From the
RNA polymerase I of Saccharomyces cerevisiae is
composed of 14 subunits. All of the corresponding genes have been
cloned with the exception of the RPA14 gene encoding A14, a
specific polypeptide of this enzyme. We report the cloning and the
characterization of RPA14. The A14 polypeptide was separated
from the other RNA polymerase I subunits by reverse-phase high pressure
liquid chromatography and digested with proteinase K. Based on the
amino acid sequence of one of the resulting peptides, a degenerate
oligonucleotide was synthesized and used to isolate the RPA14 gene from a yeast subgenomic DNA library. RPA14 is a
single copy gene that maps to chromosome IV and is flanked by CYP1 and HOM2. Disruption of RPA14 is not lethal, but
growth of the rpa14::URA3 mutant strain is impaired at 37 and
38 °C. RNA polymerase I was purified from the rpa14::URA3 strain. After two purification steps, the enzyme did not contain
the subunits A14, ABC23, and A43. This form of the enzyme was not
active in a nonspecific in vitro transcription assay. These
results demonstrate that A14 is a genuine subunit of RNA polymerase I
and suggest that A14 plays a role in the stability of a subgroup of
subunits.
As do all eukaryotic cells, the yeast Saccharomyces
cerevisiae contains three forms of nuclear RNA polymerase (I, II,
and III). These three enzymes are large multisubunit proteins composed
of 12-16 polypeptides
(1) . Nearly all of the genes
encoding these polypeptides have been
isolated
(1, 2, 3) . Each of the three enzymes is
organized around a central core composed of the two large subunits,
homologous to the subunits
The analysis of the
conserved domains of the
Among the 14 polypeptides
present in a purified preparation of RNA polymerase I from S.
cerevisiae, five are enzyme-specific (A49, A43, A34.5, A14, and
A12.2). A comparison of the polypeptide content of RNA polymerase I
from different yeasts revealed that the subunit pattern is conserved in
different Saccharomyces subspecies. However, in the RNA
polymerase I preparation obtained from the yeasts
Schizosaccharomyces pombe and Candida tropicalis,
components homologous to the A49, A43, A34.5, A14, and A12.2 subunits
were not detected
(10) .
The enzyme-specific subunits A49,
A43, and A34.5 are loosely associated with the enzyme. A43 is present
in substoichiometric amounts, and A49 and A34.5 are easily dissociated
from the multisubunit complex generating a simplified A* form of the
enzyme
(11, 12) . The A* form has altered enzymatic
properties in a nonspecific in vitro transcription assay and
is more sensitive to
We report here the isolation and the characterization of the
RPA14 gene encoding the RNA polymerase I specific A14
polypeptide. Disruption of RPA14 shows that this gene is not
essential for cellular growth. Biochemical studies of the RNA
polymerase I isolated from the rpa14::URA3 strain shows that
the absence of A14 results in a marked instability of the enzyme,
demonstrating that A14 is a genuine subunit of RNA polymerase I.
After each purification
step, RNA polymerase I in the fractions was detected by immunoblot
analysis. Samples were run on a 13% SDS-polyacrylamide gel and then
transferred onto a nitrocellulose membrane (Schleicher and
Schüll). The membrane was incubated with anti-yeast RNA polymerase
I antibodies (3 µg/ml) supplemented with anti-AC19 (3 µg/ml)
and anti-A14.5/A14 (1 µg/ml) subunit-specific
antibodies
(32) . After incubation with an anti-rabbit
horseradish peroxidase-conjugated secondary antibody, protein-antibody
complexes were visualized using an enhanced chemoluminescence Western
blotting detection system (ECL; Amersham). RNA polymerase I activity
was assayed in a nonspecific in vitro transcription assay as
described by Buhler et al.(32) using
poly[d(A-T)].
In
vivo synthesis of small RNAs in an rpa14::URA3 strain was
assayed by pulse labeling with [5,6-
In the present work, we have cloned the gene, named
RPA14, that encodes the RNA polymerase I specific subunit A14.
This work completes the cloning of the 14 RNA polymerase I subunit
genes
(1) . RPA14 is a single copy gene located on
chromosome IV, which encodes an acidic protein of a predicted molecular
mass of 14.6 kDa. A strain containing a disrupted RPA14 gene
exhibits a thermosensitive phenotype. The gene disruption removed most
of the RPA14 coding sequence including nine nucleotides of a
HSE-like sequence that was presumed to belong to the CYP1 promotor
(35) (see Fig. 3). However, we assume that
the thermosensitivity of the mutant rpa14::URA3 strain is
related to the absence of the A14 polypeptide and not to a lower
expression level of the nearby CYP1 gene, since the deletion
of CYP1 confers no detectable phenotype at 24, 30, or 37
°C
(41) .
The first piece of evidence that A14 is a
subunit of the RNA polymerase I was obtained by in vivo labeling of RNAs, which indicated that the level of 5.8 S rRNA in
the rpa14::URA3 strain was slightly reduced compared with the
level of the 5 S and tRNAs. Analysis of the RNA polymerase I enzyme
purified from the rpa14::URA3 strain confirmed this
assumption. Phosphocellulose chromatography and Western blot analysis
from crude extracts of wild-type and rpa14::URA3 strains
indicated that the mutant produced the same amount of RNA polymerase I
as the wild type. Yet, we were not able to purify an active enzyme from
the rpa14::URA3 strain when using the classical purification
procedure. Only an inactive form lacking A43, ABC23, and A14 subunits
was obtained. Together, these results indicated that identical amounts
of RNA polymerase I are assembled in mutant and wild-type cells but
that the mutant RNA polymerase I is less stable compared with the
wild-type enzyme.
The inactive form of the enzyme lacking subunits
A43, ABC23, and A14 is likely not the enzyme form present in vivo in the rpa14::URA3 mutant strain, since both A43 and
ABC23 are required for cell viability
(42) .
An interesting question is
to determine which of the lost subunits, A43, ABC23, or A14, was
responsible for the loss of enzyme activity. Although the subunit A43
is essential in vivo,
The nucleotide
sequence(s) reported in this paper has been submitted to the
GenBank
We thank Isabelle Treich and Christine Conesa for
discussions and suggestions in cloning strategies, Pierre Thuriaux for
help in the course of the gene disruption experiment, and Yves Barral
for help in tetrad dissection. We thank Cathy Jackson and Pierre
Thuriaux for a critical reading of the manuscript.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
` and
of the bacterial RNA
polymerase, and of subunits related to the
subunit of the
prokaryotic enzyme. This conserved core is associated with a set of
subunits common to all three RNA polymerases (ABC27, ABC23, ABC14.5,
ABC10
, and ABC10
) and a variable set of enzyme-specific
polypeptides (for reviews see Refs. 1-3).
`- and
-like subunits indicated that
these subunits are involved in the basic functions of the RNA
polymerases such as binding of the initiator
nucleotides
(4, 5, 6) , the interaction with the
DNA template, and the interaction with the nascent RNA (see Refs. 1 and
2). So far, few data are available that pertain to the function of the
smaller subunits. The subunits shared by the three RNA polymerases or
common to enzymes I and III (AC40 and AC19) are strictly essential for
the cell viability. These subunits probably provide basic functions in
the catalytic process or in the enzyme assembly
(7) . The
importance of the enzyme-specific subunits for the structure and/or the
function of each RNA polymerase is more difficult to assess. One can
imagine that some of them are involved in functions that are
characteristic of the mechanism of transcription by each RNA polymerase
such as the interaction with specific transcription factors. Indeed, it
has been shown that the C34 subunit of RNA polymerase III interacts
with TFIIIB (8) and that the C31 subunit is involved in the formation
of the initiation complex
(9) .
-amanitin
(11, 12, 13) . These results
suggested that A49 and A34.5 are part of the RNA polymerase I enzyme.
The A49 polypeptide has been found to copurify with an RNaseH
activity
(14, 15, 16) . The genes encoding the
A49, A43, A34.5, and A12.2 subunits have been cloned and
sequenced
(17, 18) .
(
)(
)
Except for the A34.5 subunit, their specific importance for
the RNA polymerase I transcription machinery has been demonstrated by
different approaches. The disruption of the RPA43 gene is
lethal, whereas the other three genes, RPA49, RPA34,
and RPA12, are not strictly required for cell viability. The
importance of the A49 subunit for rRNA synthesis was demonstrated by
in vivo RNA labeling experiments, which revealed that the rate
of synthesis of 5.8 S rRNA versus 5 S RNA and tRNAs was
reduced in a strain deleted for the RPA49 gene
(17) .
The results of biochemical and genetic experiments with a
thermosensitive rpa12::LEU2 strain suggest that A12.2 at least
plays a role in the assembly of the largest subunit, A190
(18) .
Yeast Strains
Yeast strain S288C (MAT Suc2 mal mel gal2 CUP1) was from the yeast genetic stock center
(Berkeley). Yeast strains YPH499 (MATa ade2-101° lys2-801a ura3-52 trp1-
63 his3-
200
leu2-
1) and YPH500 (MAT
ade2-101 uaa
ura3-52 lys2-801 uag trp1-
63 his3-
200
leu2-
1) were provided by Sikorski and Hieter
(19) .
Purification of A14 and Microsequencing of
Peptides
Yeast RNA polymerase I was purified according to Buhler
et al. (20). The A14 subunit was isolated from the purified
enzyme by reverse-phase high pressure liquid chromatography
(RP-HPLC)(
)
using a chromatograph 130A (Applied
Biosystems). The conditions of chromatography described by Carles
et al.(21) were modified as follows. Purified RNA
polymerase I (200 µg) in aqueous solution was applied to an
Aquapore RP300 column (2.1
30 mm; Brownlee Labs) at a flow rate
of 200 µl/min and at a temperature of 35 °C. The column was
developed in solvent A (0.1% trifluoroacetic acid in water) with a
linear gradient from 40 to 52% of solvent B (0.075% trifluoroacetic
acid in 40% acetonitrile, 40% isopropyl alcohol, 20% water) for 60 min
followed by a gradient from 52 to 100% of solvent B within 10 min.
Absorbance peaks at 214 nm were collected, and the proteins present in
the different fractions were analyzed by SDS-polyacrylamide gel
electrophoresis and silver staining according to Blum et al.
(22). Fractions containing the A14 subunit purified from 1.2 mg of RNA
polymerase I were pooled, diluted with 1.3 volumes of water, and
adjusted to pH 8.0 with 0.25 volume of 1 M ammonium carbonate,
pH 8.0. Digestion of the protein was performed overnight at 37 °C
in the presence of proteinase K (1.5 µg), and then 1 µg of
proteinase K was added and incubation was continued for 16 h at 37
°C. Finally, 0.1 mM dithioerythreitol and 0.5 mM
EDTA were added, and the sample was incubated at 40 °C for 3 h. The
proteolytic peptides were separated by RP-HPLC on an Applied Biosystems
130-A microbore HPLC apparatus according to Lefebvre et
al.
(23) , and the purified peptides were microsequenced by
using an Applied Biosystems model 477A protein sequencer.
Cloning and Sequencing of the RPA14 Gene
Based on
the amino acid sequence of the largest peptide, FKGLPPAQDF, a
degenerate oligonucleotide was synthesized, reducing the degeneracy by
including deoxyinosine at the third position of the most degenerate
codons: TT(C/T)-AA(A/G)-GGI-(C/T)TI-CCI-CCI-GCI-CA(A/G)-GA(C/T)-TT. The
oligonucleotide was used to probe a Southern blot of genomic DNA
prepared from the S288C strain as described by Dequard-Chablat et
al.(24) . Based on the result of the Southern blot
analysis, two subgenomic libraries were constructed as follows. After
digestion of 200 µg of yeast genomic DNA with EcoRI or
XbaI, the DNA was separated by electrophoresis in a 1% agarose
gel. For the EcoRI subgenomic library DNA fragments in the
range of 2200 bp and for the XbaI subgenomic library DNA
fragments in the range of 1000 bp were isolated from the gel by
digestion of the gel with -Agarase I (New England Biolabs). The
gel-purified DNA was cloned in the Bluescript vector (Stratagene)
cleaved with the appropriate restriction enzyme. Escherichia coli strain XL1-Blue (Stratagene) was transformed with both subgenomic
libraries, and positive transformants were identified by colony
hybridization with the degenerate oligonucleotide using the same
conditions as for the Southern blot hybridization. One positive clone
of each subgenomic library was selected and called pE2200 (containing a
2200-bp EcoRI fragment) and pX1000 (containing a 1000-bp
XbaI fragment), respectively. pE2200 and pX1000 were used to
construct the subclones pX1300 and pXE500 (Fig. 2B).
These clones were used to determine the nucleotide sequence of the
RPA14 locus by the dideoxynucleotide chain termination method
(25) using the T7 sequencing kit from Pharmacia Biotech Inc. To
determine the nucleotide sequence of the RPA14 gene around
+187 and +197 (Fig. 3), the sequencing reaction was
performed in the presence of 7-deaza-dITP included in the Deaza T7
sequencing kit from Pharmacia. Sequence analysis was performed with the
``DNA strider'' program
(26) . GenBank
and
GenPept
data base searches were performed using the NCBI
BLAST'E-Mail server
(27) .
Figure 2:
Strategy for cloning and sequencing
RPA14. A, genomic Southern blot hybridization.
Genomic DNA from S288C yeast strain (25 µg) was digested with the
indicated restriction enzymes (E, EcoRI; X,
XbaI), electrophoresed in 1% agarose gel, transferred to nylon
Hybond N membrane (Amersham Corp.), and probed with the
P-end-labeled 29-mer oligonucleotide as described under
``Experimental Procedures.'' The size of the hybridizing DNA
fragments is indicated. B, restriction map of the RPA14 locus and subcloned fragments. The restriction map of the
RPA14 locus was deduced from the Southern blot analysis in
A. Fragments indicated in black and listed on the
right were subcloned in the Bluescript vector to give plasmids
used for DNA sequencing. Localization of RPA14 on chromosome
IV between the genes CYP1 and HOM2 is represented.
Arrows indicate the direction of the open reading frame of the
genes.
Figure 3:
Nucleotide sequence of RPA14 coding and flanking regions. The coding sequence begins at the ATG
at position +1. The translation of the nucleotide sequence is
given in the one-letter amino acid code. The underlined amino
acids indicate the peptides determined by microsequencing. The sequence
of the doubleunderlined peptide was used to deduce
the sequence of the 29-mer oligonucleotide that was used to isolate the
RPA14 gene (see ``Experimental Procedures''). The
HSE box is overlined. The restriction sites used for the gene
disruption are indicated.
Disruption of the RPA14 Gene
By screening 10,000
colonies of a genomic library from the yeast strain FL100
(28) using the EcoRI insert of pXE500 as a probe
(Fig. 2B), eight clones were obtained, which contained
an uninterrupted fragment with the RPA14 gene including 5`-
and 3`-terminal sequences. One of the clones was chosen for the
substitution of the RPA14 gene by the URA3 gene from
the plasmid YDp-U
(29) as follows (Fig. 4A).
After digestion of the clone with AflII, filling in the
cohesive 3`-ends with Klenow DNA polymerase and cleavage of the plasmid
DNA with SacI, a 1400-bp SacI-AflII fragment
was gel-purified and subcloned into the vector Bluescript KS digested with SacI and HincII. The
XbaI-SgrAI fragment of the resulting recombinant
plasmid was replaced by the BamHI fragment from YDp-U
containing the URA3 gene
(29) . The disrupted gene was
released by cleavage with SacI and AflII, and the DNA
was used to transform the YPH499xYPH500 diploid strain by the lithium
acetate procedure according to Gietz and Schiestl
(30) . URA3 colonies were selected, the genomic DNA of several transformants
was prepared by rapid extraction according to Hoffman and
Winston
(31) , and the DNA digested by EcoRI was
analyzed by Southern blot analysis with a
P-labeled
1400-bp PstI-AflII fragment (Fig. 4, A and B) as probe. One transformant was induced to
sporulate and subjected to tetrad analysis. The auxotrophy for uracil
of the spores was tested by replica plating on minimal medium with or
without uracil.
Figure 4:
RPA14 disruption. A, restriction
map of the RPA14 locus and structure of the rpa14::URA3 disruption mutation. A 1100-bp BamHI fragment containing
URA3 was inserted in replacement of the
XbaI-SgrAI RPA14 fragment. Direction of
translation of the RPA14, CYP1, and HOM2 open reading frames is indicated by an arrow. Restriction
sites are as follows: A, AflII; B,
BamHI; E, EcoRI; P, PstI;
S, SacI; Sg, SgrAI. The dottedlinearrows represent the EcoRI
fragments. B, Southern blot analysis of the gene disruption.
The ura diploid strain YPH499xYPH500 was transformed
with a SacI-AfIII fragment containing RPA14 disrupted
by URA3. Genomic DNA of YPH499xYPH500 (lane Wildtype)
or one URA
transformant (lane rpa14::URA3)
was restricted with EcoRI and subjected to Southern blot
analysis by hybridization with the 1400-bp PstI-AflII
fragment. The size of the hybridizing DNA fragments (bp) is
indicated. C, tetrad analysis from a diploid transformant
containing one copy of RPA14 and one copy of rpa14::URA3 (rpa14::URA3/RPA14). The spores were grown on YPD medium.
The slow growing spores were always
URA
.
Purification and Analysis of RNA Polymerase I
RNA
polymerase I from the haploid wild-type strain (YPH499) or the
rpa14::URA3 strain were purified as described by Riva et
al.(10) with the following modifications. Crude extracts
prepared from 40 g of wild-type or rpa14::URA3 cells were
incubated with phosphocellulose (Whatman P11) in buffer I (20
mM Tris-HCl, pH 8, 10 mM 2-mercaptoethanol, 0.5
mM EDTA, 10% glycerol) containing 50 mM ammonium
sulfate. Bound proteins were eluted with buffer I containing 400
mM ammonium sulfate. The eluate was dialyzed against buffer I
until the ammonium sulfate concentration reached 150 mM,
further diluted by the addition of 2 volumes of buffer I and then
chromatographed on a 6-ml Resource Q fast protein liquid chromatography
column (Pharmacia) equilibrated in buffer I containing 50 mM
ammonium sulfate. RNA polymerase I was recovered from this column by a
linear gradient from 50 to 525 mM of ammonium sulfate in 180
ml of buffer I at a flow rate of 12 ml/min.
Purification of the A14 Subunit and Characterization of
the RPA14 Gene
In order to clone the RPA14 gene, the
A14 polypeptide was purified from the other RNA polymerase I subunits
by RP-HPLC (Fig. 1). N-terminal sequence analysis of the purified
14-kDa polypeptide demonstrated that this polypeptide is N-terminally
blocked. Whether or not this N-terminal blocking is natural or
artefactual was not determined. Internal sequence information was
obtained by digesting the purified A14 polypeptide with proteinase K
and submitting the proteolytic peptides to Edman degradation after
RP-HPLC purification. Four peptidic sequences were determined. A
degenerate oligonucleotide was synthesized according to the sequence of
the largest peptide (FKGLPPAQDF) and was used to probe a Southern blot
of yeast genomic DNA digested with EcoRI, XbaI, or
both enzymes together. In each case, the probe hybridized to a single
DNA fragment (Fig. 2A), which indicated that the gene
RPA14 encoding the A14 polypeptide is present in one copy per
haploid genome. On the basis of the result of the Southern blot
analysis, two subgenomic libraries were constructed and screened with
the degenerate oligonucleotide (see ``Experimental
Procedures''). In each case, one of the positive clones was
selected, and the nucleotide sequence of the RPA14 gene was
determined from the insert of appropriate subclones
(Fig. 2B). As shown in Fig. 3, the nucleotide
sequence of the RPA14 gene revealed an open reading frame of
414 bp encoding an acidic polypeptide of 137 amino acids with a
predicted molecular mass of 14.6 kDa and a calculated pI of 4.9. The
four peptidic sequences that were determined were found within this
open reading frame (Fig. 3), which demonstrated that RPA14 encodes the protein purified by RP-HPLC. Comparison with protein
sequences in different data bases did not reveal any significant
homology of the A14 polypeptide with any protein. In particular, no
homology was found with the subunits of yeast RNA polymerases I, II,
and III so far characterized. Additionally, no DNA consensus motifs
were identified in the 5`- or 3`-flanking regions of RPA14 (Fig. 3). Southern blot analysis of filters containing the
whole yeast genome in ordered clones (33) indicated that RPA14 is located on chromosome IV (data not shown). This result was
confirmed by computer analysis of nucleotide sequences in data banks,
which indicated that the RPA14 open reading frame is located
upstream of CYP1(34, 35) and HOM2(36) on chromosome IV
(37, 38) (Fig. 2B). In particular, the coding region
of the RPA14 gene is contained in the fragment from -943
to -493 upstream of the CYP1 coding region published by
Sykes et al.(35) . The reported sequence of the open
reading frame of RPA14 within this fragment was partly
incorrect since the nucleotide sequence corresponding to +189 to
+197 in RPA14 was wrongly determined. The sequence of the
same region, from +187 to +193, was also incorrect in the
report of Thomas and Surdin-Kerjan (36). This region is surrounded by a
G/C-rich DNA sequence extending from position +178 to +213,
which is able to form a strong stem-loop structure. The sequence of
this region was finally resolved by using 7-deaza-dITP instead of dGTP
in the sequencing reaction. The sequence of RPA14 that we have
determined is likely to be correct since all the peptides
microsequenced are found in the RPA14 open reading frame,
which was not the case in the previously described sequences.
Figure 1:
Isolation of the RNA polymerase I
specific polypeptide A14 by RP-HPLC. A, RNA polymerase I (25
µg) was chromatographed on a RP-HPLC column as described in
``Experimental Procedures.'' Peaks of absorbance at 214 nm
are numbered from 1 to 7. B, purified RNA
polymerase I (lane L) and proteins present in the fraction 6
(lane 6) were analyzed by SDS-polyacrylamide gel
electrophoresis and silver-stained. The apparent molecular mass of RNA
polymerase I subunits ( 10
kDa) and the
position of the isolated A14 polypeptide are
indicated.
Disruption of the RPA14 Gene Is Not Lethal
To test
whether the RPA14 gene is essential for cell growth, the
chromosomal RPA14 gene was disrupted by homologous
recombination using the one-step gene disruption technique
(39) .
The XbaI-SgrAI fragment of RPA14 that
contains most of the coding sequence was replaced by a 1100-bp URA3 containing BamHI fragment (Fig. 4A). All
but 4 and 10 codons at the 5`- and 3`-ends of the RPA14 open
reading frame were deleted in this construction. The resulting plasmid
was used to transform the homozygous ura diploid
strain YPH499xYPH500. Southern blot analysis of several transformants
confirmed that in each case one of the two chromosomal copies of
RPA14 had been replaced by the URA3-disrupted
construct (Fig. 4B). Tetrad analysis was performed on
rich medium at 30 °C. Each tetrad of the transformant contained two
slow growing spores and two spores growing at the same rate as the
wild-type spores (Fig. 4C). Replica plating on minimal
medium without uracil indicated that, in each case, the slow growing
cells were prototrophic for uracil and thus contained the
RPA14-disrupted allele. This result indicated that RPA14 was not essential for yeast cell viability but that its disruption
resulted in a mutant strain, rpa14::URA3, which exhibited a
small growth defect at 30 °C. The doubling time at 30 °C was
120 ± 10 min for the parental wild-type strain and 140 ±
10 min for the rpa14::URA3 strain. At 37 °C, this
difference was amplified, since the doubling time of rpa14::URA3 was 160 ± 10 min while the wild-type growth rate was not
affected. These effects were stronger when the cells were grown for 1
or 2 days at 37 °C before determining the size of colonies on a YPD
plate or the doubling time in YPD medium at 37 °C. On YPD plates at
38 °C, wild-type cells formed small visible colonies within 1 week,
while mutant cells stopped growing after several divisions. Therefore,
RPA14 encodes a protein that is not strictly required for
growth, but whose absence causes a detectable thermosensitivity at
37-38 °C (but no cryosensitivity at 15 °C).
H]uracil of
mutant or wild-type cells. Cells were grown at 37 °C overnight,
diluted to the same cell density, further grown at 37 °C, and
pulse-labeled in the exponential phase for 30 min at 37 °C
according to Stettler et al.(40) . Analysis of the RNAs
by polyacrylamide gel electrophoresis showed that synthesis of the 5.8
S rRNA relative to the 5 S RNA and tRNAs was slightly reduced in the
mutant strain (data not shown). Since in vivo labeling in a
30-min pulse reflects primarily the synthesis of rRNA
species
(40) , it appears that disruption of RPA14 caused a slight but detectable decrease in the synthesis of rRNA.
The Gene Product of RPA14 Is a Genuine Subunit of RNA
Polymerase I
The reduced growth rate of the rpa14::URA3 strain indicated that the mutant RNA polymerase I activity was
sufficient to support suboptimal cell growth but probably had some
altered properties. In order to study the RNA polymerase I from the
rpa14::URA3 strain in vitro, the enzyme was purified
according to the classical purification procedure
(10) .
Surprisingly, no enzyme activity was obtained, whereas a parallel
purification starting from the same amount of wild-type cells yielded
the usual amount of active RNA polymerase I. Analysis of the protein
content of the glycerol gradient fractions from the rpa14::URA3 strain by silver staining and Western blot analysis indicated that
a small amount (approximately 20% compared with the wild-type strain)
of an inactive form of enzyme depleted of subunits A43, ABC23, and A14
was present. This result suggested that RNA polymerase I purified from
the rpa14::URA3 strain had lost several subunits essential for
activity. This low purification yield could be explained either by a
lower amount of mutant RNA polymerase I assembled in vivo or
by a poor purification yield. Thus, we analyzed by Western blot, using
anti-RNA polymerase I antibodies
(32) , the amount of enzyme in
mutant and wild-type cells after adsorption of the cell extracts on
phosphocellulose at low ionic strength (50 mM ammonium
sulfate). The amounts of enzyme eluted stepwise at 400 mM
ammonium sulfate were almost identical (data not shown), suggesting
that the low purification yield observed for the RNA polymerase I from
rpa14::URA3 was not due to a lower amount of assembled RNA
polymerase I in vivo. Therefore, the low yield of mutant
enzyme was probably related to a different chromatographic behavior
and/or to a decreased stability during the purification procedure.
Interestingly, after purification on phosphocellulose, the A43 subunit
that was present in a submolar amount in wild-type RNA polymerase I was
barely detectable in the enzyme purified from the rpa14::URA3 strain. Proteins eluted of the phosphocellulose at 400 mM
ammonium sulfate (see ``Experimental Procedures'') from both
mutant and wild-type extracts were then chromatographed on a Resource Q
column. The presence of RNA polymerase I in the elution fractions was
monitored by Western blot analysis. In the case of the wild-type
extract, a major form of enzyme that contained all of the subunits
classically described for RNA polymerase I eluted at 220 mM
ammonium sulfate (Fig. 5, fractions17-22), whereas a quantitatively minor form of RNA
polymerase I lacking subunits A43, ABC23, and A14 eluted at the
beginning of the gradient (Fig. 5, fractions
13-15). On the other hand, chromatography under the same
conditions of the rpa14::URA3 cell extract resolved only one
form of RNA polymerase I that did not contain the subunits A43, ABC23,
and A14 and that was totally inactive (Fig. 5, fractions
12-14). The trailing fractions of this enzyme were
contaminated by subunits A43 and ABC23 that coeluted at high ionic
strength (Fig. 5, fractions 15-19). To confirm
that the two subunits A43 and ABC23 were not associated with the
enzyme, proteins in fractions 15-19 were sedimented on a glycerol
gradient. Analysis by SDS-polyacrylamide gel electrophoresis of the RNA
polymerase I present in the glycerol gradient fractions indicated that
subunits A43 and ABC23 did not sediment with the enzyme (data not
shown). These results clearly demonstrate that the lack of the A14
polypeptide causes a major instability of two other, essential
subunits.
Figure 5:
RNA polymerase I analysis from wild-type
and rpa14::URA3 strains. RNA polymerase I from wild-type
strain or rpa14::URA3 mutant was purified as described under
``Experimental Procedures.'' After batch chromatography of
the crude extract on phosphocellulose, the enzyme was applied onto a
Resource Q column and eluted by a salt gradient. The same volume (5
µl) of the different elution fractions indicated by their number
were analyzed by Western blot with anti-yeast RNA polymerase I
antibodies (3 µg/ml) supplemented with anti-AC19 (3 µg/ml) and
anti-A14.5/14 (1 µg/ml) subunit-specific antibodies. Lane
I, 50 ng of purified wild-type RNA polymerase I. The positions of
the different RNA polymerase subunits are
indicated.
A unique
form of mutant RNA polymerase I lacking subunits A43, ABC23, and A14
was isolated by chromatography on a Resource Q column. Interestingly,
when wild-type RNA polymerase I was isolated in the same manner,
besides the major form of enzyme with the full set of subunits, a minor
form was detected that does not contain the A43, ABC23, and A14
subunits. This enzyme was inactive and therefore passed unnoticed in
previous work that did not use an immunodetection method and relied
only on enzyme activity. We note, however, that the same incomplete
form has been previously obtained by J.-M. Buhler
(43) after
phosphocellulose chromatography of purified RNA polymerase I
preincubated in 4 M urea at 0 °C. Therefore, subunits A43,
ABC23, and A14 can also dissociate simultaneously from wild-type RNA
polymerase I, leading to an inactive form of the enzyme. In the absence
of A14, subunits A43 and ABC23 are much more labile and are completely
dissociated. These observations suggest the existence of a subcomplex
of the three subunits A43, ABC23, and A14, the latter being important
for RNA polymerase I stability. Free subunits A43 and ABC23 were
coeluted in the same fractions after chromatography on the Resource Q
column (Fig. 5, fractions 15-19), suggesting that
these two subunits were associated as a binary complex. This
possibility has yet to be investigated.
this subunit is not
necessary for enzyme activity in a nonspecific transcription assay
in vitro(12) . The common subunit ABC23 is also
essential in vivo(42) , and it has been shown that
antibodies directed against ABC23 subunit inhibit the enzyme activity
(32). Moreover, Valenzuela et al.
(44) suggested that
RNA polymerase I activity was correlated with the amount of the ABC23
subunit present in the enzyme. Thus, the absence of solely ABC23 could
be responsible for the loss of activity. Since we were not able to
purify a form of RNA polymerase I missing only the subunits A43 and
ABC23, we cannot completely exclude the possibility that the loss of
nonspecific in vitro transcriptional activity is due to the
absence of both ABC23 and A14 subunits. The role of these individual
subunits will be assessed by in vitro reconstitution
experiments with recombinant A14, A43, and ABC23 subunits.
/EMBL Data Bank with accession number(s) U23208.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.