©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Association of Three Subunits with Yeast RNA Polymerase Is Stabilized by A14 (*)

Amke Smid (§) , Michel Riva (¶) , Franoise Bouet (1), André Sentenac , Christophe Carles

From the (1) Service de Biochimie et de Génétique Moléculaire and Laboratoire d'Ingénierie des Protéines, CEA Saclay, F-91191 Gif-sur-Yvette Cédex, France

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

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 ` 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).

The analysis of the conserved domains of the `- 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) .

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 -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) .

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.


EXPERIMENTAL PROCEDURES

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.

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)].


RESULTS

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).

In vivo synthesis of small RNAs in an rpa14::URA3 strain was assayed by pulse labeling with [5,6-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.




DISCUSSION

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) . 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.

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, 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.


FOOTNOTES

*
This research was supported in part by Grant CSD 92408 from the Human Frontier Sciences Program Organization. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank/EMBL Data Bank with accession number(s) U23208.

§
Supported by Human Capital and Mobility Grant ERBCHBICT 940 992.

To whom correspondence should be addressed. Tel.: 331 69 08 84 17; Fax: 331 69 08 47 12; E-mail: biochimi@jonas.saclay.cea.fr.

P. Thuriaux, unpublished result.

O. Gadal and P. Thuriaux, unpublished result.

The abbreviations used are: RP-HPLC, reverse-phase high pressure liquid chromatography; YPD, yeast/peptone/dextrose; bp, base pair(s).


ACKNOWLEDGEMENTS

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.


REFERENCES
  1. Sentenac, A., Riva, M., Thuriaux, P., Buhler, J.-M., Treich, I., Carles, C., Werner, M., Ruet, A., Huet, J., Mann, C., Chiannilkulchai, N., Stettler, S., and Mariotte, S.(1992) Transcriptional Regulation, pp. 27-54, Cold Spring Harbor Press, Cold Spring Harbor, NY
  2. Thuriaux, P., and Sentenac, A.(1992) in The Molecular and Cellular Biology of the Yeast Saccharomyces: Gene Expression (Broach, J., Pringle, J. R., and Jones, E. W., eds) pp. 1-48, Cold Spring Harbor Press, Cold Spring Harbor, NY
  3. Young, R. A.(1991) Annu. Rev. Biochem. 60, 689-715 [CrossRef][Medline] [Order article via Infotrieve]
  4. Riva, M., Schäffner, A. R., Sentenac, A., Hartmann, G. R., Mustaev, A. A., Zaychikov, E. F., and Grachev, M. A.(1987) J. Biol. Chem. 262, 14377-14380 [Abstract/Free Full Text]
  5. Riva, M., Carles, C., Sentenac, A., Grachev, M. A., Mustaev, A. A., and Zaychikov, E. F.(1990) J. Biol. Chem. 265, 16498-16503 [Abstract/Free Full Text]
  6. Treich, I., Carles, C., Sentenac, A., and Riva, M.(1992) Nucleic Acids Res. 20, 4721-4725 [Abstract]
  7. Mann, C., Buhler, J.-M., Treich, I., and Sentenac, A.(1987) Cell 48, 627-637 [CrossRef][Medline] [Order article via Infotrieve]
  8. Werner, M., Chaussivert, N., Willis, I. A., and Sentenac, A.(1993) J. Biol. Chem. 268, 20721-20724 [Abstract/Free Full Text]
  9. Thuillier, V., Stettler, S., Sentenac, A., Thuriaux, P., and Werner, M. (1995) EMBO J. 14, 351-359 [Abstract]
  10. Riva, M., Buhler, J.-M., Sentenac, A., Fromageot, P., and Hawthorne, D. (1982) J. Biol. Chem. 257, 4570-4577 [Abstract/Free Full Text]
  11. Huet, J., Buhler, J.-M., Sentenac, A., and Fromageot, P.(1975) Proc. Natl. Acad. Sci. U. S. A. 72, 3034-3038 [Abstract]
  12. Hager, G. L., Holland, M. J., and Rutter, W. J.(1977) Biochemistry 16, 1-8 [Medline] [Order article via Infotrieve]
  13. Huet, J., Dezélée, S., Iborra, F., Buhler, J.-M., Sentenac, A., and Fromageot, P.(1976) Biochimie (Paris) 58, 71-80 [Medline] [Order article via Infotrieve]
  14. Huet, J., Wyers, F., Buhler, J.-M., Sentenac, A., and Fromageot, P. (1976) Nature 261, 431-432 [Medline] [Order article via Infotrieve]
  15. Huet, J., Buhler, J.-M., Sentenac, A., and Fromageot, P.(1977) J. Biol. Chem. 252, 8848-8855 [Medline] [Order article via Infotrieve]
  16. Iborra, F., Huet, J., Bréant, B., Sentenac, A., and Fromageot, P. (1979) J. Biol. Chem. 254, 10920-10924 [Abstract]
  17. Liljelund, P., Mariotte, S., Buhler, J.-M., and Sentenac, A.(1992) Proc. Natl. Acad. Sci. U. S. A. 89, 9302-9305 [Abstract]
  18. Nogi, Y., Yano, R., Dodd, J., Carles, C., and Nomura, M.(1993) Mol. Cell. Biol. 13, 114-122 [Abstract]
  19. Sikorski, R. S., and Hieter, P.(1989) Genetics 122, 19-27 [Abstract/Free Full Text]
  20. Buhler, J.-M., Sentenac, A., and Fromageot, P.(1974) J. Biol. Chem. 249, 5963-5970 [Abstract/Free Full Text]
  21. Carles, C., Treich, I., Bouet, F., Riva, M., and Sentenac, A.(1991) J. Biol. Chem. 266, 24092-24096 [Abstract/Free Full Text]
  22. Blum, H., Beier, H., and Gross, H. J.(1987) Electrophoresis 8, 93-99
  23. Lefebvre, O., Carles, C., Conesa, C., Swanson, R. N., Bouet, F., Riva, M., and Sentenac, A.(1992) Proc. Natl. Acad. Sci. U. S. A. 89, 10512-10516 [Abstract]
  24. Dequard-Chablat, M., Riva, M., Carles, C., and Sentenac, A.(1991) J. Biol. Chem. 266, 15300-15307 [Abstract/Free Full Text]
  25. Sanger, F., Nicklen, S., and Coulson, A. R.(1977) Proc. Natl. Acad. Sci. U. S. A. 74, 5463-5467 [Abstract]
  26. Marck, C.(1988) Nucleic Acids Res. 16, 1829-1836 [Abstract]
  27. Altschul, S. F., Gish, W., Miller, W., Myers, E. W., and Lipman, D. J. (1990) J. Mol. Biol. 215, 403-410 [CrossRef][Medline] [Order article via Infotrieve]
  28. Stettler, S., Chiannilkulchai, N., Hermann-Le Denmat, S., Lalo, D., Lacroute, F., Sentenac, A., and Thuriaux, P.(1993) Mol. & Gen. Genet. 239, 169-176
  29. Berben, G., Dumont, J., Gilliquet, V., Bolleand, P.-A., and Hilger, F. (1991) Yeast 7, 475-477 [Medline] [Order article via Infotrieve]
  30. Gietz, R. D., and Schiestl, R. H.(1991) Yeast 7, 253-263 [Medline] [Order article via Infotrieve]
  31. Hoffman, C. S., and Winston, F.(1987) Gene (Amst.) 57, 267-272 [CrossRef][Medline] [Order article via Infotrieve]
  32. Buhler, J. M., Huet, J., Davies, K. E., Sentenac, A., and Fromageot, P. (1980) J. Biol. Chem. 255, 9949-9954 [Abstract/Free Full Text]
  33. Link, A. J., and Olson, M. V.(1991) Genetics 127, 681-698 [Abstract/Free Full Text]
  34. Haendler, B., Keller, R., Hiestand, P. C., Kocher, H. P., Wegmann, G., and Movva, N. R.(1989) Gene (Amst.) 83, 39-46 [CrossRef][Medline] [Order article via Infotrieve]
  35. Sykes, K., Gething, M.-J., and Sambrook, J.(1993) Proc. Natl. Acad. Sci. U. S. A. 90, 5853-5857 [Abstract]
  36. Thomas, D., and Surdin-Kerjan, Y.(1989) Mol. & Gen. Genet. 217, 149-154
  37. Mortimer, R. K., Contopoulou, C. R., and King, J. S.(1991) The Molecular and Cellular Biology of the Yeast Saccharomyces: Genome Dynamics, Protein Synthesis, and Energetics, pp. 737-812, Cold Spring Harbor Press, Cold Spring Harbor, NY
  38. McLaughlin, M. M., Bossard, M. J., Koser, P. L., Cafferkey, R., Morris, R. A., Miles, L. M., Strickler, J., Bergsma, D. J., Levy, M. A., and Livi, G. P.(1992) Gene (Amst.) 111, 85-92 [Medline] [Order article via Infotrieve]
  39. Rothstein, R. J.(1983) Methods Enzymol. 101, 202-211 [Medline] [Order article via Infotrieve]
  40. Stettler, S., Mariotte, S., Riva, M., Sentenac, A., and Thuriaux, P. (1992) J. Biol. Chem. 267, 21390-21395 [Abstract/Free Full Text]
  41. Heitman, J., Movva, N. R., Hiestand, P. C., and Hall, M. N.(1991) Proc. Natl. Acad. Sci. U. S. A. 88, 1948-1952 [Abstract]
  42. Woychik, N. A., Liao, S.-M., Kolodziej, P. A., and Young, R. A.(1990) Genes & Dev. 4, 313-323
  43. Buhler, J.-M.(1979) Structural Studies of Yeast RNA Polymerases A and B. Ph.D. thesis, University of Paris VII
  44. Valenzuela, P., Bell, G., and Rutter, W.(1976) Biochem. Biophys. Res. Commun. 71, 26-31 [Medline] [Order article via Infotrieve]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.