 |
INTRODUCTION |
RNA polymerase (RNAP)1
II is a large multisubunit enzyme responsible for catalyzing the
transcription of protein coding genes in eukaryotes. The largest
subunit of RNAP II contains at its C terminus a unique and highly
conserved domain composed of tandem repeats of the consensus sequence
YSPTSPS (for a review, see Ref. 1). Genetic analysis of the C-terminal
domain (CTD) has established that it is essential for viability (for a
review, see Ref. 2), although it is dispensable for transcription from
some promoters in vitro. Two forms of the enzyme exist
in vivo that differ with respect to the phosphorylation of
the CTD. RNAP IIA is unphosphorylated, whereas RNAP IIO is highly
phosphorylated (3). RNAP IIA assembles into preinitiation complexes
with the general transcription factors (4-7). Although multiple
protein kinases can phosphorylate the CTD (for a review, see Ref. 1),
the initial phosphorylation is catalyzed by a protein kinase intrinsic
to the preinitiation complex (8, 9). The idea that the phosphorylation
of the CTD results in a disruption of the protein-protein interactions that initially brought RNAP IIA to the preinitiation complex remains an
attractive but unproven hypothesis. Transcript elongation is catalyzed
by RNAP IIO. Completion of the transcription cycle is dependent on the
dephosphorylation of RNAP IIO, a reaction that may be coupled to
transcript termination. CTD phosphatase (CTDP) has been characterized
in yeast and mammalian cells (10-16). Although active in the
dephosphorylation of free RNAP IIO, its activity with respect to
dephosphorylating RNAP IIO in an elongation complex has not been
studied in detail. A recent report by Cho et al. (15)
demonstrates that, when transcription is initiated in a defined system,
the ternary elongation complex can be dephosphorylated by CTD phosphatase.
Increasing evidence suggests that an interplay of positive and negative
factors regulate transcript elongation. Although the role of the CTD in
elongation remains unclear, the CTD appears to be the regulatory focus
of many protein factors. Distinct structural changes occur in early
elongating RNAP II between +25 and +40 (17, 18). Furthermore, in this
same region, the elongation factor P-TEFb can phosphorylate the CTD,
resulting in a stabilization of the elongation complex (19). This
reaction is inhibited by the nucleotide analogue DRB. The failure of
P-TEFb to act can lead to abortive transcription. In addition to
P-TEFb, several negative factors coordinately regulate the transition
from abortive to productive elongation. DSIF was initially
characterized as a protein factor required to reconstitute DRB
sensitivity in vitro (20). In the absence of DRB, DSIF
represses transcription and antagonizes the positive action of P-TEFb
(21). The negative effect of DSIF depends on the state of CTD
phosphorylation. Prior phosphorylation of the CTD by TFIIH or P-TEFb
should result in transcription complexes that are resistant to the
effects of DSIF (20, 22). Interestingly, both NELF and DSIF are
required to repress transcript elongation, although neither functions
to repress transcription by RNAP IIO. This indicates that if RNAP
becomes dephosphorylated during the course of transcription, DSIF and NELF can repress transcription (21).
Recent results suggest that CTDP can play a direct role in the
regulation of transcript elongation. The human immunodeficiency virus
type 1 transcriptional activator, Tat, interacts with and inhibits the
activity of CTD phosphatase (23). Conversely, P-TEFb is recruited by
Tat (24-27). Accordingly, the presence of Tat leads to a high level of
CTD phosphorylation, resulting in a highly processive RNAP II and the
efficient expression of the viral genome.
These results indicate that the elongation efficiency of RNAP II is
regulated at least in part by protein kinases and phosphatase(s) that
establish the level of CTD phosphorylation. The objective of these
studies is to gain insights into the factors that regulate the
sensitivity of RNAP IIO in elongation complexes to dephosphorylation. The results presented suggest that RNAP IIO in early elongation complexes is more sensitive to dephosphorylation than is RNAP IIO in
elongation complexes that have cleared the promoter. Resistance to
dephosphorylation appears to involve both a conformational change in
RNAP II and the association of a specific factor(s).
| |
EXPERIMENTAL PROCEDURES |
Buffers
Buffer A contained 50 mM Tris-HCl, pH 7.9, and 0.1 mM EDTA. Buffer B contained 50 mM Tris-HCl, pH
7.9, 0.1 mM EDTA, 5 mM MgCl2, 0.5 mM dithiothreitol, 20 mM KCl, 0.025% Tween 80, and 20% glycerol. Buffer C contained 50 mM Tris-HCl, pH
7.9, 0.1 mM EDTA, 5 mM MgCl2 0.5 mM dithiothreitol, 20% glycerol, and KCl as indicated.
Buffer D contained 25 mM Tris, pH 7.9, 5 mM
MgCl2, 0.5 mM dithiothreitol, 0.025% Tween 80, 20% glycerol, and KCl as indicated.
Purification and Labeling of RNAPs IIA and IIO
RNAP IIA was purified from calf thymus by the method of Hodo and
Blatti (28) with the modifications described by Kang and Dahmus (29).
RNAP IIA was labeled with 32P as described in Chambers
et al. (13). Casein kinase II was obtained from Upstate
Biotechnology, Inc. (Lake Placid, NY). 32P-Labeled RNAP IIO
for dC-tailed transcription and control CTDP reactions was prepared
in vitro by the phosphorylation of 32P-labeled
RNAP IIA as described by Marshall and Dahmus (23).
Preparation of DNA Templates
To produce the DNA templates, 0.5 µg of pUC HTXB (30) was
subjected to 30 rounds of polymerase chain reaction with 2 µM concentrations of the following primers: forward
5'-TTCCCAGTCACGACGTTGTA-3' and reverse 5'-CACAGGAAACACGTATGACC-3'. The
reaction buffer was 20 mM Tris, pH 7.9, 50 mM
KCl, 1.5 mM MgCl2, 200 µM dATP,
dCTP, dGTP, and dTTP in a 2-ml reaction divided into 100-µl aliquots. This results in a template DNA 945 base pairs in length that will give
a promoter-dependent run-off transcript of 622 nucleotides. The polymerase chain reaction was loaded onto a 1-ml MonoQ column (Amersham Pharmacia Biotech) and eluted with a 20-ml linear gradient of
0.1-1 M NaCl in buffer A. The HTXB DNA fragment elutes at
~0.75 M NaCl. Promoter-independent transcription on
dC-tailed templates was performed using templates produced with a
biotinylated reverse primer. Promoter-dependent
transcription was carried out on templates biotinylated on the forward primer.
Promoter-independent DNA templates (dC-tailed) were generated from 5 µg of HTXB. The template was cut with SacI (Life
Technologies, Inc.) following the manufacturer's instructions, after
which the DNA was phenol/chloroform- and chloroform-extracted, and
ethanol-precipitated. After resuspension in buffer A, a tailing
reaction was assembled with 38 units of terminal deoxynucleotide
transferase (Promega) and 1 mM dCTP (Amersham Pharmacia
Biotech) following the manufacturer's instructions in a reaction
volume of 100 µl. After 15 min of incubation (sufficient to add
60-90 dCMPs to the 3'-OH generated by SacI), the tailed DNA
reaction was adjusted to 750 mM NaCl and bound to Dynabeads
(Dynal) as described by the manufacturer. 4.2 µg of DNA was added per
100 µl of Dynabeads previously equilibrated in buffer A. The reaction
was incubated at 22 °C for 60 min in buffer A containing 750 mM NaCl while turning on a rotator. Dynabeads were washed
free of unbound DNA with buffer A and resuspended at a DNA
concentration of 20 ng/µl in buffer A. About 95% of input DNA was
bound to the beads.
Preparation of Transcription Extracts
Transcription extracts were purified as described by Laybourn
and Dahmus (31) with the following changes. HiTrap heparin (Amersham
Pharmacia Biotech) was substituted for heparin-Sepharose, and the
primary transcription extract (DE0.25) and RNA polymerase II (DE0.6)
containing fractions were eluted from the DEAE-5PW (Waters) with 0.25 and 0.6 M KCl, respectively. TFIIA does not bind to the
heparin column (HS0.24 fraction) and was added back separately from the
other general transcription factors found in the DE0.25 fraction.
In Vitro Transcription
dC-tailed Templates--
Standard dC-tailed transcription
reactions were initiated by the addition of 0.25 pmol of
32P-labeled RNA polymerase IIO in ~2 µl of buffer D
containing ~500 mM KCl to 100 ng of dC-tailed HTXB DNA
immobilized on Dynabeads (as described above) in the presence of 0.5 µg/µl BSA in buffer C containing no MgCl2. Final
reaction conditions prior to the addition of nucleotides were
equivalent to buffer C containing 56 mM KCl, 0.5 µg/µl
BSA, and no MgCl2 in a 20-µl reaction volume. After
incubation at 30 °C for 30 min, ATP, UTP, and GTP were added to 0.6 mM, and CTP and MgCl2 were added to 10 µM and 6 mM, respectively. The final reaction
volume was 25 µl. Five µCi of [
-32P]CTP (Amersham
Pharmacia Biotech) was included in reactions carried out to determine
the size distribution of RNA transcripts and to quantify their amount.
RNAP IIO was allowed to elongate for 10 min and then washed free of
nucleotides with buffer C containing 50 mM KCl. If
digestion of nascent RNA was required, 1 unit of RNase H (Life
Technologies, Inc.) was added, and reactions were incubated for 10 min
at 37 °C. RNA products or RNAP II was visualized as described below.
Ad2-MLP Pulse Elongation Complexes--
Preinitiation complexes
were formed in a standard reaction by the addition of 1 µl of HiTrap
heparin flow-through in buffer C with 100 mM KCl (contains
TFIIA), 5 µl of DE0.25 in buffer C with 50 mM KCl
(contains remaining general transcription factors), 0.25 pmol of
32P-RNAP IIA in ~1 µl of buffer D containing ~500
mM KCl, and 100 ng of template DNA conjugated with
Dynabeads. The reaction was adjusted to 56 mM KCl in a
final volume of 20 µl in buffer C prior to incubation for 30 min at
30 °C. After incubation, 0.6 mM ATP and UTP, 250 nM CTP, 6 mM MgCl2, and 50 mM KCl were added for a total reaction volume of 25 µl.
Five µCi of [-32P]CTP was added when radiolabeled
RNA was to be analyzed. Complete reactions were incubated for 2 min at
30 °C. Transcription was stopped as specified in the figure legends
either by the addition of 15 mM EDTA or by twice washing
the transcription complexes to remove free nucleotides.
Ad2-MLP Chase Elongation Complexes--
Beginning with washed
pulse complexes in a 20-µl volume, chase elongation complexes were
formed by the addition of ATP, UTP, and GTP to 0.6 mM and
CTP to 10 µM in buffer C with 200 mM KCl and
0.5 mg/ml BSA in a final volume of 25 µl. Complexes were allowed to
elongate for 1 min at 30 °C before being washed in buffer C with 50 mM KCl. Radiolabeled RNAP II in transcription complexes was
visualized by SDS-PAGE carried out according to the method of Laemmli
(32) with a 5% polyacrylamide resolving gel. Radiolabeled RNA
transcripts were analyzed by urea-PAGE as described by Chesnut et
al. (5) utilizing 12.5% polyacrylamide. Specific RNA transcripts were quantitated on a Fuji phosphor imager by comparing radioactive signal intensity of transcript bands to known amounts of
[-32P]CTP standard.
CTDP Assays
The sensitivity of RNAP IIO in elongation complexes was
determined by a modification of the assay described by Chambers
et al. (13). Unless otherwise specified, RAP74 was included
in all CTDP assays. Input radiolabeled RNAP IIO in transcription complexes on immobilized DNA was washed twice in buffer B. CTDP was
purified as described previously (23). Assays were quantitated on a
Fuji phosphor imager in the following fashion. The region of the gel
ranging from just above the subunit IIo band to just below the subunit
IIa band was subdivided into approximate thirds. These thirds were
designated IIo, int, and IIa for the regions corresponding to the
subunit IIo and the intermediate and subunit IIa bands and were
quantitated each as a percentage of the whole after subtraction of a
lane-specific background (sample taken from beneath the subunit IIa
band). Ten fmol of RNAP (~1 µl) was removed from the reaction mix
and suspended in Laemmli buffer just prior to the first wash to serve
as a quantitation reference standard. The reference standard is removed
prior to the first wash to account for the ~15% nonspecific loss of
CKII label during the preincubation of the 32P-labeled RNAP
with transcription extract.
| |
RESULTS |
The CTD Phosphatase Sensitivity of RNAP IIO in Elongation Complexes
on a dC-tailed Template Is Comparable with That of Free RNAP
IIO--
To establish a base line for the sensitivity of RNAP IIO,
purified 32P-labeled RNAP IIO was incubated with increasing
concentrations of CTD phosphatase (Fig.
1A, lanes
1-5). The amount of subunits IIo and IIa were quantitated
from phosphor imager scans and plotted against mU of CTD phosphatase
(Fig. 1B, left panel). The 50%
conversion of RNAP IIO to IIA occurs at 9.5 mU of CTD phosphatase.
Transcription was then initiated on a dC-tailed template immobilized on
paramagnetic beads, and the CTD phosphatase sensitivity of RNAP IIO in
elongation complexes was established. Analysis of RNA transcripts
indicates that at 10 min RNAP IIO is distributed from about 150 to 650 nucleotides downstream from the site of initiation (Fig.
1C). The CTD phosphatase sensitivity of RNAP IIO in these
elongation complexes does not differ appreciably from that of free RNAP
IIO (Fig. 1A, compare lanes 1-5 with
lanes 11-15, and in Fig. 1B, compare
left and right panels). Accordingly,
the formation of an elongation complex per se does not
result in protection of the CTD from dephosphorylation. Furthermore,
the addition of free DNA and paramagnetic beads does not appreciably
alter the sensitivity of RNAP IIO (Fig. 1, A and B, lanes 6-10 and center
panel, respectively). Lanes 1-10
contained 0.06 pmol of RNAP IIO, whereas lanes
11-15 contained about 0.13 pmol of RNAP IIO in elongation
complex. The amount of CTDP required for 50% dephosphorylation of free
RNAP IIO, free RNAP IIO in the presence of template DNA and Dynabeads,
and RNAP IIO in transcription complexes is 9.5, 17, and 23 milliunits,
respectively (Table I).
View larger version (35K):
[in this window]
[in a new window]
|
Fig. 1.
CTDP sensitivity of free RNAP IIO and
elongating complexes on dC-tailed templates. A,
increasing amounts of CTDP were incubated with RNAP IIO, and the
resultant products were analyzed as described under "Experimental
Procedures." Lane 0 is a 10-fmol RNAP II reference
standard of RNAP IIO. All CTDP reactions contained 16 pmol of RAP74 and
either 0, 0.4, 4, 40, or 400 mU of CTDP as indicated. To establish the
sensitivity of free RNAP IIO, 60 fmol of 32P-labeled RNAP
IIO was incubated with increasing amounts of CTDP as indicated and
resolved by 5% SDS-PAGE (lanes 1-5). Lanes
6-10 are identical to lanes 1-5 except that reactions
also contained 25 ng of dC-tailed template DNA bound to Dynabeads.
Lanes 11-15 contain paused elongation complexes on a
dC-tailed template obtained as described under "Experimental
Procedures" and incubated with CTDP. Reactions in lanes 1-10
and 16-17 were terminated by the addition of Laemmli
buffer. Reactions on dC-tailed templates (lanes 11-15) were
washed once to remove RNAP II that dissociated from the template during
the CTDP reaction and were then terminated by the addition of Laemmli
buffer. B, quantitation of the amount of subunits IIo and
IIa, as well as the intermediate region (int) as described
under "Experimental Procedures." The results are graphed as
percentage of each of IIo, int, and IIa against mU of CTDP.
Numbers on the x axis above
the graphs refer to the reaction in A from which
the data were obtained (lane number).
C, dC-tailed template transcription reactions were assembled
as described under "Experimental Procedures." After the addition of
the nucleotide mix containing 5 µCi of [-32P]CTP,
aliquots were removed and stopped at 0, 2, 5, 10, and 15 min,
extracted, precipitated, and resolved on 12.5% urea-PAGE.
|
|
View this table:
[in this window]
[in a new window]
|
Table I
The CTDP sensitivity of RNAP IIO
The left-hand column denotes the source of the RNAP IIO. All CTDP
reactions were carried out in the presence of RAP74 except where
indicated ( RAP74). The middle column presents the amount of RNAP IIO
either free or in elongation complex calculated from the reference
standards. Data in the middle column are calculated from the 0-mU CTDP
reaction. The column on the right displays the calculated mU of CTDP
required to dephosphorylate 50% of the input RNAP IIO. The relative
amount of subunit IIo at 0 mU CTDP was taken as 100%.
|
|
Promoter-independent transcription on dC-tailed templates produces
transcripts in a RNA-DNA hybrid, which prevents the DNA strands from
reannealing behind RNA polymerase (33). To account for differences in
the sensitivity of dC-tailed complexes that might arise from these
RNA-DNA hybrids, RNase H was employed to digest the hybridized RNA,
thereby allowing the reannealing of the DNA template. Digestion of the
nascent RNA does not alter the CTDP sensitivity of RNAP IIO (data not shown).
CTD Phosphatase Sensitivity of Elongation Complexes That Initiate
Transcription on the Ad2-MLP in the Presence of a Nuclear
Extract--
Promoter-dependent transcription was carried
out utilizing a DNA fragment containing the Ad2-MLP immobilized on
Dynabeads. Preinitiation complexes were formed with
32P-labeled RNAP IIA in the presence of a partially
fractionated nuclear extract free of endogenous RNAP II. ATP, CTP, and
UTP were added, and the reaction was incubated for 2 min to allow the
production of short transcripts. Although the first G is at position
11, the predominant product is 11 nucleotides in length and corresponds
to a pause due to limited CTP (see Fig. 3B, lane 1, for an equivalent reaction). Following incubation, 10 fmol of RNAP II was removed from the reaction (Fig.
2A, lane
0). The bead-bound complexes were then washed in phosphatase
buffer to remove both unbound extract and unbound RNAP II. The RNAP II
bound to immobilized DNA is shown in lane 1. Less
than 1% of the input RNAP II is recovered in complex. Furthermore,
although the major fraction of RNAP II in the reaction is IIA
(lane 0), the major fraction of RNAP II
associated with the DNA has been converted to RNAP IIO (lane
1). Quantitation of the amount of transcript produced
relative to the amount of RNAP II bound indicates that 12-50% of RNAP
II bound under these conditions produces a transcript.
View larger version (38K):
[in this window]
[in a new window]
|
Fig. 2.
CTD phosphatase sensitivity of early
elongation complexes initiated from the Ad2-MLP. To provide a
reference point for the sensitivity of RNAP IIO in elongation
complexes, transcription was initiated in the presence of a nuclear
extract on the Ad2-MLP. Early elongation complexes, prepared in the
absence of GTP, were purified and assayed for sensitivity to CTDP.
A, lane 0 is a 10-fmol RNAP II reference
standard. Lane 1 is a reaction stopped prior to incubation
with CTDP. Lanes 2 and 3 are the bound and wash
fractions, respectively, resulting from a 30-min control incubation in
buffer B. Lanes 4-9 are identical to lanes 2 and
3 except for the presence of increasing amounts of CTDP as
indicated and 35 pmol of RAP74. Lanes 10 and 11 are identical to lanes 8 and 9 except for the
inclusion of exogenous radiolabeled RNAP IIO as a control for CTDP
activity. Lanes 12 and 13 are purified reference
RNAP IIO incubated in the absence and presence of CTDP as indicated.
B, quantitation of the amount of subunits IIo and IIa, as
well as the intermediate region (int) as described under
"Experimental Procedures." Numbers above each
graph correspond to the lane in A from
which each set of numbers was obtained.
|
|
The washed RNAP II complexes were incubated with increasing amounts of
CTDP, in the presence of 35 pmol of RAP74. At the end of the CTDP
reaction, the complexes were washed, resulting in two populations of
RNAP. Both populations were resolved by SDS-PAGE, and the sensitivity
of the bound population was determined by quantitation of subunits IIo
and IIa (Fig. 2B). The bound fraction (Fig. 2A,
even lanes 2-10) remains attached to
the template DNA while the wash fraction (odd
lanes 3-11) is released. Only RNAP IIA is found
in the wash fraction even in the absence of CTDP (Fig. 2A,
lane 3). Although some dephosphorylation is seen,
complete dephosphorylation is not obtained with the highest
concentrations of CTDP tested under these conditions (Fig.
2A, lane 8). As a control, the
reaction containing 400 mU of CTDP was run in duplicate in the absence
(lanes 8 and 9) and presence
(lanes 10 and 11) of exogenous
32P-RNAP IIO. The finding that free RNAP IIO is not
protected from dephosphorylation, as indicated by the absence of a
subunit IIo band in the wash fraction (lane 11),
indicates that protection is not conferred by a transacting factor.
These results suggest that RNAP II in complexes formed in the presence
of a nuclear extract is about 50-fold more resistant to
dephosphorylation than complexes formed on tailed templates (Table I).
However, the interpretation of these results is complicated by the fact
that the substrate for dephosphorylation is a mixed population of RNAP IIO, only some of which is in functional elongation complexes.
Sarkosyl Treatment Removes Nontranscribing RNAP II--
A
characterization of the CTD phosphatase sensitivity of RNAP IIO in
elongation complexes is dependent on the analysis of a homogenous
population of functional complexes. This is assured only if the molar
amount of transcript produced equals the molar amount of RNAP II in
complex. In an effort to remove nonproductively bound RNAP II,
complexes were treated with Sarkosyl. Transcription was initiated on
immobilized DNA in the presence of an RNAP II-depleted nuclear extract
supplemented with 32P-labeled RNAP IIA. Furthermore, in
reactions to determine the size distribution of RNA and to quantify the
amount of transcript produced, nascent transcripts were labeled by a
2-min incubation in the presence of [-32P]CTP, ATP,
and UTP. Pulsed complexes were washed with buffer C or 1% Sarkosyl in
buffer C. The reaction scheme is shown in Fig.
3A. One pair of samples was
assayed immediately following the pulse (Fig. 3, A-C,
lanes 1 and 2 and lanes
5 and 6), whereas a second pair of samples was
assayed after a 1-min chase in the presence of ATP, CTP, GTP, and UTP
(Fig. 3, A-C, lanes 3 and
4 and lanes 7 and 8). To
serve as a reference standard in the quantitation of RNAP II, an
aliquot containing 10 fmol of RNAP was removed prior to the first wash
step and run on SDS-PAGE (Fig. 3, A and C,
lane 0).
View larger version (25K):
[in this window]
[in a new window]
|
Fig. 3.
Effect of Sarkosyl on transcript elongation
and the molar ratio of RNAP IIO and transcript in purified elongation
complexes. The stoichiometry of RNA transcript to RNAP II was
quantitatively and qualitatively analyzed in a
promoter-dependent assay. Reactions were carried out with
32P-labeled RNAP IIA utilizing a HeLa cell transcription
extract depleted of RNAP II. Transcription was initiated on the Ad2-ML
promoter in the presence of either [-32P]CTP
(B) or unlabeled CTP (C) in the absence of GTP
(pulse conditions) and stopped by the addition of EDTA to 15 mM. Certain reactions were incubated further for 1 min in
the presence of NTPs and 200 mM KCl (chase conditions).
A, a schematic representation of the experiment.
Filled circles, control wash steps;
unfilled circles with an
S, Sarkosyl wash steps. Reactions were assembled and
preincubated for 30 min followed by a 2-min pulse and in some cases a
1-min chase as described under "Experimental Procedures." Reactions
indicated with lane numbers 1-8 refer
to the corresponding lanes in the transcription and protein
gels in B and C, respectively. Lane
0 refers to C. B, 12.5% urea-PAGE of RNA
transcripts produced under different reaction conditions (4× standard
reaction). Transcripts were labeled with [-32P]CTP as
described above. The left two lanes
correspond to reactions carried out in the absence of RNAP IIA. The
right lane, designated M, is a DNA
size marker generated by digestion of pBR322 with MspI.
C, the distribution of RNAPs IIO and IIA corresponding to
the transcription reactions in B. Lanes 1-8 in B
and C correspond to equivalent points in the transcription
reaction. Lane 0 is a 10-fmol RNAP II reference standard.
Lanes 1 and 2 are the bound and first wash
fractions from a control pulse reaction. Only 5% of the total wash
fraction was loaded in lanes 2 and 6. Lanes
3 and 4 are the corresponding chase fractions.
Lanes 5-8 are the corresponding 1% Sarkosyl pulse and
chase fractions. D, a graphical representation of the
calculated fmol of RNAP II and transcript derived from the bound lanes
in B and C. Each pair of bars is
derived from equivalent portions of the urea-PAGE and
SDS-polyacrylamide gels and quantitated as described under
"Experimental Procedures." The numbers are the average
of three independent experiments, and the error
bars represent one S.D. nt, nucleotides.
|
|
The size distribution of transcripts produced under pulse and chase
conditions is shown in Fig. 3B. Transcripts up to 24 nucleotides in length are observed in pulse conditions. Treatment of
complexes with 1% Sarkosyl does not appreciably change either the
amount or distribution of bound nascent transcripts (Fig.
3B, compare lanes 1 and 5).
Although slight variations in the relative amount of specific
transcripts between control and Sarkosyl-treated complexes are
sometimes observed, no consistent pattern has emerged. Following a
1-min chase, transcripts range in size from about 50 to 150 nucleotides
in length (Fig. 3B, lanes 3 and
7). Furthermore, neither the amount nor distribution of
transcripts differs appreciably between control or Sarkosyl-treated
complexes. The absence of transcripts in the wash fractions
(lanes 2, 4, 6, and
8) indicate that elongation complexes are stable under the
conditions of the experiment. Furthermore, essentially all complexes
paused after the initial labeling period are efficiently chased into
longer transcripts (Fig. 3B, compare lanes
1 and 3, and compare lanes 5 and 7).
The amount and state of phosphorylation of the largest RNAP II subunit
associated with the various elongation complexes is shown in Fig.
3C. Comparison of the subunit IIo bands retained after the
first wash reveals that substantially less RNAP remains attached to the
template DNA after treatment with Sarkosyl (Fig. 3C, compare
lanes 1 and 5). Although treatment of
pulse complexes with Sarkosyl removes a major fraction of the bound
RNAP II, it does not significantly alter the amount of bound transcript
(Fig. 3B, compare lanes 1 and
5).
Following the pulse step, paused elongation complexes were chased by
the addition of NTPs and 200 mM KCl. Chase conditions result in a substantial loss of RNAP IIO from control complexes (Fig.
3C, compare lane 1 with
lanes 3 and 4) and no significant change in Sarkosyl-treated complexes (Fig. 3C, compare
lane 5 with lanes 7 and
8). Quantitation of bound subunit IIo and transcript produced reveals that, in contrast to the control pulse reactions, approximately equimolar amounts of transcript and RNAP II are present
in chase complexes (Fig. 3D). The observation that the chase
step in control reactions results in the dissociation of nontranscribing RNAP II can be attributed to nucleotide and KCl destabilization of RNAP in the nonfunctional preinitiation complexes (34, 35).
Quantitation of the molar amount of RNAP II and RNA transcript was
carried out as described under "Experimental Procedures." Values in
Fig. 3D are averages of three independent data sets. Although under pulse conditions with control complexes there is a molar
excess of RNAP II, chase control complexes and both pulse and chase
complexes treated with 1% Sarkosyl contain equimolar amounts of RNAP
II and nascent transcript. The fact that Sarkosyl-treated pulse
complexes contain equimolar amounts of transcript and RNAP II indicates
that Sarkosyl efficiently and selectively dissociates nontranscribing
RNAP II.
These results demonstrate that stoichiometric amounts of RNAP and
transcript exist in pulse and chase complexes that have been treated
with 1% Sarkosyl and that control chase complexes contain equimolar
amounts of RNAP II and transcript. These complexes are ideally suited
to assess the sensitivity of elongation-competent RNAP II to
dephosphorylation by CTDP.
CTD Phosphatase Sensitivity Is Influenced by both the Position of
the Elongation Complex and Sarkosyl Treatment--
The objectives of
experiments described in this section are 2-fold: first, to examine the
CTD phosphatase sensitivity of RNAP IIO as a function of its position
downstream of the transcriptional start site, i.e. promoter
proximal (pulse) and distal (chase) elongation complexes; second, to
examine the effect of Sarkosyl treatment on CTDP sensitivity.
Elongation complexes were prepared as described above and incubated
with increasing concentrations of CTD phosphatase. The experimental
protocol is shown schematically in Fig.
4A. Although transcription was
initiated by the addition of 32P-labeled RNAP IIA,
transcripts were not labeled. The sensitivity of pulse and chase
elongation complexes under both control and Sarkosyl wash conditions
was examined. The distribution of subunits IIo and IIa is shown in Fig.
4B, whereas the quantitation is shown in Fig. 4C.
The amount of CTD phosphatase required for 50% conversion of RNAP IIO
to IIA differs for each complex and is shown in Table I. Pulse control
complexes are more sensitive than chase complexes (Fig. 4C,
compare upper left and right
panels). Similarly, Sarkosyl pulse complexes are more
sensitive than chase complexes (Fig. 4C, compare
lower left and right
panels). Accordingly, under both sets of conditions, RNAP
IIO in elongation complexes at positions 50-150 nucleotides downstream
from the start site is more resistant to dephosphorylation than is RNAP
IIO at positions 11-24. Although it is difficult to quantify the
magnitude of the difference in control complexes given the extreme
resistance of RNAP IIO, the difference in sensitivity between the
Sarkosyl-treated complexes is about 8-fold.
View larger version (28K):
[in this window]
[in a new window]
|
Fig. 4.
CTD phosphatase sensitivity of
promoter-proximal and -distal elongation complexes in the presence and
absence of Sarkosyl. Elongation complexes were prepared as
described in the legend to Fig. 3, and their sensitivity to CTD
phosphatase was determined. A, a schematic representation of
the experiment. Filled circles, control wash
steps; unfilled circles with
an S, 1% Sarkosyl wash steps. Reactions were
assembled and preincubated for 30 min followed by a 2-min pulse and in
some cases a 1-min chase. B, an SDS-polyacrylamide gel
displaying only the final template-bound portion of each reaction. The
reaction was assembled as a 16× standard reaction. Lane 0 is a 10 fmol RNAP II reference standard. Reactions in lanes
1-8 are control complexes, whereas reactions in lanes
9-16 are treated with 1% Sarkosyl. Lanes 1-4 and
9-12 are pulse complexes incubated with 0, 4, 40, and 400 mU of CTDP, respectively. Lanes 5-8 and lanes
13-16 are chase complexes. Dynabead-based assays (lanes
1-16) were washed once prior to the addition of Laemmli buffer.
C shows a quantitation of the amount of subunit IIo, IIa,
and int in each complex as a function of increasing amounts of CTDP.
Numbers above each graph correspond to
the lane in B from which each set of numbers was
quantitated. Pulse reactions are on the left, and chase
reactions are on the right. Control complexes are on the
top, and Sarkosyl-washed complexes are on the
bottom. The numbers presented in Fig. 4C are
average values derived from two experiments.
|
|
Given that only a fraction of RNAP IIO in control pulse complexes is in
functional elongation complexes, it is also difficult to directly
compare the sensitivity of these complexes to control downstream
complexes or Sarkosyl-treated complexes at the same position. Despite
this qualification, it is clear that Sarkosyl treatment dramatically
increases the sensitivity of RNAP IIO contained in elongation complexes
(Fig. 4C, compare upper and lower
panels). The sensitivity of RNAP IIO in promoter-proximal
complexes treated with 1% Sarkosyl approaches that of RNAP IIO in
elongation complexes initiated on dC-tailed templates (Table I, also
compare right panel of Fig. 1C with
lower left panel of Fig.
4C).
Transcription Extract Contains a Factor That Protects RNAP IIO in
Elongation Complexes from Dephosphorylation--
The finding that
elongation complexes washed with 1% Sarkosyl exhibit an increased
sensitivity to CTDP suggests that Sarkosyl either dissociates a
factor(s) that contributes to resistance or induces a conformational
change that results in increased sensitivity. To test this hypothesis,
pulsed complexes were washed with 1% Sarkosyl and then incubated for
10 min with transcription extract or one of several different protein
fractions. After incubation with transcription extract, elongation
complexes were washed into buffer B and assayed in a standard CTDP
reaction. The reaction scheme is shown in Fig.
5A. The presence of increasing
amounts of transcription extract results in increased protection of the CTD to dephosphorylation by CTDP (Fig. 5, B and
C, compare lanes 3-6). The addition
of BSA at a 33-fold higher concentration did not affect phosphatase
sensitivity (Fig. 5, B and C, lanes
7 and 8). Similarly, the addition of the HS0.24
fraction containing TFIIA, which was present in the original reaction,
had no effect on CTDP sensitivity although it was present at a 4-fold
higher concentration than in transcription reactions (Fig. 5,
B and C, compare lanes 6 and 10). These results suggest that the sensitivity of RNAP
IIO in elongation complexes is determined in part by a specific
factors(s) present in the transcription extract.
View larger version (33K):
[in this window]
[in a new window]
|
Fig. 5.
Effect of nuclear extract on the CTDP
sensitivity of RNAP IIO. In an effort to identify factors that
influence CTDP sensitivity, purified Sarkosyl-treated elongation
complexes were incubated with increasing amounts of nuclear extract,
and the effect on CTDP sensitivity was determined. A, a
schematic representation of the experiment (lanes 1-6).
B, pulse Sarkosyl complexes were formed as described above,
washed twice to remove free nucleotides, and incubated for 10 min with
various protein fractions. Complexes were extensively washed and
assayed for CTDP sensitivity by incubation with 200 mU of CTDP and 16 pmol of RAP74. The reactions were loaded onto a 5% SDS-polyacrylamide
gel as described above. Lane 0 is a 10-fmol RNAP II
reference standard. Lanes 1 and 2 contain
complexes incubated without and with DE0.25 transcription extract,
respectively, and incubated with buffer B. Lanes 3-6 were
incubated with increasing amounts of DE0.25 transcription extract and
then washed and incubated with CTDP. Lanes 7 and
8 were incubated with BSA, and lanes 9-10 were
incubated with HS0.24 fraction. Lanes 11 and 12 are 32P-labeled RNAP IIO incubated without and with 40 mU
of CTDP and 16 pmol of RAP74, respectively. C, a
quantitation of the amount of subunit IIo, IIa, and int plotted as a
function of the amount of protein present in the transcription extract
or other fractions added. Numbers at the top of
the graph refer to the lane of origin in
B.
|
|
RAP74 Stimulates the Dephosphorylation of RNAP IIO in Elongation
Complexes--
The RAP74 subunit of the general transcription factor
TFIIF stimulates the dephosphorylation of free RNAP IIO by CTDP (13). Since TFIIF influences both the initiation and elongation phase of
transcription (36-39), it was of interest to determine if TFIIF also
stimulates the dephosphorylation of RNAP IIO in elongation complexes.
To assess the effect of RAP74 on dephosphorylation, the sensitivity of
pulse Sarkosyl-treated elongation complexes was determined in the
absence (Fig. 6A,
lanes 1-4) or presence (lanes
5-8) of RAP74. The major fraction of subunit IIo was
converted to subunit IIa in the presence of 40 mU of CTDP and RAP74,
whereas in the absence of RAP74, very little dephosphorylation was
observed even in the presence of 400 mU CTDP (Fig. 6A,
compare lanes 4 and 7). RAP74 also
stimulates the dephosphorylation of pulse control complexes (data not
shown).
View larger version (40K):
[in this window]
[in a new window]
|
Fig. 6.
Effect of RAP74 on CTDP activity on
Sarkosyl-treated elongation complexes. A, lane
0 is a 10-fmol RNAP II reference standard. Lanes 1-4
are pulse Sarkosyl-treated elongation complexes incubated with
increasing amounts of CTDP as indicated. Lanes 5-8 are
identical to lanes 1-4 except for the inclusion of RAP74 as
indicated. Lanes 9-11 contain exogenous RNAP IIO alone,
RNAP IIO with 40 mU of CTDP, and RNAP IIO with 40 mU of CTDP and 50 pmol of RAP74, respectively. B shows a quantitation of
subunits IIo, IIa, and int plotted as a function of mU of CTDP.
Numbers at the top of the graph refer
to the lane of origin in A.
|
|
| |
DISCUSSION |
The first indication that RNAP IIO was involved in elongation came
from experiments in which nascent transcripts were shown to cross-link
to subunit IIo but not subunit IIa (3, 40). Furthermore, the finding
that no transcripts were cross-linked to subunits with an
electrophoretic mobility between that of subunit IIo and IIa indicates
that the elongating enzyme is fully phosphorylated. The simplest
interpretation of these results is that the CTD is fully phosphorylated
by TFIIH at the time of initiation and that there is no turnover of CTD
phosphate during transcript elongation. However, the finding that the
synthesis of long transcripts in vitro is inhibited by the
nucleotide analogue DRB suggested that a protein kinase might play an
important role in processive transcript elongation (41, 42). These
studies resulted in the identification of P-TEFb, which was
subsequently shown to be a CTD kinase (19). These results are
consistent with a model in which TFIIH catalyzes CTD phosphorylation
during the initiation phase of transcription, whereas P-TEFb catalyzes
the phosphorylation of RNAP II in elongation complexes. It is not
presently possible to distinguish between models in which RNAP II
cleared the promoter before it was fully phosphorylated as opposed to
being dephosphorylated during early stages of transcript elongation.
For this reason, as well as those noted below, it is important to
understand the parameters that govern the sensitivity of RNAP IIO in
elongation complexes to dephosphorylation by CTD phosphatase.
Results presented here indicate that the sensitivity of RNAP IIO to
dephosphorylation can be dramatically influenced by its assembly into
an elongation complex. The observation that RNAP IIO that initiates
transcription on a dC-tailed template has a sensitivity comparable with
that of free RNAP IIO indicates the CTD is readily accessible to
dephosphorylation in elongation complexes that are free of other
factors. Early elongation complexes that have initiated transcription
on the Ad2-MLP in the presence of a nuclear extract and have been
treated with 1% Sarkosyl also have a sensitivity comparable with that
of free RNAP IIO. However, as RNAP IIO clears the promoter it becomes
more resistant to dephosphorylation. This is supported by the
observation that the resistance to dephosphorylation of
Sarkosyl-treated elongation complexes increases 8-fold as RNAP IIO
moves from positions +11-24 to positions +50-150. Since both promoter
proximal and distal complexes have been treated with 1% Sarkosyl, this
change in sensitivity is most likely the result of a conformational
change as opposed to the association of specific proteins.
RNAP IIO in elongation complexes on the Ad2-ML template that have been
purified in the absence of Sarkosyl is highly resistant to
dephosphorylation. It is difficult to compare directly the sensitivity
of control and Sarkosyl-treated early elongation complexes, because in
the absence of Sarkosyl, a significant fraction of RNAP IIO bound to
the template is not in functional elongation complexes. Accordingly,
the sensitivity determined is an average value of different complexes
and is not necessarily an accurate reflection of the sensitivity of
early elongation complexes. However, the chase conditions for the
production of promoter distal elongation complexes result in the
dissociation of nonproductively bound RNAP IIO. Accordingly, the
sensitivity of RNAP IIO at positions +50-150 downstream from the start
site can be accurately assessed. Control downstream complexes are at
least 5-fold more resistant to dephosphorylation than complexes at the
same position treated with 1% Sarkosyl. This result suggests that a
factor(s) present in the nuclear extract can associate with the
elongation complex and confers protection against dephosphorylation.
Presumably, the dissociation of this factor(s) by 1% Sarkosyl results
in the increase in sensitivity. Alternatively, treatment with Sarkosyl may induce a conformational change that alters the sensitivity of RNAP
IIO. The finding that incubation of Sarkosyl-treated elongation complexes with the nuclear extract can selectively reestablish resistance to CTDP suggests that a factor(s) is present that associates with the elongation complex to modulate the sensitivity of RNAP IIO.
The stabilization of RNAP IIO against dephosphorylation as it clears
the promoter is seen in both control and Sarkosyl-treated elongation
complexes. The mechanism for this decrease in sensitivity with
increasing transcript length is unknown. However, RNAP is known to go
through several transitions during the early stages of transcript
elongation. Samkurashvili and Luse (17, 18) have described a
conformational change that alters the footprint of RNAP II relative to
the nucleotide addition site. As the RNA lengthens, the footprint of
RNAP II becomes more trailing up to nucleotide +25. Beyond +25, the
footprint becomes more centered on the site of nucleotide addition. The
pulse (+11-24 nucleotides) and chase (+50-150 nucleotides) reactions
bracket this transition zone. Since the decrease in sensitivity of
chase complexes occurs in both control and Sarkosyl-washed complexes,
it is likely to be the result of a conformational change associated
with the early stages of transcript elongation.
A variety of biochemical studies establish that the CTD plays an
essential role in co-transcriptional processing of the primary transcript (for a review, see Refs. 43 and 44). Since the phosphorylated CTD appears to play a key role in RNA processing, dephosphorylation of the CTD during the elongation phase of
transcription would probably lead to dissociation of the processing
machinery. This uncoupling could in principle lead to the arrest of
transcription. These studies establish the importance in maintaining a
high level of CTD phosphorylation during elongation and add
significance to studies on the regulation of CTD phosphatase activity.
The observation that RAP74 stimulates the ability of CTDP to
dephosphorylate RNAP IIO in an elongation complex suggests that TFIIF
might function as a negative regulator of transcript elongation. This
is not unprecedented in that the ability of elongin to stimulate
transcript elongation is inhibited by the presence of TFIIF (45).
Recent studies also establish that the level of CTD phosphorylation can
be regulated during transcript elongation by a protein factor that
regulates both CTD kinase and CTD phosphatase. The observations that
the human immunodeficiency virus type 1 transcriptional activator, Tat,
is a positive regulator of P-TEFb and a negative regulator of CTDP
support the idea that a high level of CTD phosphorylation is essential
for processive elongation (23, 24, 46, 47). Furthermore, Tat is known
to increase the ratio of RNAP IIO/IIA and activate transcription of the
viral genome by increasing the processivity of RNAP II (48-50).
The assembly of preinitiation and elongation complexes in defined
systems results in complexes that are sensitive to dephosphorylation by
CTDP (15). Although it is difficult to directly compare these results
with those presented here, transcription carried out in the presence of
purified transcription factors probably results in complexes that are
more similar to Sarkosyl-treated complexes than those formed in a
nuclear extract.
These results suggest that the sensitivity of RNAP IIO in elongation
complexes is influenced by a number of factors. First, there appears to
be a conformational change that accompanies promoter clearance and
results in the establishment of an elongation complex that is
relatively resistant to dephosphorylation. Second, factors in the
nuclear extract probably associate with the elongation complex,
resulting in an increased stabilization against dephosphorylation. There are several potential mechanisms that could account for the
factor-induced protection. These include (a) interference with the association of CTDP with the elongation complex,
(b) direct protection by the association of protein with the
phosphorylated CTD, and (c) direct inhibition of CTDP
activity. CTDP interacts with a docking site on free RNAP IIO that is
distinct from the CTD (14). The observation that CTDP does not
dephosphorylate either recombinant CTDo or free RNAP II subunit IIo
suggests that the association of CTDP with the docking site on the
native enzyme is essential. Accordingly, the association of factors
with the elongation complex could regulate the accessibility of this
site. Second, both the elongator complex (51) and proteins involved in
processing of the primary transcript have been shown to directly interact with the phosphorylated CTD (43, 44). The association of these
proteins with the CTD could in principle block dephosphorylation although CTDP is able to associate with the elongation complex. Finally, cellular proteins, such as Tat, may associate with the elongation complex and directly inhibit the activity of CTDP. As noted
above, the human immunodeficiency virus type 1 transcriptional activator, Tat, is able to influence the state of phosphorylation of
RNAP II by directly inhibiting the activity of CTDP.
The observation that the sensitivity of RNAP IIO in an elongation
complex can be influenced by its position relative to the promoter and
the association of a factor(s) in the nuclear extract supports the
hypothesis that an interplay between positive and negative factors
including CTD kinases and CTDP works to regulate the transcription
efficiency of RNAP II. It is now important to identify and characterize
the factor(s) that influence CTD phosphatase sensitivity and to
establish their relationship to known elongation factors.
TOP
ABSTRACT
INTRODUCTION
To whom correspondence should be addressed. Tel.: 530-752-3551;
Fax: 530-752-3085; E-mail: medahmus@ucdavis.edu.
-D-ribofuranosylbenzimidaze;
DSIF, DRB
sensitivity-inducing factor;
NELF, negative elongation factor;
P-TEFb, positive transcription elongation factor b;
RAP74, the 74-kDa subunit
of TFIIF;
PAGE, polyacrylamide gel electrophoresis;
BSA, bovine serum
albumin;
mU, milliunits.
CiteULike 
Complore 
Connotea 
