcription by increasing the
amount of RNAP IIA available for recruitment to the promoter. The
effect of CTD phosphatase on transcription from the Ad2-MLP was
determined as described under ``Experimental Procedures.''
The addition of CTD phosphatase at low concentrations had no effect on
transcription in either a crude or reconstituted transcription
reaction. However, when the molar ratio of CTD phosphatase relative to
RNAP II exceeded 5, transcription was repressed (Fig. 3A). The inhibitory activity co-chromatographs
with CTD phosphatase on Mono Q, the last column in the purification of
CTD phosphatase (data not shown). The inhibition of transcription is,
therefore, likely a direct consequence of CTD phosphatase and not a
contaminating activity.
Figure 3:
CTD phosphatase inhibits preinitiation
complex formation. Panel A, increasing amounts of CTD
phosphatase were added to a reconstituted transcription reaction with a
fixed amount of RNAP IIA (0.035 pmol) as described under
``Experimental Procedures.'' Lanes 1-4, 0,
0.18, 0.35, and 0.7 pmol of CTD phosphatase added at time 0. Panel
B, preinitiation complexes were assembled in the presence of the
DE0.25 extract, 0.05 pmol of P-labeled RNAP IIA, and
either 1 pmol of CTD phosphatase or in the absence of CTD phosphatase.
Reactions were chromatographed on a Sepharose CL-4B column and the
fractions analyzed on a 5% polyacrylamide-SDS gel as described under
``Experimental Procedures.'' Panel C, the amount of
P-labeled RNAP IIA in each lane was quantitated using a
PhosphorImager, the proportion in each lane was calculated relative to
the total amount, and plotted versus fraction number. Samples
from the reaction with no CTD phosphatase are indicated with filled
circles, and samples from the reaction containing CTD phosphatase
are indicated with open circles.
Since CTD phosphatase appears to bind
directly to RNAP II, the possibility that this interaction interfered
with the entry of RNAP II into the preinitiation complex was examined.
Preinitiation complexes were formed with P-labeled RNAP
IIA in the presence and absence of CTD phosphatase, in the absence of
ribonucleotides, and the complexes purified by chromatography on
Sepharose CL-4B (Fig. 3B). Preinitiation complexes are
excluded from the column and recovered in fractions 10-11 whereas
free RNAP II is included and recovered in fractions 13-20. The
presence of CTD phosphatase results in a substantial decrease in the
assembly of RNAP IIA into preinitiation complexes (Fig. 3, B and C). These results indicate that the association of
CTD phosphatase with RNAP II prevents the entry of RNAP II into the
preinitiation complex either directly or by preventing RNAP II from
binding TFIIF, a factor necessary for the efficient recruitment of RNAP
II(32) . Alternatively, CTD phosphatase may bind and sequester a
general transcription factor necessary for formation of the
preinitiation complex.
Figure 4:
CTD phosphatase interacts with RNAP II. Panel A, increasing amounts of CTD phosphatase were added to
transcription reactions assembled in the presence of the DE0.25 extract
and a fixed amount of RNAP IIA (0.025 pmol). CTD phosphatase was added
either prior to the formation of a preinitiation complex (lanes
1-3) or after preinitiation complex formation and 1 min
after the addition of nucleotides (lanes 4-6). The
amount of CTD phosphatase present is indicated at the top of each lane,
and the position of the 560 nucleotide runoff transcript is indicated
on the left. Panel B, increasing amounts of CTD phosphatase
were added to transcription reactions in the presence of the DE0.25
extract and either RNAP IIA (0.025 pmol in lanes 1-3) or
RNAP IIB (0.03 pmol in lanes 4-6). CTD phosphatase was
added before preinitiation complex formation at a concentration
indicated at the top of each lane. Panel C, eluates from an
RNAP II (lane 1) and control (lane 2) column were
dialyzed against buffer A and assayed for CTD phosphatase activity as
described under ``Experimental Procedures.'' The positions of
RNAP subunits IIo and IIa are indicated on the
left.
To directly test if CTD phosphatase can
form a stable complex with RNAP II, a HeLa cell extract was loaded onto
an RNAP II affinity column and CTD phosphatase activity determined in
salt-eluted fractions. CTD phosphatase activity was detected in the 0.5 M KCl eluate from the RNAP II column but not in the 0.5 M KCl eluate from a control column that did not contain RNAP II (Fig. 4C).
CTD Phosphatase Is Stimulated by TFIIF
In an
effort to identify general transcription factors that might influence
the activity of CTD phosphatase, highly purified or recombinant general
transcription factors were tested individually in a CTD phosphatase
assay. The presence of recombinant (baculovirus) TFIIF dramatically
stimulates the activity of CTD phosphatase (Fig. 5A).
None of the other general transcription factors at the concentrations
examined have a stimulatory effect on CTD phosphatase activity. TFIIF
does not stimulate the dephosphorylation of RNAP IIO in the absence of
CTD phosphatase. The effect of increasing concentrations of TFIIF on
CTD phosphatase activity is shown in Fig. 5B. The
presence of only 1.3 fmol of TFIIF results in a 2-fold increase in CTD
phosphatase activity. Quantitation of the results in this experiment
demonstrates that the addition of TFIIF leads to a 3-fold stimulation
of activity (Fig. 5C). This is an underestimate due to
the assay proceeding past its linear range. In the presence of lower
amounts of CTD phosphatase, TFIIF stimulates about 5-fold. Partially
purified HeLa cell TFIIF and recombinant TFIIF expressed in E. coli also stimulate CTD phosphatase 5-fold (data not shown). Titration
of TFIIF shows that approximately equimolar ratios of TFIIF to RNAP II
are sufficient to achieve significant stimulation (Fig. 5C).
Figure 5:
TFIIF
stimulates CTD phosphatase activity. CTD phosphatase was assayed in the
presence of purified transcription factors as described under
``Experimental Procedures.'' Panel A: lane
1, no CTD phosphatase; lanes 2-8, contain CTD
phosphatase; lane 2, no transcription factor; lane 3,
1.4 pmol of TFIIB; lane 4, 1.6 pmol of TBP; lane 5,
0.5 pmol of TFIIE; lane 6, 0.4 pmol of TFIIF; lane 7,
0.5 µg of TFIIH; and lane 8, 1 µg of TFIIJ. The amount
of the transcription factors chosen was empirically determined as that
being optimal for a reconstituted transcription reaction. Panel
B, all lanes contain CTD phosphatase; lane 1, no TFIIF; lanes 2-6, 0.5, 1.3, 2.6, 5.2, and 13 fmol of TFIIF,
respectively. Panel C, the CTD phosphatase activity from panel B was quantitated on a PhosphorImager and the
photo-stimulated luminescence values plotted versus the
TFIIF/RNAP IIO molar ratio. The assay contained 3 fmol of RNAP IIO. For
simplicity, moles of TFIIF were calculated assuming a dimeric
structure.
Since CTD phosphatase activity is observed
in the absence of exogenous TFIIF, TFIIF appears to stimulate a basal
level of CTD phosphatase activity. Alternatively, TFIIF may be
essential for CTD phosphatase activity and be present in suboptimal
levels in the CTD phosphatase and/or RNAP II preparations. To test
this, aliquots of CTD phosphatase, RNAP IIA, and the CTD kinase used in
the preparation of RNAP IIO were analyzed on an immunoblot using an
antisera raised against TFIIF. No TFIIF was detected in the CTD
phosphatase, RNAP IIA, or the CTD kinase preparations even though the
assay was capable of detecting less than 1 ng (data not shown).
Therefore, reactions assembled in the absence of exogenous TFIIF must
contain less than 0.1 fmol of TFIIF. This result indicates that TFIIF
is not essential for a basal level of CTD phosphatase activity.
The COOH-terminal Domain of RAP74 Is Sufficient to
Stimulate CTD Phosphatase
TFIIF is composed of two subunits,
RAP30 and RAP74. The ability of individual subunits to influence CTD
phosphatase activity was determined. The RAP74 subunit stimulates CTD
phosphatase activity whereas the RAP30 subunit has no effect (Fig. 6). Furthermore, RAP30 has no effect on the activity of
RAP74 or TFIIF, even if added in a 200-fold molar excess prior to the
addition of RAP74 or TFIIF (data not shown). RAP74 stimulates CTD
phosphatase activity more than 20-fold in contrast to TFIIF which
stimulates only 5-fold. However, TFIIF is more stimulatory at low
concentrations than is RAP74 (data not shown). For full stimulation by
TFIIF, approximately 20 fmol is required, whereas 500 fmol of RAP74
still does not show saturation.
Figure 6:
The RAP74 subunit of TFIIF stimulates CTD
phosphatase activity. CTD phosphatase was assayed in the presence of
TFIIF, RAP30, and RAP74 as described under ``Experimental
Procedures.'' All lanes contain CTD phosphatase. Lane 1,
CTD phosphatase alone; lane 2, TFIIF (0.2 pmol); lane
3, RAP30 subunit (3 pmol); lane 4, RAP74 subunit (4.3
pmol).
The sequence of RAP74 suggests it is
organized into a NH- and COOH-terminal domain and a highly
charged central domain(33) . A panel of RAP74 proteins deleted
in various regions was tested to locate the region in RAP74 responsible
for stimulating CTD phosphatase (Fig. 7). CTD phosphatase
activity was determined in the presence of 500 fmol of each of the
RAP74 deletion proteins. Like RAP74, this concentration does not
saturate the assay. The smallest protein able to stimulate CTD
phosphatase contains residues 358-517 and stimulates about
5-fold. All active deletion proteins contain this region, and proteins
from which this region is deleted (1-409, 407-517) are inactive. However, two of the deletion
proteins that contain residues 358-517 are inactive (205-517,
136-258), both of which
contain portions of the central charged region. Deletion of the central
charged region (
137-356) results in a fully active
molecule. Construct
137-356 is even more active than the
wild type RAP74 at lower concentrations resulting in 20-fold
stimulation in the presence of only 50 fmol (data not shown). The
NH
-terminal region of RAP74 is also important for CTD
phosphatase stimulatory activity. Deletion of residues 1-74 has
little effect, whereas deletion of a further 13 residues, encompassing
a highly conserved region, results in a dramatic loss of activity (Fig. 7, compare lanes 15 and 16). Combination
of two inactive fragments encompassing the entire subunit of RAP74 (1-409 and 407-517, 1-205 and 207-517) failed to reconstitute CTD phosphatase
stimulatory activity (data not shown).
Figure 7:
Stimulatory activity of RAP74 deletion
constructs. CTD phosphatase was assayed in the presence of 500 fmol of
RAP74 deletion proteins as described under ``Experimental
Procedures.'' Lane 1, no CTD phosphatase; lane
2, CTD phosphatase alone; lane 3, CTD phosphatase and
TFIIF; lane 4, CTD phosphatase and RAP74; lanes
5-20, CTD phosphatase and deletion proteins as designated in
the left margin. The numbers on the left refer to the amino
acid residues present except where the numbers are preceded by a ,
in which case the numbers reflect the deleted region. CTD phosphatase
activity was quantitated on a PhosphorImager and the fold stimulation
calculated relative to CTD phosphatase alone. Due to the small linear
range of the assay, stimulatory values for 1-517, 74-517,
and
137-356 were measured in independent experiments with
the CTD phosphatase titrated into the linear
range.
TFIIB Inhibits the Ability of TFIIF to Stimulate CTD
Phosphatase
To further investigate the effect of the general
transcription factors on CTD phosphatase activity, each general
transcription factor was added to assays containing TFIIF and CTD
phosphatase. TFIIB, and to a lesser extent TBP, inhibits the TFIIF
stimulated activity (Fig. 8, A and B). TFIIB
had no effect on CTD phosphatase activity in the absence of TFIIF (Fig. 8B, compare lanes 2 and 3). In
the presence of TFIIF, TFIIB inhibits CTD phosphatase activity to basal
levels (Fig. 8B, compare lanes 2 and 3 with 8). TFIIB also inhibits the RAP74 stimulated assay (Fig. 8A, compare lanes 7 and 8). High
amounts of TBP (1600 fmol) partially inhibit CTD phosphatase activity
in the presence or absence of TFIIF. The inhibitory effect of TBP may
be a consequence of its binding to the CTD (34). Little effect is seen
with less than 500 fmol of TBP. The presence of 30 fmol of TFIIB in an
assay containing 100 fmol of TFIIF results in a 75% reduction in the
stimulatory activity of TFIIF (Fig. 8C).
Figure 8:
TFIIB
inhibits the stimulatory effect of TFIIF. Panel A, CTD
phosphatase and 50 fmol of TFIIF or 500 fmol of RAP74 were assayed in
the presence of the general transcription factors as described under
``Experimental Procedures.'' Lane 1, CTD phosphatase
and TFIIF alone; lane 2, plus 1.4 pmol of TFIIB; lane
3, plus 1.6 pmol of TBP; lane 4, plus 0.5 pmol of TFIIE; lane 5, plus 0.5 µg of TFIIH; lane 6, plus 1
µg of TFIIJ; lane 7, CTD phosphatase and RAP74 alone; and lane 8, plus 270 fmol of TFIIB. Panel B: lane
1, no CTD phosphatase; lane 2, CTD phosphatase; lane
3, CTD phosphatase and 270 fmol of TFIIB; lanes
4-8, contain 0, 8, 27, 80, and 270 fmol of TFIIB,
respectively, 100 fmol of TFIIF and have half as much CTD phosphatase
as lanes 2 and 3. Panel C, the CTD
phosphatase activity from lanes 4 to 8 of panel B was quantitated on a PhosphorImager, the fold stimulation
calculated, and plotted versus fmol of TFIIB. Assays contained
3 fmol of RNAP IIO.
Figure 9:
Model of CTD phosphatase action. CTD
phosphatase is represented by the white oval, RNAP II by the shaded area, and CTD by the wavy line. Details of the
model are described in the text.
The results presented above indicate that TFIIF
may be involved in the regulation of CTD phosphatase activity. TFIIF is
a heterotetramer of RAP30 and RAP74 and both subunits can interact with
RNAP II directly (22). During initiation, TFIIF suppresses the
non-selective binding of RNAP II to DNA, facilitates the recruitment of
RNAP II to the preinitiation complex, and stabilizes the preinitiation
complex(22) . In addition, TFIIF stimulates elongation by
reducing pausing of RNAP II (36). Results presented above show that
TFIIF is also capable of stimulating the dephosphorylation of RNAP IIO
by CTD phosphatase. The mechanism by which TFIIF stimulates CTD
phosphatase is not known but could affect any of several steps, such as
the binding of CTD phosphatase to RNAP II or the subsequent
dephosphorylation reaction.
-terminal and central regions of RAP74.
(
)Central sequences of RAP74 mask RNAP II binding while
NH
-terminal sequences counteract this effect. This
COOH-terminal region of RAP74 also stimulates accurate transcription in vitro although it is not necessary for
initiation(36, 38) .
The presence of the
NH
-terminal region increases CTD phosphatase stimulation
from 5- to 20-fold and is absolutely required for activity if the
central region is present. This observation is consistent with the
effect of NH
-terminal sequences in reducing the masking
effect of central sequences on RNAP II binding.
Particularly critical are residues 74-87 that encompasses a
highly conserved region containing mostly charged residues.
Interestingly, the region involved in binding RAP30 (residues
1-172) spans the region which is critical for the 20-fold
stimulation and may explain the different levels of stimulation by
TFIIF and RAP74.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.