(Received for publication, August 14, 1995; and in revised form, December 18, 1995)
From the
Two subunits in RNA polymerase II (e.g. RPB3 and RPB11
in yeast) and two subunits common to RNA polymerases I and III (e.g. AC40 and AC19 in yeast) contain one or two motifs
related to the subunit in prokaryotic RNA polymerases. We have
sequenced two different cDNAs (AtRPB36a and AtRPB36b), the two
corresponding genes from Arabidopsis thaliana that are
homologs of yeast RPB3, and an Arabidopsis cDNA
(AtRPB13.6) that is a homolog of yeast RPB11. The B36a subunit
is the predominant B36 subunit associated with RNA polymerase II
purified from Arabidopsis suspension culture cells, and this
subunit has a stoichiometry of about 1. Results from protein
association assays showed that the B36a and B36b subunits did not
associate, but each of these subunits did associate with the B13.6
subunit in vivo and in vitro. Two motifs in the B36b
subunit related to the prokaryotic
subunit were shown to be
required for the in vitro interactions with the B13.6 subunit.
Our results suggest that the B36 and B13.6 subunits associate to form
heterodimers in Arabidopsis RNA polymerase II like the AC40
and AC19 heterodimers reported for yeast RNA polymerases I and III but
unlike the B44 homodimers reported for yeast RNA polymerase II.
Eukaryotes contain three classes of nuclear RNA polymerase,
referred to as RNA polymerases I or A, II or B, and III or C. Each
class of RNA polymerase is a multimeric enzyme composed of two unique
large subunits in excess of 100 kDa that are related to ` and
subunits of Escherichia coli RNA polymerase and 10 or
more smaller subunits (reviewed in (1, 2, 3, 4, 5) ). In the
yeast Saccharomyces cerevisiae five of these smaller subunits
are common to RNA polymerase I, II, and III, and seven subunits are
common to RNA polymerases I and III(3, 4) . Subunits
of about 40 kDa (e.g. yeast AC40 and B44 or RPB3) and
12.5-19 kDa (e.g. yeast AC19 and B12.5 or RPB11) in RNA
polymerase I, II, and III have limited amino acid sequence homology
with the
subunit of the prokaryotic RNA
polymerase(6, 7, 8) . The localized amino
acid sequence homology between the eukaryotic
-like subunits and
the
subunit in prokaryotic RNA polymerases has been referred to
as the ``
motif''(2, 9) . The yeast
AC40 and AC19 subunits are common to RNA polymerase I and III, and the
related subunits, B44 or RPB3 and B12.5 or RPB11, are unique to RNA
polymerase II(6, 8, 10, 11) .
Bacterial RNA polymerase has an
subunit with a stoichiometry of
2, and the core enzyme is composed of
`(12) . The yeast B44 subunit is
reported to have a stoichiometry of 2 in RNA polymerase
II(13) , but the AC40 and AC19 subunits in RNA polymerases I
and III have apparent stoichiometries of 1(3, 14) .
The stoichiometry of the yeast B12.5 subunit has not been reported.
Yeast RNA polymerase II contains a total of 12 subunits, and each of these is encoded by a single copy gene (reviewed in (3) and (4) ). All of the RNA polymerase II subunit genes in yeast have been sequenced. Only a limited number of RNA polymerase II subunit genes in other eukaryotes have been cloned and sequenced(4) . With the exception of genes encoding the largest subunit of RNA polymerase II in soybean and trypanosomes(15, 16, 17) , those RNA polymerase II subunit genes that have been identified in organisms besides yeast are reported to be single copy genes.
Nuclear RNA
polymerase subunit-subunit interactions, subunit functions, and
assembly pathways are only beginning to be unraveled. For example, the
AC40 and AC19 subunits of yeast RNA polymerase I and III have been
shown to associate with one another in a yeast two-hybrid
system(9) . Extragenic suppression of mutations in the AC40 and
AC19 subunit genes confirmed the interaction between these two subunits
and a third subunit, ABC10(9) . Studies on mutations in
the three largest subunits of yeast RNA polymerase II indicate that the
B44 subunit associates with second largest subunit (i.e. B150
or RPB2), which in turn complexes with the largest subunit (i.e. B220 or RPB1) to facilitate assembly of the enzyme(18) .
Here, we report on the cloning and sequencing of genes and/or cDNAs for the 36-kDa (B36a and B36b) and 13.6-kDa (B13.6) subunits in Arabidopsis RNA polymerase II, which are homologs to yeast B44 and B12.5 (i.e. encoded by the RPB3 and RPB11 genes in S. cerevisiae), respectively, to determine the stoichiometry of the B36 subunit in the enzyme and investigate its self-association and its association with the B13.6 subunit.
An EST cDNA clone (GenBank(TM) accession number Z47635) from an Arabidopsis cell suspension library (22) was identified which had homology to yeast RPB11(8) . The EST sequence was reported as a partial sequence of a full-length cDNA clone. The complete sequence of this cDNA clone was obtained from the EST cDNA clone, which was provided by Dr. Gabriel Phillips (Laboratoire de Biologie Moleculaire de Plantes, CNRS, Strasbourg Cedex, France). We refer to this clone as AtRPB13.6.
Both
AtRPB36a and AtRPB36b cDNA clone inserts were used to screen an A.
thaliana (ecotype Columbia) EMBL3 genomic library (provided
by Harry Klee, Monsanto Chemical Company, St. Louis, MO). Genomic
clones were selected, purified, and mapped with restriction enzymes.
Restriction fragments corresponding to genomic fragments of AtRPB36a and AtRPB36b were subcloned into pBluescript
(Stratagene, La Jolla, CA) vectors or pMOB (23) and sequenced
using a Tn1000 kit (Gold Biotechnology, St. Louis, MO).
Northern blotting was carried out with 2 µg of
poly(A) RNA isolated from Arabidopsis suspension culture cells(27) . RNA was isolated by a
standard protocol(25) , denatured with glyoxal and
Me
SO, subjected to electrophoresis on 1.4% agarose gels,
and transferred to a nylon membrane(26) . AtRPB36a and AtRPB36b
cDNAs were labeled with
P using the Prime-a-Gene labeling
system (Promega Corp., Madison, WI). A mixed probe was used for
hybridization in 6
SSPE(26) , 1% nonfat dry milk, 1%
SDS, and 0.5 mg/ml denatured herring sperm DNA at 68 °C. Washings
were in 2
SSC and 0.1% SDS for 15 min at 25 °C, 0.5
SSC and 0.1% SDS for 15 min at 25 °C, and 0.2
SSC and 1%
SDS for 30 min at 50 °C
For purification of RNA polymerase II, 200 g of cells
were thawed and suspended in 200 ml of grinding buffer (50 mM Tris-HCl (pH 8.0), 0.1 mM EDTA, 60 mM ammonium
sulfate, 0.5 mM dithiothreitol, and 20% (v/v) ethylene glycol)
containing 1 mM Pefabloc SM (Boehringer Mannheim) 10 µg/ml
aprotinin, 1 µg/ml pepstatin, 300 µg/ml benzamidine, and 10
µg/ml leupeptin. All purification steps were carried out at 4
°C. Cells were broken by grinding for 2 min using full speed with a
Polytron PT20ST and subsequently with 15 30-s bursts and 90-s
intermittent periods with a Bead-Beater and 100 g of acid-washed glass
beads (425-600 microns; Sigma). The homogenate was filtered
through two layers of Miracloth and centrifuged at 10,000 g for 20 min. The supernatant was collected, and RNA polymerase II
was purified by precipitation with and elution from Polymin P, ammonium
sulfate precipitation, and chromatography on DEAE cellulose and
phosphocellulose as described by Jendrisak and Burgess(34) .
The phosphocellulose fraction was dialyzed against 20 mM HEPES
(pH 7.8), 0.1 mM EDTA, 0.5 mM dithiothreitol, and 50%
glycerol, and dialysate was stored frozen at -80 °C. Wheat
germ RNA polymerase II was purified using the methods of Jendrisak and
Burgess (34) with final chromatography on
heparin-Sepharose(21) .
The purified RNA polymerase II was
judged to be nearly homogeneous on 15% SDS-polyacrylamide gels (29) when compared with purified wheat germ RNA polymerase II.
Subunit stoichiometries were determined for the three largest subunits
(205 + 175-, 135-, and 36-kDa subunits) in purified Arabidopsis RNA polymerase II using 7.5 and 15% SDS gels. Peak
areas for the three largest subunits were measured for Coomassie
Blue-stained gels using Image I Software (Universal Image, Corp.,
Westchester, PA). Quantitation of S incorporation into the
three largest subunits was carried out with a Fuji BAS1000 instrument
and MacBAS1000 software (Fuji Medical Systems, Stamford, CT).
Figure 1:
Comparison of amino acid sequences
derived from Arabidopsis AtRPB36a and AtRPB36b cDNA clones
with homologous subunits in yeast (B44 or RPB3) and human (B33).
Identical amino acids that are shared by more than one species are shaded in each subunit. Two domains with homology to the
subunit of E. coli RNA polymerase are shown with a double
underline.
-motif 1 is the N-terminal ``
motif''(2, 7, 9) , and
-motif 2 is the leucine-rich C-terminal
-like
motif(7, 36) . Asterisks indicate the
positions of cysteines in the putative metal-binding motifs of B36a and
B36b. Positions of N-terminal and C-terminal truncations made in the
B36b subunit are indicated with arrows above the sequence
alignments.
Southern blot analysis of Arabidopsis genomic DNA suggested
that more than one copy of this subunit gene was present in the Arabidopsis genome because a variety of restriction
endonucleases produced multiple restriction fragments (of varying
intensities) that hybridized to the AtRPB36a cDNA probe (Fig. 2A). To determine if more than one gene encoded the
36-kDa RNA polymerase II subunit, we rescreened 5 10
plaque-forming units of the
YES cDNA library with the
AtRPB36a cDNA and selected six positive clones. Each purified clone was
partially sequenced. Five of these were identical in sequence to
AtRPB36a with the exception of the position of the poly(A) tail in the
3`-untranslated region (data not shown), reflecting heterogeneity in
the site selection for poly(A) addition. One of the clones contained a
full-length cDNA that was related, but distinct from AtRPB36a. This
1.2-kb cDNA clone, AtRPB36b, contained an ORF encoding 319 amino acids
with 88% identity to the amino acid sequence in AtRPB36a and 37% identical to yeast RPB3 (Fig. 1). The predicted pI of the B36b
protein was 4.7. Within the ORFs, AtRPB36a and AtRPB36b showed 91% identity in nucleotide sequence, and in the untranslated regions, the
two cDNA clones were 82% identical (data not shown). A Northern blot
with a mixed AtRPB36a and AtRPB36b probe revealed only one size mRNA of
1.5 kb (Fig. 2B). We have not attempted to quantitate
the relative amounts of the individual AtRPB36a and AtRPB36b mRNAs.
Figure 2: Southern and Northern analyses. A, the Southern blot was carried out with an AtRPB36a cDNA probe. Genomic DNA (0.5 µg/lane) was digested with the following restriction enzymes: BamHI (lane 1), HindIII (lane 2), EcoRI (lane 3), EcoRI + BglII (lane 4), NcoI (lane 5), and NcoI + NsiI (lane 6). B, the Northern blot was carried out with a mixed AtRPB36a and AtRPB36b cDNA probe. Molecular mass markers are indicated in kilobases to the left.
The ORFs in AtRPB36a and AtRPB36b encode putative metal-binding
motifs (i.e. ``zinc-fingers''),
CXCX
CX
C,
starting at position Cys
in B36a (Fig. 1). The
motif in B36b differs from that in B36a because the B36b clone contains
an N-terminal extension of this motif,
CX
CX
CX
CX
C.
These putative metal-binding motifs differ slightly from those found in
the homologous RNA polymerase II subunit in S. cerevisiae(10) , Schizosaccharomyces pombe(36) ,
human(35) , and Tetrahymena thermophila(7) ,
which are conserved as
CXCX
CX
C. The yeast
RPB3 subunit has been reported to bind
Zn using a
zinc-blotting technique(37) , and we have preliminary evidence
that the Arabidopsis B36a and B36b subunits bind zinc using
the methods of Treich et al.(37) . (
)The
B36a and B36b subunits contain two motifs related to the prokaryotic
RNA polymerase
subunit (Fig. 1). One of these motifs (the
more N-terminal) consists of a stretch of amino acids that is referred
to as the ``
motif'' ( (7) and (9) ;
reviewed in Refs. 3 and 4). The second
-like motif consists of a
leucine-rich C-terminal region including amino acids Leu
to Leu
in B36a and B36b. Both of these
-like
motifs have been previously identified in S. cerevisiae, S. pombe, Tetrahymena, and human subunit
homologs(7, 36) .
Figure 3: Genomic structure of AtRPB36a and AtRPB36b. Exons are indicated by the closed boxes, 3`- and 5`-untranslated regions by the open boxes, and introns, promoters, and 3` regions of the genes by the thick lines. The percentage identity of nucleotide sequences in different regions of the two genes is indicated. ATG and TGA indicate the translation start and termination codons, respectively. A 1-kb size marker is shown below the two genes.
Figure 4:
Arabidopsis RNA polymerase II
purified from cell suspension cultures. A, Coomassie
Blue-stained 15% SDS-polyacrylamide gel (left panel) and
autoradiogram of the gel (right panel). Cells were labeled
with [S]methionine as described under
``Materials and Methods.'' Lanes 1 and 2 show Arabidopsis RNA polymerase II subunits resolved on
an SDS-polyacrylamide gel from two independent purifications. Lane
3 is wheat germ RNA polymerase II. Subunit molecular masses are
shown to the left. B, Coomassie Blue-stained 7.5%
SDS-polyacrylamide gel (left panel) and autoradiogram of the
gel (right panel). Lane 1, Arabidopsis RNA
polymerase II purified from a cell suspension culture labeled with
[
S]methionine; lane 2, Arabidopsis RNA polymerase II purified from unlabeled cells; lane 3:
wheat germ RNA polymerase II. His-tagged B36a (lower band) and
B36b (upper band) subunits expressed in and purified from E. coli are shown next to lane 3 in the Coomassie
Blue-stained gel. In vitro translated,
S-labeled
B36a and B36b subunits are shown to the left of the RNA
polymerase in the autoradiogram. RNA polymerase subunit molecular
masses are shown to the left. Subunit stoichiometries
determined for
S-labeled 205 + 175-, 135-, and 36-kDa
subunits in three independent purifications are shown to the right of the autoradiogram.
The third largest subunits in S. pombe and S. cerevisiae RNA polymerase II are
reported to have a stoichiometry of 2 in the purified
enzymes(13, 36) , while the homologous subunits in
plant and animal RNA polymerase II are reported to have a stoichiometry
of
one(39, 40, 41, 42, 43) .
To determine the stoichiometry of the B36a subunit in Arabidopsis RNA polymerase II, we measured the peak areas for the 205 +
175-, 135-, and 36-kDa subunits by imaging (Image I Software) 7.5%
Coomassie Blue-stained gels (Fig. 4B, left
panel). This analysis indicated that the stoichiometries of the
three largest subunits were 1, 1.1, and 1.3 (i.e. using the
largest subunit, 205 + 175, as the base line), for the 205 +
175-, 135-, and 36-kDa subunits, respectively. As a second method for
determining stoichiometry of these subunits, we quantitated the S incorporation in the three largest subunits in Arabidopsis RNA polymerase II that had been labeled in
vivo with [
S]methionine. Since the amino
acid sequences for the 205-(15) , 135-(38) , and 36-kDa
subunits (see Fig. 1) are known, the number of methionines in
each subunit could be divided into the
S incorporated into
each subunit (i.e. determined by phosphor imaging) to
determine the subunit stoichiometries. The stoichiometry for each
subunit was near unity. The relative stoichiometries obtained for three
independent assessments are shown to the right of each subunit
in Fig. 4B (right panel). With results from
Coomassie Blue staining and
S labeling taken together, the
best estimate for stoichiometry of the B36 subunit in Arabidopsis RNA polymerase II is 1.
Figure 5:
Comparison of amino acid sequences derived
from Arabidopsis AtRPB13.6 with homologous subunits in yeast
(B12.5 or RPB11) and human (B14). Identical amino acids found in more
than one species are shaded in each subunit. The N-terminal
`` motif'' (
-motif 1) and a leucine-rich
C-terminal
-like motif (
-motif 2) with homology to
subunit of E. coli RNA polymerase are shown with a double underline.
Figure 9:
The
N-terminal `` motif'' and a leucine-rich C-terminal
-like motif in B36a, B36b, B13.6, and homologous subunits in other
eukaryotic RNA polymerases. A, The N-terminal ``
motif'' (
motif 1) in large and small
-like subunits in
RNA polymerases I, II, and III from Arabidopsis, yeast, and
human. B, the leucine-rich C-terminal
-like motif (
motif 2) in large and small
-like subunits in RNA polymerases I,
II, and III from Arabidopsis, yeast, and human. Amino acid
alignments are shown with conserved amino acids shaded black for identity (the predominant amino acid at each position) and gray for similarity (BOXSHADE program, Kay Hofman,
Bioinformatic group, Lausanne, Switzerland). Leucines and isoleucines
that predominate at specific positions are indicated at the top. B36a, B36b, B13.6, AC42, AC43, and AC14 are Arabidopsis subunits. Yeast subunits are indicated by a y, and human subunits by an h. EcRpoA is the E.
coli
subunit, and TobCt is the tobacco chloroplast
-like subunit. The N-terminal amino acid position for the
conserved domain in each subunit is indicated to the left of
the sequences.
Figure 6: B36a, B36b, and B13.6 subunit interactions assayed by immunoprecipitation with epitope-tagged subunits. A, B36a and B36b subunit interactions. Lanes 1, 2, 4, and 5 are autoradiograms of in vitro translated subunits. Lane 1, B36a; lane 2, co-translated B36a and HA epitope-tagged B36b; lane 4, B36b; lane 5, co-translated B36b and HA epitope-tagged B36b. Lanes 3 and 6 are autoradiograms of immunoprecipitates with co-translated subunits (shown in lanes 2 and 5) using an HA epitope-tag and HA monoclonal antibody. In vitro translation products were resolved on 10% SDS-polyacrylamide gels. B, B36a and B36b interactions with HA epitope-tagged B13.6. Odd-numbered lanes are autoradiograms of in vitro translated subunits used in immunoprecipitation assays. Lane 1, co-translated B36a and B13.6; lane 3, co-translated B36b and B13.6; lane 5: co-translated Arabidopsis AC42 and B13.6; lane 7, co-translated B36b and epitope-tagged IAA4/5 (i.e. IAA4/5 is an auxin-induced cDNA from pea and is not related to any RNA polymerase subunit). Even-numbered lanes are autoradiograms of immunoprecipitates of co-translated subunits (shown in odd-numbered lanes). The B13.6 subunit was epitope-tagged in lanes 1-6, and the IAA4/5 polypeptide was epitope-tagged in lanes 7 and 8. In vitro translation products were resolved on 10% Tricine-SDS-polyacrylamide gels. Positions of subunits are indicated adjacent to the autoradiograms.
A second in vitro approach that showed B36b interaction with B13.6 was obtained by resolving in vitro co-translated subunits by electrophoresis in polyacrylamide gels under nondenaturing conditions. When the B36b subunit was co-translated with the B13.6 subunit, the B36b subunit showed a mobility shift in the gels (Fig. 7). The gel lane (lane 2) containing a band with decreased mobility was excised and subjected to electrophoresis in an SDS-polyacrylamide gel. The SDS gel showed that the mobility shift in the nondenaturing gel was due to association of the B13.6 subunit with the B36b subunit.
Figure 7: Interaction of the B13.6 kDa subunit with B36b subunit in a gel mobility shift assay. Panel A, autoradiogram of in vitro translated subunits resolved on a nondenaturing 7.5% polyacrylamide gel. Lane 1, B36b; lane 2, co-translated B36b and B13.6; lane 3, B13.6 (i.e. the B13.6 subunit has run off the gel). The B13.6 subunit was HA epitope-tagged. F, position of free B36b; C, position of B36b-B13.6 complex. Panel B, autoradiogram of in vitro translated subunits resolved on a two-dimensional polyacrylamide gel. The B36b and B13.6 subunits were co-translated and subjected to nondenaturing gel electrophoresis as in panel A, lane 2. The gel lane was excised, laid on its side (lane 2), and subjected to SDS-polyacrylamide gel electrophoresis. Lane 1, co-translated B36b and HA epitope-tagged B13.6 (i.e. an aliquot of the translation products was applied to the first dimension, nondenaturing gel); lane 3, immunoprecipitate of translation products shown in lane 1 using an HA monoclonal antibody. The arrow indicates the position of the B13.6 subunit that migrates in the B36b-B13.6 complex (C) resolved from free (F) B36b on the nondenaturing gel. Some smearing or tailing of the B36b-B13.6 complex is evident in the nondenaturing gel.
Figure 8:
C-terminal and N-terminal truncations that
prevent B36b and B13.6 interactions. Panel A, schematic
diagrams of the N-terminal and C-terminal truncations in the B36b
subunit. The position of the putative zinc-finger is indicated above construct 1. The hatched box and black box indicate the positions of the N-terminal `` motif''
(
motif 1) and leucine-rich C-terminal
-like motif (
motif 2), respectively. The stippled box represents the GH2/4
GST fusion protein. HA-13.6 is a schematic of the HA epitope-tagged
B13.6 subunit showing the N-terminal position of the epitope-tag (open diamond) and the
-like motifs (hatched and black boxes). The N refers to N-terminal, and the C refers to C-terminal truncations at the amino acid positions
indicated by the number. Panel B, autoradiograms of in vitro translation products used for immunoprecipitation
assays. HA epitope-tagged B13.6 was co-translated with B36b or a B36b
C-terminal truncation, and translation products were resolved on 10%
Tricine-SDS-polyacrylamide gels. The B36b subunit or truncations tested
were untruncated (319 amino acids) (lane 1); C314 (314 amino
acids with 5 amino acids missing from C terminus (lane 2) (see Fig. 1); C310 (lane 3); C300 (lane 4); C288 (lane 5); C244 (lane 6); N47 (272 amino acids with 47
amino acids missing from N terminus) (lane 7); C terminus
(amino acids 249-319) fused to the C terminus of the GST protein
GH2/4 (39) (lane 8). Panel C,
immunoprecipitations of samples shown in panel
B.
We did
not observe interactions between the leucine-rich C-terminal -like
motif in B36b and B13.6 when the C-terminal motif of B36b was tested as
a fusion protein in isolation from the remainder of the B36b subunit
(GST-N249) (Fig. 8C, lane 8). In this case,
amino acids 249-319 in B36b were fused to a GH2/4 protein at its
C terminus(32) . The GH2/4 cDNA encodes a glutathione S-transferase(33) . Failure to observe interaction
between the fused B36b C-terminal motif and B13.6 could result if more
than one interaction motif or a more extensive interaction motif is
required for the stable association of B36b and B13.6. Since the
``
motifs'' in the N-terminal regions of RPB3 and AC40
in yeast appear to be important for subunit interactions and enzyme
assembly(9, 18) , we made an N-terminal truncation
that removed a portion of the ``
motif'' in B36b. This
truncated subunit was not immunoprecipitated with epitope-tagged B13.6
in co-translation experiments (Fig. 8C, lane
7). This result is consistent with there being two motifs or one
extended motif (i.e. including both
-like motifs in B36)
involved in the B36 subunit interaction with the B13.6 subunit. While
it is possible that the N-terminal and C-terminal truncations in B36b
resulted in conformational changes (e.g. incorrect folding of
the in vitro translated truncations), which indirectly
prevented interaction with B13.6, the truncation experiments suggest
that both
-like motifs in B36a may be required for interaction
with B13.6.
In addition to the ``
motif,'' Arabidopsis B36 subunits ( Fig. 1and Fig. 9B) and AC42/43 subunits (Fig. 9B)
contain the leucine-rich C-terminal
-like motif originally pointed
out by Martindale (7) and Azuma et al.(36) for S. pombe RPB3, S. cerevisiae RPB3 and AC40, human RPB33, and Tetrahymena CnjC
subunits. Inspection of the B13.6 subunit in Arabidopsis RNA
polymerase II, the homologous subunits in RNA polymerase II from other
organisms, and the related 12.5-19-kDa subunits in RNA
polymerases I and III suggests that the leucine-rich C-terminal
-like motif is also conserved in these small
-like subunits (Fig. 9B). Thus, both the larger 36-44-kDa
-like subunit and the smaller 12.5-19-kDa
-like subunit
in RNA polymerases I, II, and III contain two motifs with similarity to
the prokaryotic RNA polymerase
subunit. One of these domains has
been previously referred to as the ``
motif''(2, 4, 6, 7, 9) ,
and the second is a more C-terminal motif that is rich in
leucine(7, 36) . While these two
-like motifs are
spaced apart from one another in the 36-44-kDa subunits, they are
nearly contiguous in the 12.5-19-kDa subunits (see Fig. 1, Fig. 5, and Fig. 8A).
Our results have shown that Arabidopsis contains two
genes that encode the third largest subunit of RNA polymerase II. This
differs from other RNA polymerase II genes in Arabidopsis and
in most other organisms studied, where the genes are single copy. The
two genes are expected to encode the third largest subunit in RNA
polymerase II, based on stronger homology to the third largest subunit
in yeast and human RNA polymerase II and lesser homology to a related
subunit in yeast and mouse RNA polymerases I and III. This is supported
by our cloning of two additional cDNAs from Arabidopsis that
encode proteins that show stronger homology to the yeast and mouse AC40
subunits in RNA polymerases I and III than to the yeast RPB3 or human
hRPB33 subunit in RNA polymerase II(46) . Therefore, it appears
that Arabidopsis expresses two genes for the 36-kDa subunit in
RNA polymerase II and two genes for the 42/43-kDa subunit in RNA
polymerase I and III. All four polypeptides encoded by these genes
contain the N-terminal `` motif'' (7) and a
leucine-rich C-terminal
-like motif(7, 36) ,
which are related in amino acid sequence to the prokaryotic RNA
polymerase
subunit. Two other cDNAs from Arabidopsis that contain these
-like motifs in their ORFs have been
cloned. One of these, described above, encodes a protein of 13.6 kDa
(B13.6) that is a homolog of the yeast RNA polymerase II B12.5 (RPB11)
subunit. The second encodes a protein of 14 kDa, which is a homolog of
the yeast RNA polymerase I and III AC19 subunit. (
)
The
different mobilities of the B36 subunits in SDS-polyacrylamide gel
electrophoresis allowed us to distinguish the B36a from the B36b
subunit. RNA polymerase II purified from Arabidopsis suspension culture cells contains a third largest subunit with an
apparent molecular mass of 40 kDa, which migrates identically to in
vitro translated B36a in SDS-polyacrylamide gel electrophoresis.
If the B36b subunit is associated with purified RNA polymerase II, then
it is present in amounts not detectable by Coomassie Blue staining or
by autoradiography in S-labeled enzyme. The reason for
predominance of the B36a subunit in the purified enzyme is not clear
because the promoters for both AtRPB36 genes are active in
transgenic tobacco tissues undergoing cell division and in transfected
protoplasts from carrot suspension culture cells.
Furthermore, based on cDNA cloning, both AtRPB36 genes
are expressed in the rapidly dividing Arabidopsis suspension
culture cells from which the RNA polymerase II was purified. While we
have not quantitated the relative amounts of B36a and B36b mRNAs in
suspension culture cells, the fact that six out of seven cDNA B36 cDNA
clones isolated were B36a suggests that the B36a mRNA is more abundant
than the B36b. Our results on in vivo and in vitro protein-protein interactions suggest that the B36b subunit is not
defective in assembly (i.e. at least assembly with the B13.6
subunit). The high conservation in amino acid sequence between B36a and
B36b suggests that both subunits should be capable of assembly into RNA
polymerase II. It is possible that the B36b subunit is expressed at
much lower levels or assembles into RNA polymerase less efficiently
than the B36a subunit in cell suspension cultures and that this subunit
has simply escaped our detection. The B36b subunit may be more abundant
in Arabidopsis RNA polymerase II found in specific tissues or
specific developmental stages. It is worth noting that heterogeneity in
the size of the third largest subunit in RNA polymerase II has been
reported for enzymes purified from wheat and rye
embryos(39, 42) , suggesting that more than one gene
encodes this subunit in other plant species.
Based upon the yeast
two-hybrid system and immunoprecipitation experiments with
epitope-tagged in vitro translated subunits, the B36a and B36b
subunits fail to stably associate with themselves or one another but do
associate with the B13.6 subunit. Our in vivo results are
similar to the in vivo results of Lalo et
al.(9) , who used the yeast two-hybrid system to show that
the yeast RNA polymerase AC40 and AC19 subunits associate with one
another as a heterodimer, but that the AC40 subunit fails to
homodimerize. Our in vitro protein-protein interaction results
confirm the in vivo results. Based upon these in vivo and in vitro protein-protein interaction results and the
apparent unit stoichiometry of the Arabidopsis B36 and yeast
AC40 subunits, it is possible that heterodimers, Arabidopsis B36/B13.6 and yeast AC40/AC19, are the equivalent of an
homodimer in prokaryotic RNA polymerase. A
stoichiometry of 1 for the third largest subunit of other plant and
animal RNA polymerase II enzymes has been documented previously, based
upon the intensity of subunit staining with Coomassie
Blue(39, 40, 41, 42, 43, 47) .
Unit stoichiometry for the third largest subunit in Arabidopsis RNA polymerase II is supported by the relative intensity of
Coomassie Blue staining and by the ratio of
S incorporated
to the number of methionines in this subunit compared with the two
largest subunits. In contrast to our results with Arabidopsis RNA polymerase II, the RPB3 subunits in S. pombe and S. cerevisiae RNA polymerase II are reported to have a
stoichiometry of 2(13, 36) , and evidence has been
presented that is consistent with a S. cerevisiae RNA
polymerase II assembly pathway initiating with the homodimerization of
RPB3 and subsequent interaction with RPB2 and RPB1 (18) . While
this proposed assembly pathway for yeast RNA polymerase II resembles
that reported for bacterial RNA polymerase(48) , there is no
direct evidence for the homodimerization of yeast B44 (RPB3) subunits.
It remains possible that yeast B44 and B12.5 (RPB11) form heterodimers
like Arabidopsis B36 and B13.6. On the other hand, it is
possible that the subunit stoichiometries and assembly pathways differ
in RNA polymerase II from yeast and plants.
Lalo et al.(9) showed that a number of mutations in the ``
motif'' (see Fig. 9) of yeast AC40 were lethal. On the
other hand, similar mutations within the ``
motif'' of
yeast AC19 produced only minor growth defects. These results suggest
that the ``
motif'' in yeast AC40 and AC19 may not be
functionally equivalent (i.e. in enzyme assembly or activity).
Other results have indicated that mutations in the ``
motif'' of the
subunit of E. coli RNA polymerase
and the yeast RNA polymerase II RPB3 subunit produce defects in enzyme
assembly(18, 49, 50) . Our results with the Arabidopsis B36b subunit suggest that the N-terminal
``
motif'' and a second
-like motif in the C
terminus of this subunit may both be important for association with the
B13.6 subunit. Similar to the N-terminal ``
motif,'' the
second C-terminal
-like motif appears to be conserved in the
larger (i.e. 36-44-kDa) and smaller (i.e,
12.5-19-kDa) RNA polymerase subunits related to the prokaryotic
RNA polymerase
-subunit. It is possible that both
-like
motifs (i.e. the ``
motif'' and the C-terminal
leucine-rich motif) may contribute to subunit-subunit interactions and
enzyme assembly. Recent results with the
subunit in E. coli RNA polymerase indicate that both
subunit motifs (shown in Fig. 9) that are conserved in eukaryotic
-like subunits are
important for
dimerization(51, 52) . It is
interesting that the C288 truncation (see Fig. 1, Fig. 8,
and Fig. 9) in the B36b subunit, which is the shortest
truncation tested that resulted in a loss of association between the
B36b andB13.6 subunits, is located within two amino acids (i.e. see the alignment of leucine-rich C-terminal
-like motifs in Fig. 9) of an insertion mutant that renders the E. coli
subunit inactive in dimerization(52) . Additional
experiments will be required to confirm the importance and specificity (i.e. specificity of AC subunit interactions versus B
subunit interactions, specificity of plant subunit interactions versus animal or yeast subunit interactions) of the
-like
motifs in these subunit interactions.