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(Received for publication, May 24, 1995; and in revised form, August 9, 1995) From the
Yeast RNA polymerase I contains 14 distinct polypeptides,
including A43, a component of about 43 kDa. The corresponding gene, RPA43, encodes a 326-amino acid polypeptide matching the
peptidic sequence of two tryptic fragments isolated from A43. Gene
inactivation leads to a lethal phenotype that is rescued by a plasmid
containing the
In the yeast Saccharomyces cerevisiae, as in other
eukaryotes, the 25 S, 18 S, and 5.8 S ribosomal RNAs (rRNAs) derive
from the post-transcriptional cleavage of a large pre-rRNA precursor,
the Biochemical studies
have shown that purified preparations of yeast pol I contain 14
distinct polypeptides (Buhler et al., 1976; Valenzuela et
al., 1976; Carles et al., 1991). Five of them (ABC27,
ABC23, ABC14.5, ABC10 In
addition to this conserved or even common core of subunits shared by
all three RNA polymerases, four polypeptides, A49, A43, A34, and A14,
are found in pol I preparations only. The role of these polypeptides in
the synthesis of rRNA is still poorly understood, but there is evidence
that they are not strictly required for the catalytic activity of yeast
pol I. Indeed, disrupting the A34- or A14-encoding genes hardly affects
cellular growth (Smid et al., 1995), (
RPA43 was also isolated from a genomic DNA
library as a plasmid able to complement two rrn mutants
originally classified in the complementation group called RRN12. Mutants defective in Strain D101
was constructed by disrupting one RPA43 allele of the diploid
strain YPH499
Mutational alteration in the original rrn12(rpa43) mutant strains NOY639 and NOY640 (these strains
harbor the same mutation) was determined by recovering the mutant
allele by gap repair (Orr-Weaver et al., 1983) and DNA
sequencing. pNOY272 (Fig. 1), which complemented the mutation,
was digested with NsiI and BglII, and a 3.5-kb
fragment including the proximal two-thirds of the RPA43 coding
region was removed. The linearized plasmid containing the remaining
portion was transformed into the mutant strains, generating
Leu
Figure 1:
Organization of the RPA43-RPA190 region. Restriction map of the 17-kb region corresponding to the
pNOY16 insert (Mémet et al., 1988;
Wittekind et al., 1988) bearing RPA190 and RPA43 and isolated from a genomic library of strain AB320, using colony
hybridization against
Figure 2:
Predicted amino acid sequence,
hydrophilicity pattern, and distribution of acidic/basic amino acid
residues of A43. Amino acid sequences determined by analyzing two
tryptic peptides derived from purified A43 are underlined. The vertical arrows denote sites of truncation corresponding to
the lethal rpa43-1 nonsense mutation eliminating the last 110
amino acids of A43, and to the viable mutant form encoded by pNOY273
and its derivative, where the last 18 amino acids are replaced by other
amino acids derived from the relevant vectors (see text for
explanation). DNA sequence accession number
U22949.
In the course of
the sequence analysis of RPA43, we found that its initiator
ATG is separated by 805 base pairs from a divergently transcribed
reading frame, that turned out to be RPA190 (Mémet et al., 1988), encoding the
largest subunit (A190) of pol I (Fig. 1). As previously noted,
the RPA43-RPA190 intergenic region contained putative RPG and
PAC boxes (Mémet et al., 1988;
Dequard-Chablat et al., 1991) that could be involved in
regulation of the transcription of these genes. This physical linkage
cannot be due to an artifact generated by some in vitro recombination event during the preparation of the genomic library,
because the three RPA43 clones mentioned above, which were
derived from three distinct yeast libraries (see ``Materials and
Methods''), all have the same restriction enzyme sites covering
this region. In addition, the linkage of RPA43 to RPA190 was also directly demonstrated by nucleotide sequencing of the DNA
derived from AB320 (
Figure 3:
Tetrad analysis of D101 (rpa43
The complementation data described above show that A43
is essential for cell growth, but do not prove that this protein is
required for rRNA synthesis and thus is a functional component of pol
I. The proof was obtained by the demonstration that the lethal
phenotype of the RPA43 deletion can be rescued by the presence
of pNOY102, which carries the GAL7-
The mutation carried by mutant NOY639 was
recovered by gap repair using plasmid pNOY272 carrying the cloned RPA43 (missing the COOH-terminal 18 amino acids), as described
under ``Materials and Methods.'' The mutation carried by both
NOY639 and NOY640 was identified to be a G to A transition at
nucleotide +650 that changes the UGG Trp codon at amino acid
position 217 to a UAG nonsense codon. The results demonstrate that the rrn12 mutation is in fact in the RPA43 gene, and that
truncation of A43 (326 amino acids) at the position distal to His-216
by a nonsense mutation leads to lethality to yeast cells. The presence
of the truncated A43 protein fragment in the mutant cells (under
permissive growth conditions, that is, mutant cells growing in
galactose medium using the GAL7-
Figure 4:
Polyacrylamide-agarose gel electrophoresis
of RNA synthesized in a rpa43-1 mutant (NOY639) and the parent
(NOY418) strains growing in galactose with and without prior glucose
addition. Parent strain NOY418 (lanes 1 and 2) and rpa43-1 mutant NOY639 (lanes 3 and 4) were
grown at 30 °C in galactose medium. At a cell density (A
It has been known that highly purified preparations of yeast
pol I, but not pol II or pol III, contained the A43 protein. However,
some pol I preparations lacking both A49 and A43 retained catalytic
activity as assayed with nonspecific DNA template (Hager et
al., 1977). Although A49 has since then been shown to be a genuine
subunit of pol I (Liljelund et al., 1992), the possibility of
a fortuitous co-purification of A43 with pol I could not be ruled out.
We have now sequenced and characterized the corresponding gene, RPA43. Its inactivation by a nonsense mutation or by an
extended internal deletion is lethal, but cells carrying these
mutations can be rescued by introduction of the GAL7- Genes encoding distinct subunits of the same heteromultimeric
enzyme are usually dispersed on the yeast genome, and this also applies
to RNA polymerase subunits (Young, 1991; Thuriaux and Sentenac, 1992).
Surprisingly, however, RPA43 is physically linked to RPA190. The two genes are divergently transcribed, and their
coding regions are separated by 805 base pairs containing putative RPG
and PAC boxes (Mémet et al. 1988;
Dequard-Chablat et al., 1991). Thus, as in the case of the
L46-L24 ribosomal protein gene pair (Kraakman et al., 1989),
these two genes might be co-regulated using common upstream activating
sequences such as the putative RPG and PAC boxes, thereby helping
balance synthesis of these two essential pol I subunit proteins. Early studies showed that A43 is not immunologically related to
yeast pol II or pol III or their purified subunits, suggesting that A43
is unique to pol I (Huet et al., 1982). The present study
extends this conclusion by showing that A43 has no significant sequence
homology to any other entries in current data banks and is, in
particular, unrelated to any of the 12 subunits of yeast pol II and to
the 14 genetically characterized subunits of yeast pol III (Young
(1991), Thuriaux and Sentenac(1992), and Sadhale and Woychik(1994), and
references therein). However, there is a poorly characterized
polypeptide of 37 kDa, C37, that is present in some catalytically
active preparations of yeast pol III and might thus be a genuine
subunit of that enzyme (Huet et al., 1985). The corresponding
gene has not yet been identified. As mentioned in the Introduction,
there are three other subunits, A49, A34, and A14, in addition to A43,
which are unique to pol I. However, the genes for A49, A34, and A14 can
be disrupted with only limited adverse effects on cell growth
(Liljelund et al., 1992; Nogi et al., 1993; Smid et al., 1995),
The nucleotide
sequence(s) reported in this paper has been submitted to the
GenBank(TM)/EMBL Data Bank with accession number(s)
U22949[GenBank].
Volume 270,
Number 41,
Issue of October 13, 1995 pp. 24252-24257
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
S ribosomal RNA gene fused to the GAL7 promoter, which allows the synthesis of
S rRNA by RNA
polymerase II in the presence of galactose. A screening for mutants
rescued by the presence of GAL7-
SrDNA identified
a nonsense rpa43 allele truncating the protein at amino acid
position 217. [
H]Uridine pulse labeling showed
that this mutation abolishes S rRNA synthesis without
significant effects on the synthesis of 5 S RNA and tRNAs. These
properties establish that A43 is an essential component of RNA
polymerase I. This highly hydrophilic phosphoprotein has a strongly
acidic carboxyl-terminal domain, and shows no homology to entries in
current sequence data banks, including all the genetically identified
components of the other two yeast RNA polymerases. RPA43 mapped next to RPA190, encoding the largest subunit of
polymerase I. These genes are divergently transcribed and may thus
share upstream regulatory elements ensuring their co-regulation.
S rRNA. The synthesis of that transcript accounts for
about 60% of the total transcriptional activity of rapidly growing
yeast cells. It takes place in the nucleolus and is catalyzed by RNA
polymerase I (pol I), (
)the more abundant of the three
eukaryotic nuclear RNA polymerases (Warner, 1989; Nomura et
al., 1993). In vertebrates, pol I operates in conjunction with two
major transcription factors, UBF1 and SL1, forming a stable
preinitiation complex (Reeder, 1992; Eberhard et al., 1993;
Radebaugh et al., 1994; Zomerdijk et al., 1994). It
has been established that pre-rRNA synthesis is the only essential
function of yeast pol I, since the growth defect of pol I-defective
mutants can be bypassed by placing the gene encoding the S
rRNA under the control of a RNA polymerase II (pol II) promoter (Nogi et al., 1991a). This specialization of pol I enzyme is
presumably a general property of eukaryotes.
, and ABC10
) are common to all three
nuclear RNA polymerases and the corresponding genes (RPB5, RPB6,
RPB8, RPB10, and RPC10) have been characterized (Woychik et al., 1990; Woychik and Young, 1990; Treich et al.,
1992; Lalo et al., 1993). Two other subunits (AC40 and AC19)
are shared by pol I and pol III and are encoded by RPC40 and RPC19, respectively (Mann et al., 1987;
Dequard-Chablat et al., 1991). Of the remaining seven pol I
subunits, the two large ones, A190 and A135 (encoded by RPA190 and RPA135), have a sequence homology to the two large
subunits of pol II, pol III, and bacterial RNA polymerases
(Mémet et al., 1988; Yano and Nomura,
1991). These conserved or common nine subunits are essential for
growth, as shown by the mutational inactivation of their genes
(Young(1991) and Thuriaux and Sentenac(1992), and references therein).
Remarkably, all but two of them (ABC14 and ABC10) also have a
structural equivalent in archeal RNA polymerases (Langer et
al., 1995). This conservation obviously suggests that the
corresponding polypeptides fulfill functions that are common to the
central mechanism of transcription itself. Another subunit A12 (encoded
by RPA12 or RRN4) has some homology to the B12.6
subunit of pol II (encoded by RPB9). RPA12 and RPB9 have been shown to be only conditionally essential
(Woychik et al., 1991; Nogi et al., 1993).
)whereas a
pol I preparation lacking both A49 and A43 retained catalytic activity in vitro as tested on nonspecific DNA templates (Hager et
al., 1977). A49 has since then been shown to be a genuine pol I
subunit that is partly dispensable in vivo (Liljelund et
al., 1992). In the case of A43, the possibility of a fortuitous
co-purification of that polypeptide with pol I could not be ruled out
so far. The present work establishes that A43 is indeed an essential
and specific component of yeast pol I, as shown by cloning and
genetically characterizing the corresponding gene (RPA43) and
by discovering that a mutation directly isolated as defective in
ribosomal rRNA synthesis maps in RPA43.
Plasmids and Strains
The RPA43 inserts
isolated in the present work originated from three distinct laboratory
strains of S. cerevisiae, X2180, AB320, and DBY476.
The A43-1 clone was previously isolated by screening of a
gt11 genomic library prepared from strain X2180 using anti-A43
antibodies (Riva et al., 1986). A 2.4-kb HindIII-EcoRI fragment of
A43-1 was subcloned in
pBluescriptKS+ (Stratagene) to generate pA43, whereas YCpA43-3 and
YCpA43-6 were constructed by subcloning a BamHI fragment of
1.9 kb in YCp50. pA43
-LEU was generated by creating an additional BglII site a few base pairs downstream of the putative
initiator triplet of RPA43 using polymerase chain reaction
amplification (a GCC triplet encoding the Ala in position 7 of the
predicted A43 amino acid sequence was replaced by TCT), and
substituting the 0.6-kb BglII fragment thus produced by a
1.6-kb BamHI LEU2 cassette. pNOY16 (Wittekind et
al., 1990) carries a genomic insert containing RPA190 and RPA43 derived from strain AB320 (Mémet et al., 1988). The 4.2-kb BamHI-SalI
fragment of pNOY16 containing RPA43 was subcloned on
pBluescriptKS+ and YCp50 to generate pBA43-12 and YCpA43-12,
respectively.S rRNA synthesis were
isolated from strain NOY418 (MATa ade2 ade3 leu2 ura3 lys2
CAN1/pNOY103) as described previously (Nogi et al.,
1991b). These mutants can grow on galactose, but not on glucose.
Mutants NOY639 and NOY640, corresponding to two distinct isolates but
bearing the same nonsense allele (rpa43-1) were classified in
complementation group RRN12. The yeast genomic library used
for cloning of the RRN12(RPA43) gene contained DNA
fragments obtained from partial MboI digestion of the
chromosomal DNA (from strain DBY476) inserted into the BamHI
site of the YCpN1 vector (CEN3 ARS1 TRP1; see Nogi et al. (1993)). The mutant was transformed with this library and
Trp
transformants that can grow on glucose were
isolated. One of the corresponding plasmids (pNOY286) contained an
insert which was recovered as a 5.6-kb SalI-SmaI
fragment using the two outside restriction sites in the vector DNA. The
fragment was then inserted between the SalI and SmaI
sites of pRS315 to give pNOY272. The 5.6-kb insert contained a single BamHI site and a single PvuII site, and both the
1.8-kb BamHI-SmaI fragment and 1.2-kb PvuII-SmaI fragment were subcloned and sequenced. The
latter fragment was sufficient to complement the rrn12 mutation when subcloned into pRS314(pNOY273). The DNA sequence
covering the RPA43 coding region was determined for pNOY273.
it showed one base deletion within the vector region adjacent to the
insert (a deletion of Cys, presumably due to nibling during plasmid
construction using the SmaI site), resulting in the open
reading frame encoding a fusion protein with 4 amino acids derived from
the vector portion replacing the missing 18 amino acids.
YPH500 (see Smid et al. (1995)) into the rpa43::LEU2 null allele using the technique of
Rothstein(1983). The 3.2-kb HindIII-PstI fragment
containing the disrupted rpa43 gene (rpa43::LEU2) was prepared from p43A-LEU and
transformed into a diploid strain constructed by crossing YPH499 with
YPH500 (ade2-1 lys2-801 ura3-52 trp1-63 his 3-200
leu2-1) and Leu transformants were selected.
The presence of the expected rpa43
::LEU2/RPA43 heterozygous structure was examined by genomic Southern
hybridization using a digitoxin-labeled pA43 DNA probe. One of the
transformants that showed the correct structure was retained. Strains
NOY639 and NOY640 were obtained by ethylmethane sulfonate mutagenesis
of strain NOY418 as previously described (Nogi et al., 1991b).
Growth media were previously described (Mosrin et al., 1990;
Nogi et al., 1991b). Briefly, YPD is a complete medium with 2%
(w/v) glucose as carbon source, YPGal is YPD where glucose has been
replaced by the same amount of filter-sterilized galactose, SD is a
glucose-containing synthetic minimal medium that was supplemented with
appropriate amino acids and bases to make the uracil, tryptophan, or
leucine-omission media. Uracil auxotrophic clones were obtained by
selection on the 5-fluororotic acid-containing medium (Boeke et
al., 1984).DNA Sequence Analysis
DNA sequencing was done on
alkali-denatured double-stranded DNA by the dideoxy method using a
modified T7 DNA polymerase (Sequenase II from U. S. Biochemical Corp.)
or using the Taq DyeDeoxy Terminator Cycle Sequencing Kit
(Applied Biosystems, Foster City, CA). Both strands were sequenced for
each DNA sample./Ura
transformants which were
selected on galactose medium and screened for non-growth phenotype on
glucose medium. Plasmids were prepared from transformants that were
unable to grow on glucose medium, and pNOY272 derivatives that failed
to complement the rpa43-1 mutation were recovered in E.
coli. The rpa43-1 mutation was identified by sequencing
both strands of the entire open reading frame of the RPA43 gene in these plasmids.
A43-1 (Mémet et
al., 1988). The horizontal thin line above the map
indicates the region that covers both the sequence previously published
for RPA190 (J03530; Mémet et
al., 1988) and the one determined in the present work (accession
number U22949). Shaded areas correspond to the RPA190 and RPA43 coding sequences, with arrowheads giving their transcriptional orientation. The insets corresponding to the various constructions of the present work are
symbolized by horizontal boxes in the lower half of
the figure. The open boxes (
A43-1, pA43, YCpA43-3,
YCpA43-6) denote an inset originating from strain X2180 and
isolated by antigenic screening in E. coli (Riva et
al., 1986). The filled box (YCpA43-12, pBA43-12) denotes insets subcloned from pNOY16 (see above). The striped
boxes (pNOY286, pNOY272, and pNOY273) correspond to insets originating from strain DBY476 and isolated by genetic
complementation of the rpa43-1 mutation (see text for
explanation). The position of the restriction sites is shown by the short vertical bars (HindIII, ClaI, NsiI, and BamHI are, respectively, abbreviated in H, C, N, and B). Sites denoted by a star originate from the vector cloning sites or from in
vitro mutagenesis, as in the case of the SalI* site
generated by mutagenizing a pre-existing ClaI site on pNOY16,
Wittekind et al.(1988)). MboI sites are shown only
for those bordering the pNOY286 insert.
Analysis of RNA Labeled in Vivo
Cells were grown
in synthetic galactose medium supplemented with casamino acids,
tryptophan, and adenosine at 30 °C to a cell density (A) of approximately 0.2. Each culture was
divided into two parts. Glucose was added to one culture to a final
concentration of 2%, and the other culture received water, serving as
control. After incubation at 30 °C for 1 h,
[
H]uridine (174 mCi/mmol; 100 mCi/ml) was added,
and after a 30-min incubation the labeling was stopped by immersing
culture flasks into a dry ice-ethanol bath. RNA was isolated and
analyzed by electrophoresis on a 2% polyacrylamide, 0.5% agarose
composite gel followed by autoradiography as described previously (Nogi et al., 1991a).
Sequence Determination and Physical Linkage between
RPA43 and RPA190
RPA43 was first isolated as a
A43-1 clone constructed from the S. cerevisiae strain
X2180. This clone, obtained by immunological screening of the
gt11
bank with polyclonal antibodies raised against A43, produced a
polypeptide of 43 kDa (as determined by SDS-polyacrylamide gel
electrophoresis) when expressed in Escherichia coli (Riva et al., 1986). A 2.4-kb EcoRI-HindIII
fragment of the
A43-1 insert was subcloned in pBluescriptKS+,
yielding plasmid pA43 (Fig. 1). The DNA sequence of this
fragment and of the corresponding regions derived from two other yeast
strains, AB320 and DBY476 (see legend of Fig. 1), was determined
(see below for the sequence obtained from pNOY272 and pNOY273). The
contiguous 2097-base pair DNA sequence (accession number U22949)
generated from these determinations contained a 978-base pair
uninterrupted open reading frame encoding a 326-amino acid polypeptide.
The predicted amino acid sequence is identical in all three strains,
but AB320 differs from the other two by a silent G
A transition
at nucleotide 546 of the open reading frame. There is a perfect match
with the amino acid sequences of two peptides (SQAESLPIVSNK and
ILDADPL, underlined in Fig. 2) which were isolated from
the purified A43 protein after tryptic digestion and whose amino acid
sequences were directly determined. (
)This confirms the
original identification of the gene (hereafter called RPA43)
based on immunological methods using specific antibodies against A43.
The absence of the (A/T)ACTAAC consensus motif present at the branch
point of all S. cerevisiae introns identified so far strongly
argues that RPA43 is an intron-less gene.
A43-1) and from DBY476 (pNOY286). RPA190 (McCusker et al., 1991) and RPA43 (Riva et
al., 1982) were already known to map to chromosome XV.
Properties of the Predicted Amino Acid Sequence of
RPA43
The RPA43 open reading frame encodes a protein of
326 amino acids. Its calculated molecular mass (36.2 kDa) is notably
smaller than the 43 kDa estimate on SDS-polyacrylamide gel
electrophoresis analysis (Buhler et al., 1976), a difference
that could merely reflect a somewhat atypical electrophoretic migration
of this phosphoprotein (Bréant et al.,
1983). The predicted amino acid sequence of A43 has no significant
homology to entries of current data banks. Moreover, no significant
homology was detected when aligning A43 with known subunits of yeast
pol II and pol III in pairwise alignments. There is some similarity
between A43 and the pol III-specific subunit C31 (Mosrin et
al., 1990) but this is restricted to a hyperacidic COOH-terminal
end present in both subunits. In the case of A43, this acidic
COOH-terminal domain corresponds to the last 46 residues (with 39%
acidic residues). As a matter of fact, A43 is a very hydrophilic
protein, with abundant polar residues accounting for 55% of the total
sequence. The pI (4.8) calculated from the deduced amino acid of A43 is
close to the experimentally determined value of 5.1 (Buhler et
al., 1976). It should be noted, however, that A43 is a
phosphoprotein and contains an average of four phosphates per molecule
(Bréant et al., 1983). The acidic
COOH-terminal domain has several phosphorylation sites for casein
kinase II. Furthermore, Ser-244 is potentially phosphorylatable by
three distinct kinases (cAMP-dependent protein kinase,
calmodulin-dependent protein kinase, and phosphorylase kinase).RPA43 Is an Essential Gene Required for rRNA
Synthesis
The rpa43::LEU2 deletion, removing most
of the RPA43 coding sequence (Fig. 1), is recessive
lethal. This conclusion was first obtained by tetrad analysis using a
diploid strain (D101) carrying rpa43::LEU2 on one
chromosome and RPA43 on another chromosome. Tetrad dissection
showed the 2:2 segregation of viable (RPA43) and non-viable (rpa43::LEU2) spores in the offspring (Fig. 3, left panel). As such, this lethal phenotype could be limited
to a defective spore germination phenotype, or due to a negative effect
of the deletion on the expression of adjacent RPA190. Both
interpretations were ruled out by showing that viable rpa43::LEU2 segregants can be recovered in the D101
offspring when the latter strain bears the centromeric plasmid
YCpA43-12 (URA3 RPA43) (Fig. 2, middle panel).
As expected, the segregants carrying rpa43::LEU2 invariably harbored the URA3 RPA43 plasmid, and could not
lose it as evidenced by their inability to form colonies on
5-fluororotic acid medium which selects for the loss of the URA3 plasmid.
::LEU2/RPA43). Left panel, tetrads derived
from diploid D101. Spores were isolated by micromanipulation and
germinated on YPD. Note the mendelian pattern of two viable and two
lethal spores (12 tetrads analyzed and four examples shown). Central panel, tetrads derived from D101 bearing the YCpA43-12 (CEN4 URA3 RPA43) plasmid. More than two viable spores were
recovered in most asci, indicating the complementation of rpa43::LEU2 by the YCpA43-12 (CEN4 URA3 RPA43)
plasmid (see text for further explanations). Right panel,
tetrads derived from D101 bearing pNOY102 (URA3,
GAL7-SrDNA). Spores were germinated on YPGal. Note
the mendelian segregation of two fast-growing (RPA43) and two
slow-growing segregants, the latter bearing the rpa43
::LEU2 with partial rescue of growth defect due to the (inefficient)
synthesis of S rRNA from the GAL7-
SrDNA fusion gene (Nogi et al.,
1991a).
SrDNA fusion
gene (Nogi et al., 1991b). This fusion gene allows the
synthesis of the
S rRNA by pol II from the GAL7 promoter in the presence of inducer galactose. As shown in Fig. 3(right panel), spores isolated from strain
D101(pNOY102) and germinated on YPGal have a 2:2 segregation of two
fast-growing RPA43 spores, and two slow growing spores
corresponding to the rpa43
::LEU2 segregants rescued by
the GAL7-SrDNA fusion. The latter segregants
failed to grow when replica plated on glucose containing YPD medium and
were also unable to grow on galactose medium in the presence of
5-fluororotic acid, confirming that their growth depends on the
presence of this plasmid.
Isolation of the rpa43-1 Mutation
Mutants that
depend on the presence of the GAL7-SrDNA gene for
growth were isolated and classified into complementation groups by
genetic crosses (Nogi et al., 1991b) and the genes
representing some of these complementation groups were cloned and
characterized (Nogi et al., 1993; Nomura et al.,
1993; Keys et al., 1994). Complementation group RRN12 was previously defined by two such mutants, NOY639 and NOY640. (
)To clone and characterize the corresponding gene, a yeast
genomic library (prepared from strain DBY746) was introduced into
mutant NOY639, and transformants able to grow on glucose media (where
the GAL7 promoter is repressed) were recovered. A plasmid
recovered from one of them (pNOY286) was analyzed further. Sequencing
of the subcloned DNA fragments (pNOY272 and pNOY273; see Fig. 1)
responsible for the complementation revealed the presence of the
amino-terminal portion of the RPA190 gene (in pNOY272) and an
open reading frame containing the first 308 codons of the RPA43 coding sequence, but ending with GATC (nucleotide 921-924;
the Ala of the first ATG of the RPA43 open reading frame is
the +1) followed by the sequence of the original cloning vector
adjacent to the BamHI site (GGATCC) used for construction of
the DNA library. Thus, the RPA43 insert carried by the
original complementing plasmid (pNOY286) as well as its derivatives are
actually bearing a mutant form of RPA43, that encodes A43 down
to the isoleucine residue at position 308 but lacks the
carboxyl-terminal 18 amino acids. In pNOY273, the missing 18 amino
acids are replaced by four amino acids, PGIH, translated from
nucleotide sequence derived from the multicloning site of the vector
(pRS314) used to construct this plasmid. This generated a small
decrease (about 1.5 kDa) in the size of A43 which was confirmed by
Western immunoblot analysis of extracted prepared from rrn12 mutants carrying pNOY273 (data not shown). The growth rates of the rrn12 mutants carrying pNOY273 was only slightly (about 10%)
slower than the growth rate of the parent wild type strain, and the
carboxyl-terminal 18-amino acid portion is therefore not critical to
the function of A43.SrDNA fusion gene
and pol II) was, in fact, demonstrated by Western immunoblot analysis
of extracts using antiserum against A43, and its size relative to the
size of A43 was roughly consistent with the position of the nonsense
codon (data not shown). We have not examined the question of whether
(defective) pol I missing the intact A43 exists stably in these mutant
cells, and if so, the observed truncated A43 fragment is associated
with the defective pol I. In any event, we renamed RRN12 as RPA43 and the mutation carried by NOY639 and NOY640 is now
designated as rpa43-1.
Direct Demonstration of Defects in
As described above, mutants carrying rpa43S rRNA Synthesis in the rpa43-1 Mutant
Strain
::LEU2 or rpa43-1 fail to grow on glucose
media, but were able to grow on galactose media, if they carried the GAL7-SrDNA fusion gene on a suitable multicopy
plasmid. This indicated that A43 is essential for
S rRNA
synthesis by pol I in vivo. We confirmed this conclusion
directly by [
H]uridine pulse-labeling experiments
using a rpa43-1 mutant strain. Both the mutant (NOY639) and
the control wild-type (NOY418) strains were grown in synthetic
galactose medium, and [H]uridine pulse labeling
experiments were carried out with and without prior repression of the GAL7 promoter by glucose, as was done in previous studies
(Nogi et al., 1991a, 1991b). As shown in Fig. 4,
synthesis of large rRNA (18 S, 25 S, 5.8 S, and other precursor rRNAs)
was strongly inhibited by glucose relative to synthesis of 5 S RNA and
tRNAs, showing that rpa43 -1 abolishes the synthesis
of large rRNA by pol I specifically without significant effect on 5 S
RNA and tRNA synthesis.
) of approximately 0.2, cultures were divided
into two parts: and glucose (final concentration 2%) was added to one
part (lanes 2 and 4), the other received water,
serving as control (lanes 1 and 3). At 1 h after
glucose addition, cells were pulse-labeled with
[
H]uridine for 30 min. RNA was isolated from each
culture, and samples containing equal amounts (approximately 1
10
cpm) of [H]RNA were analyzed by
electrophoresis on a polyacrylamide-agarose composite gel. An
autoradiogram of the dried gel is shown. We note that autoradiograms
obtained after longer gel exposures indicated that 5.8 S rRNA synthesis
in the mutant was inhibited by glucose, as was that of 18 S and 25 S
rRNAs.
SrDNA fusion gene and its transcription by
pol II in the presence of inducer galactose. The results indicate that
the absence of intact A43 leads to failure to synthesize
S
rRNA using pol I, and this conclusion was confirmed directly by
[
H]uridine pulse labeling experiments using a rpa43 nonsense mutant. Thus, A43 is essential for S rRNA synthesis by pol I in vivo, and hence,
must represent an essential subunit of pol I, even if it does not
appear to be required for pol I-dependent transcription of nonspecific
DNA templates (Hager et al., 1977). It will be interesting to
re-investigate the pol I preparations lacking A43 using in vitro transcription assays that allow specific transcription initiation
of the natural pol I promoter (Lue and Kornberg, 1990; Riggs and
Nomura, 1990; Schultz et al., 1991; Keys et al.,
1994).
while the gene for A43 is
essential. Therefore, A43 appears to be special and may have some
important function(s) specific to pol I. For example, it may directly
interact with the yeast equivalents of the SL1 and UBF1 initiation
factors that are well characterized in vertebrate in vitro transcription systems (Reeder, 1992; Eberhard et al.,
1993; Radebaugh et al., 1994; Zomerdijk et al.,
1994), or to the products of several RRN genes (Keys et
al., 1994) that are known to be specifically required for pol I
activity in vivo or some other unidentified pol I-specific
regulatory factors. A similar role was proposed for a complex of three
pol III-specific subunits, C82, C34, and C31 (Thuriaux and Sentenac,
1992; Werner et al., 1993). Curiously, A43 and the C31 subunit
of yeast pol III (Mosrin et al., 1990), although otherwise
unrelated, share a strongly acidic COOH-terminal domain of about 40
residues. Deleting the last 16 amino acids of C31 leads to a
conditional growth phenotype and produces a mutant enzyme that is
affected in transcription initiation, but not termination or
elongation, suggesting that it could be altered in its interaction with
components of the pol III-specific initiation factor TFIIIB (Thuillier et al., 1995). In the case of A43, the functional significance
of the acidic tail is unclear. Removing the COOH-terminal 18 amino
acids representing nearly half of the acidic tail gives only a weak
negative effect on its function as judged by the ability of pNOY273
(and other related plasmids) to complement the rpa43-1 mutation. Another interesting feature of A43 is that it is a
phosphoprotein with an average of four phosphorylated residues per
molecule (Bréant et al., 1983). With the
complete amino acid sequence of A43 now deduced from the nucleotide
sequence, it becomes possible to identify the phosphorylated residues
and then design experiments to examine the functional and/or regulatory
significance of the phosphorylation, thereby clarifying the function of
this essential component of the rRNA synthesis machinery.
))))
We thank Karen Sutton for her participation in
isolation and complementation analysis of two rrn12(rpa43)
mutants described in this paper, Claire Boschiero, Catherine Doira, and
Eric Quémeneur for their contribution to DNA
sequencing, and Michel Riva for communicating the peptidic sequence of
the two trypic fragments of A43.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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