(Received for publication, October 24, 1996, and in revised form, April 2, 1997)
From the Cancer Research Group, Institute for Molecular Biology and
Biotechnology, McMaster University, Hamilton, Ontario L8N 3Z5,
Canada and the Department of Molecular and Cell
Biology, University of California, Berkeley, California 94720
The role of yeast RNA polymerase II (pol II)
subunit RPB9 in transcript elongation was investigated by examining the
biochemical properties of pol II lacking RPB9 (pol II9). The maximal
rate of chain elongation was nearly identical for pol II and pol
II
9. By contrast, pol II
9 elongated more efficiently through DNA
sequences that signal the elongation complex to pause or arrest. The
addition of purified recombinant RPB9 to pol II
9 restored the
elongation properties of the mutant polymerase to those of the
wild-type enzyme. Arrested pol II
9 complexes were refractory to
levels of TFIIS that promoted maximal read-through with pol II.
However, both pol II and pol II
9 complexes stimulated with TFIIS
undergo transcript cleavage, confirming that transcript cleavage and
read-through activities can be uncoupled. Our observations suggest that
both TFIIS and RPB9 are required to stimulate the release of RNA
polymerase II from the arrested state.
The mRNA transcription machinery in eukaryotes is a complex of more than 30 different polypeptides of which 12 polypeptides are tightly associated with RNA polymerase (pol)1 II. The largest two subunits of pol II, which are thought to harbor catalytic activity, are related to the largest subunits of the other nuclear RNA polymerases (1, 2). Each of the remaining pol II subunits is conserved throughout the Eukarya and more than half are also conserved in the Archae (3).
In Saccharomyces cerevisiae, the genes encoding the twelve subunits of pol II have been cloned and sequenced. Five of the ten small subunits of yeast pol II (RPB5, RPB6, RPB8, RPB10, and RPB12) are common to all three nuclear RNA polymerases (4, 5). Several remaining pol II subunits have homologues in pol I and pol III. RPB11 (6) is related to AC19, a subunit shared by pols I and III (7). RPB7 is similar to the pol III subunit C25 (8). RPB9 is related to the pol I subunit, A12.2 (9). At least six pol II small subunits from S. cerevisiae are functionally interchangeable with human subunits (RPB6, RPB7, RPB8, RPB9, RPB10, and RPB12) (10-12). Genetic analysis indicates that only two yeast pol II subunits, RPB4 and RPB9, are not essential for cell viability (13, 14).
In yeast, deletion of RPB9 results in mild temperature sensitivity and relatively normal levels of transcription in vivo. However, for most genes examined, the selectivity of the site of transcription initiation is altered, with new start sites shifted upstream relative to wild-type sites (15-17). The transcription initiation phenotype can be recapitulated in vitro, and addition of recombinant purified RPB9 (rRPB9) restores wild-type start site selection (15). RPB9 from S. cerevisiae is a 122-amino acid polypeptide that contains two zinc binding domains (14). The COOH-terminal zinc binding domain shares 25% sequence identity with that of the general transcript elongation factor TFIIS (18) and is predicted to adopt a zinc ribbon fold (17-19). This domain is required for the function of RPB9 in start site selection2 and is required within TFIIS for elongation stimulation (20).
The homology of RPB9 to TFIIS suggested that RPB9 may play a role in
transcript elongation. In addition, the altered start site selection of
pol II lacking RPB9 (pol II9) suggested some alteration in
positioning the catalytic center of the polymerase, and a mobile
catalytic center is a prominent feature of several recent models for
transcript elongation (21-24). Thus the elongation properties of
highly purified pol II and pol II
9 were compared. The availability
of purified rRPB9 and TFIIS was exploited to characterize the
functional interactions among pol II and these factors during the
transcription elongation process.
RNA polymerase
II lacking RPB9 (pol II9) was purified from the S. cerevisiae strain WY9 (14). pol II and pol II
9 were purified as
described previously (25), except that 10 µM
ZnCl2 was included in all buffers. The polymerase
preparations were stored at
80 °C in 10 mM Tris-HCl,
pH 7.9, 40 mM ammonium sulfate, 10% glycerol, 2.5 mM DTT, 100 µM EDTA, and 10 µM
ZnCl2.
The RPB9
coding region (14) was subcloned using polymerase chain reaction
amplification to generate the coding region with an NdeI
restriction site at the 5 end of the sequence and a BamHI site at the 3
end. The amplified insert was then cloned between the
NdeI and the BamHI sites of the pET15b bacterial
expression vector (Novagen, Madison, WI) to generate a fusion protein
between RPB9 and an NH2-terminal hexahistidine tag.
BL21(DE3) cells containing the pET15bRPB9 plasmid were grown to
A600 of 0.8 at 37 °C. The RPB9 protein
expression was induced at 30 °C by the addition of 0.5 mM isopropyl-1-thio-
-D-galactopyranoside.
The cells were harvested 3 h postinduction by centrifugation,
resuspended in 50 mM Hepes, pH 7.5, 10% sucrose, 100 mM NaCl, 10 µM ZnCl2, 1 mM PMSF, and 1 mM benzamidine (2 ml/g of
cells), frozen, thawed, and then lysed by sonication at 4 °C. All
subsequent steps were performed at 4 °C. The lysate was clarified by
centrifugation at 55,000 × g for 30 min in a Beckman
SW28 rotor, and then NaCl was added to a final concentration of 500 mM. The supernatant was passed through a DE52 column
(2.5 × 5 cm; Whatman, Maidstone, UK) and loaded directly onto
His-bind resin (2.5 × 2.5 cm; Novagen, Madison, WI), previously
charged with 200 mM NiSO4 and equilibrated in
purification buffer (50 mM Hepes, pH 7.5, 10 µM ZnCl2, 10% glycerol, 1 mM
PMSF, and 1 mM benzamidine) containing 5 mM
imidazole. The His-bind column was washed with 5 column volumes of
purification buffer containing 500 mM NaCl and 50 mM imidazole, pH 7.5. RPB9 was eluted from the column with
purification buffer containing 500 mM NaCl and 300 mM imidazole, pH 7.5. The eluate was dialyzed for 8 h
against purification buffer containing 100 mM NaCl and 10 mM DTT but lacking PMSF and benzamidine. The dialysate was treated with bovine thrombin (3 µg of thrombin/mg of RPB9 fusion) for
4 h at room temperature, diluted 2-fold with purification buffer
containing 10 mM DTT, passed through a Poros S column
(0.75 × 10 cm; PerSeptive Biosystems, Cambridge, MA), and loaded
onto a 2-ml hydroxyapatite fast protein liquid chromatography column (Bio-Rad). The column was developed with a 5 mM to 150 mM gradient of sodium phosphate, pH 7.5 in 50 mM NaCl, 10 mM DTT, 10 µM
ZnCl2, 1 mM PMSF, and 1 mM
benzamidine, and RPB9 eluted at 50 mM phosphate. The
purified protein was dialyzed against 5 mM Hepes, pH 7.5, 100 mM NaCl, 10 µM ZnCl2, and 10 mM DTT and stored frozen at
70 °C.
To generate a fusion protein of GST and RPB9, the RPB9
coding region (14) was subcloned using polymerase chain reaction amplification to generate the coding region with a BamHI
restriction site at both the 5 and 3
ends. The amplified insert was
then cloned into the BamHI site of the pGEX-2T bacterial
expression vector (Pharmacia Biotech Inc.). The GST-RPB9 fusion protein
was expressed as described above for recombinant RPB9 using HB101 cells. The cells were harvested and lysed as described previously, with
the exception that the NaCl concentration was maintained at 100 mM. The supernatant was passed through a DE52 column
(2.5 × 2 cm; Whatman) and loaded directly onto a
glutathione-Sepharose-4B column (2.5 × 3 cm; Pharmacia)
previously equilibrated with Buffer A (10 mM sodium
phosphate, 1.8 mM potassium phosphate, 2.7 mM KCl, 140 mM NaCl 10 mM DTT, 1 mM
PMSF, and 1 mM benzamidine). The glutathione column was
washed with 10 column volumes of Buffer A followed by 5 column volumes
of Tris buffer (50 mM Tris, pH 8.0, 10 mM DTT,
1 mM PMSF, and 1 mM benzamidine). The GST-RPB9 fusion protein was eluted from the column with Tris buffer containing 10 mM glutathione. The purified protein was dialyzed
against 5 mM Hepes, pH 7.5, 100 mM NaCl, 10%
glycerol, 10 µM ZnCl2, and 10 mM
DTT and stored frozen at
70 °C.
Yeast TFIIS was cloned and
expressed as a fusion protein containing an amino-terminal
hexahistidine tag in bacterial cells (26), and the expressed protein
was purified as described below. All procedures were performed at
4 °C. Bacterial cells (2L) expressing the yeast TFIIS protein were
lysed using a French pressure cell in Buffer A (20 mM
Hepes, pH 7.5, 10% glycerol, 10 µM ZnCl2, 1 mM PMSF, and 1 mM benzamidine) containing 100 mM NaCl, 10 mM DTT, and 1 mM EDTA.
The lysate was clarified by centrifugation at 55,000 × g for 30 min in a Beckman SW28 rotor, and the supernatant
was loaded onto a DE52 column (2.5 × 2.5 cm; Whatman). The flow
through was loaded onto an SP Fast Flow column (1 × 8 cm;
Pharmacia), washed with Buffer A containing 100 mM NaCl,
and eluted with Buffer A containing 500 mM NaCl. The eluate
was loaded directly onto a Poros MC column (0.75 × 10 cm;
PerSeptive Biosystems, Cambridge, MA) equilibrated with
NiSO4. The column was washed with Buffer A containing 500 mM NaCl and 5 mM imidazole. Yeast TFIIS was
eluted with Buffer A containing 500 mM NaCl and 500 mM imidazole, immediately dialyzed against Buffer A
containing 50 mM NaCl and 10 mM DTT, and stored
frozen at 70 °C.
These procedures were carried out as
described previously (26). Briefly, transcription by purified pol II
was initiated from a 3 deoxycytidine tailed template containing a
human histone H3.3 gene fragment, which contains well characterized
blocks to elongation (27). The RNA transcript was pulse-labeled at the 5
end by incubating in the presence of 20 µCi of
[
-32P]CTP (3000 Ci/mmol, DuPont NEN), 0.8 mM GTP, ATP, and UTP for 75 s, followed by elongation
to the blocks to elongation in the presence of 0.1 mM
unlabeled CTP for 75 s. Ternary complexes stalled at these sites
were treated in two different ways. For the read-through assay, yeast
TFIIS was added, transcription was allowed to proceed for the
designated intervals, and the resulting transcripts were collected by
ethanol precipitation and resolved by electrophoresis on a 6%
polyacrylamide (19:1 acrylamide:bisacrylamide), 8.3 M urea,
Tris borate-EDTA gel. The transcripts were quantified by phosphorimaging (Molecular Dynamics, Sunnyvale, CA). For the cleavage reaction, the stalled ternary complexes were purified from
unincorporated nucleotides by two sequential Bio-gel 30 spin columns
(Bio-Rad). TFIIS was then added to the purified complexes, and
incubation was continued at 30 °C. At designated time points,
aliquots were removed, and the reaction was stopped by the addition of
stopping buffer. The transcripts were resolved and quantified as
described above.
For analysis of intrinsic cleavage, the standard cleavage assay described above was modified by including a third Bio-gel 30 spin column equilibrated with 70 mM CAPS, K+, pH 9.5, 100 mM KCl, 5 mM MgCl2, and 10% glycerol. Reactions were incubated at 30 °C, and aliquots were removed to an equal volume of stop buffer and resolved as described above.
Binding Assay between Yeast TFIIS and RNA Polymerase IIAn
oligonucleotide encoding the heart myosin kinase (HMK) consensus
phosphorylation sequence (RRASVDF) was inserted into the NdeI site of the plasmid pET15bPPR2 (26), between the
sequences encoding the thrombin cleavage site and TFIIS. The resulting
TFIIS fusion protein was expressed and purified as described for the purification of yeast TFIIS. This protein was phosphorylated at the HMK
consensus site using HMK (Sigma) and [-32P]ATP
according to the manufacturer's instructions. The labeled protein was
dialyzed against Buffer A containing 10 mM DTT and 50 mM NaCl to remove the free radiolabel. The
32P-labeled TFIIS and unphosphorylated TFIIS were equally
active in assays of read-through and transcript cleavage. For the
binding assay, 0.85 pmol of 32P-HMK-TFIIS were incubated in
a 5-µl volume for a minimum of 20 min with 0.1-4.5 pmol of pol II in
buffer containing 50 mM Tris, pH 7.5, 10% glycerol, and 5 mM DTT and a final salt concentration between 40 and 50 mM KOAc. The binary TFIIS-polymerase complex was resolved
from free yeast TFIIS by electrophoresis for 2-3 h at 100 V at 4 °C
on a 5% polyacrylamide (30:0.8 acrylamide:bisacrylamide) gel
containing 50 mM Tris, 50 mM borate, 10 µM ZnSO4, and 1% glycerol. The amount of
bound 32P-HMK-TFIIS was quantified by phosphorimaging.
pol II9
was isolated using a combination of heparin, immunoaffinity, and anion
exchange chromatography. This method yielded a purified enzyme devoid
of RPB9 but containing all of the remaining subunits (Fig.
1). The yield of pol II
9 enzyme was similar to that
of pol II, approximately 0.8 mg/100 g of packed yeast. Purified pol
II
9 consistently demonstrated a 2-3-fold higher specific activity
than pol II in assays of promoter-independent RNA chain elongation on
denatured DNA templates. However, the maximal elongation rate was
indistinguishable between the two polymerases. The enzymes were also
similar in the rate and extent of pyrophosphorolysis and the
temperature dependence of promoter-independent assays of RNA synthesis
(data not shown).
rRPB9 was expressed and purified from Escherichia coli. The yield of rRPB9 was 5 mg/liter of bacterial culture. We judged rRPB9 to be functional on the basis of two observations. First, the zinc content of rRPB9 was determined by atomic absorption spectroscopy to be 2.4 ± 0.4 mol of zinc/mol of rRPB9, in keeping with the predicted stoichiometry of zinc binding by RPB9.3 Second, this RPB9 preparation restores accurate transcription start site utilization in nuclear extracts prepared from yeast cells lacking RPB9.4
Although rRPB9 was transcriptionally active, it readily formed aggregates. Dynamic light scattering studies of rRPB9 at 3 mg/ml revealed a polydisperse preparation whose average size corresponded to a pentamer. In an attempt to reduce the propensity to aggregate, another recombinant version of RPB9 was prepared, in the form of a fusion to GST. The purified GST-RPB9 was dimeric (in keeping with the dimeric nature of native GST), monodisperse in solution, and also active in assays of promoter-dependent transcription. However, if the GST moiety was removed from the purified GST-RPB9, the RPB9 that was generated once again aggregated. We could not prepare a version of RPB9 that was unaggregated. Therefore we used RPB9 fusion proteins for reconstitution experiments. In each such experiment, the source of the RPB9 is indicated, and the corresponding state of aggregation was therefore known.
pol IIThe observation that pol II and pol
II9 have the same maximal elongation rate yet pol II
9 synthesizes
more RNA in chain elongation assays suggested that pol II
9 pauses
less frequently during elongation. To test this idea, we examined the
behavior of pol II
9 at well characterized blocks to elongation.
Purified pol II stops in vitro at several sites within the
human histone H3.3 first intron (27). The strongest block, TIa, has
been used extensively to study the elongation properties of pol II from yeast and mammalian cells (26, 28-33). We observed that pol II
9 transcribes through TIa with higher efficiency than pol II; 50-65% of
pol II
9 elongates past TIa compared with 15-30% for pol II. The
differences in elongation efficiency are moderate but reproducible; these values reflect the range of TIa read-through in more than a dozen
independent experiments with at least two different preparations of
each enzyme.
To exclude the possibility that the purification of pol II9 for some
reason results in a more efficiently elongating enzyme and that the
elongation differences were indeed a property of RPB9, we tested
whether purified RPB9 could restore the wild-type elongation properties
of pol II
9. The addition of increasing amounts of rRPB9 or GST-RPB9
to pol II
9 resulted in decreased (wild-type) levels of read-through
at the TIa intrinsic block to elongation (Fig.
2A). The decrease in transcription likely results from the reconstitution of wild-type polymerase activity by
RPB9 and pol II
9 because addition of greater than 200-fold molar
excess of rRPB9 to either pol II
9 or pol II did not decrease read-through below that observed with pol II alone. Wild-type elongation levels were restored to pol II
9 by a 3-5-fold molar excess of GST-RPB9 and a 10-fold molar excess of rRPB9.
The addition of purified RPB9 to pol II9 not only decreased
read-through activity but also decreased the total level of
transcription from the dC-tailed templates (Fig. 2B). We
observed that pol II
9 synthesized 3-15 times more transcripts from
the dC-tailed template than did either pol II
9 + RPB9 or the
wild-type enzyme, even though pol II
9 was only 1.5-2-fold more
active than the wild-type enzyme in the normal assays for
promoter-independent chain elongation. RPB9 must inhibit transcription
from these templates at an early stage in the reaction because the
transcripts are pulse-labeled during the formation of the first 20 or
so nucleotides. Using native gel electrophoretic mobility shift assays,
we have shown that the two enzymes bind the tailed template with equal
affinity (data not shown). Therefore, the inhibitory effect of RPB9
takes place after binding of the polymerase and before the synthesis of
the first 20 nucleotides. The mechanism of inhibition remains to be
established.
The addition
of rRPB9 to pol II9 causes more polymerase to arrest at TIa. The
effect of this subunit apparently contrasts with that of the elongation
factor TFIIS, which stimulates transcription through TIa, as well as
several other elongation blocks. To explore the functional interactions
between RPB9 and TFIIS, we compared read-through stimulation by TFIIS
with both pol II and pol II
9-containing arrested complexes. For the
wild-type enzyme, a 3-fold molar excess of TFIIS maximally stimulates
read-through of TIa by 30 min, at which time nearly two-thirds of the
initially arrested complexes are reactivated (Fig.
3A). By contrast, arrested pol II
9 ternary complexes were completely unresponsive to this level of TFIIS during a
30-min incubation (Fig. 3B, lanes 6-10; also
Fig. 3D for quantification). The apparent decrease in the
levels of the TIa transcript in Fig. 3B (lane 5)
reflects a decreased level of radioactivity in the entire lane and not
a TFIIS-independent read-through event.
Once again, to ensure that the inability of pol II9 to respond to
these levels of TFIIS was caused by the lack of RPB9, we attempted to
restore wild-type TFIIS responsiveness by the addition of purified
RPB9. A 3-fold molar excess of TFIIS was added to pol II
9 ternary
complexes formed in the presence of rRPB9. The reconstituted polymerase
(pol II
9 + RPB9) responded to TFIIS with the same kinetics of
read-through as did pol II (Fig. 3C, lanes
6-10). It was not necessary to preincubate the RPB9 with pol
II
9 to observe reconstitution of activity; identical results were
obtained if RPB9 was added with TFIIS.
We tested if the pol II9 arrested complexes were completely
refractory to TFIIS treatment or whether a large molar excess of TFIIS
was able to promote read-through by pol II
9-containing ternary
complexes. pol II
9 arrested complexes were incubated with a 100-fold
molar excess of TFIIS, and at these levels the arrested complexes could
be marginally stimulated to read-through the TIa arrest site, as
determined by PhosphorImager analysis (Fig. 3B, lanes
11-15).
TFIIS-stimulated read-through requires a nascent
transcript cleavage event (34-36). Because pol II9 appears to be
deficient in responding to TFIIS, we tested whether pol II
9
displayed aberrant cleavage activity by comparing cleavage by pol II
and pol II
9 complexes in response to TFIIS. As had been observed
previously (26), the addition of a 3-fold molar excess of TFIIS to
arrested wild-type elongation complexes rapidly generated shortened
transcripts (C1 and C2) that were stably associated with active ternary
complexes as shown by their ability to elongate upon the addition of
nucleotides (Fig. 4A). The cleaved
transcripts appear to be generated sequentially. The first cleaved
product (C1) appeared within 1 min (Fig. 4A, lane
3), and over the course of 30 min a smaller product (C2) appeared,
followed then by a range of shorter RNA species. Such a pattern of
digestion has been observed previously for both yeast and mammalian
enzymes (26, 34).
The pattern and kinetics of TFIIS-stimulated transcript cleavage by pol
II9 differ significantly from that of pol II. Like pol II, pol
II
9 complexes also cleave the nascent RNA chain to C1 in the
presence of a 3-fold molar excess of TFIIS. The pol II
9-generated C1
cleavage products appear more slowly than do the pol II-generated C1
cleavage products (compare Fig. 4A, lanes 3-6,
and Fig. 4B, lanes 3-7). In contrast to the pol
II complexes, pol II
9 complexes did not generate the second and
subsequent cleavage products, even after 30 min or longer in the
presence of TFIIS. To ensure that the differences in the cleavage
reaction were due to the presence or the absence of RPB9, we tested if purified RPB9 would restore the wild-type cleavage pattern and kinetics. A 10-fold molar excess of rRPB9 was added to pol II
9 complexes either prior or subsequent to arrest complex formation. The
resulting enzyme (pol II
9 + RPB9) displayed an identical response to
TFIIS compared with pol II (Fig. 4C, lanes 1-5).
The requirement for RPB9 in read-through could be diminished by the addition of a 100-fold molar excess of TFIIS (Fig. 3). Similarly, the
addition of a 100-fold molar excess of TFIIS to arrested pol II
9
complexes returned the cleavage patterns and kinetics to that seen in
complexes formed with the wild-type enzyme (Fig. 4D).
Therefore, in the absence of RPB9, the polymerase requires substantially higher TFIIS levels to effect cleavage and
read-through.
The observed results would be explained if pol II9 was defective in
catalyzing transcript cleavage. Thus the intrinsic cleavage activities
of pol II and pol II
9 were compared. We found that pol II
9 did
not appear to have any intrinsic defect in catalyzing transcript
cleavage. Moderate elevation in pH can stimulate intrinsic cleavage by
pol II,5 similar to the intrinsic cleavage
reaction observed with E. coli RNA polymerase (37). At basic
pH, yeast pol II complexes halted at TIa cleave their transcripts in
the absence of TFIIS. Although such intrinsic cleavage can occur at
physiologic pH under some solution conditions, the conditions used to
assay TFIIS-stimulated cleavage demonstrated no intrinsic cleavage over
the course of 30 min (Fig. 4, A and B,
lanes 1 and 2). In comparing intrinsic cleavage
by pol II and pol II
9 under mildly alkaline conditions, we observed
no difference in the size of the cleavage products nor in the rate of
transcript cleavage (Fig. 5). The intrinsic cleavage
products correspond approximately to the mobilities of the
TFIIS-stimulated cleavage products; however, direct determination of
the cleavage increment has not been established. Extension of the
intrinsic shortened transcripts at pH 9.5 can result in synthesis back
to the original TIa site. Some intrinsic shortened transcripts are
elongated slightly past the TIa site (see Fig. 5, Nuc lane);
the mechanism of this elongation event remains to be established, and
further characterization of the intrinsic cleavage activity of pol II
will be presented elsewhere.5 In summary, the similarity of
pol II and pol II
9 in the intrinsic cleavage reaction suggests that
pol II
9 is deficient in some other aspect of TFIIS-mediated
read-through.
pol II and pol II
A trivial explanation for our observations would be that
the reduced response of pol II9 to TFIIS arises from a reduced
affinity of pol II
9 for TFIIS. Therefore, the affinity of pol II and
pol II
9 for TFIIS was examined using an electrophoretic mobility shift assay that measures the association of radiolabeled TFIIS with
polymerase in nondenaturing polyacrylamide gels (38). In this assay,
both pol II and pol II
9 bind TFIIS with equivalent affinity
(approximately 80 nM) (Fig. 6). Binding of
TFIIS to either polymerase saturates at a 1:1 molar ratio and is
competed by the addition of unlabeled TFIIS (data not shown). In this
gel system, pol II and the pol II-TFIIS complex enter the gel, whereas
free TFIIS migrates toward the cathode.
Wild-type RNA polymerase II and the enzyme lacking RPB9 have
interesting differences in elongation properties, and these differences suggest that this subunit is important for transmitting signals to the
elongating ternary complex. The behavior of ternary elongation complexes containing pol II9 and the wild-type enzyme differ in two
ways. First, pol II
9 does not arrest at intrinsic blocks to
elongation with the frequency of the wild-type enzyme. Second, when pol
II
9 does arrest during elongation, the arrested pol II
9 complex
is much less responsive than wild-type complexes to the read-through
and cleavage stimulatory factor, TFIIS. These altered properties have
also been reported for two different mutants in the
Drosophila RNA polymerase II largest subunit (39). The association of these properties with a single subunit in yeast RNA
polymerase II sheds additional light on the mechanism of arrest site
utilization.
The process of arrest is thought to occur in at least two steps. First, the elongating RNA polymerase pauses transiently at blocks to elongation. Blocks can be either DNA sequences or physical blocks such as bound proteins. Pausing defines a state of the elongating enzyme from which there are three outcomes. Many paused complexes, after some period, continue chain elongation. A proportion terminates transcription (i.e. dissociate from the template and release the RNA), and the remainder converts to an arrested state, in which the ternary complex is intact but incapable of further transcription in the absence of accessory factors. The amount of the complexes undergoing arrest also can be correlated with the length of time the complexes remain blocked during elongation (40-42).
The decreased propensity of pol II9 to arrest at intrinsic blocks to
elongation might be explained if pol II
9 complexes either (i) had an
overall elongation rate faster then the wild-type enzyme or (ii) had
reduced ability to recognize signals that lead to an intrinsic block to
elongation (31). Our experiments suggest that the latter explanation is
the case, namely that pol II
9 complexes do not recognize intrinsic
blocks to elongation or other pause sites as efficiently as the
wild-type complexes. Paradoxically, although pol II
9 complexes do
not appear to respond to DNA sequences that trigger transcription
arrest, the pol II
9 complexes that do arrest appear to be more
stable as judged by their reduced responsiveness to TFIIS. These
results highlight the complexity of the behavior of RNA polymerases at
arrest sites.
RNA polymerases likely adopt many different conformations during the
elongation process (21, 39). If arrest site recognition may be mediated
by only a subset of the RNA polymerase conformations (arrest-competent), then the efficiency of transcription arrest would
be governed by the proportion of the elongating enzymes in an
arrest-competent conformation. The relative amounts of each conformation might be regulated by accessory factors, substrate levels
and composition, solution conditions, and polymerase subunits. The
behavior of pol II9 is consistent with a role for RPB9 in increasing
the amount of RNA polymerase in an arrest competent conformation. In
the absence of RPB9, the read-through conformation might be favored,
and as a result a smaller proportion of the elongating enzymes arrest.
In the presence of RPB9, the arrest competent conformation would be
favored, and consequently a greater proportion of wild-type complexes
arrest compared with pol II
9. This proposed role for RPB9 in
regulating the interaction of pol II with the nucleic acid is
consistent with the location of the RNA polymerase I RPB9 homologue
(A12.2) (9) in the three-dimensional structure of yeast RNA polymerase
I. Subunit A12.2 is located near the region thought to include the
sites for DNA interaction (43) and in a region proposed to envelope the
DNA template in an elongation complex. If RPB9 occupies an analogous
position in RNA polymerase II, then the subunit is situated perfectly
to modulate the structure of the elongation complex.
pol II and pol II9 elongation complexes also differ in the ability
to respond to TFIIS, which promotes reactivation of transcription after
arrest. Arrested pol II
9 complexes fail to read-through the TIa site
in response to levels of TFIIS that completely stimulate read-through
by arrested wild-type complexes. We showed that the defect in pol
II
9 complexes is not in binding to TFIIS or in intrinsic cleavage
activity. Rather, in pol II
9 complexes, TFIIS stimulated
read-through occurs much more slowly than with the wild-type enzyme.
The reactivation of arrested pol II
9 complexes is therefore affected
at a step after TFIIS binding and transcript cleavage. Perhaps RPB9
facilitates the conversion of an arrest-competent conformation to a
read-through competent conformation, and this interconversion is a
obligatory step in the read-through process. Thus, our observations
define at least a three-step process for the release from the arrested
state; binding to TFIIS, induction of transcript cleavage, and then a
reactivation step(s) that likely includes a conformational change. This
multistep model is consistent with the observations of
Ciprés-Palacín and Kane, who also showed, using mutants
of TFIIS, that transcript cleavage could be uncoupled from read-through
(44).
There is a sequential appearance of transcript cleavage products in
both mammalian (34) and yeast RNA polymerase II arrest complexes at the
T1a site. For the mammalian enzyme, only cleavage to the first site was
required for read-through (35). The cleavage to C1 can occur in pol
II9 complexes in response to low levels of TFIIS, although the
kinetics are slower than with the wild-type enzyme. However, at these
levels of TFIIS, the subsequent transcript cleavage events are not
detected in the absence of RPB9, nor is read-through detected even when
the first cleavage event has occurred in the absence of RPB9. Our
observations raise the possibility that transcript cleavage by purified
pol II at the TIa site can be distinguished into two steps. The first
cleavage event, to C1, is not sufficient for read-through. Rather,
cleavage to the C2 site, and perhaps an accompanying conformational
change, is correlated with the read-through process.
Transcription arrest, according to the model described above, is mediated by a particular conformation(s) of the polymerase that interacts specifically with the DNA in arrest sites. Elongating polymerase is proposed to contain two DNA binding domains, one upstream and one downstream of the position of the active site (21, 45). The downstream DNA binding domain likely plays a significant role in recognition of the arrest site because in many instances the sequences that trigger arrest lie downstream of the catalytic site in regions yet to be transcribed (23). The downstream DNA binding activity of RNA polymerase may also play a role in the selection of the transcription start site. In yeast, start site selection is thought to involve specific recognition of DNA sequences ahead of the actual start site (46) because the distance from the TATA box to the start site in yeast can vary anywhere from 40 to 120 bases (47). The most frequently used start site(s) therefore cannot be chosen based on a set distance from the TATA box. Rather, the DNA sequences encompassing the start site are likely selected by pol II, perhaps via a scanning mechanism (48, 49).
In cells lacking RPB9, the selection of the start site appears less
stringent than in wild-type cells. In these cells, pol II9 uses a
wider range of start sites, which are located upstream of the major
sites used in wild-type cells (15, 17). The pol II
9 enzyme also uses
these altered start sites in vitro (15). Perhaps this start
site alteration is related to the altered elongation properties of the
pol II
9 enzyme. RNA polymerase, once recruited to the promoter, may
need to recognize and bind specific sequences to position the catalytic
site for accurate initiation. pol II
9 may be less capable of
effective recognition and binding to the start site because an
increased proportion of the enzyme is in the read-through conformation.
In other words, the pol II
9 enzyme does not efficiently arrest to
allow the correct positioning of the catalytic site at the
transcription start site. RPB9 would allow pol II to adopt a
conformation compatible with selective binding to the sequences that
constitute the start sites of transcription.
A necessary feature of this model is that promoter sequences and arrest sites share common features recognized by RNA polymerase. Although sequence similarities are not readily apparent, promoter release and release from intrinsic arrest sites share many characteristics. First, the release of polymerase from both a promoter and intrinsic arrest sites appears to be unfavorable in that the polymerase utilizes accessory factors to facilitate each process (50-52). Second, the polymerase is catalytically active within both initiation and elongation complexes and is able to perform abortive synthesis of small transcripts while remaining in place. For example, when bound to a promoter, RNA polymerases catalyze the formation of small RNAs that are abortively terminated and ejected from the complex (53-55). Similarly in arrested ternary complexes, the enzyme can also catalyze several cycles of transcript cleavage and re-extension with ejection of the small cleaved RNAs (29, 34, 36, 56). Third, for bacterial RNA polymerase, escape from both promoter sequences and the movement through blocks to elongation is accompanied by a downstream movement of the forward edge of the enzyme, as detected by a significant increase in the size of nuclease footprint ahead of the position of the catalytic center of the enzyme (24, 53, 57). Fourth, the biochemical analysis of yeast RNA polymerase III harboring a point mutation in the C160 subunit has also revealed functional connections between promoter clearance, arrest site recognition, and transcript cleavage (58). The C160 mutant polymerase more efficiently recognized arrest sites, less efficiently cleared the promoter, and showed increased transcript cleavage. Fifth, recent studies with E. coli RNA polymerase demonstrate that the bacterial elongation factors, GreA and GreB, also stimulate the conversion from an abortive synthesis complex at a promoter to an elongation competent ternary complex (55).
RNA polymerase lacking subunit RPB9 clearly has altered initiation and
elongation properties. The ability to reconstitute wild-type pol II
activity from purified pol II9 and RPB9 will facilitate the
comparison of the in vivo and in vitro behavior of RPB9 mutants. Finally, the stability of the pol II
9 arrested complexes, even in the presence of TFIIS, provides a means to derive
structural information about the complex of an elongating RNA
polymerase bound to TFIIS.
We thank Drs. M. Hull and N. Woychik for providing the WY9 strain and D. Reines and M. Chamberlin for helpful discussions.