From the Department of Pharmacology, Robert Wood Johnson Medical School, and Molecular Biosciences Graduate Program at Rutgers University, Piscataway, New Jersey 08854
Received for publication, July 12, 2000, and in revised form, November 30, 2000
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ABSTRACT |
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Control of transcription elongation requires a
complex interplay between the recently discovered positive
transcription elongation factor b (P-TEFb) and negative transcription
elongation factors, 5,6-dichloro-1- Transcription in eukaryotic cells is a complex process and
involves three major steps including initiation, elongation, and termination. Although it was thought originally that regulation occurs
primarily at the level of initiation, it is now recognized that the
elongation step of transcription is a critical target for regulation of
gene expression (1-3). Density analysis of RNA
pol1 II in cells led to the
identification of a number of genes including c-myc
(4), c-fms (5), hsp70 (6), and HIV (7), which are
potentially regulated at the elongation stage of transcription. Shortly
after initiation, RNA pol II faces a barrier of negative transcription
elongation factors and enters abortive elongation. The action of
positive transcription elongation factors (P-TEF) lowers the barrier of
negative transcription elongation factors and helps RNA pol II to
escape from this transition phase which could lead to premature
termination of transcription (for an excellent review, see Ref. 8). A
positive elongation factor, P-TEFb, allows the transition into
productive elongation producing longer mRNA transcripts (8).
Proteins involved in positive and negative regulation of elongation
were discovered during studies aimed at understanding the mechanism of
transcription inhibition by a nucleoside analog, 5,6-dichloro-1- Recent studies using DRB as a transcription inhibitor led to the
discovery of positive and negative elongation factors. P-TEFb was
originally identified as an activity that released RNA pol II from an
elongation pause in a DRB-sensitive manner (13, 14) and is proposed to
facilitate the transition from abortive to productive elongation by
phosphorylating the CTD of the largest subunit of RNA pol II (15).
P-TEFb is composed of two subunits: the catalytic subunit
cyclic-dependent kinase CDK9 (previously named PITALRE) and
the regulatory subunit cyclin T1 (16-18). Complexes containing CDK9
and cyclin T1-related proteins, cyclin T2a or cyclin T2b, are also
active for P-TEFb activity (19). Two negative transcription elongation
factors, DSIF (DRB sensitivity-inducting factor) and NELF (negative
elongation factor), have recently been identified and characterized
(12, 20). DSIF is composed of two subunits, p160 and p14, which are
homologs of the Saccharomyces cerevisiae transcription
factors Spt5 and Spt4, respectively (12, 21). NELF is composed of five
polypeptides, named as NELF-A to -E, and contains a subunit identical
to RD, which is a putative RNA-binding protein of unknown function.
DSIF and NELF function cooperatively and strongly repress RNA pol II
elongation (20).
One elegant example of transcription elongation control is the
mechanism of HIV-1 gene expression (for recent reviews, see Refs.
22-26). 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 long terminal repeat (LTR) region.
Tat activates transcription by binding to a highly structured RNA
element, TAR RNA, which is located at the 5'-end of nascent viral
transcripts (27). Tat functions through TAR RNA to control an early
step in transcription elongation that is sensitive to protein kinase
inhibitors and requires the C-terminal domain (CTD) of the large
subunit of RNA pol II (22). Mutational analysis of HIV-1 Tat protein
has identified two important functional domains: an arginine-rich
region that is required for binding to TAR RNA, and an activation
domain that mediates the interactions with cellular machinery (28, 29).
Recent studies showed that Tat transactivation function is mediated by
a nuclear Tat-associated kinase, TAK (22-26). The transactivation
domain of Tat interacts with TAK (30, 31), which was recently shown to
be identical to the kinase subunit of P-TEFb (17, 32). Tat interacts
with cyclin T1 subunit of P-TEFb and recruits the kinase complex to TAR
RNA. Recruitment of P-TEFb to TAR has been proposed to be both
necessary and sufficient for activation of transcription elongation
from the HIV-1 long terminal repeat promoter (33).
P-TEFb is a component of preinitiation transcription complexes and
functions by phosphorylating the CTD of the largest subunit of RNA pol
II during elongation steps (34-36). The CTD of the largest subunit of
RNA pol II contains tandem repeats of the consensus sequence (YSPTSPS)
that is differentially phosphorylated during the transcription cycle
and phosphorylation of the CTD is critical for transcription regulation
(37). Cellular kinases and phosphatases may contribute in transcription
regulation based on their ability to alter the phosphorylation status
of the RNA pol II CTD (37). For example, TFIIH phosphorylates the CTD
of RNA pol II and assists in promoter clearance (38, 39).
It is not well understood when DSIF and NELF are recruited to the
active elongation complexes and how they function. We were intrigued by
the studies reporting that Spt5, one of the subunits of DSIF, is
involved in Tat-mediated transactivation and functions as a positive
elongation factor in HIV-1 LTR promoter in the presence of Tat (40,
41). Since DSIF was identified as a negative elongation factor, these
results suggested that there is a critical transition step which
converts Spt5 from a negative to a positive elongation factor during
Tat transactivation. We reasoned that DSIF could be a substrate for
P-TEFb phosphorylation and the phosphorylation status of Spt5 was a key
element to determine the Spt5 function in transcription.
In this paper, we used a stepwise transcription approach to isolate
homogeneous populations of active RNA pol II elongation complexes to
study the interaction between negative elongation factors and RNA pol
II transcription complex. In addition, we carried out kinase
experiments during elongation to analyze the function of Spt5 in HIV-1
Tat transactivation. Our results demonstrate that DSIF and NELF
associate with RNA pol II complexes during early transcription
elongation and travel with elongation complexes as the nascent RNA is
synthesized. Our results also show that HIV-1 Tat protein stimulated
Spt5 and RNA pol II phosphorylation by P-TEFb. We propose a model for
the regulation of elongation in HIV-1 LTR by Tat and Spt5.
Tat Protein Purification--
Recombinant HIV-1 Tat protein
expressed in Escherichia coli as a glutathione
S-transferase fusion protein was purified by glutathione-Sepharose affinity chromatography according to previously described procedures (42). HIV-1 glutathione
S-transferase-Tat expression vectors were obtained from AIDS
Research and Reference Reagent Program, Division of AIDS, NIAID,
National Institutes of Health, which were made available by Dr. Andrew Rice.
DNA Templates--
The DNA templates used for preparing
transcription ternary complexes were generated by polymerase chain
reaction. Polymerase chain reaction was carried out by using plasmid
p10SLT containing HIV-1 LTR promoter as a template (43), and three
primers based on p10SLT plasmid sequence (5'-ACCAGTTGAACCAGAGC,
5'-CACACTGACTAAAAGGGT, and 5'-CACTTTATGCTTCCGGCT). The first primer
contained a biotin at the 5'-end which was used to immobilize DNA to
magnetic beads. Polymerase chain reaction products were purified on
1.0% agarose gels.
Immobilization of DNA on Magnetic Beads--
DNA templates (0.25 µg) with biotin at 5'-end was bound to 25 µl of streptavidin-coated
magnetic beads (Dynal Inc.) by incubating DNA and beads in TE buffer
(10 mM Tris-HCl, pH 8.0, 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 (44, 45). Stepwise
transcriptions were performed as described previously (35).
Preinitiation complexes (PICs) were assembled 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. For RNA pol II labeling
experiments at the PIC formation step, nuclear extracts were first
incubated with 10 units of casein kinase II and 10 µCi of
[ Western Blotting--
For the isolation of ternary complexes on
immobilized DNA template, the PICs and TECs stalled at different steps
were cleavaged by restriction enzymes at 30 °C. The solution phase
containing ternary complexes was mixed with 1 × SDS loading
buffer and heated at 100 °C for 5 min. Released proteins were
fractionated by SDS-PAGE, and transferred onto polyvinylidene
difluoride membrane (Bio-Rad) to detect protein compositions. DSIF and
NELF antibodies were kindly provided by Dr. Hiroshi Handa (Tokyo
Institute of Technology, Japan). P-TEFb and RNA pol II antibodies were
kind gifts from Dr. David H. Price (University of Iowa, Iowa City) and
Dr. Michael Dahmus (University of California, Davis), respectively. The
anti-[HA] biotin mouse antibodies were purchased from Roche Molecular
Biochemicals. Protein contents were visualized with either ECL system
(Amersham Pharmacia Biotech) or BM chemiluminescence Blotting Kit
(Roche Molecular Biochemicals). The blots were exposed to x-ray film for various times (between 15 s and 10 min).
Kinase and Immunoprecipitation Assays--
TECs stalled at
A-22 were incubated with additional nuclear extract in the
absence or presence of 100 ng of HA-Tat at room temperature for 10 min.
After removing the unbound proteins, TECs were chased by adding 10 µCi of [ DSIF and NELF Are Not Components of Preinitiation Complex--
To
determine whether DSIF and NELF are present in PICs, we prepared RNA
pol II complexes on immobilized DNA templates containing HIV-1 LTR.
Experimental design is shown in Fig.
1A. We prepared PICs
containing RNA pol IIA and RNA pol IIO forms as described under
"Experimental Procedures." To visualize the formation of RNA pol
IIA and RNA pol IIO forms in the presence of dATP, the largest subunit
of RNA pol II was labeled by phosphorylation of the most C terminus
residue (position 1928) by incubation with casein kinase II in the
presence of [
Next, we analyzed the composition of PICs by Western blotting. PICs
containing RNA pol IIA and IIO forms were formed as described above and
released from immobilized DNA templates by cleaving with restriction
enzyme and subjected to SDS-PAGE. Protein contents were transferred to
polyvinylidene difluoride membrane, and detected by Western blotting
with various antibodies raised against RNA pol II, a subunit of DSIF
(Spt5), a subunit of NELF (NELF-E), and a subunit of P-TEFb (CDK9).
Antibodies against RNA pol II were kindly provided by Dr. Michael
Dahmus (University of California, Davis). DSIF and NELF antibodies were
a kind gift from Dr. Hiroshi Handa (Tokyo Institute of Technology,
Japan). P-TEFb antibodies were a kind gift from Dr. David H. Price
(University of Iowa, Iowa City). Results of this analysis are shown in
Fig. 1C. Regardless of the phosphorylation states of RNA pol
II, DSIF and NELF are clearly not present in the PICs (Fig. 1C,
lanes 2 and 3). However, P-TEFb was detected in PICs
containing RNA pol IIA and IIO forms (Fig. 1C). P-TEFb as a
component of PIC has been previously reported (35), and these results
show that the phosphorylation status of RNA pol II did not affect the
P-TEFb-PIC association. In addition, these results also indicate that
there is no nonspecific interaction between DSIF, NELF, and DNA
templates. To normalize the amount of transcription complexes, RNA pol
II was detected as an internal standard. RNA pol IIA is converted to
pol IIO form in the presence of dATP (Fig. 1B), 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 (45). 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 nuclear extracts. These
results indicate that DSIF and NELF do not interact with the RNA pol
IIA and IIO forms in the PICs.
DSIF and NELF Associate with RNA Pol II Transcription Complexes
during Elongation--
Since DSIF and NELF are not components of the
PICs (Fig. 1C), it is likely that these factors are
recruited to the transcription complexes during the elongation stage.
To test this hypothesis and determine the stage of elongation where
DSIF and NELF interact with the transcription complex, we isolated
homogeneous populations of TECs by a stepwise transcription approach
(35). 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
U-14. Further initiation was inhibited by Sarkosyl wash as
described under "Experimental Procedures." Stepwise walking of the
TECs stalled at U-14 was accomplished by repeated incubation
with different sets of 3 NTPs. The viability of the stalled complexes
was confirmed by adding all 4 NTPs, which produced runoff products of
expected lengths indicating that 100% of the complexes were
transcriptionally active (data not shown).
We isolated TECs at A-22 and C-61 positions which display
different sequence and structure of RNA transcripts (Fig.
2). TECs were incubated with HeLa nuclear
extracts to provide with DSIF and NELF since these factors were not
present in the PICs. After removing the unbound proteins from nuclear
extracts, TECs were released by cleaving the DNA template with
PvuII, which is located downstream of TATA box sequence and
this restriction digest avoids further initiation events. Protein
contents were separated by SDS-PAGE and detected by Western blotting
using RNA pol II, DSIF, and NELF antibodies. As shown in Fig. 2, DSIF
and NELF can be detected in TECs after incubation with the nuclear
extracts (lanes 2 and 4). As expected, there was
no DSIF and NELF present in TECs without the addition of nuclear
extract (Fig. 2B, lanes 1 and 3). Since there was
no detectable binding of DSIF and NELF to the DNA templates or PICs
(Fig. 1C), the associations of DSIF and NELF with TECs
represent that these factors specifically interact with the elongation
machinery. In agreement with our previous studies, P-TEFb was present
in the elongation complexes (35). It is important to note that these
experiments are not quantitative, therefore, the relative stoichiometry
of P-TEFb, DSIF, NELF, and RNA pol II 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 the elongation
complexes. For example, DSIF bands are more intense than CDK9 and RNA
pol II (Fig. 2B) and do not necessarily represent the
stoichiometry of these proteins. Our results showing the coexistence of
DSIF and NELF in TECs are in agreement with previous findings
indicating that NELF acts cooperatively with DSIF (20). Taken together,
these results demonstrate that DSIF and NELF associate with RNA pol II
complexes during the elongation stage and form a functional DSIF-NELF
complex.
DSIF and NELF Association with RNA Pol II Elongation Complexes Does
Not Require RNA--
NELF is composed of five polypeptides, the
smallest of which is identical to RD, a putative RNA-binding protein of
unknown function (20). RD contains a tract of alternating Arg-Asp
residues (RD motif). Since NELF interacts with elongation complexes
(Fig. 2), it is possible that it recognizes RNA and this RNA binding activity of NELF recruits DSIF-NELF to the elongation complex. To
address this question, we treated TECs with RNase A to remove the RNA
moiety extruded outside of RNA pol II complexes and examined the
binding of DSIF-NELF to these elongation complexes.
Before testing the RNA dependence of DSIF/NELF-TEC interactions, it was
necessary to demonstrate that RNase A can hydrolyze RNA exposed out of
the TECs and these TECs containing short nascent transcripts are
competent to carry out transcription elongation. The experimental
outline to remove nascent RNA and to test elongation is shown in Fig.
3. TECs stalled at A-22 were
prepared by stepwise transcription reactions. The isolated TECs
(A-22') were treated with RNase A to digest the extruded 5'-end
of the RNA. After removing the enzyme, TECs were further walked to
G-26. RNA transcripts were isolated by phenol extraction and
ethanol precipitation, and analyzed on 15% polyacrylamide-7
M urea gels. Compared with RNA in TECs stalled at
A-22 (Fig. 3B, lane 1), RNA isolated from RNase
A-treated TECs, A22', was ~7 nucleotides shorter than a 22-nucleotide
transcript (Fig. 3B, lane 2). The protected 15-nucleotide RNA represents the RNA which is part of the RNA-DNA hybrid as well as
single-stranded RNA inaccessible to the RNase A. The addition of cold
ATP-free nucleotide mixture was able to move TECs from A-22 to
G-26 (Fig. 3B, lane 3). These results demonstrate that nascent RNA exposed from the TECs can be removed by RNase A digestion and RNase A-treated TECs are competent to carry out further
transcription elongation.
We next analyzed the DSIF/NELF interaction with TECs that were treated
with RNase A (Fig. 3C). TECs were stalled at A-22 and RNA was digested with RNase A. After incubating the TECs with nuclear
extracts and removing the unbound proteins, the TECs were released from
the beads by PvuII cleavage. Proteins were separated by
SDS-PAGE and detected by immunoblotting as described above. As shown in
Fig. 3C, RNase A treatment did not affect the DSIF/NELF interaction with TECs (lanes 2 and 3). There was
no DSIF/NELF detected in TECs which were not incubated with nuclear
extracts (lane 1). Based on these results, we conclude that
DSIF and NELF associate with elongation complexes and this interaction
is RNA-independent.
DSIF/NELF Travels with RNA Pol II Complexes through the Process of
Transcription Elongation--
We next determined whether DSIF/NELF is
released from TECs or it remains attached with TECs during elongation
(Fig. 4). TECs stalled at A-22
were incubated with or without nuclear extract, washed extensively with
buffers to remove unbound proteins, and chased by the addition of NTPs
mixture for short periods of time. After TECs were released by
restriction enzyme digestion, protein contents of TECs were separated
on SDS-PAGE and detected by immunoblotting. RNA transcript analysis
showed that the TECs stalled at A-22 could be chased to G-646
after incubation with NTPs for short periods of time. DSIF and NELF are
present in TECs stalled at A-22 only when complexes are
incubated with nuclear extracts (Figs. 2 and 3). DSIF and NELF were
also detected in TECs stalled at G-646 (Fig. 4B, lane
2). Since there is no further addition of cellular factors during
chase from A-22 to G-646, these results indicate that DSIF/NELF
remains attached to the TECs during the elongation process.
HIV-1 Tat Does Not Interfere with the Association of DSIF/NELF with
RNA Pol II Elongation Complexes--
HIV-1 Tat protein stimulates
elongation through the positive effects of P-TEFb. It is quite
reasonable to postulate that Tat could dislodge negative elongation
factors such as DSIF-NELF complex from the elongating polymerase. To
determine the effect of Tat on the interaction of DSIF/NELF with RNA
pol II elongation complexes in HIV LTR promoter, we prepared TECs
stalled at the C-61 position which would expose a functional
TAR RNA structure for Tat binding. TECs stalled at C-61 were
incubated with HIV-1 Tat containing HA-tag in the presence and absence
of HeLa nuclear extracts. After removing the unbound proteins, TECs
were isolated by PvuII restriction digest and analyzed by
immunoblotting using antibodies against RNA pol II CTD, DSIF, and HA.
Fig. 5 shows that Tat can bind to TECs in
the absence and presence of nuclear extracts (lanes 2 and
4) and DSIF also interacts with the TECs in the presence of Tat (lanes 3 and 4). RNase A digestion of TECs
stalled at C-61 in the presence of Tat did not release Tat
protein from the elongation complexes2 indicating that
Tat binds TAR RNA and then transfers to the elongation machinery (48).
DSIF was identified as one of the negative transcription elongation
factors by Handa and co-workers (12, 49) and P-TEFb could alleviate the
negative effects of DSIF. Our previous studies have shown that P-TEFb
is a component of the preinitiation and elongation complexes (35).
Therefore, the presence of Tat, P-TEFb, and DSIF/NELF in the elongation
complexes suggests that Tat could be involved in regulating the
function of negative elongation factors through activation of P-TEFb
(see below).
HIV-1 Tat Stimulates P-TEFb-mediated Phosphorylation of RNA Pol II
and Spt5--
Spt4 and Spt5 inhibit DRB-mediated transcription
elongation (12, 49). Recent studies also showed that Spt5 is involved in Tat transactivation which is a positive elongation activity (40,
41). Since our results indicate that DSIF, P-TEFb, and Tat are present
in elongation complexes, we proposed two hypotheses. 1) In addition to
CTD of RNA pol II, Spt5 is the other cellular target for P-TEFb. 2) The
phosphorylation status of Spt5 may play a key role in DRB-mediated
repression and Tat-dependent transactivation. To address
these questions, we set up a protein phosphorylation experiment during
elongation (Fig. 6). TECs stalled at A-22
were isolated, incubated with nuclear extracts in the absence
or presence of HIV-1 Tat protein, washed with buffers to remove unbound
proteins, and chased to G-646 by the addition of nonradioactive
NTPs and [
Fig. 6A shows that RNA pol II and Spt5 were phosphorylated
during elongation from position A-22 to G-646 (lane
1). Tat stimulated phosphorylation of RNA pol II and Spt5 (compare
lanes 1 and 3). DRB inhibited phosphorylation of
these proteins and Tat was unable to overcome this effect (lanes
2 and 4). Enhanced phosphorylation of RNA pol II by Tat
and DRB effect on phosphorylation are in agreement with previous
reports showing that DRB inhibits the kinase activity of CDK9 which is
responsible for RNA pol II hyperphosphorylation by Tat (17, 32, 36).
Tat-mediated phosphorylation of RNA pol II showed that Tat bound to
elongation complexes was functional. One interesting observation from
these experiments was that Spt5 phosphorylation was also enhanced
2 ± 0.1-fold by Tat during elongation and inhibited by DRB
(lanes 3 and 4). These results show that Tat
stimulates Spt5 phosphorylation which is inhibited by DRB and P-TEFb
activity suggesting that Spt5 is a target of P-TEFb phosphorylation and
the status of Spt5 phosphorylation determines the function of Spt5 in
transcription elongation.
We have utilized a stepwise transcription approach and Western
blotting to demonstrate the interaction of negative elongation factors,
DSIF and NELF, with active RNA pol II transcription machinery during
various stages of transcription. Our results also show that DSIF/NELF
associates with the elongation complex through protein-protein
interactions and does not depend upon the nascent RNA sequence. In
addition, we demonstrate that Tat does not dislodge negative elongation
factors from the RNA pol II complex and stimulates RNA pol II and Spt5
phosphorylation. Our results provide new insights into the mechanisms
of transcription elongation and the role of Tat protein in the
regulation of HIV-1 gene expression.
Recent studies have uncovered that shortly after postinitiation, RNA
pol II comes under the control of negative and positive elongation
factors (for a recent review, see Ref. 8). DSIF and NELF are two
negative elongation factors which are able to impede RNA pol II
elongation (12, 20). The kinase activity of P-TEFb may be required to
overcome their negative effects (49). DSIF interacts with RNA pol II
and may directly modulate its elongation activity (49). Since P-TEFb
phosphorylates the CTD of the largest subunit of RNA pol II, it was
suggested that CTD phosphorylation by P-TEFb somehow alleviates the
negative effect of DSIF (49). Our results show that the Spt5 subunit of
DSIF is phosphorylated by P-TEFb in the presence of Tat suggesting a
mechanism for overcoming the negative effect of DSIF by P-TEFb.
Spt4 and Spt5 are present in a variety of species from yeast to humans
(12, 21, 40, 50). The C-terminal domain of human Spt5 (hSpt5) is rich
in Ser, Pro, and Tyr and contains two C-terminal repeat motifs, CTR1
and CTR2 (51). The consensus sequence of CTR2 is similar to the CTD of
the largest subunit of RNA pol II. CTR1 and CTR2 contain multiple Ser
and Thr residues and may provide phosphorylation sites for cellular
kinases. In vivo phosphorylation of hSpt5 during mitosis has
recently been reported (51). Recent studies showed that Spt4 and Spt5
function during early transcription elongation process which is
regulated by P-TEFb (12, 49). Immunodepletion of DSIF and P-TEFb could restore transcription to normal levels, and addition of recombinant DSIF was able to repress transcription in a dose-dependent
manner (49). In addition, in the presence of P-TEFb, DSIF had no effect without DRB (49). These results indicated that DSIF in the absence of
P-TEFb plays a role of negative regulator in transcription (49). Ivanov
et al. (52) reported that the CTR1 domain of Spt5 is
important in transcription elongation in the presence of DRB or the
HIV-1 Tat protein. In vitro kinase assays using Spt5 as a
substrate showed that the addition of CDK9 resulted in phosphorylation
of Spt5 (52, 53). Spt5 and TFIIF are phosphorylated by P-TEFb, however,
no functional significance of the phosphorylation of these factors has
been observed.3
How do these negative and positive transcription regulator proteins
work together to control the processivity of RNA pol II that is
enhanced by viral Tat protein? On the basis of our results, combined
with previous reports, we propose a model for the regulation of
transcription elongation by Tat (Fig. 7).
RNA pol II containing nonphosphorylated CTD of the largest subunit
(IIA) assembles on the HIV LTR promoter to form a preinitiation
complex. TFIIH binds to nonphosphorylated RNA pol II and plays a
critical role in transcription initiation and promoter clearance (38,
54-56). TFIIH phosphorylates the CTD of the largest subunit of RNA pol
II and assists in promoter clearance. The TFIIH complex dissociates
from TECs 30 to 50 nucleotides after initiation and is not part of the
elongation complexes (35, 57). P-TEFb, composed of CDK9 and cyclin T1,
is a component of PICs, however, it may not be an active kinase at this
stage (14, 35). After promoter clearance, DSIF and NELF associate with
transcription complex during early elongation stage. Under standard
physiological conditions and non-HIV-1 LTR promoters, Spt5 is
phosphorylated by CDK9 once DSIF/NELF associate with early elongation
complex and this phosphorylation of Spt5 may sufficiently support
regular transcription elongation. In the presence of DRB, the kinase
activity of CDK9 is inhibited and Spt5 cannot be phosphorylated by
P-TEFb. The unphosphorylated form of Spt5 acts as a negative regulator
and causes inhibition of RNA pol II elongation. In contrast to cellular
promoters, transcription from HIV-1 LTR promoter is not efficient and
CDK9 is activated by Tat protein. In the absence of Tat, elongation
complexes originated at HIV-1 promoter meet DSIF and NELF and CDK9 is
unable to efficiently phosphorylate Spt5 and as a result elongation is
not processive. After the transcription of a functional TAR RNA
structure, Tat binds to TAR and repositions P-TEFb in the vicinity of
the CTD of RNA pol II and Spt5. Hyperphosphorylation of the CTD is
carried out by P-TEFb after the formation of Tat·TAR·P-TEFb complexes (23). In addition to CTD phosphorylation, Tat also enhances
the phosphorylation of Spt5 mediated by P-TEFb and phosphorylated form
of Spt5 turns DSIF into a positive regulator of transcription elongation. It is still unclear how P-TEFb gets activated. It is quite
possible that autophosphorylation of P-TEFb or phosphorylation by a
cellular kinase changes P-TEFb conformation and functional properties
during transcription stages. During elongation on HIV-1 LTR, assembly
of the P-TEFb·Tat·TAR ternary complex could be critical for
activation of CDK9 kinase function.
-D-ribofuranosylbenzimidazole (DRB)
sensitivity inducing factors (DSIF) and the negative elongation factor
(NELF). Activation of HIV-1 gene expression is regulated by a nascent RNA structure, termed TAR RNA, in concert with HIV-1 Tat protein and
these positive and negative elongation factors. We have used a stepwise
RNA pol II walking approach and Western blotting to determine the
dynamics of interactions between HIV-1 Tat, DSIF/NELF, and the
transcription complexes actively engaged in elongation. In addition, we
developed an in vitro kinase assay to determine the
phosphorylation status of proteins during elongation stages. Our
results demonstrate that DSIF/NELF associates with RNA pol II complexes
during early transcription elongation and travels with elongation
complexes as the nascent RNA is synthesized. Our results also show that
HIV-1 Tat protein stimulated DSIF and RNA pol II phosphorylation by
P-TEFb during elongation. These findings reveal a molecular mechanism
for the negative and positive regulation of transcriptional elongation
at the HIV-1 promoter.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-D-ribofuranosylbenzimidazole (DRB). DRB
was discovered as an inhibitor of hnRNA and mRNA synthesis in HeLa
cells (9, 10). DRB inhibits the production of full-length RNA and
increases the amount of short transcripts from a variety of genes,
suggesting that RNA pol II elongation was affected (10). In
addition, DRB has no effect on promoter-independent RNA pol II
transcription and on transcription reconstituted by purified general
transcription factors and RNA pol II (11, 12). These studies
suggested that there are cellular proteins other than the RNA pol II
and general transcription factors that confer DRB sensitivity on elongation.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]ATP for 15 min at 30 °C. Then immobilized DNA
templates and poly(dA-dT) were added into the mixture with 20 µM DRB, and incubated for an additional 15 min at
30 °C. RNA pol IIO complexes at the PIC stage were formed by adding
200 µM dATP during incubation. To remove unbound
materials, 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). PICs were walked to position U-14 by incubation with 12.5 mM phosphocreatine, 20 µM CTP, GTP, UTP, and dATP for 5 min at room temperature.
Transcription elongation complexes (TECs) stalled at U-14 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 were walked stepwise
along the DNA templates by incubating with different combinations of
NTPs. For all experiments that required additional cellular factors
supplied by nuclear extract, TECs were incubated with nuclear extract
at room temperature for 10 min and washed three times with wash buffer C. For chase experiments, stalled TECs were incubated with 4 NTP mixtures and incubated at room temperature for 5 min.
-32P]ATP, 20 µM CTP, GTP, UTP,
and 2 µM ATP, in the absence or presence of 20 µM DRB. To reduce nonspecific phosphorylation by other
kinases such as DNA-dependent protein kinase, 50 µM inhibitor LY294002 (Sigma) was included in the chase
reactions. TECs were washed three times with 25 µl of wash buffer C
to remove unincorporated radioisotope and then cleaved by restriction
enzyme to isolate the ternary complexes. The isolated TECs were
incubated with Protein G-Sepharose beads (Amersham Pharmacia Biotech),
which were pre-bound with Spt5 antibodies (Transduction Laboratories)
in RIPA buffer (20 mM Tris-HCl, pH 8.0, 0.5% Nonidet P-40,
1% Tritone X-100, and 150 mM KCl), at 4 °C for
overnight. The beads were washed with 300 µl of RIPA buffer
containing 0.015% Sarkosyl three times and once with 300 µl of RIPA
buffer. The bead phase was resuspended in 1 × SDS loading buffer
and subjected to 7.5% SDS-PAGE. The radiolabeled protein contents were
visualized by autoradiography. The composition of TECs were also
detected by immunoblotting as described above.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]ATP and this phosphorylation of RNA
pol IIA did not alter the electrophoretic mobility of the enzyme (46,
47). Kinase activity of TFIIH at this step was inhibited by the
addition of DRB. PICs containing labeled RNA pol IIA were washed to
remove excess radioisotope, DRB, and unbound proteins. RNA pol IIA was
observed in the absence of dATP (Fig. 1B, lane 3) which was
converted to IIO form in the presence of dATP (lane 4).
These results show that PICs containing RNA pol IIA and IIO can be
isolated and both forms of PICs are stable on immobilized DNA
template.
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Fig. 1.
DSIF and NELF do not interact with RNA pol II
in preinitiation complexes. A, experimental outline to
form RNA pol IIA in the preinitiation complex and conversion of RNA pol
IIA to RNA pol IIO. PICs containing 32P-labeled RNA pol II
were prepared by incubating immobilized DNA templates containing HIV-1
LTR with HeLa nuclear extract, casein kinase II, and
[ -32P]ATP (see "Experimental Procedures"). Kinase
activity of TFIIH was inhibited by the addition of DRB. PICs containing
labeled RNA pol IIA were washed extensively to remove unbound proteins,
DRB, and unincorporated radioisotope. Further incubation of the PICs
with dATP converted RNA pol IIA to RNA pol IIO by TFIIH
phosphorylation. B, isolation of PICs and analysis of RNA
pol II. To isolate PICs, DNA was cleaved at BspEI
restriction site. Released proteins were analyzed by 5% SDS-PAGE and
visualized by PhosphorImaging. RNA pol IIA (lane 3) and RNA
pol IIO (lane 4) were detected in the absence and presence
of dATP, respectively. Marker lanes 1 and 2 show
purified RNA pol II forms prepared as described earlier (46, 47).
C, DSIF and NELF are not present in PICs containing RNA pol
IIA and RNA pol IIO. PICs were formed on immobilized DNA templates in
the absence (lane 2) or presence (lane 3) of
dATP. The PICs were isolated by the cleavage of restriction enzyme and
analyzed on 10% SDS-PAGE. Proteins were detected by using antibodies
against RNA pol II CTD, P-TEFb, DISF, and NELF. Lane 1, 5%
of the nuclear extract used in the PIC formation.
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Fig. 2.
DSIF and NELF associate with RNA pol II
transcription complexes during elongation.
A, isolation of homogeneous population of RNA pol
II elongation complexes. RNA pol II elongation complexes stalled at
specific sites during transcription were prepared by stepwise
transcription on immobilized DNA templates (35). RNA transcripts were
labeled using [ -32P]CTP during transcription.
Transcription elongation complexes stalled at A-22 and C-61
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.
B, TECs stalled at A-22 and C-61 were
isolated without (lanes 1 and 3) and with nuclear
extract incubation (lanes 2 and 4). Unbound
proteins were removed by extensive washing with transcription buffer.
TECs were isolated by PvuII cleavage and analyzed on 10%
SDS-PAGE and proteins were detected by immunoblotting using antibodies
against RNA pol II CTD, P-TEFb, DSIF, and NELF.
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Fig. 3.
DSIF and NELF association with elongation
complex does not require RNA. A, experimental outline
to remove nascent RNA and to test elongation. TECs were formed at
A-22 position by stepwise transcription. The isolated TECs
(A22') were treated with RNase A to digest the extruded
5'-end of the RNA. After removing the enzyme, TECs were further walked
to G-26. RNA transcripts were isolated by phenol extraction and
ethanol precipitation, and analyzed on 15% polyacrylamide, 7 M urea gels. B, RNA transcripts isolated from
different TECs are shown: radiolabeled RNA transcript from TECs at
A-22 position before RNase A treatment (lane 1),
after RNase A digestion (lane 2), and TECs walked to
G-26 after RNase A digestion (lane 3). C,
the TECs were stalled at A-22 position by stepwise
transcription and RNA was digested by RNase A treatment. TECs were
incubated with nuclear extract and unbound proteins were removed by
extensive washing with transcription buffer. TECs were isolated by
PvuII cleavage and analyzed on 10% SDS-PAGE and proteins
were detected by immunoblotting using antibodies against RNA pol II
CTD, DSIF, and NELF. TECs stalled at A-22 were isolated without
and with nuclear extract incubation (lanes 1 and
2). TECs stalled at A-22 were treated with RNase A prior to
nuclear extract incubation (lane 3).
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[in a new window]
Fig. 4.
DSIF travels with RNA pol II elongation
complexes. A, transcript analysis for the elongation
complexes stalled at A-22 and G-646. DNA templates containing
HIV-LTR were used in transcription reactions and the RNA transcripts
were analyzed on denaturing gels as described in the legend to Fig. 2.
Lane M is a 50-base pair DNA marker. B, TECs
stalled at A-22 were prepared by stepwise transcription. TECs
incubated with or without nuclear extract were chased to G-646
by adding NTPs mixtures. TECs were isolated by PvuII
cleavage and analyzed on 10% SDS-PAGE and proteins were detected by
immunoblotting using antibodies against RNA pol II CTD, DSIF, and NELF.
TECs stalled at G-646 were isolated without (lane 1) and
with nuclear extract incubation at A-22 (lane 2).
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Fig. 5.
HIV-1 Tat does not affect DSIF interaction
with RNA pol II elongation complexes. TECs stalled at C-61
were prepared as described above and incubated with HIV-1 Tat protein
containing HA-tag in the presence and absence of nuclear extract. After
removing the unbound proteins, TECs were isolated by PvuII
restriction digest and analyzed by immunoblotting using antibodies
against RNA pol II CTD, DSIF, and HA.
-32P]ATP. DRB (20 µM) was
included during elongation reactions to specifically inhibit
Tat-dependent P-TEFb activity in HIV-1 LTR promoter (17).
TECs were isolated by BglII cleavage, immunoprecipitated with Spt5, analyzed on 7.5% SDS-PAGE, and visualized by
autoradiography. Protein contents were normalized by immunoblotting
using antibodies against RNA pol II and Spt5 (as described in Figs.
2-5). Therefore, the different intensity of radiolabeled proteins in
Fig. 6A represents the degree of phosphorylation of these
proteins.
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Fig. 6.
HIV-1 Tat stimulates phosphorylation of RNA
pol II and SPT5 by P-TEFb. A, phosphorylation of RNA
pol II and SPT5 during elongation. Elongation complexes stalled at
A-22 were incubated with nuclear extract and Tat protein. After
removing the unbound proteins, TECs were chased to G-646 by the
addition of nonradioactive NTPs and [ -32P]ATP in the
absence or presence of 20 µM DRB. TECs were isolated by
BglII cleavage, immunoprecipitated with Spt5, and analyzed
on 7.5% SDS-PAGE. Phosphorylation of RNA pol II and SPT5 was detected
by autoradiography. TECs were treated with (lanes 2 and
4) or without (lanes 1 and 3) DRB in
the absence (lanes 1 and 2) or presence of
(lanes 3 and 4) Tat protein. B,
protein contents of TECs in lanes 3 and 4 were
also visualized by immunoblotting using antibodies against RNA pol II
CTD and SPT5.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (33K):
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Fig. 7.
A model for the negative and positive
regulation of transcriptional elongation at HIV-1 promoter. See
text for details.
It is interesting that after joining the elongation complexes at early
stages, negative elongation factors travel with the transcription
machinery. There are three possible explanations for postinitiation
interaction of DSIF/NELF with the RNA pol II complexes: (a)
departure of initiation factors from the complex allows more accessible
surfaces for protein-protein interaction, (b) a
conformational change in the structure of RNA pol II occurs that is
recognized by DSIF/NELF, (c) DSIF/NELF are recruited through interaction with other proteins such as Tat-SF1 (41, 58) and this
interaction is only possible during elongation. These results provide a
number of intriguing possibilities related to the function of negative
regulators of elongation. 1) DSIF associates with the early elongation
complex to achieve a kinetic delay in elongation so that RNA processing
machinery can load onto the RNA pol II complex. 2) Negative elongation
factors in conjunction with P-TEFb could be involved in altering the
phosphorylation status of the RNA pol II CTD which is important in
recruitment of RNA capping and polyadenylation factors (59, 60). It has
been reported that phosphorylation of specific residues in the CTD has
a differential effect on recruitment and activation of the capping
enzyme (61). 3) These factors could directly play a role in RNA
processing. For example, Spt5 stimulates mRNA capping (62). 4) DSIF
can be converted into a transcription repressor during elongation by
dephosphorylation of Spt5. Future studies on P-TEFb and DSIF/NELF would
provide exciting insights into the mechanisms of gene expression.
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ACKNOWLEDGEMENTS |
---|
We thank Drs. David H. Price, Michael Dahmus, and Hiroshi Handa for the generous gift of antibodies, and Dr. Jonathan Karn for HIV-1 LTR containing plasmids. We also thank AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, National Institutes of Health, for providing various HIV-1 glutathione S-transferase-Tat expression vectors which were made available by Dr. Andrew Rice.
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FOOTNOTES |
---|
* This work was supported in part by National Institutes of Health Grant AI 43198.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Recipient of a Research Career Development Award from the National
Institutes of Health. To whom correspondence should be addressed. Tel.:
732-235-4082; Fax: 732-235-3235; E-mail: rana@umdnj.edu.
Published, JBC Papers in Press, December 8, 2000, DOI 10.1074/jbc.M006130200
2 C. Zhou and T. M. Rana, unpublished results.
3 D. Price, personal communications.
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ABBREVIATIONS |
---|
The abbreviations used are:
pol II, polymerase II;
P-TEFb, positive transcription elongation factor b;
DRB, 5,6-dichloro-1--D-ribofuranosylbenzimidazole;
DSIF, 5,6-dichloro-1-
-D-ribofuranosylbenzimidazole sensitivity
inducing factors;
NELF, negative elongation factor;
HIV, human
immunodeficiency virus;
LTR, long terminal repeat;
CTD, C-terminal
domain;
PIC, preinitiation complexes;
TEC, transcription elongation
complexes;
PAGE, polyacrylamide gel electrophoresis;
NTP, nucleotide triphosphate(s).
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