From the Department of Microbiology and
§ Graduate Program of Molecular and Cellular Biology,
State University of New York at Stony Brook, Stony Brook, New York
11794, and the ¶ Howard Hughes Medical Institute, Cold Spring
Harbor Laboratory, Cold Spring Harbor, New York 11724
Received for publication, January 4, 2001, and in revised form, February 8, 2001
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ABSTRACT |
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The human U6 small nuclear (sn) RNA core promoter
consists of a proximal sequence element, which recruits the
multisubunit factor SNAPc, and a TATA box, which
recruits the TATA box-binding protein, TBP. In addition to
SNAPc and TBP, transcription from the human U6 promoter
requires two well defined factors. The first is hB", a human homologue
of the B" subunit of yeast TFIIIB generally required for transcription
of RNA polymerase III genes, and the second is hBRFU, one of two human
homologues of the yeast TFIIIB subunit BRF specifically required for
transcription of U6-type RNA polymerase III promoters. Here, we have
partially purified and characterized a RNA polymerase III complex that
can direct transcription from the human U6 promoter when combined with
recombinant SNAPc, recombinant TBP, recombinant hB", and
recombinant hBRFU. These results open the way to reconstitution of U6
transcription from entirely defined components.
The nuclear eucaryotic RNA polymerases cannot recognize their
target promoters without the help of transcription factors. In the case
of RNA polymerase II, basal transcription from a TATA-containing mRNA promoter can be reconstituted in vitro with a set
of recombinant or entirely defined factors, both in Saccharomyces
cerevisiae and in mammalian systems (1-4). In the case of RNA
polymerase III, however, this has been achieved only in the yeast
system, for transcription of the U6 snRNA gene (5).
The yeast U6 snRNA promoter consists of a TATA box located upstream of
the transcription start site and A and B boxes located downstream of
the transcription start site (6, 7). The A and B boxes recruit TFIIIC
which, together with the TATA box, then recruit TFIIIB (8). In
vitro, however, the yeast U6 snRNA promoter can be transcribed in
the absence of the B box and TFIIIC because the TATA box is sufficient
to recruit TFIIIB (9, 10). S. cerevisiae TFIIIB is a
three-subunit complex consisting of the TATA box-binding protein
(TBP)1 (11), the
TFIIB-related factor BRF (TDS4/PCF4) (12-14), and the protein B"
(TFIIIB90/TFC5/TFC7) (5, 15, 16). All three components have been
cloned, and TFIIIB has been reconstituted from recombinant subunits
(5). Thus, in the case of the yeast U6 promoter, basal RNA polymerase
III transcription can be reconstituted in vitro with
recombinant TFIIIB and highly purified RNA polymerase III (5).
The human U6 snRNA promoter is, unlike the yeast U6 snRNA promoter,
entirely located upstream of the transcription start site (see Ref. 17
and references therein). The core promoter consists of a proximal
sequence element and a TATA box, and both elements are required
for efficient transcription in vitro. The proximal sequence
element recruits a multisubunit complex known as SNAPc (18)
or PTF (19), which has been reconstituted from recombinant subunits
(20). The TATA box recruits TBP (21, 22). Until recently, however,
little was known about which other TFIIIB components were required for
human U6 transcription because mammalian TFIIIB was only partially
characterized. We recently isolated cDNAs encoding hB", a human
homologue of yeast B", and showed that this factor is required for RNA
polymerase III transcription from the U6 promoter as well as from
gene-internal promoters (23). We also isolated cDNAs encoding
hBRFU, a novel homologue of yeast BRF. Unlike hBRF, a previously
characterized homologue of yeast BRF that is functional for
transcription from gene-internal promoter but not from the U6 promoter,
hBRFU is specifically required for U6 transcription (23). Thus, in a
extract depleted of both BRF and BRFU, addition of recombinant
BRF (and recombinant TBP) specifically reconstituted transcription from
the adenovirus 2 VAI gene-internal promoter, but not from the human U6
promoter (17, 24). Reciprocally, addition of recombinant hBRFU
reconstituted transcription from the human U6 promoter, but not from
the VAI promoter (23).
A protein identical to hBRFU (called TFIIIB50) was very recently
isolated by Teichmann et al. (25) as part of a complex containing several other polypeptides. In the transcription system used
by these authors, addition of recombinant hBRFU to a depleted fraction
did not reconstitute U6 transcription, but addition of a
BRFU-containing complex isolated from HeLa cells expressing a tagged
BRFU did, suggesting that the factors associated with BFRU were
absolutely required for U6 transcription. In addition, a protein called
BRF2 and corresponding to a splice variant of hBRF was also reported to
be involved in U6 transcription (26). In this case also, addition of
recombinant BRF2 could not reconstitute U6 transcription to a depleted
extract, but addition of a BRF2-containing complex immunoprecipitated
from human culture cells expressing tagged BRF2 could.
Here we show that we can purify an RNA polymerase III complex that,
together with recombinant SNAPc, TBP, hB", and hBRFU, can
reconstitute transcription from the human U6 promoter. These results
confirm the essential role of hB", and indicate that all factors
absolutely required for U6 transcription in vitro are present in the RNA polymerase III complex.
Sources of Proteins--
Whole cell extract was prepared from
HeLa suspension cells as described (27) and dialyzed against buffer
D50 (50 mM HEPES, pH 7.9, 0.2 mM
EDTA, 20% glycerol, 0.1% Tween 20, 50 mM KCl, 3 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl
fluoride). Glutathione S-transferase-TBP was expressed in
Escherichia coli BL21(DE3) cells with the T7 expression
system, as previously described (28). The protein was then bound to
glutathione-agarose beads and TBP was released from the beads by
cleavage with thrombin, which cleaved just after the glutathione
S-transferase moiety of the fusion protein. Mono-Q
SNAPc was purified as described in Ref. 29, and recombinant
SNAPc was produced in insect cells and purified as
described in Ref. 20. Recombinant hB" and hBRFU were produced in
E. coli and purified as described (23).
For immunoprecipitation of an RNA polymerase III complex, rabbit
polyclonal anti-BN51 antibodies (CS682) directed against the last 14 amino acids of BN51 (see Fig. 1), either crude or affinity purified,
were cross-linked to protein A-agarose beads (Roche Molecular
Biochemicals) with the dimethyl pimelimidate method as described in
Ref. 30. The beads were mixed with whole cell extract at a 1:1 ratio,
incubated at room temperature for 2 h with agitation, and
collected by centrifugation. The beads were then washed 4 times with
120 bead volumes of buffer D100. The washed beads were then
tested directly in transcription assays. Alternatively, and with
similar results, material bound to the beads was eluted by incubation
with 1 bead volume of buffer D100 containing 100 µg/ml of
the synthetic peptide against which the antibody was raised, and the
eluate was tested in transcription assays.
The RNA polymerase III complex active in U6 transcription was purified
as follows. 500 ml of HeLa whole cell extract (5785 mg of protein) was
first fractionated by an 18-40% ammonium sulfate precipitation. The
precipitate was resuspended in 100 ml of HEDPG (25 mM
HEPES, pH 7.9, 15% glycerol, 0.1 mM EDTA, 0.1% Tween 20, 3 mM dithiothreitol, 0.5 mM
phenylmethylsulfonyl fluoride). The fraction was then diluted with
~400 ml of HEDPG until the conductivity of the sample was equivalent
to that of a 200 mM KCl solution. The sample (1650 mg of
protein) was then loaded onto a 240-ml P11 phosphocellulose column
(Whatman). The column was washed with 3 column volumes of
HEDPG200 (HEDPG with 200 mM KCl) and bound proteins were eluted with a 5-column volume HEDPG gradient extending from 200 to 1000 mM KCl. The P11 fractions containing U6
transcription activity (eluting between 550 and 850 mM KCl)
were pooled (126 mg of protein), diluted with 1400 ml of buffer Q (20 mM HEPES, pH 7.9, 5% glycerol, 0.5 mM EDTA, 10 mM MgCl2, 0.1% Tween 20, 3 mM
dithiothreitol, and 0.5 mM phenylmethylsulfonyl fluoride) to give a final concentration of 50 mM KCl.
The diluted sample was then loaded onto a 10-ml Mono S column (Amersham
Pharmacia Biotech). The column was washed with 3 column volumes of
Q50 (Q buffer containing 50 mM KCl) and bound
proteins were eluted with a 10-column volume Q gradient extending from 50 to 1000 mM KCl. The Mono-S fractions containing the peak
of U6 transcription activity (eluted between 200 and 450 mM
KCl) were pooled (20 mg of protein) and diluted with buffer Q to a KCl
concentration of ~100 mM. The diluted sample was loaded
onto a 1-ml Mono-Q column (Amersham Pharmacia Biotech). The column was
washed with 3 column volumes of Q100, and bound proteins
were eluted with a 10-column volume Q gradient extending from 100 mM KCl to 600 mM KCl. The peak of U6
transcription activity (3 mg of protein) eluted between 450 and 600 mM KCl. A tenth of the Mono-Q peak (300 µg of protein)
was loaded onto a 4.8-ml 10-30% (w/v) sucrose gradient in
D100 buffer. The gradient as well as a parallel gradient
loaded with protein size markers were then subjected to
ultracentrifugation for 11 h at 4 °C, 49,000 rpm, in a SW55 Ti
rotor. 100-µl fractions were withdrawn from the top of the gradient,
tested for U6 transcription, and fractionated on 5-20%
SDS-polyacrylamide gels. The fractions from the reference gradient were
analyzed on a 5-20% SDS-polyacrylamide gel. The resolved polypeptides
were visualized by staining with silver or Coomassie Brilliant Blue
(G-250), or characterized by immunoblotting with antibodies against
known proteins.
Transcription Assays--
The U6 transcription assays were
performed as described (31) in a total volume of 20 µl with 100 ng of
pU6/Hae/RA.2 DNA template. The reactions contained 8 µl of the RNA
polymerase III fractions, 10 ng of recombinant TBP, and 4-6 µl of
Mono-Q SNAPc. In Fig. 7, the reconstitutions were performed
as indicated in the figure legend.
An Anti-RNA Polymerase III Immunoprecipitate Can Direct U6
Transcription When Combined with Mono-Q SNAPc and
Recombinant TBP--
We have described before the isolation of a
cDNA clone encoding the largest subunit of RNA polymerase III and
the generation of an antibody directed against the very C terminus of
this subunit (32). To generate a second antibody directed against RNA
polymerase III, we took advantage of the published sequence of another
RNA polymerase III subunit (33), the BN51 or RPC53 subunit, to generate another anti-peptide antibody. This small subunit which is unique to
RNA polymerase III was originally cloned as the gene that complemented a mutation in a temperature-sensitive cell line that arrests in G1 at the non-permissive temperature (33). Since in
addition to generating antibodies, we were interested in
expressing recombinant BN51 to serve as a marker in immunoblots,
we generated a construct for in vitro translation of
full-length BN51 from a partial cDNA clone (34) and from polymerase
chain reaction fragments obtained from HeLa cell RNA. The resulting
open reading frame (GenBankTM AF346574) differs from
the open reading frame predicted by the BN51 sequence deposited in
GenBankTM (accession number M17754) by 10 insertions, one deletion, and three nucleotide changes. As a result,
the protein sequence predicted by our clone, which we refer to as
hRPC53, differs from the original BN51 protein sequence (accession
number AAA51838) at several positions within the N-terminal
region, as depicted in Fig.
1A. The hRPC53 amino acid
sequence is, however, identical to that predicted by a recent entry in
GenBankTM (AK026588).
We raised rabbit polyclonal antibodies against a peptide corresponding
to the last 14 amino acids of the hRPC53 sequence and tested whether
this antibody could co-immunoprecipitate hRPC155, the largest subunit
of RNA polymerase III. As shown in Fig. 1B, hRPC155 was
detected in HeLa whole cell extract, the starting material for the
immunoprecipitation, and in material immunoprecipitated by anti-hRPC53
antibodies, but not in material immunoprecipitated by preimmune
antibodies (compare lanes 1 and 3 to lane
2). Thus, the anti-hRPC53 antibodies co-immunoprecipitated the
largest subunit of RNA polymerase III, suggesting that they did not
disrupt the multisubunit enzyme.
Wang and colleagues (35) reported the isolation of an RNA polymerase
III holoenzyme capable of directing transcription from RNA polymerase
III genes with gene internal promoters. We tested the ability of the
RNA polymerase III complex immunoprecipitated with the anti-hRPC53
antibody to direct transcription from the gene external human U6 snRNA
promoter, either on its own or combined with recombinant TBP (rTBP), a
partially purified SNAPc fraction (Mono-Q
SNAPc, (29), or both. As shown in Fig.
2A, neither rTBP alone, Mono-Q
SNAPc alone, nor rTBP together with Mono-Q
SNAPc could direct U6 transcription (lanes
1-3). Similarly, immunoprecipitates obtained with preimmune
antibodies, or mock reactions performed with just protein A beads,
showed no or little activity, with or without added rTBP and Mono-Q
SNAPc (lanes 8-15). The anti-hRPC53 immunoprecipitate alone, or complemented with Mono-Q SNAPc
or TBP only, showed little or no activity (lanes 4-6). In
contrast, the anti-hRPC53 immunoprecipitate complemented with both rTBP and Mono-Q SNAPc resulted in high levels of U6
transcription activity (lane 7).
To exclude the possibility that factors required for U6 transcription
co-immunoprecipitated with the hRPC53 subunit of RNA polymerase III
because of indirect interactions with hRPC53 through bridging DNA
rather than because of protein-protein interactions, we performed
immunoprecipitations in the presence of the DNA intercalating agent
ethidium bromide. This agent can eliminate interactions occurring
through bridging DNA (36). As shown in Fig. 2B, the anti-hRPC53 antibodies immunoprecipitated a complex active in U6
transcription when complemented with Mono-Q SNAPc and rTBP regardless of whether the immunoprecipitation was performed in the
absence (lane 3) or presence (lane 5) of ethidium
bromide. Together, these results suggest that the anti-hRPC53
antibodies can immunoprecipitate an RNA polymerase III complex that,
together with rTBP and biochemically purified Mono-Q SNAPc,
is capable of directing accurate and efficient U6 transcription.
A Partially Purified RNA Polymerase III Can Reconstitute U6
Transcription When Combined with Mono-Q SNAPc and
rTBP--
The results above suggest that an immunoprecipitated RNA
polymerase III complex contains all factors required for U6
transcription except for SNAPc, factors other than
SNAPc that might be contained in the Mono-Q
SNAPc fraction, and TBP (in a form that is functional for
U6 transcription). To determine whether we might be able to purify such
a complex by another method than immunoprecipitation, we fractionated a
HeLa whole cell extract as illustrated in Fig. 3. We tested the fractions for their
ability to direct U6 transcription when complemented with Mono-Q
SNAPc and rTBP, and for the presence of the largest subunit
of RNA polymerase III. We first tested various ammonium sulfate
precipitation conditions and found that an 18-40% ammonium sulfate
cut precipitated the large majority (more than 90%) of the U6
transcription activity, while the majority of the protein (more than
65%) precipitated at ammonium sulfate concentrations higher than 40%.
Immunoblots with an anti-hRPC155 antibody showed than the 18-40%
ammonium sulfate precipitate also contained more than 90% of the total
RNA polymerase III present in the starting extract (not shown). The
proteins in the 18-40% ammonium sulfate precipitate were further
fractionated on a phosphocellulose column, which was eluted with a
linear KCl gradient extending from 200 to 1000 mM KCl. U6
transcription activity eluted in a broad peak between 550 and 850 mM KCl, and as shown in Fig.
4, the fractions containing the peak of
U6 transcription activity (Fig. 4A) also contained the peak
of RNA polymerase III as measured by the presence of the hRPC155
subunit (Fig. 4B).
The proteins present in the phosphocellulose peak of activity were
further fractionated by successive chromatography on Mono-S and Mono-Q
columns, followed by fractionation on a sucrose gradient, as described
under "Experimental Procedures." Fig.
5 shows the activity profile of the
sucrose gradient fractions. U6 transcription activity was recovered
after the 669-kDa size marker in a broad peak with maximum activity in
fraction 14. This activity profile coincided with the elution profile
of the largest subunit of RNA polymerase III (Fig.
6, A and B,
panel hRPC155) as well as with that of the hRPC53 subunit of
RNA polymerase III (Fig. 6A), suggesting co-purification
with the bulk of RNA polymerase III. Furthermore, the recovery of U6
transcription activity in a single peak after sucrose gradient
centrifugation suggested that the activity is contained within a
complex, consistent with the observation that it can be
immunoprecipitated with antibodies directed against an RNA polymerase
III subunit (Fig. 2, above).
We tested the sucrose gradient fractions, as well as the Mono-Q
SNAPc fraction used for U6 transcription, for the presence of RNA polymerase III factors, in particular factors that constitute the human TFIIIB activity. The results are shown in Fig. 6. We could
detect traces of both TBP and BRF peaking with RNA polymerase III
subunits, suggesting association of at least some of this complex with
RNA polymerase III. In contrast, hB", the human homologue of the B"
subunit of S. cerevisiae TFIIIB (23), peaked before the
activity, and BRFU, the TFIIB-related factor that functions in U6
transcription, was undetectable in all the sucrose gradient fractions
(not shown). It is noteworthy that unlike BRF, both hB" and hBRFU were
present at substantial levels in the Mono-Q SNAPc fraction
(Fig. 6, B, lane 12, and C, lane 2), which could thus provide these activities in the transcription reactions in Fig. 5.
Both the La protein, which has been implicated in RNA polymerase III
transcription termination and in recycling of RNA polymerase III
(37-43), and TFIIA, which has been reported to be required or
stimulate RNA polymerase III transcription in a mammalian system (44,
45), were detectable in the 18-40% ammonium sulfate precipitate but
not in the sucrose fractions or the Mono-Q SNAPc fraction
(note that the faint band present in all the lanes of the anti-La panel
does not co-migrate with the La signal in the 18-40% ammonium sulfate
precipitate). Finally, Oct-1, which binds to the octamer sequence in
the DSE of RNA polymerase II and RNA polymerase III snRNA promoters and
activates snRNA gene transcription, was not detected in the sucrose
gradient fractions. Together, these results suggest that U6
transcription activity co-elutes with an RNA polymerase III complex
containing RNA polymerase III and a subset of TFIIIB polypeptides,
specifically the TBP·BRF complex. We refer to this complex as
"sucrose gradient RNA polymerase III" (SG pol III).
U6 Transcription Can be Reconstituted by a Combination of SG Pol
III, rTBP, rSNAPc, rB", and rBRFU--
The SG pol III
complex was active when complemented with rTBP and biochemically
purified Mono-Q SNAPc. Since we can obtain functionally
active recombinant SNAPc (20), we asked whether we could
replace Mono-Q SNAPc with recombinant SNAPc. We
combined a peak sucrose gradient fraction as judged from hRPC155
immunoblots with either rTBP and Mono-Q SNAPc, or rTBP and
rSNAPc. Only the combination containing Mono-Q
SNAPc resulted in U6 transcription, even though both Mono-Q
SNAPc and rSNAPc were active for U6
transcription when tested by complementation of a
SNAPc-depleted extract (not shown). We then tested whether
we might be able to recover U6 transcription by addition of hB", hBRFU,
or both factors combined. The results are shown in Fig.
7.
When we combined SG pol III with rTBP and increasing amounts of Mono-Q
SNAPc, we observed U6 transcription (lanes 1 and
2), but a combination of just TBP and Mono-Q
SNAPc (lane 3), or the SG pol III fraction on
its own (lane 4), were inactive, as expected. When we
complemented SG pol III, rSNAPc, and rTBP with increasing amounts of rhB" (lanes 5-7), or increasing amounts of
rhBRFU (lanes 8-10), we obtained very low or undetectable
amounts of U6 transcription. In sharp contrast, when we complemented SG
pol III, rSNAPc, and rTBP with increasing amounts of hBRFU
together with two fixed amounts of hB", we could in each case detect
efficient U6 transcription (lanes 11-13 and
14-16). The efficiency of U6 transcription was not greatly
affected by increasing the amounts of either hBRFU or hB", suggesting
that the lowest amounts of either factors were already saturating
relative to the amounts of the other factors added in the
reconstitution. Together, these results allow us to reach several
important conclusions. First, they reveal that the Mono-Q
SNAPc fraction contains three factors functional for U6
transcription: SNAPc, hB", and hBRFU. This is consistent
with the presence of hB" and hBRFU in this fraction as determined by immunoblot (see Fig. 6 above). Second, they confirm our previous depletion results (23) that indicated an absolute hB" and hBRFU requirement for U6 transcription. And third, they show that all factors
required for basal U6 transcription besides SNAPc, TBP, hBRFU, and hB" are contained within the SG pol III fraction.
We have reconstituted U6 transcription from a combination of four
recombinant factors, TBP, SNAPc, B", and BRFU, and a
purified RNA polymerase III fraction. Our data suggest that the
activity in the RNA polymerase III fraction is constituted by RNA
polymerase III and factors associated with it, rather than by a number
of spuriously co-purifying factors. Indeed, the activity forms a single
peak in a sucrose gradient (Fig. 5), and U6 transcription could also be
reconstituted by an anti-RNA polymerase III immunoprecipitate (combined
with Mono-Q SNAPc and rTBP) (Fig. 2). Both of these observations suggest that the activity corresponds to an RNA polymerase III-containing complex. This complex is probably different from the
"holoenzyme" described by Wang et al. (35),
because unlike the holoenzyme, it was not disrupted by chromatography
on phosphocellulose or KCl concentrations above 300 mM.
The RNA polymerase III-containing complex contains all factors besides
TBP, SNAPc, B", and BRFU absolutely required for basal transcription from the U6 promoter. Its composition is, therefore, of
high interest. Our immunoblot results suggest that the complex is not
enriched in the La protein nor the transcription factor IIA. We cannot
exclude, of course, that traces of these factors are present and are
sufficient for transcription. Nevertheless, the data do not lend
support to the idea that these factors are absolutely required for
transcription in vitro. In the case of TFIIA, we showed
before that depletion of an extract with anti-TFIIA antibodies
debilitated U6 transcription, but efficient transcription was restored
by addition of rTBP (47). Thus, the anti-TFIIA depletion debilitated U6
transcription because it lowered the amounts of TBP, probably because
TBP was associated with TFIIA. This suggested that TFIIA is not
required for U6 transcription in vitro, but it also
suggested that the TBP that is functional for U6 transcription is
associated with TFIIA in the extract (47). One possible interpretation
of these results is that TFIIA may play an anti-repressor role for
basal U6 transcription, for example, by displacing from TBP a repressor
such as Mot1, which can repress transcription from the human U6
promoter in vitro (48). Such an anti-repressor function
would not be revealed in an assay lacking the repressor, such as, most
likely, the reconstitution assay used here. Similarly, it is entirely
possible that the La protein is involved in RNA polymerase III
transcription in vivo in a function that is not tested in
the reconstitution assay.
hBRFU/TFIIIB50 can be purified as part of a multisubunit complex and
functional data suggest that subunits other than BRFU in the complex
are required for U6 transcription (25). Yet, we could obtain U6
transcription by combining the SG pol III fraction with recombinant
SNAPc, TBP, B", and just BRFU (Fig. 7), consistent with our
previous results in which we could restore U6 transcription in a
BRFU-depleted extract by addition of just recombinant BRFU (23).
Addition of BRFU was required to observe U6 transcription, indicating
that this factor was not present in the SG pol III fraction, an
observation confirmed by immunoblot. Together, these results suggest
that in this assay, BRFU-associated factors are either not absolutely
required for U6 transcription, or are present, dissociated from BRFU,
in the SG pol III fraction. In any case, the absolute requirement for
BRFU in the reconstitution assay confirms the essential role of this
protein in U6 transcription.
Visualization of the proteins present in the SG pol III fraction by
silver staining reveals some 40 to 50 polypeptides, and sequencing by
mass spectrometry identified some of them as RNA polymerase III
subunits (data not shown). However, the complex will need to be
purified further to facilitate the identification of the polypeptides
relevant to transcriptional activity. Nevertheless, one can speculate
as to what these factors may be. We expect that the complex will
contain all of the RNA polymerase III subunits. S. cerevisiae RNA polymerase III consists of 17 subunits, which are
listed in Table I. For all subunits
except C128, C37, and C25, human homologues have been characterized, as
indicated in Table I, and putative homologues of C37, C25, and parts of
C128 can be found in the human ESTs and nonredundant nucleotide
databases (not shown).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
A, comparison of the open reading frame
predicted by our construct expressing full-length hRPC53 and BN51
(GenBankTM AAA51838). The hRPC53 protein sequence is
identical to that predicted from the AK026588 entry in the database
although the nucleotide sequence is not identical (see AF346574).
B, the anti-hRPC53 antibody co-immunoprecipitates hRPC155.
HeLa whole cell extract was used as the starting material for
immunoprecipitations with anti-hRPC53 or preimmune antibodies. The
material bound to the beads was peptide eluted, fractionated on a
SDS-polyacrylamide gel, and analyzed by immunoblotting with an
anti-hRPC155 antibody. Lane 1, 15 µl of HeLa whole cell
extract; lane 2, material eluted from preimmune beads;
lane 3, material eluted from anti-hRPC53 beads.
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Fig. 2.
An anti-hRPC53 immunoprecipitate is active
for U6 transcription when complemented with recombinant TBP and Mono-Q
SNAPc. A, material immunoprecipitated by
protein A-agarose beads cross-linked to either anti-hRPC53 (lanes
4-7) or preimmune (lanes 8-11) antibodies, or by
protein A-agarose beads alone (lanes 12-15) was
complemented with just buffer, rTBP alone, Mono-Q SNAPc
alone, or both rTBP and Mono-Q SNAPc, as indicated
above the lanes, and tested for U6 transcription. The
transcription activities of rTBP alone (lane 1), Mono-Q
SNAPc alone (lane 2), or rTBP together with
Mono-Q SNAPc (lane 3), as well as that of whole
cell extract complemented with rTBP and Mono-Q SNAPc
(lane 16) are shown as controls. B,
immunoprecipitations performed with anti-hRPC53 antibodies (lanes
3 and 5) or preimmune antibodies (lanes 4 and 6) either in the absence (lanes 3 and
4) or presence (lanes 5 and 6) of 75 µg/ml of ethidium bromide were tested for U6 transcription in the
presence of added rTBP and mono Q SNAPc. The transcription
activities of rTBP and Mono-Q SNAPc (lane 1) and
of whole cell extract (lane 2) are also shown as
controls.
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Fig. 3.
Purification scheme of the RNA polymerase III
complex active for U6 transcription.
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Fig. 4.
U6 transcription activity coelutes with the
peak of RNA polymerase III on a P11 phosphocellulose column.
A, whole cell extract (lane 1), or the
flow-through (lane 2) and fractions indicated at the top
(lanes 3-16) from an analytical P11 column, or just rTBP
and Mono-Q SNAPc (lane 17) were tested for U6
transcription. The reactions in lanes 2-16 were
complemented with rTBP and Mono-Q SNAPc. The band labeled
IC corresponds to a radiolabeled RNA that was added to the
transcription reactions to serve as a control for RNA handling and
recovery. B, the 18-40% ammonium sulfate fraction
(lane 1), or the flow-through (lane 2) and P11
fractions indicated above the lanes (lanes 3-16)
were fractionated on a SDS-polyacrylamide gel and analyzed by
immunoblotting with an anti-hRPC155 antibody.
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Fig. 5.
U6 transcription activity forms a single peak
in a sucrose gradient. Decreasing amounts of the Mono-Q starting
material for the sucrose gradient (lanes 2-4), or the
sucrose gradient fractions indicated on top (lanes 5-24),
were analyzed for U6 transcription by complementation with Mono-Q
SNAPc and recombinant TBP. Lane 1 shows the
transcription activity of recombinant TBP and Mono-Q SNAPc
alone. The position of the molecular mass markers (thyroglobulin, 669 kDa; catalase, 232 kDa; and aldolase, 158 kDa) are indicated by
arrows on top.
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Fig. 6.
A, the sucrose gradient fractions shown
in Fig. 5 were analyzed by immunoblotting with antibodies directed
against hRPC155, hRPC53, TBP, and Oct-1, as indicated on the
left. Lane 11 (labeled C) shows 15 µl of Mono-Q
peak fraction (the load for the sucrose gradient) except in the Oct-1
panel, where it shows 15 µl of the 18-40% ammonium sulfate
fraction. Lane 12 shows 15 µl of the Mono-Q
SNAPc fraction. The dots above the lanes
indicate the peak of U6 transcription activity. B, the
sucrose gradient fractions indicated on top were analyzed by
immunoblotting with antibodies directed against hRPC155, hBRF, hB", La,
and TFIIA, as indicated on the left. Lane 11 (labeled C) shows 15 µl of the 18-40% ammonium sulfate
fraction. Lane 12 shows 15 µl of the Mono-Q
SNAPc fraction. The dots above the lanes
indicate the peak of U6 transcription activity. C, the
Mono-Q SNAPc fraction (lane 2) and whole cell
extract (lane 3) were analyzed for the presence of BRFU by
immunoblotting with antibodies directed against BRFU. Lane 1 shows recombinant BRFU.
View larger version (45K):
[in a new window]
Fig. 7.
SG pol III can reconstitute U6 transcription
when complemented with recombinant TBP, SNAPc, hB", and
hBRFU. 8 µl of SG pol III was complemented with 10 ng of
recombinant TBP, 6 µl of recombinant SNAPc, and 1, 2, and
4 µl of recombinant hB" (4 ng/µl) (lanes 5-7), 1, 2, and 4 µl of recombinant hBRFU (5 ng/µl) (lanes 8-10), 1 µl of recombinant hB" and 1, 2, or 4 µl of recombinant hBRFU
(lanes 11-13), and 4 µl of recombinant hB" and 1, 2, and
4 µl of recombinant hBRFU (lanes 14-16), and tested for
U6 transcription. As controls, lanes 1 and 2 show
SG pol III complemented with TBP and decreasing amounts of Mono-Q
SNAPc, and lane 3 shows TBP and Mono-Q
SNAPc alone.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Subunits of Saccharomyces cerevisiae and Homo sapiens RNA polymerase
III
Although it is formally possible that RNA polymerase III itself constitutes the sole activity in the SG pol III fraction absolutely required for U6 transcription, this seems unlikely because several activities besides SNAPc and the TFIIIB activity (TBP, hB", and hBRFU) have been reported to be required for U6 transcription. Thus, human TFIIIC activity can be separated chromatographically into two activities, TFIIIC1 and TFIIIC2 (49-52). TFIIIC2, whose subunits have been cloned (53-57), corresponds to yeast TFIIIC and is not be required for U6 transcription (58, 59). In contrast, TFIIIC1, which is not well characterized, is required for transcription of all classes of RNA polymerase III promoters including U6-type promoters (58, 59).
TFIIIC1 has been shown to strengthen the weak TFIIIC2 footprint on the B box of gene internal promoters and to extend it over the upstream A box and the downstream run of T residues that constitutes the RNA polymerase III transcription terminator (60). Moreover, some components of the TFIIIC1 fraction can bind independently to the termination region (60). These components may correspond to the TFIIIC0 fraction described by Oettel et al. (59, 61), which itself contains two activities, an activity binding to the terminator region of genes with internal promoters (called TBA) and an activity required specifically for U6 transcription (TFIIIU) (61). Recently, one of the components binding to the VAI terminator region has been identified as NF1, and shown to stimulate multiple round transcription (62). Furthermore, an immunopurified TFIIIC "holocomplex," containing both TFIIIC1 and TFIIIC2, also contains the RNA polymerase II transcription coactivators DNA topoisomerase I and PC4 (63). Both factors strongly stimulate RNA polymerase III transcription from gene-internal promoters. Thus, some or all of these activities may be required for U6 transcription and may be present in the SG pol III complex.
The RNA polymerase III-containing complex was purified on the basis of
U6 transcription activity. However, we find that this complex contains
the great majority, if not all, of RNA polymerase III, indicating that
it does not correspond to a U6-specific RNA polymerase III complex.
This is further suggested by the observation that the complex contains
some BRF, which is not involved in U6 transcription (23, 24). Thus, the
fraction may contain several RNA polymerase III-containing complexes
differing by the absence or presence of just a few factors, each of
which with a different specificity for different types of RNA
polymerase III promoters. Alternatively, but perhaps less likely, it
may contain a homogenous RNA polymerase III-containing complex
competent for transcription of all types of RNA polymerase III
promoters. The characterization of the SG pol III fraction should lead
to the identification of all factors absolutely required for basal U6 transcription.
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ACKNOWLEDGEMENTS |
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We thank James Chong, Ryuji Kobayashi, Beicong Ma, Richard Maraia, Laura Schramm, Xinyang Zhao, and Xuemei Zhao for reagents, help with protocols, and discussion. We also thank Yuling Sun for technical help, and James Duffy, Michael Ockler, and Philip Renna for artwork and photography.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grant GM38810.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.
Supported by the Howard Hughes Medical Institute.
** To whom correspondence should be addressed.
Published, JBC Papers in Press, February 27, 2001, DOI 10.1074/jbc.M100088200
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ABBREVIATIONS |
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The abbreviations used are: TBP, TATA box-binding protein; rTBP, recombinant TATA box-binding protein; pol III, polymerase III.
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