From the Banting and Best Department of Medical
Research and Department of Medical Genetics and Microbiology, C. H. Best Institute, University of Toronto, Toronto, Ontario M5G 1L6, Canada
and the § Department of Biochemistry, Johns Hopkins
University, Baltimore, Maryland 21205
Received for publication, April 13, 2000, and in revised form, November 16, 2000
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ABSTRACT |
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The Rpb4 and Rpb7 subunits of yeast RNA
polymerase II form a heterodimeric complex essential for
promoter-directed transcription initiation in a reconstituted system.
Results of template competition experiments indicate that the Rpb4-Rpb7
complex is not required for stable recruitment of polymerase to active
preinitiation complexes, suggesting that Rpb4-Rpb7 mediates an
essential step subsequent to promoter binding. Sequence and
structure-based alignments revealed a possible OB-fold
single-strand nucleic acid-binding motif in Rpb7. Purified
Rpb4-Rpb7 complex exhibited both single-strand DNA- and RNA-binding
activities, and a small deletion in the putative OB-fold nucleic
acid-binding surface of Rpb7 abolished binding activity without
affecting the stability of the Rpb4-Rpb7 complex or its ability to
associate with polymerase. The same mutation destroyed the
transcription activity of the Rpb4-Rpb7 complex. A separate
deletion elsewhere in the OB-fold motif of Rpb7 also blocked
transcription but did not affect nucleic acid binding, suggesting that
the OB-fold of Rpb7 mediates both DNA-protein and protein-protein
interactions required for productive initiation.
Cellular RNA polymerases contain a core set of subunits
exemplified by the Several characteristics of a heterodimeric complex of
Saccharomyces cerevisiae Rpb4 and Rpb7 bear resemblance to
prokaryotic Rpb7 is essential for cell viability in yeast (11) but Rpb4 is not
(12). Some of the stress-sensitive phenotypes linked to an
rpb4 null mutation are suppressed by overproduction of Rpb7 (9). Biochemical evidence suggests that the yeast Rpb4 subunit may
facilitate association of Rpb7 with core polymerase (5). Taken
together, these findings suggest that the Rpb7 subunit is key to the
transcriptional activity of the Rpb4-Rpb7 complex. Here we show that
Rpb7 contains a putative OB-fold domain that seems structurally and
topologically related to a subfamily of domains typically found in
proteins involved in ssRNA and ssDNA binding. Biochemical studies with
wild-type Rpb4-Rpb7 complex and variants with targeted mutations in
this domain reveal a possible link between nucleic acid-binding
activity and an essential step in promoter utilization that occurs
after formation of a stable preinitiation complex.
Baculovirus Expression of Yeast Rpb4 and Rpb7--
Open reading
frames (ORFs) were amplified by polymerase chain reaction from yeast
genomic DNA using primers containing an NdeI restriction
site at the 5' end and a BamHI restriction site at the 3'
end of each ORF. The RPB4 gene was subcloned into the pET15b
bacterial expression plasmid (Novagen), appending a hexahistidine tag
and a thrombin cleavage site to the N terminus of Rpb4. The DNA insert
encoding His-tagged Rpb4 was excised with XbaI and BamHI and ligated between the XbaI and
BglII sites of the baculovirus transfer vector PacAB 4 (Pharmingen) to create PacAB 4/RPB4. The RPB7 gene was
cloned into PacAB 4/RPB4 between the SmaI and
BamHI sites to create PacAB 4/RPB4/7. To create recombinant
baculoviruses, PacAB 4/RPB4/7 was cotransfected into Sf9 cells
using calcium phosphate precipitation with 0.5 µg of baculovirus
GoldTM digested with Bsu 36I (New
England BioLabs). Recombinant virus containing the RPB4 and
RPB7 genes, identified by blue/white plaque screening, was
plaque-purified twice and then amplified for large-scale protein expression.
To prepare individual Rpb4 and Rpb7 recombinant baculoviruses,
RPB4 and RPB7 genes were cloned into the
baculovirus transfer vector PVL 1392 (Pharmingen). The RPB7
ORF was first cloned between the NdeI and BamHI
sites in PET11a (Novagen), and RPB4 was cloned into pET15b.
Each construct was digested with XbaI and BamHI, and the resulting ORF-containing fragments were subcloned between the
XbaI and BamHI sites of PVL 1392.
In-frame deletions in RPB7 in the PVL 1392 construct were
prepared with the QuikChange site-directed mutagenesis system
(Stratagene). After confirming each deletion by sequencing,
RPB7 alleles were subcloned between the EcoRI and
BamHI sites of pFasBac 1 (Invitrogen). Clones were
transformed into DH10Bac competent cells (Life Technologies, Inc.) to
generate recombinant baculoviruses. Rpb7 variant proteins were
coexpressed with Rpb4 in Sf9 cells by coinfection with
recombinant baculoviruses.
20 plates of Sf9 cells (150 × 20 mm; Nunc), each
containing roughly 2 × 107 cells, were infected with
250 µl of high titer virus stock. The optimal multiplicity of
infection for protein expression was determined by SDS polyacrylamide
gel electrophoresis. Infected cells were scraped from plates and
collected by centrifugation at 800 × g for 20 min.
Cell pellets were washed in 25 ml of 50 mM Hepes (pH 7.5),
150 mM NaCl to remove any remaining medium and then
resuspended in Buffer A (50 mM Hepes (pH 7.5), 750 mM NaCl, 10% glycerol, 5 mM imidazole, 0.1%
Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride, 1 mM benzamidine). Resuspended cells were flash frozen and
stored at Purification of Rpb4-Rpb7 Complexes--
Infected cells were
thawed on ice and broken by sonication (3 times for 1 min). Subsequent
steps were performed at 4 °C. The sonicate was clarified by
centrifugation at 100,000 × g for 45 min in a Beckman
Ti70 rotor. The supernatant was loaded onto a 2-ml His-Bind resin
column (Novagen) and washed with Buffer A until the eluate contained
less than 10 µg of protein per ml (Bio-Rad protein assay). The
His-Bind column was then washed with 10 columns of Buffer A containing
50 mM imidazole but lacking Nonidet P-40 and protease
inhibitors. Cleavage to remove the hexahistidine tag was performed on
the column by loading 30 µg of thrombin (Parke-Davies) in 2 ml of
buffer containing 50 mM Hepes (pH 7.5), 150 mM
NaCl, 5% glycerol (cleavage buffer) and incubating the column at
4 °C overnight. Thrombin was next eluted from the column by washing with 2 column volumes of cleavage buffer. The Rpb4-Rpb7 complex lacking
the histidine tag was then eluted by raising the NaCl and imidazole
concentrations to 500 and 50 mM, respectively (residual uncleaved His-tagged Rpb4-Rpb7 complex remained on the column and was
recovered by elution with 500 mM imidazole). The cleaved (untagged) complex was diluted to 100 mM NaCl and purified
further on a Mono Q column (5 × 50 mm; Amersham Pharmacia
Biotech) developed with a linear gradient (10 ml) of potassium acetate
(pH 7.5) from 100 mM to 1 M at 0.5 ml/min.
Fractions containing the Rpb4-Rpb7 complex (identified by SDS
polyacrylamide gel electrophoresis) were pooled, dialyzed for 12 h
against 50 mM Hepes (pH 7.6), 400 mM potassium
acetate, 10% glycerol, 1 mM dithiothreitol, and
concentrated in a Centricon 30 device (Amicon). Expression and
purification of Rpb4-Rpb7 variant complexes were carried out exactly as
described for the wild-type complex.
Biochemical Assays--
To test for single-strand nucleic
acid-binding activity, purified Rpb4-Rpb7 complex was incubated for 30 min on ice with 25 fmol of a 32P end-labeled
oligonucleotide (18-mer) in 20 µl of buffer containing 40 mM Hepes (pH 7.2), 100 mM potassium acetate,
10% glycerol. Bound and free probes were resolved by electrophoresis
in a 7.5% polyacrylamide gel containing 2% glycerol and 0.5× TBE.
Radiolabeled DNA was visualized by autoradiography and quantified using
a phosphorimager (Storm). RNA binding (65-mer) was carried out under
the same conditions except for the addition of 0.5 units of RNase
inhibitor (Promega).
For transcription assays, wild-type RNA polymerase II, TFIIF, TFIIH,
and Mediator were purified from yeast whole-cell extract (13-16). RNA
polymerase II lacking Rpb4 and Rpb7 subunits (pol II A Predicted Nucleic Acid-binding Domain in Rpb7--
The sequence
of the archaea Rpb7 homologue is similar to that of the ribosomal S1
domain from E. coli. The S1 protein exemplifies the OB-fold
(22), a structure found in many other proteins with single-stranded
nucleic acid-binding activity (23, 24) (Fig. 1a). The OB-fold motif
consists of a 5-stranded
Our study had the following three goals: to purify a transcriptionally
active recombinant Rpb4-Rpb7 complex, to test its nucleic acid-binding
activity, and to explore its role in transcription initiation. To
generate recombinant Rpb4-Rpb7 complex, subunits were first expressed
individually or together in E. coli. Although both subunits
accumulated to high levels in cells, they proved to be insoluble.
However, a soluble complex was recovered from insect cells coinfected
with recombinant baculoviruses expressing Rpb4 and Rpb7 or from cells
infected with a single recombinant baculovirus encoding both proteins.
Purification of the complex from insect cell lysates was facilitated by
adding a hexahistidine tag and thrombin cleavage site to the N terminus
of Rpb4. The complex was immobilized on a metal affinity column and
subsequently released (after extensive washing to remove unbound
contaminants) by digestion of Rpb4 with thrombin. Following subsequent
anion exchange chromatography, the purified complex was judged to be at
least 95% homogeneous by SDS polyacrylamide gel electrophoresis and
Coomassie Blue staining (Fig. 2), in a
yield of 200 µg per liter of Sf9 cells.
We designed mutant Rpb4-Rpb7 complexes containing small in-frame
deletions in the predicted OB-fold of Rpb7 using information from both
sequence alignments and detailed structural knowledge of OB-fold
interactions with nucleic acids (Fig. 1b). We constructed three Rpb7 mutants predicted to be compromised for ssDNA-binding (Fig.
1c). The first deletion,
Rpb7
The three Rpb7 variants were coexpressed with histidine-tagged Rpb4
(Fig. 2). The structural integrity of mutant proteins was tested by
monitoring the ability of each mutant to dimerize with Rpb4. The Rpb7D1
mutant was expressed but failed to copurify with Rpb4, suggesting
severe mis-folding. This mutant was not analyzed further. By contrast,
both the Rpb7D2 and Rpb7D3 variants formed a stable complex with Rpb4,
as judged by copurification with histidine-tagged Rpb4. Moreover, the
resulting mutant Rpb4-Rpb7 complexes were able to interact normally
with pol II Nucleic Acid Binding by Rpb4-Rpb7 Complexes--
To test purified
wild-type Rpb4-Rpb7 for single-strand nucleic acid-binding activity, we
performed gel mobility shift assays with a radiolabeled ssDNA
oligonucleotide probe. As predicted from its OB-fold motif, the
Rpb4-Rpb7 complex bound to ssDNA in a saturable and reversible manner
(Fig. 3) with an apparent dissociation constant of 0.7 µM (determined by half-maximal binding at
equilibrium with protein in large molar excess). Gel mobility shift
assays with an RNA oligonucleotide probe revealed similar binding to ssRNA with an apparent dissociation constant of 1.2 µM
(data not shown; see Fig. 4).
We were unable to measure the binding activity of wild-type Rpb7 in
isolation, because the protein was insoluble in the absence of Rpb4
(the same held true for N- and C-terminal Rpb7 deletion variants and
for several internal deletion variants, as well). We therefore tested
the Rpb4-Rpb7D2 and Rpb4-Rpb7D3 complexes on ssRNA and ssDNA to
identify region(s) in Rpb7 that mediate nucleic acid binding. The
Rpb7D3 mutation deleting the putative L45 loop between the predicted
Transcription Initiation in Reconstituted System--
Crude cell
extract prepared from yeast lacking the RPB4 gene fails to
support promoter-directed transcription, but activity can be restored
by adding purified Rpb4-Rpb7 complex resolved from polymerase by ion
exchange chromatography in the presence of urea (5). To confirm that
the Rpb4-Rpb7 complex is required in transcription reactions
reconstituted with purified components (and therefore lacking
nonspecific inhibitors present in crude extracts; see Ref. 13), we
assayed the activity of baculovirus-expressed Rpb4-Rpb7 complex with
the minimal set of yeast GTFs and highly purified pol II
In contrast to wild-type Rpb4-Rpb7 complex, complexes containing the
Rpb7D2 or D3 mutants were completely inactive in transcription (Fig.
5a, lanes 6-11). We performed order of addition
transcription experiments to test whether the D2 or D3 mutant complexes
could compete with wild-type complex for stable binding to polymerase. When pol II
To test the generality of the requirement for the Rpb4-Rpb7 complex, we
tried transcription with several different promoters. The complex was
required for initiation on the TEF1, CYC1, and AdML promoters (Fig. 5c) regardless of DNA template topology
(linear or supercoiled). As expected (25), addition of Mediator to the reconstituted system boosted basal transcription by pol II Rpb4 and Rpb7 Act after the Formation of a Stable Preinitiation
Complex--
Transcription initiation can be resolved biochemically
into an ordered series of steps beginning with the assembly of a
preinitiation complex and recruitment ("commitment") of polymerase
to the promoter (see Refs. 26-28; reviewed in Refs. 3 and 4). Previous
binding studies comparing the affinity of wild-type (12-subunit)
polymerase and pol II In this study we focused on the nucleic acid-binding activity of
the yeast Rpb4-Rpb7 complex and on its role in transcription initiation. Our results show that the Rpb4-Rpb7 complex binds fairly
strongly to ssDNA and RNA in vitro, as predicted from the sequence and structural homology of the OB-fold motif of Rpb7 to the S1
domain (22, 30). The complex binds RNA and DNA with nearly equal
affinity, and a mutation in the putative OB-fold abrogates binding to
both substrates, indicating a common site for interaction. Because the
OB-fold occurs in both RNA- and DNA-binding proteins, and the two types
of interaction seem structurally indistinguishable, the physiological
substrate remains to be determined.
The Rpb4-Rpb7D2 complex bearing a deletion in Endogenous pol II Reconstructions of yeast RNA polymerase II structures from electron
micrographs place the Rpb4-Rpb7 complex in the putative DNA-binding
cleft of the polymerase and suggest that the complex favors a closed
conformation of the polymerase "arm" domain around the cleft (29).
These observations led to the hypothesis that Rpb4-Rpb7 may stabilize
polymerase binding by contacting DNA in the cleft and somehow coupling
DNA entry to closure of the cleft. It is tempting to speculate that the
Rpb7D2 mutation may disrupt the DNA "sensor," whereas the D3
mutation may disrupt protein-protein interactions that transduce the
DNA signal to the polymerase arm domain. A more detailed structural
model of polymerase bound to DNA places Rpb4-Rpb7 downstream of the
catalytic site in the cleft (35), and because our biochemical findings
indicate that Rpb4-Rpb7 complex mediates a step following template
commitment, we speculate that it may stabilize the open promoter
complex prior to initiation and/or an early transcribing complex prior
to promoter escape, perhaps by binding to ssDNA in the transcript
"bubble" (in analogy to bacterial
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,
, and
' subunits of the eubacterial
enzyme. Eukaryotic nuclear RNA polymerases have an additional 8-9
accessory subunits that are shared, unique, or similar in the three
classes of polymerase (I, II, and III) (1). RNA polymerase II, which is
mainly responsible for transcribing protein coding genes, consists of
twelve subunits, named Rpb1-12, from largest to smallest (2). The
Rpb1, Rpb2, Rpb3, and Rpb11 subunits are the functional and structural
homologues of bacterial core subunits. Along with accessory Rpb
subunits, RNA polymerase II uses additional proteins called general
transcription factors (GTFs)1
to initiate transcription in a promoter-dependent fashion
(reviewed in Refs. 3 and 4). The accessory Rpbs and GTFs seem to play a
role analogous to that of the
subunits of prokaryotic RNA polymerases, which enable the core catalytic enzyme (
2
') to bind specifically to start site regions, initiate transcription efficiently, and respond to regulatory inputs.
factors; the Rbp4-Rpb7 complex is absolutely required
for accurate initiation but dispensable for RNA chain elongation (5),
and the complex can associate reversibly with core (10-subunit) RNA polymerase II (5). Moreover, the Rpb4 and Rpb7 subunits are less
abundant than other polymerase subunits in vivo (6) and appear to be critical for cellular adaptations to stress, a hallmark of
many
factors (6-9). Finally, overexpression of Rpb7 in wild-type yeast induces cell filamentation, suggesting that Rpb7 may promote gene-specific transcription (10).
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80 C.
4/7) was
purified from a rpb4
yeast strain as described (5). Yeast
TBP, TFIIB, and TFIIE were purified from recombinant
Escherichia coli strains (17, 18). Plasmid DNA
templates contained the CYC1, AdML, or TEF1
promoters fused to G-less cassettes (13, 19). Template competition
experiments employed the CYC1/G-less cassette
plasmids pJJ460 and pJJ470 (20). ATP, CTP, UTP, and [
-32P]UTP (3000 Ci/mmol) were from Amersham Pharmacia
Biotech. Preinitiation complexes were assembled during a 10-min
incubation at 23°C in reaction mixtures (20 µl) containing 20 mM Hepes-KOH (pH 7.6), 7 mM magnesium acetate,
100-110 mM potassium acetate, 250 ng of plasmid DNA, 100 ng of RNA polymerase II, 30 ng of TBP, 30 ng of TFIIB, 15 ng of TFIIE,
50-100 ng each of TFIIF and TFIIH, and (where indicated) 100 ng of
Mediator. Purified Rpb4-Rpb7 complex or dilution buffer was added as
indicated, and transcription was initiated by adding 5 µl of a
solution containing 20 mM Hepes-KOH (pH 7.6), 7 mM magnesium acetate, 2.5 mM ATP, 2.5 mM CTP, 125 µM UTP, 5 µCi of
[
-32P]UTP. For template competition experiments, this
NTP mix (5 µl) also contained 250 ng of competitor template DNA(s) as
indicated. Labeling reactions were carried out at 23°C for 15-30 min
and then terminated by adding stop mix (0.2 ml) containing 0.3 M NaCl and 2 units of T1 ribonuclease (Calbiochem).
Radiolabeled transcripts were processed for denaturing gel
electrophoresis and detected by autoradiography as described (21).
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-sheet that is coiled to form a closed
-barrel and an
-helix that connects the third and fourth strands
(Fig. 1, b and c). Some general observations about the interaction of OB-fold proteins with single-stranded nucleic
acid can be gleaned from the two known structures of OB-fold/nucleic acid complexes. First, the
-strands 1-3 (Fig. 1b,
green) form a scaffold across which nucleic acid is splayed,
making hydrogen bond and stacking contacts. Second, the loop between
the first and second strands (L12; Fig. 1b, red)
makes key hydrogen bonds with the phosphate backbone. Third, the loop
that connects the fourth and fifth
-strands (L45; Fig.
1b, yellow) clamps down on the nucleic acid
strand, making hydrogen bond and stacking interactions.
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Fig. 1.
Panel A, sequence alignment of
yeast Rpb7, an S1 domain from E. coli, and the rpoE RNA
polymerase subunit from Sulfolobus. Black and
gray boxes mark amino acid residues conserved in all three
or in two of the three proteins, respectively. Panel B,
positions of Rpb7 deletions superimposed on a typical OB-fold structure
(residues 180-290 of the largest subunit of human replication protein
A). The DNA channel in replication protein A lies between the D1 and D3
deletions when visualized in this orientation. Panel C,
boxes enclose missing amino acid residues in Rpb7 deletions
D1, D2, and D3.
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Fig. 2.
Purification of wild-type
(W.t.) and deletion mutant (D2 and
D3) Rpb4-Rpb7 complexes. Purified complexes were
resolved by denaturing gel electrophoresis and stained with Coomassie
Blue. Bands representing Rpb4 and Rpb7 subunits are
indicated.
91-97 (D1), was predicted to disrupt
-strand 1 and part of the L12 loop. The second deletion, Rpb7
108-113 (D2), was predicted to disrupt
-strand 3, which harbors an aromatic residue (Phe in this case)
critical for stacking interactions in all known structures. The third
deletion, Rpb7
151-158 (D3), was predicted
to disrupt the L45 loop, which contributes to stacking interactions in
one structure but is less well conserved among the known OB-fold proteins.
4/7 (5) as judged by comigration with the
polymerase on a size-exclusion column (data not shown) and by
competition experiments with wild-type Rpb4-Rpb7 complex (see below).
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Fig. 3.
DNA-binding activity of wild-type Rpb4-Rpb7
complex. A radiolabeled DNA probe was incubated with indicated
amounts (micrograms) of purified Rpb4-Rpb7 complex as described under
"Experimental Procedures." Resulting complexes were resolved from
unbound DNA by electrophoresis in a nondenaturing polyacrylamide gel.
Bands formed by free DNA and protein-DNA complexes
(arrows) were detected by autoradiography.
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Fig. 4.
RNA-binding activity of wild-type and
deletion mutant (D2 and D3) Rpb4-Rpb7
complexes. A radiolabeled RNA probe was incubated with indicated
amounts (micrograms) of purified Rpb4-Rpb7 complexes as described under
"Experimental Procedures." Products were analyzed as for Fig.
3.
-strands had no apparent effect on nucleic acid binding (Fig. 4). By
contrast, the affinity of the Rpb4-Rpb7D2 complex for RNA (Fig. 4) and
DNA (not shown) was at least 10-fold lower than normal. These results
suggest that this part of Rpb7 may be principally responsible for the
observed nucleic acid binding, as expected from structure predictions.
4/7. As
expected (5), pol II
4/7 was completely inactive in promoter-directed
transcription in this well defined system. Moreover, addition of
purified recombinant Rpb4-Rpb7 complex supported transcription by pol
II
4/7 at levels achieved by native (12-subunit) polymerase (Fig.
5a, lanes
1-4).
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Fig. 5.
Transcription activities of recombinant
Rpb4-Rpb7 complexes. Panel A, transcription reactions
with a CYC1 promoter template contained pol II 4/7
(lanes 1-4 and 6-11) or wild-type polymerase
(lanes 12-14) and indicated amounts (ng) of wild-type
(WT) or mutant (D2 and D3) Rpb4-Rpb7
complexes. Control reactions lacked Rpb4-Rpb7 complex (lane
1) or polymerase (lane 5). The arrow marks
bands formed by labeled RNA transcripts in a denaturing
polyacrylamide gel. Panel B, order of addition experiments
with wild-type and mutant Rpb4-Rpb7 complexes. Transcription reactions
contained 100 ng of pol II
4/7 and either 10 ng of wild-type
Rpb4-Rpb7 complex alone (WT 4/7; lane 5), or with
10 ng of mutant (D2 and D3) Rpb4-Rpb7 complex
(lanes 1-4 and 6-13) added either sequentially
or simultaneously as indicated. Molar ratios of mutant to wild-type
complexes are indicated above the lanes.
Transcripts are marked as in A. Panel C,
supercoiled (sc) or linearized (lin) plasmid
templates containing three different promoters (TEF1, AdML,
and CYC1) were transcribed with pol II
4/7 in the absence
or presence of wild-type recombinant Rpb4-Rpb7 complex (10 ng) as
indicated. Transcripts are marked as in A.
4/7 was preincubated with wild-type Rpb4-Rpb7 complex, subsequent transcription was only slightly reduced by adding excess mutant complexes (Fig. 5b, lanes 1-5). By
contrast, when polymerase was preincubated with the D2 or D3 mutant
complex first, subsequent transcription in the presence of wild-type
Rpb4-Rpb7 fell sharply (Fig. 5b, lanes 6-9);
when wild-type and mutant Rpb4-Rpb7 complexes were copreincubated prior
to transcription, the mutant complexes inhibited RNA production in a
dose-dependent manner (Fig. 5b, lanes
10-13). Taken together with size-exclusion chromatography behavior (not shown), these results suggest that both of the mutant Rpb4-Rpb7 complexes bind normally to RNA polymerase II and can even
displace wild-type Rpb4-Rpb7, as expected for reversible binding to the
same site on polymerase.
4/7 in the
presence of wild-type Rpb4-Rpb7 complex, but it did not obviate the
strict requirement for Rpb4-Rpb7 (not shown).
4/7 for a TBP/TFIIB complex implicated
Rpb4 and/or Rpb7 in the assembly of a preinitiation complex (29). To
explore this question further in the functional context of
transcription, we assessed the ability of pol II
4/7 to form a stable
preinitiation complex in the absence or presence of Rpb4-Rpb7.
Reactions contained two otherwise identical CYC1 promoter
templates encoding G-less cassette transcripts of different lengths
(20), and saturating amounts of TBP, TFIIB, TFIIE, and TFIIF, as
determined by factor titrations and by DNase I footprinting assays
showing full TATA box occupancy by TBP under identical
conditions.2 When pol
II
4/7 and the GTFs were incubated with both templates simultaneously, subsequent transcription reactions yielded equal amounts of both sets of promoter-directed transcripts (Fig.
6, lanes 1 and 2).
However, if pol II
4/7 and GTFs were preincubated with DNA I prior to
adding DNA II and NTPs, transcription ensued almost exclusively on DNA
I (lane 3). No such template commitment was observed if TBP
and TFIIB were omitted from the preincubation and added with DNA II
(data not shown). In contrast to TBP and TFIIB, Rpb4-Rpb7 complex was
not required for template commitment; the degree of commitment to DNA I
was identical regardless of whether the Rpb4-Rpb7 complex was present
in the preincubation step (compare lanes 3 and
4). These results indicate that polymerase can join a
stable, active preinitiation complex in the absence of Rpb4 and Rpb7
and therefore suggest that the essential function of the Rpb4-Rpb7
complex (Fig. 5a) comes after polymerase binds to the
promoter.
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Fig. 6.
Stable recruitment of RNA polymerase II into
preinitiation complexes in the absence of Rpb4 and Rpb7. pol
II 4/7 (100 ng) was preincubated with GTFs and Mediator for 30 min in
the absence or presence of a short G-less cassette template (DNA I) and
Rpb4-Rpb7 complex (10 ng) as indicated above the
lanes and in a schematic diagram. Rpb4-Rpb7
complex was then added to reactions in which it was lacking, followed
15 s later by simultaneous addition of DNA I and a long G-less
cassette template (DNA II; lanes 1 and 2), or by
addition of DNA II alone (lanes 3 and 4), along
with NTPs for RNA synthesis. Transcription reactions were stopped after
15 min. Specifically initiated transcripts from each template
(RNA I and RNA II) were detected as in Fig.
5.
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-strand 3 of the
putative OB-fold DNA-binding surface (Fig. 1) was indeed defective in
nucleic acid binding and moreover was completely devoid of transcription activity, raising the possibility that nucleic acid binding is key to the function of the Rpb4-Rpb7 complex. However, the
Rpb7 D3 deletion disrupting the putative L45 loop (Fig. 1) was also
completely inactive in transcription, even though it retained nucleic
acid-binding activity. Our sequence alignment may not accurately
predict the exact boundaries of the putative OB-fold, and the D3
deletion may lie outside the critical DNA-binding surfaces. Even if the
alignment is correct, the L45 loop in Rpb7 may simply be less important
for stabilizing nucleic acid interactions compared with other OB-folds.
In any case, this portion of Rpb7 evidently has a key role in
transcription distinct from DNA binding. Indeed, the ssDNA-binding
activity of the Rpb4-Rpb7 complex may be peripheral to transcription
activity, and the deleterious effect of the D2 mutation on both
activities may be coincidental. Further genetic, biochemical, and
structural studies are needed to fully delineate the role of the
OB-fold motif of Rpb7 in DNA/RNA-binding and transcription. The C25
subunit of RNA polymerase III appears to be a Rpb7 homologue (31). It
will be interesting to see whether this subunit also possesses nucleic
acid-binding activity and if so, whether it is important for
transcription initiation on class III promoters.
4/7 was incapable of promoter utilization in a
crude extract unless supplied with purified Rpb4-Rpb7 complex or
(alternatively) Gal4-VP16, a potent transcription activator (5). The
extract provided GTFs and Mediator, as well as pol II
4/7, and likely
contained Rpb7, as well (Rpb7 is essential for yeast viability). Thus,
our previous experiments did not rule out a role for Rpb7 in activated
transcription. Results reported here confirm that the Rpb4-Rpb7 complex
is indeed required for basal transcription in a completely defined
reconstituted system. We also find that the complex is required for
transcription in the presence of Mediator, indicating an important role
for the Rpb4-Rpb7 complex in transcription by RNA polymerase II
holoenzyme (16, 25, 32). Further work is needed to explore the role of
Rpb7 in activation by enhancer-binding proteins. The defining biological feature of
factors is their capacity to confer promoter class specificity on polymerase and thereby orchestrate multigene transcription programs for cellular development and responses to stress
(reviewed in Ref. 33). The real test to see whether yeast Rpb4-Rpb7
complex plays a similar role will come from whole-genome analysis with
suitable mutants (e.g. see Ref. 34).
factors) or to nascent RNA.
Sorting this out will require further biochemical studies using
cross-linking, permangenate footprinting, and kinetic measurements
along the lines used for analyzing mammalian systems (36-40).
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ACKNOWLEDGEMENTS |
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Purified yeast Mediator was a generous gift from Larry Myers and Roger Kornberg. We thank Matthew Healy, Pilar Tijerina, and Byung Ahn for providing purified transcription factors and Stefan Larsen for helping prepare some of the figures.
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FOOTNOTES |
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* This work was supported in part by grants from the Medical Research Council of Canada (MRC) (to A. M. E.) and by Grant GM50724 from the National Institutes of Health (NIH) (to M. H. S.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ Recipient of Predoctoral Research Fellowship CA73466 from the NIH.
Current address: Division of Molecular and Cellular
Mechanisms, NIH Center for Scientific Review, 6701 Rockledge Dr.,
Bethesda, MD 20892. Recipient of a Junior Faculty research award from
the American Cancer Society.
** MRC Scientist. To whom correspondence should be addressed: Banting and Best Dept. of Medical Research and Dept. of Medical Genetics and Microbiology, C. H. Best Inst., University of Toronto, 112 College St., Toronto, Ontario M5G 1L6, Canada. Tel.: 416-946-3436; Fax: 416-978-8528; E-mail: aled.edwards@utoronto.ca.
Published, JBC Papers in Press, November 21, 2000, DOI 10.1074/jbc.M003165200
2 M. S. Healy, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are:
GTFs, general
transcription factors;
ss, single-strand;
ORF(s), open reading frame(s);
pol II4/7, RNA polymerase II lacking Rpb4 and Rpb7
subunits;
TF, transcription factor;
TBP, TATA-binding protein;
D, deletion.
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