(Received for publication, September 29, 1995; and in revised form, December 20, 1995)
From the
We have examined the properties of two Drosophila RNA
polymerase II mutants, C4 and S1, during elongation,
pyrophosphorolysis, and DmS-II-stimulated transcript cleavage. The C4 and S1 mutants contain a single amino acid
substitution in the largest and second largest subunits, respectively.
Compared with wild type, C4 had a lower elongation rate and
was less efficient at reading through intrinsic elongation blocks. S1 had a higher elongation rate than wild type and was more
efficient at reading through the same blocks. During elongation, C4 and wild type responded similarly to DmS-II and
NH, whereas the S1 mutant was
less responsive to both. Differences between the two mutants also
appeared during DmS-II-mediated transcript cleavage and
pyrophosphorolysis. During extended pyrophosphorolysis, S1 polymerase was fastest and C4 polymerase was slowest at
generating the final pattern of shortened transcripts. S1 and
wild type were equal in the rate of extended DmS-II-mediated transcript
cleavage, and C4 was slower. Our results suggest that the S1 mutation increases the time spent by the polymerase in
elongation competent mode and that the C4 mutation may affect
the movement of the polymerase.
Recent studies have revealed that the control of transcription elongation is an important target for the regulation of eukaryotic gene expression(1, 2) . As the central component of the mRNA transcription machinery, RNA polymerase II itself is the final receptor of various kinds of interactions that activate or inactivate transcription elongation, and it carries out the fundamental catalysis of RNA chain elongation. However, functional roles played by its individual subunits during the process of elongation have not been thoroughly illuminated.
A number of mutations in RpII215 and RpII140, the genes encoding the two large subunits of Drosophila RNA polymerase II have been mapped at the DNA
sequence level. Among them, the C4 mutation in RpII215 changes amino acid 741 from Arg to His in the largest
subunit(3) , and the S1 mutation in RpII140 changes amino acid 728 from Ser to Cys in the second largest
subunit(4) . In vivo, the C4 mutation induces
-amanitin resistance and the Ubx effect(5, 6) ,
whereas the S1 mutation suppresses the temperature-sensitive
mutant phenotype caused by another mutation (WJK1) in the
largest subunit(7) . Little is known about what functions of
RNA polymerase II are altered and how transcription is affected by
these and other mapped mutations, except that it was shown previously
that the C4 mutant enzyme is resistant to
-amanitin and
is slower in elongation in vitro(8, 9) .
Having previously mapped mutations in both RpII215 and RpII140, we next initiated biochemical studies of a subset of
the mutant enzymes that are amenable to purification(10) . In
our studies, we found that as for the C4 mutant enzyme, the S1 mutant enzyme is also different from wild type in
elongation. Because the C4 mutation alters the largest
subunit, whereas the S1 mutation alters the second largest
subunit of Drosophila RNA polymerase II, the availability of C4 and S1 RNA polymerase II mutant enzymes provides
an opportunity possibly to identify functional roles for the two large
subunits during elongation and to dissect the molecular mechanism by
which RNA polymerase II achieves efficient elongation.
Using
cell-free transcription systems in which elongation complexes can be
assembled, a number of factors have been identified that affect
elongation properties of RNA polymerase II. For example, the Drosophila transcription factor, DmS-II, which was initially
purified from Drosophila Kc cell nuclear extracts, has been
shown to have a stimulatory effect on elongation in vitro(11, 12) . DmS-II is a 36-kDa protein that is the Drosophila counterpart for mammalian S-II (TFIIS); the gene
encoding DmS-II has been cloned and sequenced(13) . DmS-II has
been shown to reduce the time spent by RNA polymerase II at a subset of
the numerous pause sites encountered on a dC-tailed ()template, but it does not stably bind to the elongation
complex(11) . DmS-II has also been shown to be necessary and
sufficient to activate nascent transcript cleavage by Drosophila RNA polymerase II during transcription of a dC-tailed template,
and the C-terminal half of DmS-II is required for its
cleavage-activating function. A mechanism for pause suppression by
DmS-II has been proposed. DmS-II binds to the paused polymerase, causes
one cleavage event, and is then released from the complex. Elongation
by the polymerase then allows a second encounter with the pause site
and a second chance of passing the site. Complete pause suppression may
require multiple transcript shortening events for some polymerase
molecules(14) .
In the study presented here, we investigate several elongation-related properties of wild type and mutant RNA polymerase II, namely recognition of intrinsic blocks to elongation, read-through in response to DmS-II, DmS-II-stimulated cleavage of nascent transcripts, and pyrophosphorolysis. We demonstrate that the C4 and S1 mutations affect different functional processes during elongation, and we discuss the implications of our observations.
All purification steps were carried out at 0-4 °C. Frozen
dechorionated embryos (50 g) were suspended in 100 ml of embryo buffer
and homogenized using a Waring blender for 45 s at low speed and 30 s
at high speed. Following the addition of 100 ml of HGE, homogenization
was continued for 45 s each at low and high speed. The extract was
centrifuged at 10,000 rpm (16,000 g) for 30 min in a
GSA rotor (Sorvall), and the resulting supernatant was collected after
being filtered through one layer of Miracloth. One hundredth volume of
a 10% (w/v) solution of Polymin P was stirred in dropwise. After 20 min
of gentle stirring, the precipitated material was collected by
centrifugation in the GSA rotor at 8,500 rpm (12,000
g) for 20 min. The polymin P precipitate was washed in 50 ml
of 0.1 M HGKE using a glass-glass Dounce homogenizer to obtain
uniform resuspension, followed by recentrifugation at 8,000 rpm (10,000
g) for 20 min in the GSA rotor. RNA polymerase II
activity was then extracted from the pellet into 50 ml of 0.3 M HGAE using a Dounce homogenizer, followed by centrifugation at
13,000 rpm (25,000
g) for 20 min in the SA-600 rotor
(Sorvall). The resulting polymin P eluate was filtered through one
layer of Miracloth. Solid (NH
)
SO
(0.3 g/ml of polymin P eluate) was added and dissolved in by
gentle stirring over 30-40 min. The precipitated protein was
collected by centrifugation at 40,000 rpm (140,000
g)
for 30 min in a T-865 ultracentrifuge rotor (Sorvall). The pellet was
resuspended in 10-15 ml of HGE with a Dounce homogenizer to yield
ammonium sulfate enzyme, which was frozen in liquid nitrogen and stored
at -80 °C. The thawed ammonium sulfate enzyme was loaded onto
a 30-ml DE-52 column equilibrated in 0.12 M HGAE and was
eluted with a 0.3 M HGAE step. The peak of RNA polymerase II
activity was pooled and was loaded onto a 5.0 ml Econo-Pac heparin
column equilibrated in 0.18 M HGAE, and was eluted with a 0.5 M HGAE step. The RNA polymerase II activity peak was pooled
and loaded onto a 1.0 ml Mono Q fast protein liquid chromatography
column equilibrated in 0.15 M HGKE. After extensive washing of
the column with 0.15 M HGKE, the enzyme was eluted with a 22.5
ml 0.15 M to 0.6 M HGKE gradient. RNA polymerase II
activity eluted around 0.45 M KCl and was collected, frozen in
liquid nitrogen and stored at -80 °C. RNA polymerase II
activity (in units) was determined for each enzyme preparation under
standard assay conditions(16) . Note that under these
conditions wild type and S1 enzymes have almost identical specific
activities and that the C4 enzyme's specific activity is
approximately 40% lower (e.g.(9) and
``Results''). Full-length DmS-II was expressed and purified
as described in Guo and Price(14) .
Reactions containing immobilized dC-3025 were carried
out in the following manner. In order to generate comparable amounts of
the 13-mers before the elongation complexes were isolated, different
unit amounts of P2, S1, and C4 enzymes were
used. In a typical 15-µl preincubation, 178 units of P2,
238 units of S1, and 400 units of C4 RNA polymerase
II were incubated with 70 µg of dC-3025 beads (200 ng of DNA) for
10 min. in the presence of 25 mM HEPES, pH 7.6, 5 mM MgCl, 60 mM KCl, and 2 mg/ml bovine serum
albumin. Transcripts were pulse labeled by adding 35 µl of a
labeling mix containing 25 mM HEPES, pH 7.6, 5 mM MgCl
, 0.6 mM GTP and ATP, 75 mM NH
Cl, and 100 µCi of
[
-
P]CTP for 30 s. Elongation complexes were
concentrated and washed 3 times with 200 µl of HMK buffer (25
mM HEPES, pH 7.6, 5 mM MgCl
, 60 mM KCl, and 200 µg/ml bovine serum albumin). Transcripts were
chased further by resuspending the beads in 50 µl of a nonlabeling
transcription mix containing 25 mM HEPES, 5 mM MgCl
, 60 mM KCl, and 0.6 mM NTPs for
8 min. Final elongation complexes were washed and resuspended in HMK
buffer. Isolated elongation complexes were then incubated in the
presence of indicated reagents. Reactions were stopped at indicated
times, and RNAs were recovered as described above.
Figure 1:
Comparison of the elongation rates of P2 wild type and S1 mutant RNA polymerase II. A, the elongation assay with 350 units RNA polymerase II, 10
µg/ml dC-pPCP and 100 mM KCl was carried out as described
under ``Experimental Procedures,'' and transcripts were pulse
labeled for 3 min. Purified RNAs were analyzed on a 5% polyacrylamide-6 M urea-TBE gel. Time points of the chase reaction are
indicated at the top of each lane. Lengths of the DNA size standards (M) are given in nucleotides. B, the elongation assay
with 180 units RNA polymerase II, 10 µg/ml dC-pPCP, 50 mM KCl plus 80 mM (NH)
SO
was carried out as described under ``Experimental
Procedures,'' and transcripts were pulse labeled for 1 min. C, plots of the maximum lengths of RNA transcribed in the
elongation assays shown in A and B.
We found that both wild type and mutant Drosophila RNA polymerases II could be blocked at TIa, TIb, and TII in vitro. As shown in Fig. 2A, after a 5-min chase, besides the run-off transcripts (RO), transcripts with 3` ends at TII, TIb, and TIa also accumulated for both wild type and mutant enzymes. However, in the absence of any elongation factors the mutant and wild type polymerases displayed differences in the relative amounts of enzyme reaching the end of the template (RO) versus the amount held up at the intrinsic elongation blockage sites (principally TIa and TIb). Thus, the ratios of RO/(RO + TIa) and (RO + TIa)/(RO + TIa + TIb) were different for wild type and mutant enzymes. As compared in Fig. 2B, after 5 min of chase, the ratios of RO/(RO + TIa) were 34, 11, and 54% for P2, C4, and S1, respectively; after 60 min of chase, the ratios increased to 58, 49, and 62%, respectively. After 5 min of chase, the ratios of (RO + TIa)/(RO + TIa + TIb) were 64, 53, and 71% for P2, C4, and S1, respectively; after 60 min of chase, the ratios increased to 82, 78, and 82%, respectively. In these experiments, the ratios of RO/(RO + TIa) and (RO + TIa)/(RO + TIa + TIb) reflect the half-life of pausing at TIa and TIb, respectively, but they are also influenced by the timing of the arrival of polymerases from other earlier pause sites and by the number of polymerases that become arrested (unable to proceed) at TIa and TIb. The different ratios clearly indicate a functional difference between the polymerases. Compared with P2 wild type, the C4 mutant enzyme reads less efficiently through TIa and TIb, whereas the S1 mutant enzyme reads more efficiently through the same blocks.
Figure 2: Comparison of the ability of P2 wild type, C4 mutant, and S1 mutant RNA polymerase II to read through intrinsic elongation blocks. A, the read-through assay with 180 units RNA polymerase II was carried out as described under ``Experimental Procedures.'' Purified RNAs were analyzed on a 5% polyacrylamide-6 M urea-TBE gel. The run-off transcripts and transcripts with 3` ends at TIa, TIb, and TII are indicated by RO, TIa, TIb, and TII. Time points of the chase reaction are indicated at the top of each lane. B, quantitation was performed as described under ``Experimental Procedures.'' The ratios of RO/(RO + TIa) and (RO + TIa)/(RO + TIa + TIb) were calculated and plotted versus the time of chase. Symbols: filled circles, P2; open circles, S1; squares, C4.
A time course of elongation is shown in Fig. 3A and the quantitation is shown in Fig. 3B. After 5 min of chase in the presence of 2.2 nM DmS-II (note that before DmS-II was added there was a 5-min initial chase in the absence of DmS-II), the ratio of RO/(RO + TIa) for P2 was 79%, increasing from 45% after 10 min of chase in the absence of DmS-II (Fig. 2A, 10-min point); this represents a 1.8-fold stimulation. Within the period of 60 min, the presence of DmS-II resulted in an average of 1.7-fold increase in read-through of TIa by P2 (n-min points in Fig. 3versus n + 5-min points in Fig. 2). Similarly, DmS-II resulted in an average of 1.7-fold increase in read-through of TIa by C4 , although compared with P2, the C4 mutant remained less efficient at reading through TIa. In contrast, the same amount of DmS-II only resulted in an average of 1.2-fold increase in read-through of TIa by S1 mutant enzyme (compare Fig. 3and Fig. 2).
Figure 3:
Comparison of the ability of P2 wild type, C4 mutant, and S1 mutant RNA
polymerase II to read through intrinsic elongation blocks in the
presence of DmS-II. A, the read-through assay with 180 units
RNA polymerase II was set up as described under ``Experimental
Procedures.'' After 5 min of chase, DmS-II was added to a final
concentration of 2.2 nM, and incubation was continued; in a
separate titration experiment, this amount of DmS-II was nearing
saturation and stimulated P2 and C4 enzymes 1.4-fold and S1 enzyme
1.1-fold in a fixed time
incubation. Purified RNAs were analyzed on a 5% polyacrylamide-6 M urea-TBE gel. Time points of the incubation with DmS-II are
indicated at the top of each lane. B, quantitation was
performed as described under ``Experimental Procedures.'' The
ratios of RO/(RO + TIa) were calculated and plotted versus the time of chase.
The experiment shown in Fig. 4also examines the effect of DmS-II by comparing the RNA profile generated in its absence or presence. For P2 wild type, although the maximum elongation rate (determined from the leading edge of the transcript distribution at each time point) was not stimulated very much, the quantity of pulse-labeled transcripts was greater in the presence of DmS-II, indicating that DmS-II increased the number of P2 polymerase molecules that passed through early pause sites (compare lanes 5, 6, 7, and 8 with lanes 1, 2, 3, and 4). Although there was generally much more labeled RNA after an 8-min chase in the presence of DmS-II than in the absence of DmS-II, an RNA of about 140 nucleotides (Fig. 4, indicated by the arrow) diminished when DmS-II was present (compare lane 8 with lane 4), indicating pausing of P2 at this specific site was reduced by DmS-II. However, for the S1 mutant, there was not much increase in the amount of labeled RNA when DmS-II was present (compare lanes 13, 14, 15, and 16 with lanes 9, 10, 11, and 12), and there was not much decrease in the 140-nucleotide RNA (compare lane 16 with lane 12), confirming that S1 was less responsive to the action of DmS-II.
Figure 4: Comparison of the effect of DmS-II on P2 wild type and S1 mutant RNA polymerase II in elongation. Elongation reaction from dC-pPCP was set up as described for Fig. 1A with or without 2.5 nM DmS-II. Purified RNAs were analyzed on a 5% polyacrylamide-6 M urea-TBE gel. Lengths of the DNA size standards (M) are given in nucleotides, time points of the chase reaction are indicated at the top of each lane. The 140-nucleotide RNA is indicated by an arrow.
Figure 5: Comparison of DmS-II-stimulated transcript cleavage from TIa by P2 wild type, C4 mutant, and S1 mutant RNA polymerase II. Elongation complexes were formed on dC-pGEMTerm and isolated by spin columns as described under ``Experimental Procedures.'' Isolated complexes were then incubated with 4.5 nM DmS-II. Aliquots were removed after 0, 2, 5, 10, and 60 min of incubation. Finally, 600 uM GTP, 600 uM ATP, 100 uM CTP, and 450 uM UTP were added to the reaction, which had been incubated with DmS-II for 60 min, and the incubation was continued for another 10 min. RNA products were isolated and resolved by electrophoresis on a 5% polyacrylamide-6 M urea-TBE gel. The run-off transcripts and transcripts with 3` ends at TIa, TIb, and TII are indicated by RO, TIa, TIb, and TII. The major cleavage products are indicated by the arrows.
In order to look in more detail at DmS-II-mediated transcript cleavage and compare it with pyrophosphorolysis, we isolated elongation complexes stalled at a major elongation block after the incorporation of 13 nucleotides with immobilized dC-3025 template. When isolated elongation complexes were incubated with 1 mM sodium pyrophosphate or 1 nM DmS-II, transcript shortening from the 13-mer was observed for all three polymerases (Fig. 6). The pattern of the transcripts shortened by DmS-II mediated cleavage was similar to that shortened by pyrophosphorolysis, with the 13-mer decreasing and transcripts of 11, 9, 7, and 5 nucleotides appearing(14) . The amount of run-off transcripts found with the three polymerases (compare lanes 1, 6, and 11 in Fig. 6A and lanes 1, 7, and 13 in Fig. 6B) indicates that S1 passes the pause site after incorporation of 13 nucleotides more easily than wild type, whereas C4 has more difficulty, consistent with findings obtained with the dC-pGEMTerm template.
Figure 6: Comparison of pyrophosphorolysis and DmS-II-mediated transcript cleavage from the 13-mer by P2 wild type, C4 mutant, and S1 mutant RNA polymerase II. Elongation complexes were formed on immobilized dC-3025 as described under ``Experimental Procedures'' and as indicated by the diagram in the lower portion of this figure. The black circle with W symbol indicates that the elongation complexes were washed at this step. Elongation complexes were incubated for the indicated times with 1 mM pyrophosphate (A) or 1 nM DmS-II (B). RNAs were analyzed on an 18% polyacrylamide-6 M urea-TBE gel. Transcripts sizes are indicated in nucleotides.
In the presence of 1 mM pyrophosphate (Fig. 6A), 70-75% of the 13-mers in the P2 or S1 complexes were shortened after 90 min of pyrophosphorolysis, whereas 90% were shortened in the C4 complexes. In reaching the final pattern of 9-mer, 7-mer, or 5-mer, S1 was fastest, and C4 was slowest (compare lanes 4, 9, and 14 and compare lanes 5, 10, and 15). In the presence of 1 nM DmS-II (Fig. 6B), 90% of the 13-mers were shortened in the P2 or C4 complexes in 10 min, whereas 55% were shortened in the S1 complexes. In reaching the 9-mer, 7-mer, or 5-mer, S1 was similar to P2, but C4 was significantly slower (compare lanes 4, 10, and 16). These results confirm that relative to wild type and C4, S1 is different in its interaction with DmS-II. The results also suggest that C4 is altered in its ability to move backward along the template.
We have examined the properties of two Drosophila RNA polymerase II mutants in elongation, recognition of intrinsic
elongation blocks, read-through in response to DmS-II, transcript
cleavage in response to DmS-II, and pyrophosphorolysis. Our results
show that the two mutations affect distinct functions associated with
the two large subunits of RNA polymerase II. The C4 mutation
in the largest subunit probably affects the translocation of the
polymerase such that the forward and backward movement of the mutant
elongation complex is slowed down. The S1 mutation in the
second largest subunit probably affects the conformation of the
polymerase and increases the time spent by the polymerase in the
elongation competent mode such that the mutant enzyme is less
responsive to the action of DmS-II and NH.
It has been shown previously that the C4 mutant polymerase
is resistant to -amanitin in RNA polymerization due to decreased
binding affinity to the toxin(8) . We found that in the
presence of 1 µg/ml of
-amanitin, the DmS-II-stimulated
cleavage of nascent transcripts by wild type polymerase was not
observed within 60 min, confirming the inhibitory effect of
-amanitin on this
process(14, 23, 24, 25) . In
contrast, the cleavage by C4 mutant was still observed (data
not shown), consistent with C4 not binding
-amanitin
normally. Although the detailed inhibitory mechanism of
-amanitin
is yet to be elucidated, recent studies suggest that
-amanitin
inhibits pyrophosphorolysis and DmS-II-mediated transcript cleavage
differently. For example, the toxin allows (slowed) pyrophosphorolysis
within a paused elongation complex but completely blocks DmS-II
action(26) .
Because the response of C4 to DmS-II
and NH parallels that of the wild type,
the structural features involved in interacting with DmS-II and
NH
are probably not affected by the C4 mutation. Furthermore, because C4 carries out initial
DmS-II-mediated transcript cleavage and pyrophosphorolysis as well as
the wild type enzyme, the catalytic steps during these two processes
may not be affected, either. It is possible that although the C4 mutation reduces the affinity of the enzyme for
-amanitin, it
also introduces certain functional changes similar to those that would
be caused by
-amanitin binding. One possibility is that the
forward and backward translocation is slowed. This possibility is
consistent with our observations that C4 is slower in reading
through elongation blocks and is also slower in extended transcript
shortening.
Three regions of the largest subunit of RNA polymerase
II have been implicated previously in binding of TFIIS. Sawadogo et
al.(27) analyzed the interaction of yeast TFIIS with
RNAPII, RNAPII
, and RNAPII
, enzyme
whose C-terminal repeated domain (CTD) is phosphorylated, not
phosphorylated, or proteolyzed, respectively. With glycerol gradients
and electrophoresis under native conditions, they found that TFIIS
binds preferentially to II
and II
, suggesting
the CTD is involved in binding TFIIS. However, TFIIS can stimulate
elongation by RNA polymerase II in which the entire CTD has been
proteolyzed during purification. Furthermore, Christie et al.(20) showed that for Saccharomyces cerevisiae RNA
polymerase II the CTD and subunits four and seven, which are essential in vivo, are not required in vitro for read-through
of intrinsic elongation blocks and nascent transcript cleavage in
response to TFIIS. Rappaport et al.(28) showed that a
fusion protein containing a portion of the largest subunit of human RNA
polymerase II is able to inhibit the stimulatory effect of TFIIS. A
monoclonal antibody against this fusion protein can also inhibit the
stimulation by TFIIS. However, TFIIS binds only weakly to the fusion
protein they used, suggesting the protein domain around conserved
region B in the largest subunit may be only part of the TFIIS-binding
site. Archambault et al.(29) isolated seven mutations
in the gene encoding the largest subunit of S. cerevisiae RNA
polymerase II (rpo21) that confer increased growth inhibition
by the uracil analog, 6-azauracil, which is also a mutant phenotype
associated with yeast mutants lacking TFIIS. The 6-azauracil-sensitive
phenotype of RNA polymerase II mutants can be suppressed by
overexpression of TFIIS, suggesting that the region identified by the
6-azauracil-sensitive rpo21 mutations, which is located
between conserved regions G and H, may be involved in interacting with
TFIIS.
Our results with S1 mutant enzyme demonstrate the
involvement of the second largest subunit of RNA polymerase II in
responding to the action of DmS-II. One simple explanation for the S1 mutant biochemical phenotypes would be that the S1 mutation, which results in changing Ser-728 to Cys, directly
affects the binding of DmS-II. However, the observations that S1 mutant enzyme has a higher elongation rate and is more efficient
at reading through elongation blocks in the absence of DmS-II suggest
that if DmS-II binding is affected, it is not the only property
altered. The stimulatory effect of NH on
transcription has been reported in several
studies(10, 30) , and it was speculated that
NH
stimulates transcription through an
effect on the conformation of the polymerase. The observation that
NH
has a much weaker effect on S1 mutant enzyme suggests that the polymerase conformation is
probably altered by the S1 mutation.
Price et al.(12) proposed that during the elongation cycle the
polymerase can be in a paused conformation or an elongation-competent
conformation, and the conversion from a paused to an
elongation-competent conformation can be promoted by the action of an
elongation factor, such as Drosophila Factor 5 (TFIIF). From a
TFIIS mutant that was able to stimulate cleavage but that failed to
promote read-through, Cipres-Palacin and Kane (31) suggested
that conformational changes in the polymerase, in addition to
transcript cleavage, are probably necessary for efficient read-through.
It is possible that conformational changes can be induced by different
mechanisms depending on the interacting factors and that several steps
are involved, some of which may be common for different pathways. The S1 mutation may slow one of the intermediate steps, so that
even though the ternary complex can physically interact with DmS-II (or
NH) and carry out transcript cleavage, the
final conformational change can not be reached normally. At the same
time, the S1 mutation may alter the basal conformation of the
polymerase in a way that it is already more competent for elongation in
the absence of elongation factors. Comparative physical studies of
elongation complexes containing wild type or mutant polymerase should
provide additional insights into conformation changes involved in
transcript elongation and may reveal how the regions of the subunits
affected by the C4 and S1 mutations are involved in
these processes.