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INTRODUCTION |
Eukaryotic RNA polymerase II is a multifunctional and multisubunit
enzyme consisting of more than 10 putative subunits (1, 2). In contrast
to prokaryotic RNA polymerases, little is known about the molecular
architectures of eukaryotic counterparts except the following: (i) the
two largest subunits, homologous to bacterial
and
' subunits,
are involved in the binding of DNA template, the polymerization of RNA
chains, and the association of nascent RNA chains (1, 2); and (ii) the
subunit 3, a homologue of bacterial
subunit, plays a role in the
assembly of RNA polymerase (3). Knowledge of the structure and function
of the individual subunits is essential for understanding of the
molecular mechanisms of transcription and regulation of the
protein-coding genes in eukaryotes. Toward this ultimate goal, we have
studied the structure-function relationship of
Schizosaccharomyces pombe RNA polymerase II.
The S. pombe RNA polymerase II is composed of 11 subunits
lacking subunit 4 (4, 5). Analyses of the subunit-subunit contact
network using different approaches indicate that the two large subunits
provide the platform for assembly of other small subunits (6-9). In
this study, we tried to locate the active center for RNA polymerization
using the method of affinity labeling with photoreactive nucleotide
analogues. This technique has been used to identify the active sites of
Escherichia coli RNA polymerase (10-13), calf thymus RNA
polymerase II (14), Saccharomyces cerevisiae RNA polymerase
I(A), II(B), and III(C) (15-18), HeLa RNA polymerase II (19), wheat
germ RNA polymerase II (18, 20), and influenza virus RNA polymerase
(21). Depending on experimental systems such as the type of enzymes and
templates, the structure of affinity reagents and the length of nascent
RNA chains, the cross-linking has been observed at different portions
of the respective RNA polymerases, but always on one or both of the two
large subunits, i.e. prokaryotic
and
' subunits
or their eukaryotic homologues.
Here we tried to cross-link transcriptionally competent elongation
complexes of S. pombe RNA polymerase II with photoreactive nucleotides incorporated at 3' termini of growing RNA chains. The
3'-ends of nascent RNA chains were found to be cross-linked to both of
the two large subunits, Rpb1 and Rpb2, in good agreement with the
previous findings with the S. cerevisiae RNA polymerase II
(22, 23). Furthermore, after proteolytic cleavage of the subunit
proteins cross-linked with radiolabeled nascent RNA chains at the 3'
termini, we proceeded to locate the contact sites of the growing RNA
3'-ends on the primary sequences of the two large subunits of S. pombe RNA polymerase II.
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EXPERIMENTAL PROCEDURES |
RNA Polymerase--
RNA polymerase II was purified from S. pombe by either the standard procedure in this laboratory (24) or
nickel affinity column chromatography (3).
Templates--
DNA templates (Templates 1 and 2) used in this
studies have the following structure (the underlined sequences
represent the tetranucleotides which direct the incorporation of UMP or
UMP analogues and CMP residues).
Single-stranded oligodeoxyribonucleotides were synthesized by
the solid-phase phosphoramidite method with a DNA/RNA synthesizer (model 394, Applied Biosystems). Mixtures of complementary
oligonucleotides in 10 mM Tris-HCl (pH 8.4), 50 mM KCl, and 2.5 mM MgCl2 were
heated for 2 min at 95 °C, incubated for 10 min at 65 °C, and
then allowed to attain ambient temperature for annealing. Annealed
full-length duplexes were separated from shorter, incomplete chains and
unannealed oligonucleotides by non-denaturing 20%
PAGE1 and recovered from gel
slices by diffusion in 0.3 M sodium acetate (pH 5.2). The
duplexes were concentrated by repeated extraction with
n-butanol, precipitated with ethanol, and dissolved in
water. DNA concentrations were determined by measuring the absorbance at 260 nm.
Photoreactive UTP Analogues--
SOCl2 (1 ml) was
added dropwise to 5-ml methanol solutions of
-aminobutyric acid,
-aminocaproic acid, or
-aminocaprylic acid (2 mmol each), which
are the precursors of analogues 2, 3, and 4, respectively (see Fig. 1).
Methyl ethers of the corresponding acids thus obtained were evaporated
in vacuo, dissolved in the mixture of acetonitrile (5 ml)
and triethylamine (1 ml), and treated overnight with
N-hydroxysuccinimide ether of
p-azidotetrafluorobenzoic acid (334 mg, 1 mmol) which was
prepared according to the published procedure (25). The products were
separated by flash chromatography on SiO2
(hexane:chloroform:methanol, 5:5:1). The compounds obtained were
dissolved in methanol (4 ml) and treated with NaOH (1 ml of 10 M solution) for 2.5 days. The reaction mixtures were
acidified by adding 2 N HCl to pH below 1 and extracted
three times with equal volumes of chloroform. The chloroform solutions
were dried over Na2SO4 and evaporated. The
derivatives of p-azidotetrafluorobenzoic acid with the
attached residues of linear amino acids obtained were converted into
N-hydroxysuccinimide ethers. Compounds were dissolved in 6.5 ml of dry CH2Cl2 and treated with
dicyclohexylcarbodiimide (206 mg, 1 mmol) and
N-hydroxysuccinimide (115 mg, 1 mmol) overnight. The
products were purified by chromatography on silica gel
(hexane:chloroform:methanol, 5:5:1). Fractions containing the product
were pooled and evaporated in vacuo.
5-(Aminopropenyl-1)uridine-5'-triphosphate was synthesized as described
(26). The photoreactive derivatives were obtained by overnight
treatment of this compound with an excess amount of
N-hydroxysuccinimide ethers of
p-azidotetrafluorobenzoic acid derivatives described above
in a solvent of dimethylformamide:water (3:1) mixture in the presence
of triethylamine added as a catalyst. The products were subjected to
chromatography on DEAE-cellulose DE52 (250 × 10 mm) and eluted
with a 0-0.3 M linear gradient of triethylammonium
bicarbonate (pH 7.0). The final step of purification was achieved by
reverse phase chromatography (Lichroprep RP-18, 250-10 mm) with a
0-50% linear gradient of acetonitrile in 0.05 M
triethylammonium bicarbonate (pH 7.0). The products were evaporated, dissolved in the minimum volume of water, and precipitated with 2%
LiClO4 in acetone.
Chemicals and Enzymes--
[
-32P]CTP and
[
-32P]GTP were purchased from Amersham Pharmacia
Biotech. RNasin, Staphylococcus aureus V8 protease,
subtilisin, and papain were products of Takara (Japan), Sigma,
Boehringer Mannheim (Germany), and Sigma, respectively. Benzonase and
RNase T1 were from Sigma and TCN Biomedicals, respectively.
-Aminobutyric acid,
-aminocaproic acid, and
-aminocaprylic
acid were products of Sigma. All other chemicals used in this work were
commercially available products of the highest quality.
Analysis of RNA Products--
Transcription assay was carried
out at 37 °C for 10 min in 70 mM Tris-HCl (pH 8.0), 75 mM (NH4)2SO4, 6 mM MgCl2, 0.15 mM DTT, 5 mM spermidine, 2 µM template I or II, 0.4 mM each of ATP, CTP, UTP or UTP analogues, 0.05 mM GTP, 0.15 µCi/µl [
-32P]GTP, 2 units/µl RNasin, and 0.04 µg/µl RNA polymerase II. After the
reaction, an equal volume of 2× formamide loading buffer (27) was
added followed by heat treatment for 2 min at 95 °C. RNA products were analyzed on 13.5% PAGE in the presence of 6 M urea.
Affinity Labeling of RNA Polymerase II--
RNA polymerase II (3 µg) was incubated for 4 min at 37 °C in 40-µl reaction mixtures
containing 70 mM Tris-HCl (pH 8.0), 75 mM
(NH4)2SO4, 6 mM
MgCl2, 0.15 mM DTT, 5 mM
spermidine, 0.4 mM ATP, 0.4 mM one of the
photoreactive UTP derivatives, 0.4 mM GTP, and 6 µM template I or II. The transcription mixture was then UV-irradiated at 100 µJ/cm2/s for 1 min with Funa UV
Linker (
= 256 nm). After the addition of 1 µl of
[
-32P]CTP (10 µCi/µl, final concentration 0.25 µCi/µl), the incubation was continued for 15 min at 37 °C, and
then 10 µl of 10% SDS and 2.6 µl of 1 M DTT were added
to make final concentrations of 2% and 60 mM,
respectively. The mixtures were heated 6 min at 95 °C and
immediately cooled on ice. The reaction products were treated with a
mixture of nucleases (benzonase, 1.5 u/µl; RNase A, 0.02 mg/ml; and
RNase T1, 7.5 u/µl) for 3 h at 37 °C to hydrolyze RNA. After
addition of 56 µl of 2× SDS loading buffer (22), the mixtures were
heated for 3 min at 100 °C and analyzed on SDS-7.5% PAGE.
Mapping of the Labeled Sites--
Gel pieces containing
radioactive RNA polymerase II subunits were excised and subjected to
peptide mapping by limited proteolysis essentially according to the
procedure of Cleveland (28, and see Ref. 21). Gel pieces containing
labeled subunits were soaked for at least 1 h in the equilibration
buffer (0.125 M Tris-HCl (pH 6.8), 0.1% SDS, 10%
glycerol, 1 mM EDTA, 42.7 mM 2-mercaptoethanol, and 0.1% bromphenol blue). Each gel slice was put on the bottom of a
sample well of SDS-15% PAGE, and then the overlay buffer (0.125 M Tris-HCl (pH 6.8), 0.1% SDS, 20% glycerol, 1 mM EDTA, 42.7 mM 2-mercaptoethanol) was added.
Finally, 20 µl of a freshly diluted protease solution in the
equilibration buffer was overlaid. Electrophoresis for sample stacking
was performed at low voltage (1.5-2 V/cm) during the first several
hours and then at 15 V/cm for sample separation. Proteolysis took place
during the initial period when the labeled subunits and proteases
remain in 4.5% stacking gel. The duration of the proteolytic cleavage
was 4 h for S. aureus V8 protease, 2.5 h for
papain, and 3 h for subtilisin. After electrophoresis, the
peptides generated were transferred onto ProBlott membrane (Applied
Biosystems), and the membranes were stained with Coomassie Brilliant
Blue. Electroblotting was fulfilled at a constant current of 0.8 mA/cm2 at room temperature overnight using a semi-dry
blotting system. The peptide bands were excised and subjected to
NH2-terminal sequencing with a protein sequencer (model
491, Applied Biosystems).
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RESULTS |
Synthesis of Photoreactive UTP Derivatives--
To map the
catalytic site of RNA polymerization in S. pombe RNA
polymerase II, photoreactive nucleotide analogues were added only to
the 3' termini of growing RNA chains, and then transcription elongation
complexes containing such transcripts were exposed to UV light for
cross-linking. In this study, we used four types of photoreactive
base-substituted analogues of UTP with spacers of different lengths
(1.5-3 nm) connecting the base and the photoreactive arylazido group
(Fig. 1A).

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Fig. 1.
Structures and activities of the
photoreactive UTP analogues. A, chemical structures of
the photoreactive UTP analogues. X indicates a spacer
between the nucleotide and the photoreactive arylazido group.
Structures of the spacers for UTP analogues 1-4 are shown in the
table. B, RNA synthesis was carried out using the
template I and in the presence of ATP, GTP, and UTP or UTP analogues
(1-4). In addition, CTP was added in one reaction shown as
CTP+. Transcripts were analyzed by denaturing 13.5%
PAGE. The migration positions of read-through (RNART)
and stalled (RNAST) transcripts are indicated on the
left side. C, transcription was carried out by
the single nucleotide addition system using template I. In the
first-step reaction, RNA synthesis was carried out for 1 min at
37 °C in the presence of GTP and ATP, and then the second-step
reaction was continued for 4 min at 37 °C after the addition of UTP
or UTP analogues (1-4). The arrow indicates RNA
with the modified UTP incorporated at the 3'-end of transcripts.
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The synthesis of these nucleotide analogues was fulfilled starting from
5-(aminopropenyl-1) uridine-5'-triphosphate (26). This compound was
allowed to react with excess amounts of four different
N-hydroxysuccinimide ethers of
p-azidotetrafluorobenzoic acid, each containing an aliphatic
chain (X) of different length. Reactions proceeded
quantitatively in the presence of triethylamine as a catalyst and
dimethylformamide:water (3:1) as a solvent. The products were purified
by ion-exchange and reverse phase high pressure liquid chromatography
and characterized by NMR spectra (data not shown).
The substances have several characteristic features. First, the
photoreactive groups are attached at position C-5 of uracil which is
not involved in hydrogen bonding with adenine. The substitution may not
give significant effects in the conformation of ribose moiety, and
therefore the modified UTP derivatives may function as substrates in
RNA synthesis. Second, upon exposure to UV irradiation, the
p-azidotetrafluorobenzoyl group is able to generate the
highly active intermediate particle, singlet nitrene, which can react with any molecules in surroundings. The generation of singlet nitrene
proceeds very rapidly, and full decomposition of the photoreactive groups takes place within only 1 min upon exposure at 256 nm at an
energy 100 µJ/cm2/s. Probably aromatic nitrenes will be
rearranged to form inactive 7-atomic ring. The substitution of all
protons in benzene ring by fluorine atoms allows the avoidance of this
undesirable side reaction. Third, since the efficiency of affinity
labeling often depends on the structure of nucleotide analogues, the
use of spacers (X) with different lengths allows us to
choose the best compound characteristic of the enzyme to be
cross-linked.
Transcription with the Use of Photoreactive UTP
Derivatives--
In order to incorporate photoreactive UTP analogues
only at 3'-ends of transcripts, we constructed two kinds of 40-base
pair-long DNA duplexes that consist of two segments, 3'-proximal
segment with a sequence of TC mixtures and 5'-proximal segment of G
clusters, both being connected by a single A residue (Fig.
2; for detailed sequences see
"Materials and Methods"). The two single A templates carried
essentially the same sequence except that one containing A in the
middle (I) and the other near the 3'-end (II). For use as templates for
transcription in vitro, an extra sequence of 10 C residues
was attached to the 3'-ends of the template strands. On such templates,
transcription is preferentially initiated within the single-stranded
protruded region (29) at 3-5 residues upstream from the junction
between single-stranded and double-stranded regions (30). Thus,
transcripts should carry heterogeneity in the number of 5'-terminal G
residues arising from the initiation at variable positions on the
single-stranded C tail. Since both templates contain a single A
residue, UTP should be incorporated only at this unique site. In both
templates, this unique A is followed by three G residues that are
absent in the upstream regions. Thus, in the absence of CTP,
transcription should be stalled after the incorporation of UMP or UMP
analogues.

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Fig. 2.
Schematic representation of the templates and
transcripts. The nucleotide sequences of templates
I and II are the same except that the single A
residue is positioned either in the middle (template
I) or at the 3'-end (template II) of
template strand. In both cases, this single A is followed by three G
residues (underlined). In the absence of CTP, RNA polymerase
should stall after the incorporation of UMP or modified UMP residue
(Uphoto) complementary to the single A. To the 3' termini
of the template strands, 10 extra C residues were added so as to allow
efficient initiation on this C cluster.
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To test the above possibilities, RNA synthesis was carried out using
the template I and in the presence of ATP, UTP, and
[
-32P]GTP. Polyacrylamide gel electrophoresis of
transcripts indicated that in the absence of CTP, transcription
complexes become stalled after UMP incorporation, resulting in the
formation of transcripts (RNAST) of 25-30 nucleotides in
length (Fig. 1B,
CTP lane), whereas in the
presence of CTP, template-sized read-through transcripts (RNART) were synthesized (Fig. 1B, +CTP lane).
When UTP analogues were used in place of UTP, RNA synthesis was also
stalled, forming stalled transcripts with apparently the same sizes as
the transcripts observed with UTP (Fig. 1B, +UTP analogue
lanes).
To confirm the incorporation of UTP analogues into the RNA products,
the single nucleotide addition experiment was performed (Fig.
1C), in which RNA synthesis was carried out in the presence of ATP and [
-32P]GTP (without UTP) to form the stalled
complexes, and then UTP or UTP analogues were added to allow the
addition of a single U residue. The incorporation of nucleotide
derivatives bearing arylazido groups into RNA significantly decreases
the mobility in gel electrophoresis (31). We then analyzed RNA products
under improved conditions of gel electrophoresis. In fact, we could distinguish RNA products with regular UMP and UMP analogues based on
the difference in the mobility on PAGE (Fig. 1C; the
arrow indicates RNAs with modified UTP analogues). The
efficiency of UTP analogue incorporation was higher for analogues 1 and
4 (the difference in the incorporation of UTP analogues is discussed below).
Photoaffinity Labeling of Subunits of RNA Polymerase II--
For
efficient cross-linking of 3' growing ends of nascent RNA molecules to
RNA polymerase II, transcription of the test template I was carried out
in the presence of three nucleotides, ATP, [
-32P]GTP,
and photoreactive UTP analogues. After addition of CTP, however, not
all the stalled transcripts were elongated to the template-sized
read-through transcripts (data not shown), indicating that portions of
the stalled complexes tend to isomerize into dead-end complexes that
are inactive in restart of RNA synthesis after CTP is added.
In order to achieve specific cross-linking of RNA within the
catalytically active elongation complexes, the first-step RNA synthesis
was carried out in the presence of ATP, GTP, and photoreactive UTP
derivatives, and the stalled complexes thus formed were exposed to UV
light. Transcription was then continued by adding
[
-32P]CTP. Restart of RNA synthesis by the
catalytically active transcription complexes could be detected by
measuring the incorporation of radioactive CMP into stalled RNA chains.
By using this approach of catalytically competent labeling technique,
we carried out the cross-linking of nascent RNA chains to RNA
polymerase II and analyzed the cross-linked subunits by PAGE. Since the
presence of RNA chains cross-linked to the enzyme might lead to change
in the mobility of labeled subunits and thus interfere with the
identification of subunits, we tried to remove ribonucleotide moieties
by nuclease treatment of the cross-linked samples prior to SDS-PAGE. In
the course of RNA hydrolysis, the radioactive 5'-phosphate of CMP
remained attached to the 3'-hydroxy group of the UMP analogue
cross-linked to the enzyme (data not shown). This immediately indicated
that [
-32P]CTP was indeed polymerized into the stalled
RNA at the site next to the UMP analogues.
The cross-linking was found to take place with only two large subunits,
Rpb1 and Rpb2, even though the cross-linking efficiency was different
between the two subunits depending on the UTP analogues because of the
difference in spacer length and/or hydrophobicity of the reactive
moieties (Fig. 3). Without UV exposure,
the radioactivity was not detected in any RNA polymerase II subunits
(data not shown). The labeling of Rpb2 was expected because the
catalytic site of RNA polymerization in RNA polymerases is located on
the second largest subunit (prokaryotic
subunit or eukaryotic
homologues). The labeling of Rpb1 clearly indicates that this subunit
also participates in the formation of the catalytic site of RNA
polymerization or at least it is located close to the active site on
Rpb2. The most intensive patterns of labeling were obtained with the
use of analogues 4 and 1, in good agreement with the
relative level of RNA synthesis (see Fig. 1C). Since the
catalytic site of DNA polymerase is known to be located in hydrophobic
surroundings (32), the analogues with higher hydrophobicity should be
more preferable substrates for RNA polymerases. In concert with this prediction, the intensity of Rpb2 labeling increased with the increase
in hydrophobicity of UTP derivatives (2
3
1
4).

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Fig. 3.
SDS-gel electrophoresis of the RNA polymerase
II cross-linked with nascent transcripts. Transcription in
vitro by S. pombe RNA polymerase II was carried out
using template I and in the presence of ATP,
[ -32P]GTP, and the indicated UTP analogue. The RNA
polymerase with (+) and without ( ) UV irradiation was analyzed by
SDS-PAGE. Migration positions of the marker proteins are indicated on
the left side, whereas the bands of RNA polymerase subunits
with covalently cross-linked with radioactive RNA are marked on the
right side.
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When the regular UTP was used in place of the UTP analogues or when the
reaction was carried out in the absence of GTP, we detected no
radioactivity associated with the RNA polymerase II subunit bands (data
not shown).
Proteolytic Cleavage of Two Large Subunits of RNA Polymerase
II--
For identification of the cross-linking site(s) of nascent RNA
on the two large subunits of RNA polymerase II, we attempted to locate
proteolytic cleavage segments with cross-linked RNA on the primary
sequences of these subunit polypeptides. For this purpose, we first
performed limited digestion of isolated Rpb1 and Rpb2 with six
different proteases, i.e. chymotrypsin, elastase, papain,
subtilisin, trypsin, and V8 protease, among which the controlled
cleavage was possible only for three proteases, papain, subtilisin, and
V8 protease. The limited proteolysis at various protease concentrations
led to the generation of mixtures of different peptides, as observed
after SDS-PAGE analysis (Figs. 4 and
5). Since some peptides with similar
sizes co-migrate on SDS-PAGE, the complete separation of all peptides
was not always possible. Peptides thus separated were transferred onto
ProBlot membranes and subjected to NH2-terminal sequencing.
Based on the sequence knowledge, we could locate the peptides on the
primary sequence of Rpb1 and Rpb2 subunits (Figs.
6 and 7;
the numbering of peptides was only for those sequenced). Controlled
cleavage of proteins by proteases often generates domains that exist in
intact proteins. Even in the presence of SDS at the concentrations
used, only limited sites among all potential cleavage sites were
cleaved by the proteases, indicating that some structures are retained
under the conditions employed. For instance, among all the possible
cleavage sites by V8 protease (shown by vertical lines on
the primary sequences of Rpb1 and Rpb2 in Figs. 6A and
7A), only 10% sites were cleaved by V8 under such partially
denatured conditions. Taking the cleavage patterns with three different
proteases together, we concluded that Rpb2 consists of 7 domains, each
being connected by a linker that is relatively sensitive to proteolytic
cleavage. In most cases the sites of cleavage are located in the
spacers between the regions conserved among various RNA polymerases
from both prokaryotes and eukaryotes (see Fig. 10). Under the
proteolytic cleavage conditions employed, we never observed the
peptides corresponding to the NH2-terminal part of Rpb2
down to about residues 200, probably because its NH2
terminus carries a blocked structure. The specificities of proteases in
polyacrylamide gels containing SDS were different from those in
solutions without SDS. For instance, V8 protease cleaved preferentially
after Asp, whereas the specificity of papain became broader, with
nonspecific cleavage activities at Ala, Gly, Leu and Thr.

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Fig. 4.
Proteolytic cleavage of the Rpb2
subunit. Rpb2 was isolated from purified S. pombe RNA
polymerase II by 0.1% SDS-7.5% PAGE. Gel pieces containing the Rpb2
were mixed with a solution containing the indicated concentrations of
V8 protease (A), papain (B), or subtilisin
(C), subjected to 0.1% SDS-15% PAGE, and digested in the
stacking gel for 4 h (A), 2.5 h (B), or
3 h (C). After electrophoresis, proteolytic fragments
were transferred onto ProBlott membranes and stained with Coomassie
Brilliant Blue R250. Migration positions of the marker proteins are
indicated on the right side, and the Rpb2 fragments are
marked between the lines. The fragments that were sequenced
at NH2 termini are numbered in the order of
decreasing molecular weight.
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Fig. 5.
Proteolytic cleavage of the Rpb1
subunit. Rpb1 was isolated from purified S. pombe RNA
polymerase II by 0.1% SDS-7.5% PAGE. Gel pieces containing the Rpb1
were mixed with a solution containing the indicated concentrations of
V8 protease (A), papain (B), or subtilisin
(C), subjected to 0.1% SDS-15% PAGE, and digested in the
stacking gel for 4 h (A), 2.5 h (B), or
3 h (C). After electrophoresis, proteolytic fragments
were transferred onto ProBlott membranes and stained with Coomassie
Brilliant Blue R250. Migration positions of the marker proteins are
indicated on the right side, and the Rpb1 fragments are
marked between the lines. The fragments that were sequenced
at NH2 termini are numbered in the order of
decreasing molecular weight.
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Fig. 6.
Proteolytic cleavage pattern of the Rpb2
subunit. A, cleavage pattern by V8 protease. The
location of Asp (the potential cleavage site by V8) is shown on the
primary sequence of Rpb2 indicated by the horizontal thick
line. Triangles above the line indicate the
cleavage sites by V8 as analyzed in this study. The sensitivity to V8
is shown by the size of triangles. The NH2
termini of V8 fragments were determined after sequencing (the
number in brackets represent the
NH2-terminal residue), whereas the carboxyl termini were
estimated from the size of fragments. Squares under
the line represent the approximate positions of carboxyl termini
of each fragment. B, cleavage pattern by papain.
C, cleavage pattern by subtilisin.
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Fig. 7.
Proteolytic cleavage pattern of the Rpb1
subunit. A, cleavage pattern by V8 protease. The
location of Asp (the potential cleavage site by V8) is shown on the
primary sequence of Rpb1 indicated by the horizontal thick
line. Triangles above the line indicate the cleavage
sites by V8 as analyzed in this study. The sensitivity to V8 is shown
by the size of triangles. The NH2 termini of V8
fragments were determined after sequencing (the number in
brackets represent the NH2-terminal residue),
whereas the carboxyl termini were estimated from the size of fragments.
Squares under the line represent the approximate positions
of carboxyl termini of each fragment. B, cleavage pattern by
papain. C, cleavage pattern by subtilisin.
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Analysis of the proteolytic fragments of Rpb1 (Fig. 5) revealed 7 protease-sensitive regions, correspondingly generating 7 distinctive
protease-insensitive domains plus the carboxyl-terminal domain (see
Fig. 7). As in the case of Rpb2, the cleavage sites are located
approximately on the junctions between the conservative motifs (see
Fig. 11).
Identification of Segments Cross-linked with Nascent RNA
3'-End--
By using the Rpb1 and Rpb2 that were cross-linked with
nascent RNA chains containing UTP derivative 1 (with the shortest
linker) by the catalytically competent technique, we next tried to
identify the structural elements in the vicinity of the active site of RNA polymerase II. After the nuclease hydrolysis and separation on
SDS-7.5% PAGE, the radioactively labeled Rpb2 and Rpb1 subunits were
cut from the gel and subjected to the digestion by the three proteases,
V8, papain, and subtilisin, in 4.5% stacking gel in the presence of
0.1% SDS, and the peptides generated were separated on SDS-15% PAGE.
After electrophoresis, the gels were stained with a silver reagent,
dried, and exposed to phosphorimage plates. Comparison of the
silver-stained gels and the patterns of phosphorimage allowed us to
identify a set of peptides bearing the radioactive label. Some of the
results are shown on Figs. 8 and
9. The nucleotide cross-linked segments
were identified only in the case when the peptides were well separated
from other peptides on SDS-PAGE and only when the peptides gave only a
single unique amino acid sequence.

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Fig. 8.
Proteolytic analysis of the Rpb2 subunit
cross-linked with nascent transcripts. RNA polymerase II was
cross-linked with nascent transcripts by the catalytically competent
labeling method, treated with nucleases, and then subjected to subunit
separation by 0.1% SDS-7.5% PAGE. The gel band containing the Rpb2
subunit was mixed with the indicated amounts of V8 protease
(A), papain (B), or subtilisin
(C) and subjected to 0.1% SDS-15% PAGE. Protease digestion
was carried out in the stacking gel for 4 (A), 2.5 (B), or 3 h (C). After electrophoresis, the
gels were stained with a silver reagent, dried, and exposed to
phosphorimaging plates. The comparison of silver-stained gels
(Ag) with the phosphorimager-generated pictures
(IP) allowed to identify the labeled fragments of Rpb2.
Migration positions of the marker proteins are shown on the left
side, and the Rpb2 fragments are marked between the gel
patterns.
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Fig. 9.
Proteolytic analysis of the Rpb1 subunit
cross-linked with nascent transcripts. RNA polymerase II was
cross-linked with nascent transcripts by the catalytically competent
labeling method, treated with nucleases, and then subjected to subunit
separation by 0.1% SDS-7.5% PAGE. The gel band containing the Rpb1
subunit was mixed with the indicated concentrations of V8 protease
(A), papain (B), or subtilisin (C) and
subjected to 0.1% SDS-15% PAGE. Protease digestion was carried out in
the stacking gel for 4 (A), 2.5 (B), or 3 h
(C). After electrophoresis, the gels were stained with a
silver reagent, dried, and exposed to phosphorimaging plates. The
comparison of silver-stained gels (Ag) with the
phosphorimager-generated pictures (IP) allowed us to
identify the labeled fragments of Rpb1. Migration positions of the
marker proteins are shown on the left side, and the Rpb1
fragments are marked between the gel patterns.
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In the case of Rpb2, the labeled peptides include V8 segments, p1, p2,
p4, p7, and p11 (Fig. 8A), papain segment p3 (Fig. 8B), and subtilisin segments, p1, p2, and p3 (Fig.
4C). For Rpb1, the labeling was observed for V8 peptides p1
and p2 (Fig. 9A), papain segment p8 (Fig. 9B),
and subtilisin segments p4 and p5 (Fig. 9C). When the
cross-linking reaction was carried out using the template II, the
patterns of labeled peptides were essentially the same with those
obtained with the template I (data not shown).
The locations of these radioactive peptides on the primary sequence of
Rpb2 and Rpb1 polypeptides are summarized in Figs. 10 and
11, respectively. The peptides of the
most intensive labeling are located between amino acids 825 and 994 of
Rpb2, the region including the conserved sequences H and G (Fig. 10).
The less intensive labeling also occurs in the region between amino
acids 298 and 535 of Rpb2. This region contains the conserved sequences
C and D. The extent of Rpb1 labeling was lower than that of Rpb2, and all the cross-linked peptides are located at the region between amino
acids 614 and 917, which contains the conserved sequence F (Fig.
11).

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Fig. 10.
Location of the cross-linked region of Rpb2
with 3' termini of nascent transcripts. The "domains"
represent the seven segments of Rpb2 that were identified to be
resistant to V8 protease, papain, and subtilisin. The major and primary
cleavage sites by the three proteases, shown by vertical
lines, are located between these domains. The conserved
regions (A-I) represent the sequences conserved among Rpb2
homologues from prokaryotic and eukaryotic RNA polymerases. From
the collection of proteolytic fragments with cross-linked nascent
transcripts, we propose that the "cross-linked regions" are located
in two positions of Rpb2, one between residues 298 and 535 and another
between residues 825 and 994, as shown by the gray bars on
the bottom.
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Fig. 11.
Location of the cross-linked region of Rpb1
with 3' termini of nascent transcripts. The domains represent the
seven segments of Rpb1 that were identified to be resistant to V8
protease, papain, and subtilisin. The major and primary cleavage sites
by the three proteases, shown by vertical lines, are
located between these domains. The conserved regions
(A-H) represent the sequences conserved among Rpb1
homologues from prokaryotic and eukaryotic RNA polymerases. From the
collection of proteolytic fragments with cross-linked nascent
transcripts, we propose that the cross-linked region is located between
residues 614 and 917 as shown by the gray bar on the
bottom.
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DISCUSSION |
For precise identification of the catalytic site on RNA polymerase
II, we took special care by employing several new approaches. (i) The
templates were designed so as to allow the incorporation of
photoreactive substrate analogues only into 3'-ends of initiation oligonucleotide products ("initiation complex") or 3'-growing ends
of nascent RNA chains ("elongation complex"). (ii) Only the catalytically active transcription complexes were detected by labeling
with a single radioactive substrate added next to the cross-linked
nucleotides (stalled complexes tend to isomerize into dead-end
complexes where 3'-ends of RNA have different contacts with the RNA
polymerase (33)). (iii) A number of small radioactive peptide fragments
were isolated from each of the labeled Rpb2 and Rpb1 by treatment with
three different proteases to narrow down the cross-linked regions. As a
result, we found that both the initiation and elongation complexes gave
essentially the same cross-linked peptides, indicating that the same
region of RNA polymerase II is always located close to the 3' termini
of both initiation oligonucleotides and nascent RNA chains.
Several lines of evidence indicated that the cross-linking observed
under the reaction conditions employed were specific to the growing
ends of nascent transcripts, because of the following: (i) when the
transcription was carried out using UTP in place of the UTP analogues
or in the absence of GTP, we detected no radioactivity associated with
the RNA polymerase II subunit bands; (ii) likewise, we could not detect
protein-bound radioactivity when the reaction was carried out in the
absence of template DNA; and (iii) upon exposure to UV light, the UTP
analogues used are rapidly degraded with a half-life of less than 1 min
(34). It should be noted, however, that the RNA polymerase isomerizes
during the transition from initiation to elongation complexes (35, 36).
In this study, we used duplex DNA templates I and II with a tail of 10 C residues attached at 3'-end of the template strand. On such
templates, transcription is efficiently initiated on the protruded
single-stranded tails about 3-5 bases upstream from the single- and
double-strand junction (29, 30). The initiation complexes we analyzed
were associated with 3-5 G residues transcribed by this
single-stranded C tail. The template II was designed as to cross-link
the initiation complexes, in which the initiated transcripts with the
sequence of G3-5UPhotoC1-3 might
not be displaced. On the other hand, on the elongation complex formed
on the template I, the nascent transcripts with the expected sequence
of
G3-5(AG)24UPhotoC1-3
could be displaced at their 5' termini. The elongation complexes formed
on such templates as employed in this study could be different from the
typical elongation complexes formed on the complete duplex DNA. For
instance, Kadesch and Chamberlin (29) reported that approximately half
of transcripts on the elongation complexes initiated from the same type
of synthetic template remained bound to the template without being
displaced. The site of RNA displacement is, however, far from the
catalytic site of RNA polymerization, with these two sites being
separated by at least several nucleotides. Thus, the difference in the
site of RNA displacement between the synthetic and native DNA templates
might not influence the location of 3'-end of nascent transcripts.
Partial proteolysis by three proteases, V8, papain, and subtilisin,
indicated complex multidomain organizations for both Rpb1 and Rpb2.
Rpb1 contains 8 major structural domains followed by 27 repetitions of
the carboxyl-terminal domain sequence of 7 amino acid residues, whereas
Rpb2 includes 7 major domains (see Figs. 10 and 11). From the sequence
comparison, subunits 1 and 2 of RNA polymerases from both prokaryotes
and eukaryotes are known to contain 8 (A-H plus carboxyl-terminal
domain) and 9 (A-I) conserved sequences, respectively (1, 2) (see
Figs. 10 and 11). Each major structural domain of Rpb1 and Rpb2
identified in this study contains one or two of the respective
conserved sequences.
Detailed mapping of the Rpb1 and Rpb2 regions cross-linked with 3'
termini of nascent transcripts was carried out on the basis of the
cleavage map of these subunit polypeptides by these three proteases. In
the case of S. pombe Rpb2, the most intensive cross-linking was found to occur in the region between amino acids 825 and 994, including the structural domains 5 and 6 and the conserved sequences G
and H (see Fig. 10). In the case of the S. cerevisiae RNA
polymerase II, the region between amino acids 946 and 999 of the B150
(RPB2) subunit which corresponds to the region 935-988 of Rpb2 for
S. pombe is cross-linked with nucleotide analogues
polymerized into RNA chains (22). Thus, we concluded that the catalytic
site for RNA polymerization is located on the subunit 2 and that the location of the catalytic site on the subunit 2 is essentially the same
between S. pombe and S. cerevisiae RNA polymerase
II. It may be worthwhile to note that essentially the same region of
Rpb2 (amino acids 902 to 991) is involved in the molecular contact
between Rpb2 and Rpb3 (9). One possibility is that the binding of Rpb3
induces the correct folding of Rpb2 leading to expression of its
intrinsic activities. In fact, the
subunit of E. coli
RNA polymerase expresses the binding activities to rifampicin,
nucleoside 5'-triphosphates, and DNA only after the formation of
2
complex formation (37). Likewise, the Rpb2 (
homologue), Rpb3 (
homologue), and Rpb11 (
homologue) form a core
subassembly with the DNA binding activity (3).
On Rpb2, the less intensive labeling was observed in the region between
amino acids 306 and 542, including the structural domain 2 and the
conserved sequences C and D (see Fig. 10). This region also includes
the RNase-like domain (38). The isolated Rpb2 fragments containing this
RNase-like domain, however, did not show the activities of RNA cleavage
in vitro, and site-directed mutagenesis of this domain did
not affect growth of the mutant S. pombe (39).
In the case of E. coli RNA polymerase, the most sensitive
sites for proteolytic cleavage on the
subunit (Rpb2 homologue) are
located in two regions, one upstream of the conserved sequence D and
the other upstream of the conserved sequence F
(40).2 These sites may
correspond to the cleavage sites between the domains 2 and 3 and
between the domains 3 and 4 of Rpb2. The binding sites for substrates
and rifampicin of E. coli
subunit overlaps the upstream
labeling region of S. pombe Rpb2, whereas the catalytic site
for RNA polymerization of the
subunit overlaps the downstream labeling region of Rpb2 (13). The overall organization of structure and
function is similar between the prokaryotic
subunit and the
eukaryotic subunit 2, but in the NH2-terminal part of
Rpb2, there is an accessible site for proteolytic cleavage between the conserved sequences B and C, which was not observed for E. coli
subunit.
Mapping of the RNA-binding site on the subunit 1 of RNA polymerase II
has never been reported. The cross-linking site of Rpb1 with 3' termini
of nascent RNA chains was identified for the first time in the region
between amino acids 614 and 917 (see Fig. 11). This region includes the
conserved sequence F, one of the most well conserved among the
'
subunit homologues from various RNA polymerases. The F region is also
involved in the responsibility of RNA polymerase II to
-amanitin, a potent inhibitor of eukaryotic RNA polymerase II (41,
42). This amatoxin interferes with the translocation of RNA polymerase
II along the DNA template (43). Our data confirm the fact that the F
region is located close to the catalytic site of Rpb2, probably
participating in the process of RNA polymerization.
On the
' subunit of E. coli RNA polymerase, the most
available sites for trypsin are located in two regions, one between the
conservative sequences B and C and the other between the conserved sequences F and G.3 Rpb1 also
has the sites for proteolytic cleavage in these regions, but in
addition, there are three additional sites exposed for proteases. The
RNA-binding site on the E. coli
' subunit was reported to
be within the conserved sequence D (13), but our cross-linking studies
identified the RNA-binding site on Rpb1 near the conserved sequence F. This discrepancy remains to be solved. One possibility is that the
location of
' subunit relative to the catalytic site of the
subunit shifts during the process of RNA chain elongation.