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(Received for publication, June 19, 1996, and in revised form, August 13, 1996)
From the Molecular Virology Section, Laboratory of Molecular
Microbiology, NIAID, National Institutes of Health,
Bethesda, Maryland 20892-0460
ABSTRACT The carboxyl-terminal domain (CTD) of RNA
polymerase (RNAP) II contains multiple repeats with a heptapeptide
consensus: Tyr-Ser-Pro-Thr-Ser-Pro-Ser. It has been proposed that
phosphorylation of this CTD facilitates clearance and elongation of
transcription complexes initiated at the promoters. However, not all
transcribed promoters require RNAP II with full-length CTD.
Furthermore, different activators can promote capably the
transcriptional activity of polymerase II mutants deleted in the CTD.
Thus, the role of the RNAP II CTD in transcription and in response to
activators remains incompletely understood. To study the role of CTD in
the regulated transcription of human retroviruses human-T cell
lymphotropic virus I and human immunodeficiency virus 1, we used an
INTRODUCTION In the study of mRNA transcription, much attention has focused
on the unusual structure of the C-terminal domain
(CTD)1 of the large subunit of RNA
polymerase II. In mammals, the CTD is composed of 52 repeats of a
seven-amino acid consensus motif: YSPTSPS. It has been shown that
one-half of these repeats can be deleted, and cell viability is yet
preserved (2, 3). Five of the seven residues in the CTD repeats are
potential sites for phosphorylation, suggesting that this heptapeptide
could be an excellent phosphoacceptor. Indeed, the CTD of RNA
polymerase II is highly phosphorylated in cells, and the degree of
phosphorylation is such that a significant shift in migration can be
observed in SDS-PAGE (4, 5). Two RNA polymerase II forms can be clearly
visualized: pol IIO, which is hyperphosphorylated and
migrates at ~240 kDa, and pol IIA, which is
hypophosphorylated and migrates at ~210 kDa (for reviews, see Refs. 6
and 7).
The kinetics of CTD phosphorylation has been addressed with in
vitro and in vivo run-on transcription studies linking
RNA pol IIA and IIO to newly synthesized
transcripts (3, 4). In vivo, investigators have demonstrated
that paused polymerases predominantly contain the RNA pol
IIA form while elongating polymerases are of the pol
IIO form (8, 9). In vitro, other studies have
pointed to findings suggesting that transcription from selected
promoters does not necessarily require CTD-containing RNA polymerase
(10, 11, 12, 13). In a recent study, Corden and co-workers (1) have devised an
elegant protocol in which they demonstrated that expression from some
(but not all) transcription units show a striking dependence on the
length of the CTD of the transcribing RNAP II (1). These and other
studies address many aspects of CTD function and illustrate some of the
complex and unresolved questions on the various biological settings in
which CTD is (not) needed for different promoters and activators.
Understanding regulated transcription of pathogenic human retroviruses
contributes importantly to our knowledge of disease progression. HIV-1
is the etiological agent for AIDS, and HTLV-I is the virus that causes
adult T-cell leukemia. Both retroviruses encode viral activators (for
HIV-1, Tat; reviewed in Ref. 14; for HTLV-I, Tax; reviewed in Ref. 15)
that potently modulate transcription from the homologous long terminal
repeat (LTR) promoters (16). Activation of the viral promoter by HTLV-I
Tax is mediated through three imperfectly conserved 21-nucleotide
cAMP-responsive elements located in the U3 of the LTR (17, 18, 19, 20). Tax
cannot bind DNA directly and is instead recruited to the promoter via
contact with cellular proteins bound to the 21-base pair,
Tax-responsive cAMP-responsive elements (21, 22, 23, 24, 25). By contrast, Tat,
although being a similarly strong trans-activator of the HIV-1 LTR, is
targeted to the promoter by binding to a leader RNA, TAR (26, 27, 28). TAR
delivers Tat to the promoter, in which it interacts with general
transcription factors. These two contrasting activators present
comparative paradigms for exploring the settings in which
CTD-containing polymerases might be required for retroviral activity.
Here, we have characterized the requirements for RNAP II CTD in the
transcriptional functions of Tax and Tat.
EXPERIMENTAL PROCEDURES Plasmids expressing RNA polymerase II with various
lengths of CTD were graciously provided by J. Corden (Johns Hopkins
University) (1). Reporters containing chloramphenicol acetyltransferase
(CAT) cDNA driven by the minimal HIV-1 LTR TATAA box with either
six Sp1 sites or three Gal4 binding sites positioned upstream have been
described (29). Trans-activator plasmids include Tat (28), Gal4-Tax
(30), Gal4-Sp1A (31), Gal4-VP16 (32), Gal4-TBP17-56, and Gal4-CTD32x
(33). GST-Tax, GST-Tat, GST-Sp1A, and GST-TBP fusion constructs were
constructed using pGEX2T (Pharmacia Biotech). GST-CTD was a gift from
W. Dynan (University of Georgia) (34).
2 × 106 HeLa
cells were plated in 10-cm dishes. On day 1, cells were transfected
with 10 µg of polymerase II expression plasmid, 5 µg of CAT
reporter, and 5 µg of trans-activator expression plasmid using
Lipofectin (Life Technologies, Inc.). On day 2, Lipofectin-containing
media were replaced with media containing 3 µg/ml Three hours prior to
transfection, HeLa cells were treated with either 10 nM
staurosporine or mock treated with Me2SO. 3 µg of
reporter plasmid with 2 µg of trans-activator expression plasmid were
then co-transfected by calcium phosphate precipitation. In the case in
which Tat was expressed from the trans-activator plasmid, only 0.1 µg
was used because of the high efficiency of this trans-activator. Cell
lysates were assayed 48 h later for CAT activity; acetylations
were quantitated by scintillation counting of silica gel slices.
HeLa cells treated either with 10 nM staurosporine or mock treated with Me2SO
overnight were pulse-labeled for 2 h with either 200 µCi of
[35S]methionine and [35S]cysteine (ICN) or
1 mCi of [32P]orthophosphate (Amersham Corp.). Cells were
lysed with radioimmunoprecipitation assay buffer (150 mM
NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris-HCl, pH 8.0, and 1 mM
phenylmethylsulfonyl fluoride) and normalized for protein amounts
(Bio-Rad). Equivalent amounts of protein were then diluted into buffer
(150 mM NaCl and 50 mM Tris-HCl, pH 8.0) and
were immunoprecipitated overnight with 1 µg of anti-RNA polymerase II
monoclonal antibody (8WG16; Promega, Madison, WI) plus 15 µl of
protein A+G agarose (Oncogene Science, Cambridge, MA).
Immunoprecipitates were washed five times (150 mM NaCl, 50 mM Tris-HCl, pH 8.0, and 0.05% Tween 20), and samples were
solubilized (125 mM Tris-HCl, pH 6.8, 20% glycerol, 2%
SDS, 2% GST fusion proteins were immobilized
at 0.2 µg/µl onto glutathione-Sepharose beads (Pharmacia). 20 µl
of GST fusion protein-bound resins were equilibrated with 100 µg of
HeLa cell extract (prepared with modifications of the method of Dignam
et al. (35)) for at least 2 h. Unbound material was
recovered as the flow-through fraction. Resins were then washed three
times at 50 volumes with column buffer. This was followed by two
additional 50-volume washes with kinase reaction buffer containing 50 mM Tris-HCl, pH 7.5, 5 mM MnCl2,
and 5 mM dithiothreitol. To initiate the kinase reaction,
50 µl of kinase buffer supplemented with 200 ng of GST-CTD and 10 µCi of [ RESULTS To assess the CTD requirements in transcriptional activation by
HTLV-1 Tax and HIV-1 Tat, we used an approach developed by Corden and
co-workers (1). The approach hinges on the efficient expression of
The Capacity of each version of
The above findings suggest that HTLV-I Tax belongs to a class of
transcriptional activators that has a CTD-independent phenotype. To
define better this observation, we wished to compare Tax in parallel
with HIV-1 Tat and other previously described activators (Gal-TBP17-56,
Gal-Sp1A, Gal-VP16, and Gal-CTD32x; Fig. 2C). To do this we
first verified how basal expression from the two reporters, p6xSp1CAT
and p-43GalCAT (Fig. 1B), might depend on the length of the
polymerase CTD (Fig. 2B). In We next compared activator-dependent transcription (Fig.
2C). For all activators, complete experiments were performed
as shown in Fig. 2A. However, only the critical gel portions
most relevant for signal analysis are presented in Fig. 2C.
We observed that for Tat, Gal-TBP17-56, and Gal-CTD32x, relative signal
intensity differences of approximately 10-fold existed between
transcription supported by CTD It has been suggested that phosphorylation of CTD, more than the
absolute length of CTD per se, is the critical event
influencing polymerase II clearance from the promoter (reviewed in Ref.
36). We queried whether divergent requirements for phosphorylated RNA
polymerase II CTDs might be the underlying reason why Tax and Tat
exhibit differing CTD length phenotypes with the above three mutant
polymerases. If such is the case, then in the setting of wild type RNAP
II-mediated transcription, CTD-independent activators should be less
sensitive than CTD-dependent activators to inhibitors of
CTD kinase. To test this, staurosporine, a previously described
inhibitor of CTD kinase (37), was used to treat cells transiently
transfected with the reporter plus Tat, Gal-Tax, or Gal-Sp1A.
Expression in staurosporine-treated cells was compared with that in
mock-treated cells (Fig. 3A) to assess for
the relative effects of the CTD kinase inhibitor on activator function.
In these settings, we found that Tat-dependent expression
was reduced in staurosporine-treated cells to 20% of that in control
cells. Gal-Tax showed a smaller 2-fold reduction, which is in part
consistent with the fact that Tax is a phosphoprotein, and
phosphorylation of Tax is influenced by staurosporine (38). Indeed, the
activity of Gal-Sp1A, which has a CTD-independent phenotype like Tax,
actually showed a slight, but statistically insignificant, rise with
staurosporine treatment.
We verified that treatment with staurosporine did in fact affect RNAP
II phosphorylation. Mock-treated and staurosporine-treated cells were
radiolabeled in parallel with [35S]methionine and
[35S]cysteine or [32P]orthophosphate. Cell
lysates were then immunoprecipitated with a monoclonal antibody against
RNA polymerase II CTD (Fig. 3B). Essentially the same amount
of RNAP II was recovered from treated and untreated cells, as measured
by the [35S]cysteine- and
[35S]methionine-labeled signals (Fig. 3B;
lanes 1 and 2). However, when the
[32P]orthophosphate-labeled samples were analyzed, we
found that staurosporine (Fig. 3B, lane 4) reduced the
phosphorylated species of RNAP II in treated cells to approximately
10% of that in mock-treated cells (Fig. 3B, lane 3). Thus,
these results, taken together with the activity results in Fig.
3A, suggest that the Tat and Tax CTD length phenotypes might
be a secondary reflection of the relative requirements by the different
activators for interaction with phosphorylated and nonphosphorylated
RNAP II CTDs.
Many CTD kinases have been described (34, 39, 40, 41, 42). In most instances,
it is not entirely clear how the CTD kinase interacts with RNAP II. One
plausible hypothesis is that an activator that requires phosphorylated
CTD for function might carry CTD kinase activity to the promoter.
Conversely, CTD-independent activators might not show this associative
property. To test this hypothesis for Tax and Tat, we checked
biochemically for the ability of either to bind CTD kinase activity.
GST fusion ``pull-down'' protein chromatography using either Tax or
Tat as bait was performed. Retained proteins were assayed in
vitro for kinase activity.
We initially compared pull-downs from GST-Tat and GST-Tax to that from
GST alone (Fig. 4A). In these experiments,
HeLa cell extracts were equilibrated for at least 2 h with
glutathione-Sepharose beads bound with either GST or GST fusion
protein. After equilibration, each set of beads was washed extensively
with 150 column volumes of buffer. We checked the last column wash to
verify that it was free of CTD kinase activity (data not shown). We
then assayed CTD kinase activity from beads eluted with 0.25 M KCl. For comparison, CTD kinase activity in the
flow-throughs was assayed in parallel. Kinase reactions with GST-CTD
supplied as substrate showed a distinctly phosphorylated moiety with
the correct migration size in the reactions using the GST-Tat eluate
(Fig. 4A, lane 4). In comparison, this band was absent from
counterpart reactions from GST alone (Fig. 4A, lane 2) or
GST-Tax (Fig. 4A, lane 6) beads. Under these binding
conditions, we observed that virtually all of the input CTD kinase
activity could be retained by GST-Tat (note the markedly diminished
signal at the GST-CTD migration position in Fig. 4A, lane 3,
flow-through), suggesting an efficient interaction between Tat and
kinase.
We confirmed that the observed phosphorylated band was GST-CTD and not
from phosphorylation of a protein contaminant that remained after
purification. We immunoprecipitated each kinase reaction with the 8WG16
monoclonal antibody for RNAP II CTD (Fig. 4B). The
phosphoprotein in the GST-Tat kinase fraction (Fig. 4A, lane
4) was indeed efficiently recovered (Fig. 4B, lane 5),
confirming its identity as GST-CTD. Consistent with our interpretation,
no phosphorylated GST-CTD was immunoprecipitated from either GST or
GST-Tax fractions (Fig. 4B, lanes 4 and 6). We
also checked the flow-through fractions (Fig. 4A, lanes 1, 3, and 5). Because these immunoprecipitations were
performed on a larger, more diluted volume, longer exposures of gels
were done, which verified the presence of phosphorylated GST-CTD (Fig.
4B, lanes 1-3). The fact that other phosphorylated moieties
in the flow-throughs (Fig. 4A, lanes 1, 3, and
5) were not precipitated (Fig. 4B, lanes 1-3)
supports the specificity of our immunoprecipitations.
The findings from Tax and Tat suggest a correlation between CTD
independence and dependence and kinase association and lack of
association. To determine whether this might also hold for a nonviral
activator, we checked GST-Sp1A for associated CTD kinase activity.
Because Sp1A has CTD-independent activity (Fig. 2C), one
prediction is that it would not complex with a CTD kinase. In
side-by-side comparisons with GST and GST-Tat, GST-Sp1A indeed failed
to retain CTD kinase activity (Fig. 4C, lane 6).
DISCUSSION Mechanisms governing transcription and activated transcription
from polymerase II promoters hold many complex and incompletely
resolved issues. Part of the complexity of pol II promoters is perhaps
contributed by the inherent structure of the RNAP II molecule. Like
RNAP I and RNAP III, RNAP II needs to enter and dock onto a core
promoter complex to initiate transcription. However, unlike RNAP I or
RNAP III, after entry and docking additional steps are required to
facilitate the egress of RNAP II from the promoter. It has been
suggested that the pol II CTD contributes to the recruitment,
attachment, and (on modification) clearance of the polymerase from the
promoter (reviewed in Ref. 36). In this regard, it is important to note
that several investigators have shown that pol II CTD can interact
directly with general transcription factor IID (43, 44, 45, 46, 47), that this
interaction could impede departure of the polymerase from the promoter
(48, 49), and that phosphorylation of CTD after attachment of
polymerase to the nucleated transcription factor IID-IIB complex
effects conformation changes influencing disengagement of pol II from
the initiated ensemble (8, 9, 50).
The question is thus raised: how do activators affect clearance of RNAP
II from the promoter? There are at least four distinct types of
activation domains (reviewed in Ref. 51) with differences in
mechanistic properties (1, 52). It is clear that many activators
contribute directly to the assembly of RNAP II into the initiation
complex at the promoter (reviewed in Ref. 36). However, because steady
state transcription is a multicycle process in which productive
disassembly of a previous cycle permits reassembly for the next cycle,
it stands to reason that initiation events and clearance and elongation
events at the promoter are intimately (and, in some instances,
inseparably) linked (47, 53, 54). This concept has important
implications for human retrovirus transcription, since it has been a
standing paradox as to how HIV-1 Tat apparently influences initiation
and elongation of RNAP II simultaneously (reviewed in Refs. 14 and 61).
Our findings here that Tat influences polymerase II transcription in a
CTD-dependent fashion that is linked to phosphorylation,
together with genetic evidence to be presented
elsewhere,2 suggest that this activator
functions critically at the step of promoter clearance. Clearance of
the promoter in a prior round by Tat is then reflected in increased
access and efficiency of initiation complex reassembly for a subsequent
round of transcription (56).
Approximately six different CTD kinases that phosphorylate at serine,
threonine, or tyrosine residues have been identified (34, 39, 40, 41, 42, 55).
A recently suggested yet unidentified Tat-associated kinase (57), which
has activity on a CTD substrate, adds complexity to the picture.
Physiologically, it is unclear why so many kinases impinge on a common
substrate. However, it is clear that RNAP II functions in pleiotropic
settings and contacts diverse activators and adaptors; each unique
scenario might thus dictate a different activator-kinase-substrate
interaction. Similarly, HIV-1 Tat is also known to have roles beyond a
singularly focused activity at the promoter (58). In this regard, Tat
has been shown previously to bind protein kinase R (59) which likely
explains a Tat-dependent activation of nuclear factor It is intriguing that two related human retroviruses, HTLV-I and HIV-1,
have evolved very different mechanisms for transcriptional regulation,
although both conserve striking similarities in posttranscriptional
regulation of mRNA metabolism (reviewed in Refs. 61 and 62).
Conceivably, the differences in transcriptional activation are
reflected by the extreme dissimilarities between the activation domains
of Tax (30) and Tat (63) and the processes by which either is recruited
to the promoter (see the Introduction). One can only speculate on
whether these molecular differences contribute to the divergent
pathogenic characteristics between the two viruses (i.e.
HIV-1 is rapidly cytolytic, whereas HTLV-I is a transforming virus that
has an extended cellular latency).
FOOTNOTES Acknowledgment We thank Hua Xiao for discussions and critical
reading of the manuscript. We are grateful to J. Corden, W. Schaffner,
and W. Dynan for sharing reagents.
REFERENCES
Volume 271, Number 44,
Issue of November 1, 1996
pp. 27888-27894
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
Acknowledgment
REFERENCES
-amanitin-resistant system developed previously (Gerber, H. P.,
Hagmann, M., Seipel, K., Georgiev, O., West, M. A., Litingtung, Y.,
Schaffner, W., and Corden, J. L. (1995) Nature 374, 660-662). We found that transcription directed by the human T-cell
lymphotropic virus I activator protein Tax was strongly promoted by
CTD-deficient RNA polymerase II. By contrast, the human
immunodeficiency virus 1 activator Tat, which is recruited to the
promoter by tethering to a nascent leader RNA, requires CTD-containing
polymerase II for transcriptional activity. Biochemically, we
characterized that Tat associated with a cellular CTD kinase activity,
whereas Tax did not. Concordantly, we found that cellular transcription
factor Sp1, which can activate CTD-deficient polymerase II with an
efficiency similar to Tax, also failed to bind a CTD kinase. Taken
together, these observations address mechanistic corollaries between
activators with(out) a linked CTD kinase and regulated transcription by
RNA polymerase II moieties with(out) a CTD.
-amanitin
(Calbiochem). On day 3, cells were refed with fresh media containing
-amanitin. Approximately 48 h after the Lipofectin-DNA mixture
was first introduced, total RNA was isolated using RNAzol B (Tel-Test,
Friendswood, TX). Probes were prepared using a Maxiscript T7
in vitro transcription kit (Ambion, Austin, TX) and were
gel-purified. 1 × 105 cpm/10 ng of probe was
precipitated with total cellular RNA. Samples were processed using the
RNase protection assay (RPA) II procedure as described by the
manufacturer (Ambion). Briefly, RNA and probe were denatured at
95 °C, hybridized overnight at 42 °C, digested with RNase at
37 °C for 30 min, precipitated with ethanol, and resolved in an 8%
urea-polyacrylamide gel (Life Technologies). Signals were visualized by
autoradiography and/or Fuji phosphorimaging.
-mercaptoethanol, and 0.1% bromophenol blue) by boiling.
After resolution of proteins by SDS-PAGE, the gel was visualized by
Fuji phosphorimaging.
-32P]ATP were added to either 20 µl of
resin eluate or 20 µl of flow-through fraction and were incubated for
15 min at room temperature. The reaction was terminated by addition of
SDS-PAGE sample buffer. Samples were resolved by 8% SDS-PAGE and were
visualized by autoradiography. For immunoprecipitation of in
vitro kinase reactions, the samples were diluted 100-fold with
buffer A (150 mM NaCl, 50 mM Tris-HCl, pH 8.0, and 0.05% Tween 20) and immunoprecipitated overnight with 3 µg of
anti-RNA polymerase II monoclonal antibody (8WG16; Promega) plus 15 µl of protein G-agarose (Life Technologies). Immunoprecipitates were
washed three times with buffer A. Samples were solubilized and resolved
by 8% SDS-PAGE. The gel was fixed in 30% methanol and 10% acetic
acid, dried, and visualized by autoradiography.
-amanitin-resistant mutants of the large subunit of RNA polymerase
II. Versions of -amanitin-resistant mutants have been constructed to
have different numbers of CTD heptapeptides (Fig.
1A). Hence, -amanitin treatment of cells
transfected with plasmids that express mutant RNAP II results in the
``poisoning'' of the endogenous wild type RNAP II such that
subsequent transcription in the cell depends on the exogenously
expressed resistant mutant. For our studies, we tested
-amanitin-resistant polymerases that have 52, 31, or 5 copies of CTD
heptapeptides (Fig. 1A) for transcriptional responsiveness
to co-introduced Tax- or Tat-expressing plasmids. Transcription
directed by the RNA polymerase mutants was assayed by
co-introduction into cells of a third plasmid as a reporter (either
p6xSp1CAT or p-43GalCAT; Fig. 1B). p6xSp1CAT was used in
assays for Tat activity. p-43GalCAT was used as the reporter for
Gal-Tax and other Gal fusion protein-containing activators (Fig.
1C).
Fig. 1.
Schematic representations of plasmids.
A, RNA polymerase II expression plasmids with various
CTD lengths. The large subunit of the polymerase II cDNA represents
a mutated form that confers -amanitin resistance (1).
CMV, cytomegalovirus immediate early promoter;
HA, a 9-amino acid hemagglutinin tag. B, reporter
constructions containing CAT cDNA driven by the minimal HIV-1 LTR
promoter with either six Sp1 or three Gal4 binding (UAS)
sites. C, abbreviated representations of plasmids that
express unfused and Gal4 binding domain-fused trans-activator protein.
DBD, DNA binding domain.
[View Larger Version of this Image (34K GIF file)]
-amanitin-resistant polymerase
II to mediate basal and activator-dependent transcription
was assessed using an RPA. A typical experiment is shown in Fig.
2A. In this instance, HeLa cells, first
treated with -amanitin, were then transfected with reporter
(p-43GalCAT) plus a mutant polymerase-expressing plasmid (CTDwt,
CTD
31, or CTD5; Fig. 2A, lanes 5-7) or reporter plus
mutant polymerase plus Gal-Tax plasmid (Fig. 2A, lanes
9-11). Expression from p-43GalCAT was measured for each sample by
RPA for CAT mRNA (Fig. 2A, CAT, arrow). All
samples were also probed identically for actin mRNA (Fig. 2A,
lanes 14-21, actin, arrow) to normalize for input.
Residual endogenous wild type polymerase II activity that might have
escaped -amanitin poisoning was assessed by including a
co-transfection with pUC19 (which does not carry an
-amanitin-resistant polymerase) in each set of transfections
(Fig. 2A, lanes 4 and 8). A darker exposure of
the portion of the gel that contains protected mRNA signals from
lanes 4-11 is shown (Fig. 2A, right
panel). From this type of analysis, which was replicated twice, we
concluded that Gal-Tax activated transcription approximately 20-fold
over basal, and that RNA polymerase II with an extremely shortened CTD
tail (i.e. CTD5; Fig. 2A, lane 11) was
comparable to RNA polymerase II with wild type CTD (CTDwt; Fig.
2A, lane 9) and intermediate length CTD (CTD31; Fig.
2A, lane 10) in supporting this activation.
Fig. 2.
Determination by RNase protection assay of
transcription directed by RNAP II mutants. A, left panel,
Tax-mediated activation is CTD length independent. A representative RPA
analysis of Gal-Tax-activated transcription is shown. MWM,
molecular size markers consisting of 32P end-labeled,
MspI-restricted pBR322 DNA (lane 1). CAT
(lanes 2 and 3) and actin (lanes 12 and 13) input probes are shown as either untreated (
) or
treated with RNase (+). Plasmids used in the transfections are
indicated at the top. Arrows, positions of the
expected sizes of protected mRNAs for CAT or actin. Actin mRNA
was used as normalization for equivalent amounts of total RNA used in
each protection assay. Because of its abundance and stable half-life,
the amount of actin mRNA detected represents primarily transcripts
accumulated prior to the addition of -amanitin. A, right
panel, a longer exposure of the portion of the gel
(left), which contains the protected CAT mRNA signals.
B, basal expression of CAT reporters is independent of RNAP
II CTD lengths. RPA signals of CAT mRNA expressed from the
reporters in the absence of trans-activators are shown. Panels shown
represent 200-fold equivalent overexposure compared with the same
samples shown in A. Relative times of exposure are 2 days
with an intensifying screen for p6xSp1CAT and 6 days with an
intensifying screen for p-43GalCAT. C, activated expression
by some trans-activators depend on RNAP II CTD length. Tat,
Gal-TBP17-56, and Gal-CTD32x show approximately 10-fold higher RPA
signal for CAT mRNA transcribed by either CTDwt or CTD31
compared with CTD5. By contrast, Gal-Tax, Gal-Sp1A, and Gal-VP16
show essentially invariant levels of CAT mRNA expression by any of
the three forms of polymerase. The slightly higher levels of signal
from CTD5 in the Gal-Tax and Gal-Sp1A samples were found not to be
statistically significant on repetition. Relative times of exposure
using a phosphorimaging plate are 6 h for Tat, 9 h for
Gal-Tax, and 12 h for Gal-TBP17-56, Gal-Sp1A, Gal-VP16, and
Gal-CTD32x.
[View Larger Version of this Image (41K GIF file)]
-amanitin-treated HeLa
cells, co-transfection of p6xSp1CAT with a CTDwt, CTD31, or CTD5
plasmid showed identical amounts of mRNA as measured by RPA (Fig.
2B; panels shown are 200 fold-equivalent increased exposure
over the comparable basal samples presented in Fig. 2A). An
absence of signal from the co-transfection of pUC19 with p6xSp1CAT
(Fig. 2B) confirmed that we were indeed measuring
transcription mediated by each of the exogenously introduced mutant RNA
polymerases. A set of assays performed in parallel with p-43GalCAT as
the reporter (Fig. 2B) yielded a comparable pattern of
signals. From multiple replications of such assays, we concluded that
basal transcription from the reporters is insensitive to the length of
the RNA polymerase II CTD.
5 and that by CTD31 or CTDwt (Fig.
2C). On the other hand, Gal-Tax, Gal-Sp1A, and Gal-VP16
exhibited invariant transcriptional activities from each of the three
polymerases, independent of their CTD length (Fig. 2C).
Hence, statistically indistinguishable levels of CAT mRNAs were
synthesized by each of the mutant polymerases in response to the latter
three activators.
Fig. 3.
CTD kinase inhibitor staurosporine reduces
phosphorylation of RNA polymerase II and Tat-mediated activation of
transcription. A, bar summaries (mean of two experiments)
comparing relative activities by each activator in HeLa cells treated
with 10 nM staurosporine versus
Me2SO mock-treated cells. For each activator, the magnitude
of activity in mock-treated cells was arbitrarily set as 1 (not drawn
in explicitly). Relative activation reflects the amount of activity
(compared with 1) that remains after suppression by
staurosporine-mediated inhibition of phosphorylation. B,
verification of inhibition of RNAP II phosphorylation by staurosporine.
HeLa cells were treated (+, lanes 2 and 4) or
mock treated (, lanes 1 and 3) with 10 nM staurosporine overnight. Cells were then labeled
with 200 µCi of [35S]methionine and
[35S]cysteine (lanes 1 and 2) or 1 mCi of [32P]orthophosphate (lanes 3 and
4). Equivalent amounts of protein from treated and untreated
sets were used in immunoprecipitations with 1 µg of anti-RNA
polymerase II monoclonal antibody and protein A+G-agarose.
Immunoprecipitates were washed, solubilized in sample buffer, separated
on SDS-PAGE, and visualized by phosphorimaging. Arrows, two
forms of polymerase: pol IIO, hyperphosphorylated
form; pol IIA, nonphosphorylated form. Note that
staurosporine treatment did not affect the recovery by
immunoprecipitation of 35S-labeled RNAP II, which is
predominantly of the IIA form (lanes 1 and
2). However, the total immunoprecipitate from
staurosporine-treated cells has a relative paucity of the
phosphorylated IIO form (compare lanes 3 and
4) after exposure for 32P-labeled
moieties.
[View Larger Version of this Image (25K GIF file)]
Fig. 4.
Tat, but not Tax or Sp1A, associates with
cellular CTD kinase activity. A, in vitro kinase assays for
the phosphorylation of GST-CTD substrate using proteins pulled down
from total nuclear extract by GST alone (lane 2), GST-Tat
(lane 4), or GST-Tax (lane 6) beads. Lanes
1, 3, and 5, activities contained in the corresponding
flow-through fractions. Note the relative diminution of GST-CTD kinase
activity from the GST-Tat-depleted flow-through. Indicated molecular
sizes are based on co-electrophoresed 14C-protein molecular
weight markers (Amersham). FT, flow-through;
0.25, elutions with 0.25 M KCl-containing
buffer. Arrow, expected position of phosphorylated GST-CTD.
B, Phosphorylated CTD was confirmed using
immunoprecipitations with a monoclonal antibody (8WG16) directed
against the CTD epitope. Lanes 4-6 were exposed for the
same duration. Lanes 1-3 were exposed for different
durations and are presented for qualitative, not quantitative,
verification. C, GST-Sp1A does not retain a CTD kinase
activity. Experiments were conducted as in A, except that
this particular batch of extract was 10 times more concentrated.
Proteins retained by GST alone (lane 2), GST-Tat (lane
4), or GST-Sp1A (lane 6) were analyzed for CTD kinase
activity. Phosphorylated GST-CTD was observed in the GST-Tat reaction
(lane 4) but not in the GST-Sp1A (lane 6) or GST
alone (lane 2) reaction.
[View Larger Version of this Image (29K GIF file)]
B
(60, 64). Whether protein kinase R or a different kinase associates
with Tat for promoter proximal kinase activity remains to be
clarified.
To whom correspondence should be addressed: Molecular Virology
Section, Laboratory of Molecular Microbiology, NIAID, National
Institutes of Health, 9000 Rockville Pike, Bldg. 4, Room 306, Bethesda,
MD 20892-0460. Tel.: 301-496-6680; Fax: 301-402-0226; E-mail:
kjeang{at}atlas.niaid.nih.gov.
1
The abbreviations used are: CTD, C-terminal
domain; PAGE, polyacrylamide gel electrophoresis; pol, polymerase;
RNAP, RNA polymerase; HIV, human immunodeficiency virus; HTLV, human
T-cell lymphotropic virus; LTR, long terminal repeat; CAT,
chloramphenicol acetyltransferase; GST, glutathione
S-transferase; RPA, RNase protection assay; wt, wild
type.
2
H. Xiao and K.-T. Jeang, manuscript in
preparation.
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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