From the McArdle Laboratory for Cancer Research, University of Wisconsin, Madison, Wisconsin 53706
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ABSTRACT |
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Stoichiometry of the third largest subunit (Rpb3)
of the yeast RNA polymerase II is a subject of continuing controversy.
In this work we utilized immunoaffinity and nickel-chelate
chromatographic techniques to isolate the RNA polymerase II species
assembled in vivo in the presence of the
His6-tagged and untagged Rpb3. The distribution pattern of
tagged and untagged subunits among the RNA polymerase II molecules is
consistent with a stoichiometry of 1 Rpb3 polypeptide per molecule of
RNA polymerase. Deletion of either -homology region (amino acids
29-55 or 226-267) from the Rpb3 sequence abolished its ability to
assemble into RNA polymerase II in vivo.
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INTRODUCTION |
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Yeast Saccharomyces cerevisiae RNA polymerase II is a
multisubunit enzyme comprised of 12 core polypeptides (1). With minor variations its subunit composition is characteristic of all eucaryal nuclear RNA polymerases and their archaeal counterparts (1, 2). Despite
the trivial quantitative differences, a profound similarity can also be
found between these eucaryal/archaeal multisubunit enzymes and
eubacterial RNA polymerases. The latter, being heterotetramers of the
composition ()2
', resemble eucaryal RNA
polymerases in their overall appearance (3-6) and in the structural
core composition, with the two largest procaryal subunits,
and
', having fairly conserved eucaryal orthologs, represented by yeast Rpb1 and Rpb2 (reviewed in Ref. 7). Less obvious homology was noted
between eubacterial
subunit and yeast Rpb3 (8), consistent with the
reported functional equivalence of
2 and
(Rpb3)2 dimers in assembly of their respective enzymes
(9).
Young and co-authors also concluded, based on the
[S35]Met labeling of the RNA polymerase II subunits, that
two copies of the subunits Rpb3, Rpb5, and Rpb9 were present in each
RNA polymerase II molecule, whereas the rest were represented by a
single polypeptide each or else recovered in submolar amounts (10).
These inferred orthological relations between 2 and
(Rpb3)2 dimers were later questioned by the reports from
several laboratories, that failed to detect homodimerization potential
in yeast Rpb3 and its higher eucaryal homologs (11-13). Instead, in an
array of in vitro assays, Rpb3 was shown to associate with
another apparent
-subunit homolog, Rpb11, with a stoichiometry of
1:1 (11, 13). Human homologs of Rpb3 and 11 were also shown to
associate in a complex of unknown stoichiometry in the yeast two-hybrid
system (12). It was consequently suggested that the Rpb3-Rpb11
heterodimer serves as eucaryal analog of the
2-homodimer
(11); consistent with this hypothesis is the recovery of a
Rpb2-Rpb3-Rpb11 core subassembly from a partially denatured
Schizosaccharomyces pombe RNA polymerase II (14).
In this work we utilized an independent method to ascertain the stoichiometry of the yeast Rpb3, based on simultaneous expression of the wild-type (genomic) RPB3 gene and its plasmid-borne His6-tagged version followed by affinity purification of the in vivo assembled RNA polymerase II species.
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EXPERIMENTAL PROCEDURES |
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Plasmid Construction--
The RPB3 expression plasmid
p416GAL1:: RPB3.2 was created by subcloning of the
XbaI-XhoI fragment of the vector
pET33::RPB3.21 into
XbaI- and XhoI-digested vector p416GAL1 (15). The
resulting expression cassette featured the entire RPB3 coding sequence, preceded by pET33-derived sequence MGSSHHHHHHSSGLVPRGSRRASVH, under
control of galactose-inducible/glucose-repressible promoter. RPB3
deletion mutants, lacking the coding sequence for amino acids 29-55
that comprise the NH2-terminal -homology region or the COOH-terminal homology region spanning the region from position 226 to
267 were constructed using QuickChange site-directed mutagenesis kit
(Stratagene) and pET33::RPB3.2 as a template; sequences of the mutagenic oligonucleotides are available upon request. Mutagenized expression cassettes were excised using XbaI and
XhoI enzymes and subcloned into p416GAL1 to yield
p416GAL1::RPB3.5M and p416GAL1::RPB3.A2M plasmids,
respectively.
Isolation of the in Vivo Assembled RNA II Polymerase
Complexes--
Yeast S. cerevisiae strain InvSc1
(Invitrogen), transformed with the derivatives of the
galactose-inducible/glucose-repressible expression vector p416GAL1 (15)
(ATCC 87332), was grown in the yeast nitrogen base CM-URA medium (BIO
101, Inc.) supplemented with 0.5% (w/v)
(NH4)2SO4 and 2% (w/v) raffinose
with agitation at 26 °C. Cells were harvested at
A600 = 0.95 by centrifugation at 5000 × g at 4 °C for 10 min, washed with cold TE (10 mM Tris-HCl, pH 8.0, 1 mM EDTA), and
resuspended in 0.1 culture volume of cold lysis buffer (50 mM Tris-HCl, pH 8.0, 2% (v/v) glycerol, 0.1 mM EDTA, 0.1 M (NH4)2SO4,
1 mM 1,4-dithiothreitol, 0.5 mM
phenylmethylsulfonyl fluoride, Complete Mini EDTA-free protease
inhibitor mixture (Boehringer Mannheim)). Cells were disrupted by
high-speed vortexing with equal volume of acid-washed glass beads ( 0.4 mm, Thomas Scientific) for 4 min. Lysate was cleared by
centrifugation at 15,000 × g at 4 °C for 20 min.
Powdered (NH4)2SO4 was added to the
cleared lysate to the final concentration of 0.361 g/ml of the lysate, and the precipitate formed after 1-h incubation at 4 °C was pelleted by centrifugation at 15,000 × g at 4 °C for 20 min.
The pellet was used immediately for the downstream processing or stored
at
80 °C.
Analysis of the Assembled RNA Polymerase II Complexes-- Western blot analysis was performed after protein preparations were separated in 12 or 4-20% Tris-glycine SDS-PAGE gels (Novex) and electrotransferred onto Protran membrane (Schleicher & Schuell). The membranes were blocked for 2 h in 1% Blotto, rinsed in 1 × TBST (10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.1% (v:v) Tween 20), incubated for 90 min with primary murine antibodies (monoclonal antibodies 8WG16 (anti-Rpb1), 1Y27 (anti-Rpb3), 4Y11 (anti-Rpb11), and anti-Rpb2 serum) in 1% Blotto, washed three times for 5 min in 1 × TBST, incubated with goat anti-mouse IgG alkaline phosphatase conjugate (Boehringer Mannheim) in 1% Blotto, and washed five times with 1 × TBST. Alternatively, for detection of the His6-tagged proteins using Ni-NTA alkaline phosphatase conjugate (Qiagen), membranes were treated according to the manufacturer's recommendations. Blots were developed using 1 × solution prepared from the 5-bromo-4-chloro-3-indolyl phosphate-nitro blue tetrazolium tablets (Boehringer Mannheim) and scanned at 600 dpi on the Hewlett-Packard Scanjet II.
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RESULTS AND DISCUSSION |
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The previously published reports addressing the question of the Rpb3 stoichiometry for the validity of their conclusions implicitly relied either on accuracy and efficiency of RNA polymerase labeling in vivo (10) or upon the actuality and biological relevance of the interactions detected in vitro (11, 13). In this work we extended the elegant combinatorial approach of Guilfoyle and co-authors (11, 13) to the in vivo assembly of RNA polymerase. To that end we utilized a yeast host-vector system allowing simultaneous expression of the wild-type RPB3 gene from its native promoter and the His6-tagged RPB3 cassette from the vector-borne GAL1 promoter. Initial assumptions made in this work were: (i) stoichiometric incorporation of the Rpb3-tagged and -untagged subunits into RNA polymerase II and (ii) equally efficient incorporation and independent assortment of the tagged and untagged subunits during the polymerase assembly. These assumptions would yield different predictions depending on the stoichiometry of Rpb3 in the assembled polymerase. In the case of 1:1 stoichiometry of the third largest subunit relative to the polymerase core, only two classes of RNA polymerase II molecules should be present in the cell: one with tagged and another with untagged subunit. These two classes can be separated from each other using Ni-NTA affinity chromatography. If the alternative (2:1) proposition were true, independent assortment of the His6-tagged and -untagged subunits would result in appearance of three distinct classes of RNA polymerase II molecules: two homogeneous classes with both Rpb3 subunits either tagged or untagged and the third class of heterogeneous RNA polymerase II, comprising one tagged and one untagged subunit. Consequently, these heterogeneous molecules would be recovered in the eluate off the Ni-NTA column at nondenaturing conditions, resulting in the appearance of the untagged subunit in the Ni-NTA binding fraction.
By analyzing the relative abundance of the tagged and untagged Rpb3
protein in the crude and fractionated extracts of the yeast cells grown
on different carbon sources, we established that RPB3 expression from
the plasmid-borne GAL1 promoter is nearly identical to that from its
native promoter in the cells grown on 2% raffinose (Fig. 1, lane
1, and data not shown). Western blot
analysis of the immunoaffinity-purified RNA polymerase II confirmed our
initial assumptions regarding comparable efficiencies of incorporation
of the tagged and untagged versions of the third largest subunit into
the assembled enzyme, as the relative levels of these subunits in the
crude extract were similar to those in the anti-Rpb1 CTD mAb-binding
fraction (Fig. 1, lanes 1 and 3). The latter was
then subjected to the Ni-NTA column chromatography; unbound RNA
polymerase II material (pooled from the flow-through and wash
fractions) and nickel-bound retentate from the 160 mM imidazole eluate were probed with anti-Rpb3 antibodies (Fig. 1, lanes 4 and 5). Analysis of these data
unambiguously supports the conjecture of the third largest subunit
being present in a single copy per molecule of the S. cerevisiae RNA polymerase II, since not only was the untagged
subunit not recovered in the nickel-bound fraction, the heterologous
(one tagged + one untagged subunit) class of molecules, expected to be
the most prominent in the nearly equimolar mixes of the two different,
freely assorted subunits, was not found even among the unbound material
(Fig. 1, lane 4). Our experiment clearly distinguishes
between the two proposed Rpb3 stoichiometries in favor of the presence
of only one such subunit per RNA polymerase molecule. This finding is
consistent with the Rpb3-Rpb11 heterodimer being an ortholog of the
2-homodimer in eubacterial RNA polymerases (13).
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Having established an efficient system of incorporation of the
engineered Rpb3 into the in vivo assembled RNA polymerase
II, we next investigated the role of the two segments of this
polypeptide (amino acids 29-55 and 226-267), exhibiting some limited
homology to the Escherichia coli -subunit (13).
The Rpb3 mutant lacking amino acids 226-267 (Rpb3C,
calculated molecular mass 33,428 Da), when expressed from the
GAL1 promoter in presence of raffinose, accumulated in the
cell at the wild-type levels (Fig. 2A, lane
1), but in contrast to its
full-length counterpart Rpb3* (Fig. 1) was not found in the RNA
polymerase II-containing fractions that eluted from the 8WG16-Sepharose
(Fig. 2A, lanes 4 and 5). Instead, it appears to
be present only in the flow-through fraction (Fig. 2A, lane
2).
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The NH2-terminal -fragment (8) of Rpb3 (amino acids
29-55) corresponds to the region in the
-subunit where a number of mutations were isolated that affected
-
dimerization (17-20). In
order to evaluate the importance of this fragment for the assembly of
the RNA polymerase II in vivo, we constructed a deletion
mutant of RPB3, lacking the coding sequence for amino acids 29-55
(Rpb3
N), and cloned it into the p416GAL1 expression
vector. Unlike the His6-tagged derivative of the wild-type
Rpb3, whose calculated molecular mass was significantly larger than
that of its untagged counterpart (38,034 versus 35,300 Da),
His6-tagged mutant subunit comigrated with the untagged
wild type polypeptide during SDS-PAGE (their calculated molecular
masses being 35,077 and 35, 300 Da, respectively). In order to
distinguish between the two, we utilized a combination of Rpb3-specific
antibodies and His6-specific Ni-NTA-alkaline phosphatase
conjugate. When subjected to the immunoaffinity chromatography on the
8WG16 column, all the Rpb3-associated Ni-NTA-binding potential eluted
in the flow-through (data not shown), whereas a majority of the RNA
polymerase II (the fraction containing nonproteolyzed Rpb1 CTD) eluted
only after addition of propylene glycol (data not shown). Conversely,
when the crude lysate from the cells, expressing both the untagged
wild-type and the His6-tagged
29-55 mutant subunits,
was passed through the Ni-NTA-agarose column, the bound material
included the mutant His6-tagged protein, but not other
Rpbs, such as the two largest, Rpb1 and Rpb2, and another
-ortholog,
Rpb11 (Fig. 2, lane 1, and data not shown), that copurified in similar conditions with the wild-type His6-tagged Rpb3
(Fig. 2B, lane 2, and data not shown).
Both mutant Rpb3 proteins accumulated in the cell at approximately the same level as the wild type and did not significantly affect growth even when overexpressed in the medium containing both raffinose and the inducing carbon source, galactose (data not shown).
Thus, deletion of either -homology region in the yeast Rpb3
effectively abolished its incorporation into the RNA polymerase II (in
presence of the comparable amount of the wild-type protein). This
finding is consistent with the relatively innocuous phenotype of these
mutants in the merodiploid (relative to the RPB3 gene) yeast. The exact
nature of the interactions disrupted by these mutations will be
addressed in a series of the pairwise far-Western blot and pull-down
experiments involving RNA polymerase II subassemblies and individual
subunits. Our experimental approach serves as a complement to the
techniques already in use in the research of RNA polymerase II (11, 13,
14). Among the substantial advantages of this approach is its
operational simplicity, compared with the gene transplacement method
used by Ishihama and co-workers (14) to produce His6-tagged
yeast RNA polymerase II. The fact that expression of the engineered
subunit in yeast can be regulated over several orders of magnitude by
changing the carbon source in the growth media or by switching to
another of the many "sister" plasmids (15) allows not only the
adjustment of the expression to the wild-type level, but also staging
the in vivo "competition" experiments and overproduction
of the His6-tagged subunits in yeast and E. coli
using the same expression
cassette.3
Our results together with data from other laboratories (11-13)
strengthen the hypothesis that the homology between the -subunit of
the eubacterial RNA polymerase and eucaryal subunits 3 and 11 reflects
not only the past common origin, but also persisting similar functional
roles in determining the architecture of their respective RNA
polymerases. The question remains unanswered of the evolutionary
bifurcation that separated eucaryal and archaebacterial enzymes with
their
-like heterodimers (subunits 3/11 and D/L, respectively (Fig.
3)) from eubacterial RNA polymerases,
built around
2-homodimer. Based on the phylogenetic
clustering of the polypeptide sequences for the cloned
-homologs
(Fig. 3), we find it plausible that a heterodimeric enzyme, similar in
architecture to the eucaryal/archaebacterial RNA polymerases, existed
prior to the separation of the three evolutionary lineages, with
subsequent loss of the smaller (Rpb11- or L-like) subunit
in the early history of eubacteria. This thesis is in agreement with
previously proposed hypothesis of the reductionist evolution leading to
the modern eubacterial RNA polymerases (2).
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FOOTNOTES |
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* 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.
1 M. de Arruda, Svetlov, V., Chambers, K. J., and Burgess, R. R., manuscript in preparation.
2 The abbreviations used are: CTD, carboxyl-terminal domain; Ni-NTA, nickel-nitrilotriacetic acid; PAGE, polyacrylamide gel electrophoresis; mAb, monoclonal antibody.
3 V. Svetlov, unpublished observation.
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REFERENCES |
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