 |
INTRODUCTION |
Transcription elongation in eukaryotic genes is a complex process
that involves a number of regulatory factors. It is becoming increasingly clear that the elongation stage of RNA pol
II1 is a major regulatory
process of gene expression (1-6). After successful initiation of RNA
synthesis, RNA pol II can pause, get arrested, pass through terminator
sequences, or terminate transcription. Release of RNA pol II from
stalled complexes is a rate-limiting step in transcription of inducible
eukaryotic genes (3, 4, 7). In the absence of inducer protein, RNA pol
II elongation complexes pause 20-60 nucleotides downstream of the
promoter. Promoter-proximal pausing is released by DNA- or RNA-binding
activators that recruit or stimulate positive-acting transcription
elongation factors. General transcription factors such as TFIIH play a
key role in promoter clearance and the promoter-proximal release of RNA
pol II (8, 9).
Human immunodeficiency virus, type 1 (HIV-1) encodes a small regulatory
protein, Tat, which is required for efficient transcription of viral
genes. Tat enhances the processivity of RNA pol II elongation complexes
that initiate in the HIV LTR region. Tat activates transcription by
binding to a highly structured RNA element, TAR RNA, that is located at
the 5' end of nascent viral transcripts (for review see Refs. 10 and
11). Tat functions through TAR RNA to control an early step in
transcription elongation that is sensitive to protein kinase inhibitors
and requires the carboxyl-terminal domain (CTD) of the large subunit of
RNA pol II (12). The Tat protein contains two important functional
regions: an arginine-rich region that is required for binding to TAR
RNA and an activation domain that mediates the interactions with
cellular machinery (11). Tat has been reported to interact with a
number of cellular proteins associated with transcription, including
TFIID (13), SP1 (14), TAFII-55 (15), TFIIH (16-18), and RNA pol II
(19).
Recent studies indicate that Tat transactivation function is mediated
by a nuclear Tat-associated kinase, TAK (12, 20, 21). The
transactivation domain of Tat interacts with TAK (22, 23), which was
recently shown to be identical to the kinase subunit of P-TEFb, a
positive-acting transcription elongation factor (24, 25). P-TEFb is
required for transcription elongation at many genes (26, 27) and is
proposed to facilitate the transition from abortive to productive
elongation by phosphorylating the CTD of the largest subunit of RNA pol
II (28). Experimental evidence has established that the CTD is required
for Tat transactivation (29-31). It is possible that P-TEFb kinase
activities are also involved in regulation of other cellular
proteins. P-TEFb is composed of at least two subunits: the catalytic
subunit cyclic-dependent kinase CDK9 (previously named
PITALRE) and the regulatory subunit cyclin T1 (24, 32, 33).
Complexes containing CDK9 and cyclin T1-related proteins, cyclin T2a or
cyclin T2b, are also active for P-TEFb activity (34). It has been
demonstrated that cyclin T1 interacts directly with the activation
domain of Tat and mediates its high affinity and specific binding to
TAR RNA (33). Tat-cyclin T1 interaction is distinctive in nature
because other Tat-binding proteins identified so far have not been
shown to enhance Tat affinity and specificity for TAR RNA. Therefore,
cyclin T1 interaction with Tat could be responsible for P-TEFb
recruitment to the RNA pol II elongation complexes stalled after TAR RNA.
In this paper, we provide the evidence that P-TEFb is a component of
the preinitiation complex and travels with the RNA pol II elongation
complex as the nascent RNA is synthesized. As outlined in Fig. 1, we
have used an experimental strategy to walk RNA pol II in stepwise
transcription and to stall elongation complexes at specific sites. The
stalled transcription complexes were purified by first magnetic beads
and then by releasing the template DNA by cleaving it with restriction
enzymes. The protein composition in the released complexes was then
measured by Western blotting. Our results demonstrate that P-TEFb is a
component of the preinitiation complex and travels with the elongating
RNA pol II, whereas TFIIH is released from the elongation complexes
before the TAR RNA is synthesized. Our results suggest that two
cellular kinases, TFIIH and P-TEFb, are involved in the clearance of
promoter-proximal pausing of RNA pol II on the HIV-1 LTR at
different stages.
 |
EXPERIMENTAL PROCEDURES |
Template DNAs--
The test plasmids (pWT2 and pPT529) used in
this study were derived from the p10SLT plasmid, which contains HIV-1
5'-LTR (35). Plasmid pWT2 was constructed by inserting a synthesized
DNA fragment containing a triplex target sequence (5'-AAA AGA AAA GGG
GGG-3') between the HindIII and NarI sites of
plasmid p10SLT. Plasmid pPT529 contains a triplex target sequence at
+529 between XbaI and XbaI sites of p10SLT.
Oligonucleotide Synthesis and
Purification--
Oligodeoxyribonucleotides were synthesized on an
automated DNA/RNA synthesizer (ABI 392). Psoralen was conjugated to the
5' end of the triplex probes (see Fig. 1) by using phosphoramidite derivatives of psoralen. Biotin-modified oligonucleotides were prepared
on biotin containing support. Cytosine was replaced by 5-methyl-cytosine. All the oligonucleotides were deprotected in ammonium hydroxide at 55 °C for 8 h and purified on 20%
polyacrylamide, 7 M urea gels.
Triplex Formation and Cross-linking--
Linearized DNA (final
concentration, 0.1 µM) was incubated with excess
psoralen-oligonucleotide probe (×250) in a buffer containing 10 mM Tris-HCl (pH 6.5), 50 mM NaCl, 10 mM MgCl2, and 0.5 mM spermine for
30 min at 37 °C and then cooled down slowly to room temperature. The
mixture was irradiated with UV (360 nm) in a Photochemical Reactor for
8 min. For magnetic bead binding, template DNA was separated from
unreacted psoralen-oligonucleotide probe on 1.2% agarose gels.
Immobilization of DNA on Magnetic Beads--
Template DNA (0.5 µg) cross-linked to a psoralen probe modified with biotin was bound
to 25 µl of streptavidin-coated magnetic bead (250 µg, Dynal Inc.)
by incubating DNA and beads in TE buffer (10 mM Tris-HCl,
pH 7.4, 1 mM EDTA) containing 1 M NaCl at room temperature overnight on a shaker.
Stepwise Walking of RNA pol II--
HeLa cell nuclear extracts
were prepared according to published procedures (36, 37) with minor
modifications (38). Preinitiation complexes (PICs) were formed by
incubating the immobilized DNA templates (200 ng) in a volume of 25 µl containing 12 µl of nuclear extract, 6 mM
MgCl2, and 0.5 µg of poly(dA·dT) for 15 min at
30 °C. PICs were washed with 25 µl of washing buffer A (20 mM HEPES, pH 7.9, 100 mM KCl, 20% (v/v)
glycerol, 0.2 mM EDTA, 0.5 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, and 6 mM
MgCl2) to remove unbound proteins. The PICs were walked to
position U14 by incubation with 12.5 mM phosphocreatine, 20 µM CTP, GTP, and UTP, 20 µM dATP, and 10 µCi of [
-32P] CTP (25 Ci/mmol, ICN; final
concentration, 160 µM) for 5 min at room temperature.
Transcription elongation complexes (TECs) stalled at U14 were washed
with 25 µl of wash buffer B (wash buffer A containing 0.05% Nonidet
P-40 and 0.015% Sarkosyl) and twice with wash buffer C (wash buffer A
containing 0.05% Nonidet P-40). The TECs stalled at U14 were walked
stepwise along the DNA by repeated incubation with different sets of
three NTPs. Unincorporated NTPs were removed by washing the immobilized
complexes with buffer C. To isolate RNA products from stalled TECs, 175 µl of stop solution (0.3 M Tris-HCl, pH 7.4, 0.3 M sodium acetate, 0.5% SDS, 2 mM EDTA) was
added. The mixture was extracted with 200 µl of
phenol-chloroform-isoamyl alcohol (50:48:2) and then with chloroform
(200 µl). RNA transcripts were precipitated with ethanol and analyzed
on 15% polyacrylamide, 7 M urea gels.
Western Blotting--
For the isolation of ternary complexes,
the PICs and TECs stalled at different steps on immobilized DNA
templates were digested with restriction enzymes at 30 °C. The
solution phase containing ternary complexes was mixed with 1 × SDS gel loading buffer (50 mM Tris-HCl, pH 6.8, 100 mM dithiothreitol, 2% SDS, 0.1% bromphenol blue, 10%
glycerol) at 100 °C for 3 min. Released proteins were separated from
beads, fractionated by 6% SDS-polyacrylamide gel electrophoresis, and
transferred onto nitrocellulose membranes to detect protein
composition. The RNA pol II CTD antibody was a gift from Michael E. Dahmus (University of California, Davis), and the CDK9 antibody was a
gift from David H. Price (University of Iowa, Iowa City). The
polyclonal antibodies to TFIIH (p89 and p62) and TBP were from Santa
Cruz Biotechnology (Santa Cruz, CA). Complexes were visualized with the
ECL system (Amersham Pharmacia Biotech) by using anti-mouse or
anti-rabbit horseradish peroxidase-labeled secondary antibodies. The
blots were exposed to x-ray film for various times (between 10 s
and 10 min).
 |
RESULTS |
Stepwise Transcription--
To determine the protein composition
of transcription elongation complexes at different stages, it is
important to isolate homogeneous populations of RNA pol II ternary
complexes stalled at specific sites. We used triplex DNA structure and
psoralen photochemistry to immobilize DNA templates and stall
elongation complexes (38). The experimental strategy for site-specific psoralen cross-links in the DNA template containing HIV-1 promoter to
isolate the stalled RNA pol II elongation complexes is outlined in Fig.
1. This method involves the following
five steps: 1) insertion of a target sequence for triple helix
formation at a predetermined position in the DNA template; 2)
restriction digest with an enzyme to yield the triplex site upstream or
downstream of the promoter sequences; 3) synthesis of a third strand
for triplex formation containing a psoralen at its 5' end and a biotin
at the 3' end; 4) triplex formation between the DNA template and the
third strand followed by nearly ultraviolet irradiation (360 nm); and
5) immobilization of the cross-linked DNA template on
streptavidin-conjugated magnetic beads and in vitro
transcription. Immobilized DNA templates with beads upstream of the
promoter were used for stepwise walking of RNA pol II, and DNA
templates with beads downstream of the promoter were used to stall
elongation complexes chased with all four nucleotides. Preinitiation
complexes were formed on immobilized DNA templates, and elongation was
initiated by adding dATP, UTP, CTP, and GTP. These elongation complexes
were starved for ATP and therefore stalled at U14. Further initiation
was inhibited by sarkosyl wash as described under "Experimental
Procedures." Stepwise walking of the TECs stalled at U14 was
accomplished by repeated incubation with different sets of three NTPs
(39). A typical gel of our RNA pol II walking experiments is shown in Fig. 2. TECs stalled at A-22, C-30, G-36,
U-46, A-51, and C-61 are shown. Viability of the stalled complexes was
confirmed by adding all four NTPs, which produced run-off products of
expected lengths indicating that 100% of the complexes were
transcriptionally active (data not shown).

View larger version (19K):
[in this window]
[in a new window]
|
Fig. 1.
A, experimental design to walk RNA pol
II in stepwise transcription (left) and to stall elongation
complexes at specific sites (right). A target sequence for
triple helical DNA is inserted into the DNA template. Plasmid pWT2 is
linearized with restriction enzymes (HindIII or
AccI). A psoralen- and biotin-containing oligonucleotide is
used to form triplex DNA, and UV irradiation covalently cross-links
psoralen to the template. Psoralen cross-linked template is immobilized
on streptavidin-conjugated magnetic beads, and noncross-linked DNA is
washed away with buffer. For stepwise RNA pol II walking, DNA templates
containing psoralen cross-links upstream of the promoter are used
(left). To stall elongation complexes at positions +185 and
+529, psoralen cross-links at downstream sequences are used
(right). The RNA transcripts are analyzed by electrophoresis
on 15% polyacrylamide, 7 M urea gels, and the protein
compositions of the transcription complexes are analyzed by Western
blotting. B, DNA sequence of the early transcribed region
(+1 to +71) in the HIV-1 promoter.
|
|

View larger version (13K):
[in this window]
[in a new window]
|
Fig. 2.
Stepwise walking of RNA pol II elongation
complexes. Preinitiation complexes were formed on immobilized
templates (Fig. 1, left), and transcription elongation
complexes were walked stepwise along the DNA by repeated incubation
with different sets of three NTPs (see "Experimental Procedures").
RNA transcripts were labeled with [ -32P]CTP during
transcription. Transcription elongation complexes stalled at various
positions were isolated and the RNA transcripts were analyzed on 15%
polyacrylamide, 7 M urea gels. Length of the RNA
transcripts was confirmed by molecular weight markers. Numbers on the
top show various positions where TECs were stalled.
|
|
Isolation of Preinitiation and Elongation Complexes--
We formed
preinitiation and stalled transcription elongation complexes on DNA
templates immobilized on streptavidin beads, and the DNA was cleaved
with restriction enzymes to isolate the RNA pol II complexes. The
strategy for these experiments is outlined in Fig.
3 (A and B). To
isolate PICs, DNA was cleaved at the BspE1 restriction site.
For isolation of TECs, RNA pol II complexes were stalled at different
sites and then cleaved with BglII. RNA contents were
analyzed on 7 M urea, 15% polyacrylamide gels (as shown in
Fig. 2), and proteins were separated on denaturing SDS gels.

View larger version (22K):
[in this window]
[in a new window]
|
Fig. 3.
Isolation of preinitiation
(A) and elongation complexes
(B). PICs and stalled TECs were formed on DNA
templates immobilized on streptavidin beads, and the DNA was cleaved
with restriction enzymes to isolate the RNA pol II complexes. To
isolate PICs, DNA was cleaved at BspE1 restriction site. For isolation
of TECs, RNA pol II complexes were walked to different sites and then
cleaved with BglII. C, CDK9 is a component of
preinitiation and elongation complexes, whereas TFIIH is released
during elongation. Transcription complexes isolated from preinitiation
and stalled during elongation at two positions, C-30 and C-71, were
analyzed on SDS-PAGE, and proteins were detected by using antibodies
against RNA pol II CTD, CDK9, and TFIIH (p89 or ERCC3 and p62)
(lanes 3, 4, and 5). Lanes
3, 4, and 5 contain 3, 10, and 15 standard
transcription reactions, respectively. Lane 1, 5% of the
nuclear extract present in the transcription reaction. Lane
2, PICs formed in the presence of sarkosyl.
|
|
Analysis of Proteins in Preinitiation and Elongation
Complexes--
Recently, it has been reported that P-TEFb is involved
in Tat transactivation (12, 24, 25). A number of studies also suggest
that TFIIH kinase complex plays an important role in Tat activation
(16-18). We planned to investigate the role of TFIIH and P-TEFb in
transcription using HeLa nuclear extracts and DNA templates containing
HIV-1 promoter. We used immobilized templates and isolated RNA pol II
preinitiation and elongation complexes stalled at different sites
during transcription as described above. Complexes were released by
cleaving DNA templates with restriction enzymes and subjected to
SDS-PAGE. Proteins were transferred to nitrocellulose membrane, and
various proteins were detected by Western blotting with antibodies
raised against TBP, two subunits of TFIIH (p89 or ERCC3 and p62), RNA
pol II, and a subunit of P-TEFb (CDK9). Affinity purified antibodies
against TFIIH and TBP were purchased from Santa Cruz Biotechnology. RNA
pol II and CDK9 antibodies were kindly provided by Drs.
Michael Dahmus (University of California, Davis) and David Price
(University of Iowa, Iowa City), respectively. Results of
this analysis are shown in Fig. 3C. P-TEFb is a component of
RNA pol II preinitiation complex and remains attached to the elongation
complexes stalled at C-30 and C-71. On the other hand, TFIIH is
detected in the PICs and not in the TECs stalled at C-30 and C71. To
confirm that sarkosyl treatment of TECs did not remove TFIIH and other
transcription factors, we isolated TECs stalled at A-15 and washed with
sarkosyl containing buffer and analyzed for protein contents as
described above. Western analysis showed that TFIIH was present in TECs stalled at A-15 (data not shown). TBP analysis was performed as a
control experiment showing that TBP was present in the PICs and not in
the stalled elongation complexes (data not shown).
Our findings about the assembly and release of TBP and TFIIH are in
agreement with previous studies by Zawel et al. (40) who
used a defined reconstituted transcription system containing adenovirus
2 major late promoter. It is interesting to note that using nuclear
extracts and DNA templates containing HIV-1 promoter sequences did not
change the properties of TFIIH. To normalize the amount of
transcription complexes at each step, we detected RNA pol II by
immunoblotting in the PICs and the TECs. It is now generally accepted
from previous studies that the PIC contains nonphosphorylated CTD of
RNA pol II (IIa) and TECs contain phosphorylated CTD of RNA pol (IIo)
(41). RNA pol II in elongation complexes is IIo; however, we did not
separate IIa and IIo bands in this gel because we were detecting RNA
pol II as an internal standard and wanted to detect CDK9 in the same
gel and did not run the gel for longer times to resolve proteins with
high molecular weights. We were able to separate IIa and IIo when gels
were run for longer times (38). No proteins were detected when the PIC
formation was inhibited by the addition of sarkosyl (lane 2 in Fig. 3C).
Western blotting of the nuclear extract was performed as a control
experiment for the identification of the correct proteins and to
confirm that these proteins are not modified or degraded in our stalled
complexes. It is important to note that these experiments are not
quantitative; therefore, the relative stoichiometry of P-TEFb in
elongation complexes cannot be determined from these results. The
intensity of various bands represents the immunoreactivity of the
specific antibodies and does not correspond to the amount of proteins
present in RNA pol II complexes. For example, TFIIH bands are more
intense than CDK9 and RNA pol II (Fig. 3C, lane 3) and do not necessarily represent the stoichiometry of these proteins. During stepwise walking of RNA polymerase II elongation complexes, each wash to remove unincorporated NTPs results in a loss of
TECs. To ensure that a sufficient amount of proteins was present in
TECs stalled at longer sequences, 10 and 15 standard transcription
reactions were used to isolate TECs stalled at C-30 and C-71,
respectively. PICs contained three standard transcription reactions.
Accordingly, more CDK9 and RNA pol II were detected in TECs stalled at
C-30 and C-71 compared with the PICs (Fig. 3C, lanes
3-5). These results establish that P-TEFb is a part of
preinitiation and elongation assembly of RNA pol II, whereas TFIIH is
released from the elongation complex before full-length TAR RNA is transcribed.
P-TEFb Is a Component of the Transcription Elongation
Complex--
The above experiments show that P-TEFb associates with
preinitiation transcription complexes and remains attached to the
elongation complexes stalled at C-71. To determine whether P-TEFb is
released from the TECs after the synthesis of full-length TAR RNA or it associates with the RNA pol II during elongation, we stalled elongation complexes at specific sites by psoralen cross-link adducts (Fig. 4). Transcription complexes were
assembled on immobilized templates, and transcription was carried out
by adding four NTPs. Analysis of transcription products on denaturing
gels showed that the correct size RNA transcripts were produced (Fig.
4B). TECs were stalled on immobilized templates at positions
+185 and +529, rinsed with buffers to remove released proteins, and
isolated by cleaving DNA with HindIII. Restriction digest
with HindIII removed the PICs that could not start
transcription. Protein contents were separated by SDS-PAGE and detected
by Western blotting using CDK9 antibodies. Fig. 4C shows
that elongation complexes stalled at +185 and +529 contain P-TEFb.
There was no detectable binding of P-TEFb to the beads or the DNA
templates (data not shown). A distinguishing characteristic of P-TEFb
is its sensitivity to the kinase inhibitors DRB and H-8 (24). We
confirmed P-TEFb function by the generation of DRB-sensitive run-off
transcripts during transcription in vitro (data not shown).
These results demonstrate that P-TEFb is an integral component of RNA
pol II elongation complex and travels with the polymerase as the
nascent RNA is synthesized.

View larger version (10K):
[in this window]
[in a new window]
|
Fig. 4.
A, strategy to isolate a homogeneous
population of RNA pol II elongation complexes stalled at positions +185
and +529. DNA containing a downstream triplex target site was
immobilized on the magnetic beads (Fig. 1, right), and
cell-free transcription experiments were performed. TECs stalled at the
DNA damage site were separated by magnet. Restriction digestion was
performed to remove the promoter sequences. B, transcript
analysis for the elongation complexes stalled at DNA damage sites (+185
and +529). DNA templates containing psoralen cross-links at +185
(lane 2) and +529 (lane 3) were used in
transcription reactions, and the RNA transcripts were analyzed on
denaturing gels. Lane 1 is a 50-base pair DNA marker.
C, CDK9 travels with the elongation complexes. Transcription
complexes stalled at +185 and +529 were isolated and analyzed on
SDS-PAGE. CDK9 was detected by Western blot analysis (lanes
2 and 3). Lanes 2 and 3 contain
three and five standard transcription reactions, respectively.
Lane 1, 5% of the nuclear extract present in the
transcription reaction.
|
|
 |
DISCUSSION |
We have utilized a stepwise transcription approach and Western
blotting to determine the role of TFIIH and P-TEFb in transcription elongation in HIV-1 promoter. Our results provide new insights into the
mechanism of Tat transactivation.
Several lines of evidence suggest that Tat activation requires the CTD
of RNA pol II (16, 29-31). CTD contains a tandemly reiterated
heptapeptide sequence (YSPTSPS) that is differentially phosphorylated
during the transcription cycle (for review see Refs. 41 and 42).
Mammalian cells contain two forms of RNA pol II, phosphorylated (IIo)
and nonphosphorylated (IIa), that differ in the extent of
phosphorylation within the carboxyl-terminal domain of their largest
subunit (41). The nonphosphorylated form of RNA pol II preferentially
associates with the preinitiation complex, whereas RNA pol II derived
from isolated ternary complexes is highly phosphorylated (40, 43).
Phosphorylation of CTD is suggested to be critical for the release of
preinitiation complexes from the promoter and could disrupt
interactions between the CTD and other mediator proteins and
transcription factors, such as TBP (40, 44). Tat stimulates
hyperphosphorylation of the CTD in a transcription dependent manner
in vitro (16, 17). These findings suggest that Tat is
involved in CTD kinase steps to form processive elongation complexes.
Interaction of Tat with two cellular kinase complexes, TFIIH (16-18)
and P-TEFb (22-24), have been reported, and it has been proposed that
Tat may interact with these kinase complexes at different stages of the
transcription cycle (12). How does Tat interact with these two kinase
complexes and control the processivity of the RNA pol II elongation? On
the basis of previous findings and our results, we propose a model for
transcriptional activation by Tat (Fig.
5). Tat binds to the preinitiation
complex and interacts with TFIIH; however, its interaction with other
proteins present in the initiation complex cannot be ruled out (13-15,
19). It has been reported earlier that Tat associates with purified
transcription preinitiation complexes (45). P-TEFb is also a component
of the PIC, but it may not be involved in Tat binding at this point. TFIIH plays a critical role in transcription initiation and promoter clearance (8, 46) and is bound to nonphosphorylated RNA pol II
holoenzyme. TFIIH alone or with Tat phosphorylates the CTD and assists
in promoter clearance. The TFIIH complex dissociates from the RNA pol
II 30-50 nucleotides after initiation and is not part of the
elongation complex (40). In line with previous findings, we also
observed that TFIIH was released from the elongation complexes when
46-nucleotide-long RNA was transcribed. TFIIH is released, whereas Tat
remains in the elongation complex by interactions with cyclin T1
subunit of P-TEFb. After the transcription of a functional TAR RNA
structure, Tat binds to TAR and repositions P-TEFb kinase in the
vicinity of the CTD of RNA pol II. Hyperphosphorylation of the CTD is
accomplished by P-TEFb kinase, and processive elongation complexes are
formed. We propose that a functional Tat-P-TEFb interaction requires
TAR RNA because Tat does not become part of the elongation complexes
unless TAR RNA is synthesized (47). It is also possible that more than
one molecule of Tat participate in a single round of transactivation
and additional Tat is recruited to the elongation complex after TAR is
transcribed. Future studies to define the Tat-interacting regions of
TFIIH and P-TEFb complexes and to determine various stages of the CTD
phosphorylation will reveal mechanistic details of Tat function.

View larger version (24K):
[in this window]
[in a new window]
|
Fig. 5.
A model for the formation of processive RNA
pol II elongation complexes by Tat protein. See text for
details.
|
|