(Received for publication, February 21, 1995; and in revised form, June 7, 1995)
From the
Using defined elongation complexes formed on dC-tailed templates
with Drosophila RNA polymerase II, we have examined
elongation, pyrophosphorolysis, and DmS-II-mediated transcript cleavage
and the inhibitory effect of -amanitin on these processes.
Analysis of pyrophosphorolysis on soluble or immobilized DNA templates
confirmed that NTPs are liberated instead of dinucleotides that are
released during DmS-II-meditated transcript cleavage. 10 µg/ml
-amanitin completely inhibited DmS-II-mediated transcript cleavage
but allowed extended pyrophosphorolysis and nucleotide addition to
occur.
-Amanitin dramatically decreased the V
for nucleotide addition but only slightly affected the K
for nucleotides. Although the processes
are mechanistically distinct, both pyrophosphorolysis and
DmS-II-mediated transcript cleavage frequently resulted in similar
patterns of shortened transcript. Since polymerase molecules encounter
similar kinetic barriers during both processes, it is possible that
there is a common step in the reverse movement of the polymerase.
Transcription elongation can be viewed as a repeating cycle of
single ribonucleotide incorporation events. Ribonucleotides first bind
and then are added to the growing RNA chain, followed by the release of
pyrophosphate (PP). The cycle is completed by the
downstream translocation of the active site. Early models described
extended elongation as a simple repetition of the single cycle and
implied monotonic movement of the
polymerase(1, 2, 3) . Recent results have
demonstrated that the downstream edge of the polymerase can move
asynchronously with nucleotide
incorporation(4, 5, 6, 7) . This
suggests that besides the translocation of the active site that must
occur after every nucleotide addition there could be another kind of
polymerase movement that occurs during extended elongation (reviewed in (8) ).
In addition to elongation RNA polymerase II can
shorten its nascent transcripts in two different reactions.
Pyrophosphorolysis is the reversal of elongation utilizing PP as substrate and liberating NTPs. This reaction is dependent on
the addition of a divalent cation such as magnesium and proceeds at a
much slower rate than elongation(9) . A protein factor S-II
causes the polymerase to cleave its nascent transcripts usually in
increments greater than mononucleotides reviewed in (10) and (9) and (11) -16. S-II-mediated transcript
cleavage is also dependent on the addition of a divalent cation and
proceeds rapidly in the presence or absence of
NTPs(9, 11, 17) . After S-II-mediated
transcript cleavage the polymerase extends the new 3` end and has a
second chance of passing the block
point(9, 11, 12, 13) .
Although
both elongation and S-II-mediated transcript cleavage are sensitive to
-amanitin (18, 19) it is not clear how the fungal
toxin exerts its effect. Originally,
-amanitin was thought to
inhibit the formation of phosphodiester bonds(18) . Vaisius and
Weiland (20) later showed with calf thymus RNA polymerase II
that the first phosphodiester bond could be synthesized, but in this
study the formation of additional bonds could not be detected, and it
was concluded that the translocation step was blocked. Since relatively
short reaction times were used in this study, their results are also
consistent with the possibility that
-amanitin merely slows the
rate of nucleotide incorporation. Other studies have suggested that
nucleotide binding is affected by
-amanitin(21) . Although
it is assumed that pyrophosphorolysis is sensitive to the toxin, this
has not been demonstrated.
RNA polymerase from Escherichia coli(22) and RNA polymerase II from yeast (23) and Drosophila()are able to carry out factor-dependent
transcript shortening as well as incorporation of nucleotides into RNA
chains in the absence of template DNA. When very long reaction times
were used both types of reactions with yeast RNA polymerase II were
slowed but not completely inhibited by 100 µg/ml
-amanitin(23) . Although these results do not allow the
determination of the enzymatic step inhibited by
-amanitin, they
indicate that
-amanitin may only affect the rate of the reactions.
We show here for the first time that extended pyrophosphorolysis and
nucleotide addition can take place in a template-dependent manner in
the presence of
-amanitin levels previously thought to block the
activity of RNA polymerase II.
Figure 7:
NTP addition to PP-treated
complexes. IECs formed on immobilized dC-3025 with Drosophila RNA polymerase II were isolated as described under
``Experimental Procedures.'' Complexes were treated with 1
mM PP
for 90 min and then washed. Autoradiographs: panelA, indicated amounts
of NTPs were added to complexes for 6 s; panelB,
complexes were treated with 10 µg/ml
-amanitin 2 min prior to
the addition of the indicated amounts of NTPs for 30 min. Transcripts
were analyzed as described in the legend to Fig.1. K
and V
values
were determined by plotting 1/activity versus 1/[substrate] for each reaction (see ``Experimental
Procedures'').
Figure 1:
Pyrophosphorolysis of isolated
elongation complexes. IECs formed on soluble dC-3025 with Drosophila RNA polymerase II were isolated as described under
``Experimental Procedures.'' The IECs were incubated with the
indicated concentrations of PP or DmS-II for the indicated
times. Nucleic acids were analyzed on an 18% polyacrylamide (60:1
acrylamide/bisacrylamide), TBE, urea gel. Transcript sizes are
indicated in nucleotides.
There are two
possible explanations for the transcript pattern generated during
pyrophosphorolysis. PP could mimic the function of DmS-II,
causing the liberation of nucleotide dimers(9) . Another
explanation is that PP
might remove one nucleotide at a
time with the apparent 2-nucleotide increment being caused by a barrier
related to the removal of nucleotides in that sequence. To resolve this
issue the labeled products generated during a 30-min pyrophosphorolysis
reaction were combined with unlabeled RNA standards and analyzed by
chromatography on Mono Q. Fig.2shows that the small products
generated during pyrophosphorolysis were exclusively nucleoside
triphosphates that elute between nucleotide trimers and
tetramers(9) . Therefore, the 2-nucleotide increment reflects
the existence of a barrier to extended pyrophosphorolysis that occurs
every 2 nucleotides on this template.
Figure 2:
Analysis of the products generated during
pyrophosphorolysis. IECs formed on soluble dC-3025
([-
P]CTP-labeled) were incubated with 1
mM sodium PP
for 30 min. A control reaction
containing an equal amount of complexes was incubated with water for
the same time. The radioactive products generated were analyzed using a
Mono Q column as described under ``Experimental Procedures.''
Nucleotide monomer, dimer, etc. are indicated by numbers above the elution profile.
Figure 3:
Inhibition of pyrophosphorolysis and
DmS-II-mediated transcript shortening by -amanitin. IECs formed on
soluble dC-3025 were treated with the indicated amounts of
-amanitin for 5 min before the addition of any other factor. The
-amanitin-treated complexes were then incubated with 1 mM sodium PP
or 40 nM DmS-II for 30 min. In lane14
-amanitin-treated complexes were
incubated with 600 µM of all four nucleotides for 30 min.
Transcripts were analyzed as described in the legend to Fig.1.
In comparison with pyrophosphorolysis, DmS-II-mediated
transcript cleavage responded differently to increasing concentrations
of -amanitin (Fig.3, lanes8-14).
At low concentrations of
-amanitin some complexes were able to
carry out multiple rounds of transcript cleavage resulting in the
production of the 5-mer. At higher concentrations of
-amanitin
nearly all of the complexes were blocked for transcript cleavage. The
small percentage of ternary complexes that could undergo transcript
shortening even when 10 µg/ml of
-amanitin was used could be
due to competition between DmS-II and
-amanitin for interaction
with the polymerase. This competition also emerged when the DmS-II
concentration was titrated from 4 to 400 nM with a constant
0.5 µg/ml
-amanitin concentration (data not shown).
These
results suggest that when -amanitin is bound to the polymerase at
least one nucleotide can be removed from the nascent transcript in the
presence of PP
. Due to a significant amount of 12-mer in
the starting material (Fig.3, lane1) it was
not possible to determine if more than one nucleotide could be removed
in the presence of
-amanitin. The differential sensitivity of
pyrophosphorolysis and DmS-II-mediated transcript shortening to
-amanitin again suggests that the two processes utilize different
mechanisms.
The ability of the polymerase to carry out limited
pyrophosphorolysis in the presence of high concentrations of
-amanitin demonstrates that the active site is not rendered
inactive by the toxin. This is further supported by the reduction of
the 12-mer after readdition of all four NTPs to the complexes that were
incubated with
-amanitin (Fig.3, compare lane14 with lane1). This observation
reveals that nucleotide addition to the 12-mer is not completely
inhibited by
-amanitin. A possible explanation for these observed
results is that
-amanitin does not stop the catalytic activity of
the active center but rather slows the overall rate of nucleotide
addition or removal.
Figure 4:
Extended pyrophosphorolysis in the
presence of -amanitin. IECs formed on immobilized dC-3025 with Drosophila RNA polymerase II were isolated as described under
``Experimental Procedures'' and treated with 10 µg/ml
-amanitin 2 min before the addition of PP
. The
-amanitin-treated complexes were then incubated with 1 mM PP
for the indicated times. In lanes2 and 3 complexes were incubated with 600 µM of all four nucleotides for 90 min. Transcripts were analyzed as
described in the legend to Fig.1.
When NTPs were added back to IECs for 90 min, an
increase in runoff was obtained as well as a reduction in the amount of
13-mer and an intermediate length transcript (Fig.4, lane2, arrow). In the presence of -amanitin
nucleotide addition was not observed (Fig.4, lane3). When 1 mM PP
was added to the
complexes for increasing times the appearance of 11-mer, 9-mer, 7-mer,
and 5-mer was similar to that obtained on the soluble dC-3025 template (Fig.4, lanes4-7). In the presence of
10 µg/ml
-amanitin extended pyrophosphorolysis still occurred (Fig.4, lanes8-11). At the longest
time point most of the complexes reached the first reverse barrier
(11-mer); however, a small but significant number of complexes reached
the second and third reverse barriers (9-mer and 7-mer). These results
are consistent with pyrophosphorolysis being slowed but not blocked by
-amanitin. The autoradiograph was scanned, and the disappearance
of the 13-mer was quantitated. The disappearance rate of the 13-mer in
the presence of
-amanitin was 22 percent of the rate in the
absence of
-amanitin. The appearance of the 9-mer and 7-mer at the
90-min time points is consistent with a similar decrease in the rate of
pyrophosphorolysis at each reverse barrier. It was not possible to
determine the rate of pyrophosphorolysis at sites between the reverse
barriers because transcripts of these lengths (10, 8, etc.) were not
detectable.
Similar results are seen with an intermediate length
transcript (Fig.4, arrow). This transcript was able to
be efficiently elongated upon addition of NTPs unless -amanitin
was present (Fig.4, lanes2 and 3).
A variety of shorter transcript sizes arose from this transcript upon
addition of PP
to the reaction (Fig.4, lanes4-7). At least one of the shorter transcripts
appeared in the presence of
-amanitin (lanes8-11). Two important conclusions can be drawn from
these results. The first is that
-amanitin slows down but does not
completely inhibit the catalytic activity of the active site of the
polymerase. The second is that extended pyrophosphorolysis can take
place in the presence of
-amanitin levels known to block
elongation from forward pause sites.
Figure 5:
Nucleotide addition to
-amanitin-treated complexes. IECs formed on immobilized dC-3025
with Drosophila RNA polymerase II were isolated as described
under ``Experimental Procedures.'' 10 µg/ml
-amanitin was added 2 min prior to a 90-min incubation with 1
mM PP
. Complexes containing shortened transcripts
were then washed and incubated again with
-amanitin for 2 min as
indicated.
-Amanitin-treated complexes were incubated with 600
µM of all four nucleotides for the indicated times (see reactionline). Transcripts were analyzed as
described in the legend to Fig.1.
In order to determine if more extensive
elongation could occur in the presence of -amanitin, IECs
containing mainly the 13-mer were formed and then treated with 1 mM PP
for 90 min. After washing, the resulting complexes
contained transcripts from 5 to 9 nucleotides in length (Fig.6,
lane 1). When NTPs were added back in the absence of
-amanitin to
these complexes, transcripts were elongated to nucleotide 13 in less
than 10 min (Fig.6, lanes2-6). In the
presence of 10 µg/ml
-amanitin transcript elongation was
slowed but not blocked (Fig.6, lanes7-11). After 90 min in the presence of
-amanitin, some but not all of the polymerase molecules were able
to synthesize transcripts 13 nucleotides in length.
-Amanitin
appeared to slow the addition of every nucleotide as evidenced by an
accumulation of transcripts at bands corresponding to nucleotide
positions 8, 9, 10, 11, and 12. Similar results are seen with a longer
transcript (Fig.6, arrow). This transcript was
generated by shortening the long transcript previously described (see Fig.4, arrow). Addition of NTPs to complexes
containing this shortened transcript allowed re-elongation to the
original forward pause site (Fig.6, lane2).
Further incubation resulted in more runoff transcripts and movement
through the original pause site (Fig.6, lanes2-6). In the presence of
-amanitin,
transcripts were able to add nucleotides back to, but not through, the
original pause site in 90 min (Fig.6, lanes7-11).
Figure 6:
Extended nucleotide addition in the
presence of -amanitin. IECs were treated with 1 mM
PP
for 90 min and then washed. Where indicated, complexes
were treated with 10 µg/ml
-amanitin 2 min prior to NTP
addition. Complexes containing shortened transcripts were incubated
with 600 µM of all four nucleotides for the indicated
time. Transcripts were analyzed as described in the legend to Fig.1.
It is also possible
that extended nucleotide addition in the presence of -amanitin is
due to slippage of the RNA through the active site. We do not think
this is the case, since PP
-treated complexes incubated
first with
-amanitin and then with pairs of nucleotides for 90 min
were able to carry out only the appropriate elongation (data not
shown).
We have examined the properties of RNA polymerase II during
elongation, pyrophosphorolysis, and DmS-II-mediated transcript cleavage
and have investigated the inhibitory effect of -amanitin on these
processes. Our results show that extended nucleotide addition and
pyrophosphorolysis can take place in the presence of
-amanitin,
which was previously thought to completely block these processes. Our
results also suggest that although there are similarities in the
pattern of transcripts generated during extended pyrophosphorolysis and
DmS-II-mediated transcript cleavage, the two processes are
mechanistically distinct.
Since in the absence of
-amanitin the rate of pyrophosphorolysis is much slower than that
of elongation, the rates of the two processes are similar in the
presence of
-amanitin. Since the rate of the incorporation of a
single nucleotide is dramatically slowed by
-amanitin, the
simplest interpretation is that phosphodiester bond formation is
slowed. This interpretation is consistent with the lack of a
significant change in K
, which is
influenced by nucleotide binding and the ability to add multiple
nucleotides (translocation) in the presence of
-amanitin. It is
possible that amanitin slows the translocation rate, but this would
mean that in our studies complexes containing the 5-mer would need to
translocate before nucleotide 6 was added. It is also possible that
amanitin could slow the rate of the conformational change that occurs
after nucleotide binding found with DNA polymerases(29) .
While the two processes have significant differences, they may share a common step. The pattern of transcripts generated during pyrophosphorolysis and DmS-II-mediated transcript shortening are similar using dC-3025. Other eucaryotic enzymes and E. coli RNA polymerase give similar results(30, 31) . During extended reactions both processes require that the polymerase move in a reverse direction on the template. If there are kinetic barriers to such reverse movement both processes could be slowed at the same sites even though the reactions driving the reverse movement are different. This idea is supported by the data presented here, which show that similar kinetic barriers are found during both processes. One possibility is that barriers to the reverse movement are related to the sites of discontinuous movement seen during elongation by E. coli polymerase (32, 33) and RNA polymerase II(6) . Inability to extend the downstream contacts of the polymerase with the template can cause pausing in the forward direction(32) . It is possible that inability to release downstream contacts during reverse movement could cause similar barriers during extended pyrophosphorolysis or DmS-II-mediated transcript cleavage. Reverse barriers of this sort might be expected to occur at sites that are normally poised for elongation. Consistent with this we have observed that reverse barriers never coincide with forward pause sites.
A major question that remains to be answered is whether
the catalytic center that carries out elongation and
pyrophosphorolysis is also responsible for DmS-II-mediated transcript
cleavage. If one center carries out both reactions it must have a
substrate binding site that can change from binding the highly charged
PP molecule to binding water when S-II is present. We
showed here that pyrophosphorolysis and DmS-II-mediated transcript
cleavage are affected differently by
-amanitin. While this could
be taken as evidence for two sites it is possible that a single active
center catalyzes both reactions, and the differential effect of
-amanitin could be due to blockage of DmS-II binding by the toxin.
Unfortunately, it is difficult to determine whether
-amanitin
inhibits the binding of DmS-II since this protein does not bind Drosophila RNA polymerase II tightly. Recently, it was shown
that PP
was able to cause RNA polymerase II to cleave its
nascent transcript endonucleolytically, releasing large nucleotide
products, similar to the cleavage pattern by S-II(30) . These
data were taken as evidence for the existence of only one active site.
Circumstantial evidence for two active centers include: 1) a domain in
the large subunit of RNA polymerase II with sequence similarity to a
nuclease(34) , and 2) the existence of both an elongation site
and a hydrolysis site in functionally similar DNA polymerases.
Resolution of the problem may await genetic evidence for the separation
of the two functions in RNA polymerase.