Reconstitution of Yeast and Arabidopsis RNA Polymerase alpha -like Subunit Heterodimers*

(Received for publication, December 9, 1996, and in revised form, February 24, 1997)

Rob M. Larkin and Tom J. Guilfoyle Dagger

From the Department of Biochemistry, University of Missouri, Columbia, Missouri 65211

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES


ABSTRACT

Two subunits of about 36-44 kDa and 13-19 kDa in the eukaryotic nuclear RNA polymerases share limited amino acid sequence similarity to the alpha  subunit in Escherichia coli RNA polymerase. The alpha  subunit in the prokaryotic enzyme has a stoichiometry of 2, but the stoichiometry of the alpha -like subunits in the eukaryotic enzymes is not entirely clear. To gain insight into the subunit stoichiometry and assembly pathway for eukaryotic RNA polymerases, in vitro reconstitution experiments have been carried out with recombinant alpha -like subunits from yeast and plant RNA polymerase II. The large and small alpha -like subunits from each species formed stable heterodimers in vitro, but neither the large or small alpha -like subunits formed stable homodimers. Furthermore, mixed heterodimers were formed between corresponding subunits of yeast and plants, but were not formed between corresponding subunits in different RNA polymerases from the same species. Our results suggest that RNA polymerase II alpha -like heterodimers may be the equivalent of alpha  homodimers found in E. coli RNA polymerase.


INTRODUCTION

Escherichia coli core RNA polymerase contains three different subunits of 155 kDa (beta '), 151 kDa (beta ), and 37 kDa (alpha ). Two copies of the alpha  subunit are present in the core enzyme, resulting in a beta 'beta (alpha )2 subunit structure (1). Subunits related to the E. coli beta ', beta , and alpha  subunits are found in most multimeric RNA polymerases, including eukaryotic RNA polymerases I, II, and III. The beta ' and beta  subunits share at least eight and nine blocks of amino acid sequence similarity with their counterparts in archeal and eukaryotic nuclear RNA polymerases (2). The alpha  subunit shares two blocks of limited amino acid sequence similarity with two different subunits found in archeal RNA polymerase and eukaryotic RNA polymerases I, II, and III. The eukaryotic alpha -like subunits consist of a large alpha -like subunit, 36-44 kDa, and a small alpha -like subunit, 13-19 kDa (2-12). Blocks of amino acid sequence similarity between the E. coli alpha  subunit and the eukaryotic alpha -like subunits extend over 25-40 amino acids (2-12). Yeast (Saccharomyces cerevisiae) nuclear RNA polymerases are the most thoroughly studied of the eukaryotic nuclear enzymes (2), and yeast RNA polymerases I and III or A and C contain common alpha -like subunits of 40 kDa (AC40) and 19 kDa (AC19), while RNA polymerase II or B contains alpha -like subunits of 44 kDa (B44 or RPB3) and 12.5 kDa (B12.5 or RPB11). The alpha -like subunits of the plant Arabidopsis thaliana are comparable to those found in yeast (5-7). Arabidopsis contains AC alpha -like subunits of 42/43 and 14 kDa and B subunits of 36 and 13.6 kDa (5-7). While the yeast alpha -like subunits are encoded by single-copy genes (3, 10-12), the large AC and B alpha -like subunits are each encoded by two genes (i.e. two genes encode AC42 and AC43, and two genes encode B36a and B36b) in Arabidopsis (5, 6), but the functional significance, if any, of these redundant genes in Arabidopsis has not been determined.

The stoichiometries of the alpha -like subunits in eukaryotic RNA polymerases have not been totally resolved. Yeast AC40 and AC19 have been reported to have stoichiometries of one in RNA polymerases I and III (13), while yeast B44 has been reported to have a stoichiometry of 2 in RNA polymerase II (14). The stoichiometry of yeast B12.5 has not been determined. Lalo et al. (15) showed that yeast AC40 and AC19 subunits interact with one another, but that neither AC40 nor AC19 interacted with themselves in a yeast two-hybrid system. In a related study, Ulmasov et al. (6) used the yeast two-hybrid system and association studies with in vitro translated subunits to demonstrate that Arabidopsis B36 and B13.6 subunits interact with one another but not with themselves in vivo and in vitro. The results of Lalo et al. (15) and Ulmasov et al. (6) suggested that the large alpha -like subunit and the small alpha -like subunit might form heterodimers in yeast RNA polymerase I and III and in Arabidopsis RNA polymerase II and that these heterodimers might be the equivalent of the alpha 2 homodimer found in E. coli RNA polymerase. In contrast to the above studies, in vivo labeling studies with yeast RNA polymerase II have indicated that the large alpha -like subunit, B44, is present at a stoichiometry of 2 (14, 16). On the other hand, in vivo labeling studies with Arabidopsis RNA polymerase II are consistent with a stoichiometry of 1 for the large alpha -like subunit, B36 (6). A number of other studies in a variety of eukaryotes have suggested that the stoichiometry of the third largest subunit in RNA polymerase II is 1 (17-26).

We have used a renaturation protocol with recombinant alpha -like subunits to determine which combinations of alpha -like subunits can form stable complexes in vitro. Here, we report on renaturation studies with yeast and Arabidopsis RNA polymerase II large and small alpha -like subunits that provide information on RNA polymerase class specificity, species specificity, and stoichiometry of subunit interactions.


EXPERIMENTAL PROCEDURES

Construction of Plasmids and Strains

Plasmids encoding nuclear alpha -like RNA polymerase subunits lacking polyhistidine tags (His tags) were constructed by inserting ORFs1 into the vector pET-16b (Novagen, Madison, WI) between NcoI and BamHI sites. The ORF encoding Arabidopsis AC14 was inserted into pET-16b in two steps. First, cDNA encoding amino acids 1-117 of AC14 was excised from a plasmid in which the first codon of the AC14 ORF was converted to a NcoI site2 and the AC14 cDNA fragment was ligated into the NcoI site of pET-16b. Second, cDNA encoding amino acids 107-122 and the 3'-untranslated region of the Arabidopsis AC14 was excised from an EST clone (GenBankTM accession number Z25617[GenBank]) as an AflII-BamHI fragment. The AflII-BamHI fragment was ligated into the partial Arabidopsis AC14 in pET-16b construct between the AflII and BamHI sites. The ORFs encoding four different nuclear RNA polymerase subunits were amplified from cDNA clones using Pfu DNA polymerase according to the manufacturer's recommendations (Stratagene, La Jolla CA) with the following oligonucleotides: CATGTCATGAATGCTCCAGACAGATTCG and TCAAAATGCGTCGTCGG for yeast B12.5 (3), GCCTCATGAATGCTCCCGAACGAT and TTAAAACTGATTCGAAAACTTGG for Arabidopsis B13.6 (6), CATGCCATGGACGGTGCCACATACC and TCATCCTCCACGCATATGG for Arabidopsis B36a (6), and GTCGTCTCATGACTGAAGAAGGTCCTCAAG and CTACCAAGCATTATCATACCCTC for yeast B44 (11). Using appropriate restriction endonucleases, the ORFs encoding the yeast B12.5, Arabidopsis B13.6, and Arabidopsis B36a subunits were inserted into pET-16b between the NcoI and BamHI sites. To obtain a plasmid encoding a full-length yeast B44 subunit, the ORF encoding this subunit was isolated using oligonucleotides GTCGTCTCATGAGTGAAGAAGGTCCTCAAG and CTACCAAGCATTATCATACCCTC with PCR-mediated amplification as described above. The PCR product was digested with BspHI, and the BspHI product encoding amino acids 1-37 of the yeast B44 subunit was inserted into pET-16b at the NcoI site. The pET-16b derivative containing the partial yeast B44 subunit ORF was digested with BamHI, filled in with Klenow, digested with SalI, and ligated to a PCR product containing the yeast B44 ORF that had been digested with SalI. To obtain a construct encoding the a polyhistidine-tagged (His-tagged) Arabidopsis AC42, the AC42 ORF (5) was amplified by PCR using the oligonucleotides GGAATTCCATATGGTGACTAAAGCAGAAAAACA and TCAAAAGTCGGTTATAGCTTCAC with Pfu DNA polymerase as described above. PCR products containing the Arabidopsis AC42 subunit ORF were ligated into pET-16b between NdeI and BamHI. Plasmids encoding the His-tagged Arabidopsis B36a, B36b, and AC43 and yeast B44 subunits were constructed using a similar strategy to that described above. NdeI sites were introduced at translational start codons using specific oligonucleotides and PCR. The large alpha -like subunit ORFs were inserted into pET-16b between NdeI and BamHI sites. Each plasmid was introduced into E. coli strain BL21(DE3) and expressed using the procedure of Studier and Moffatt (27).

Renaturation and Analysis of Nuclear alpha -like Subunits

Each recombinant RNA polymerase subunit accumulated as an insoluble product in E. coli. The insoluble products were partially purified using the methods of Borukhov and Goldfarb (28) and dissolved in buffer B (6 M guanidine hydrochloride, 50 mM Tris-HCl, pH 7.9, 10 mM MgCl2, 10 µM ZnCl2, 1 mM EDTA, 10 mM dithiothreitol, 10% glycerol) (29). In most experiments (Figs. 2, 3, 4, 5), 500 µg of denatured, His-tagged large alpha -like subunit (Arabidopsis B36a, B36b, AC42, or AC43 or yeast B44) was mixed with a 5-fold molar excess of denatured small alpha -like subunit (Arabidopsis B13.6 or AC14, or yeast B12.5) and diluted to 0.5 mg/ml in buffer B. As controls to rule out nonspecific interaction of subunits with the Ni-NTA resin, the small alpha -like subunits were diluted to 0.5 mg/ml in buffer B without other alpha -like subunits and tested for binding on Ni-NTA columns (see below). In one set of experiments (Fig. 7, A and B), 250 µg of large alpha -like subunits that contained or lacked a His tag were mixed with a 5-fold molar excess of small alpha -like subunits and diluted to 0.5 mg/ml in buffer B. In another experiment (Fig. 7C), approximately equal amounts of yeast B12.5 and His-tagged yeast B12.5 were mixed with 1 molar eq of yeast B44 and diluted to 0.5 mg/ml in buffer B. Renaturation was carried out by dialysis against 500 ml of buffer C (50 mM Tris-HCl, pH 7.9, 200 mM KCl, 10 mM MgCl2, 10 µM ZnCl2, 1 mM EDTA, 5 mM beta -mercaptoethanol, 20% glycerol) (29), followed by dialysis against two 500-ml changes of buffer D (buffer C lacking EDTA). Renatured subunits were clarified by centrifugation at 14,000 rpm at 4 °C in an Eppendorf centrifuge. Clarified mixtures were applied to 0.5-ml columns containing Ni-NTA resin (QIAGEN, Chatsworth, CA) that were equilibrated in buffer D. Columns were washed with 10 volumes of buffer D, followed with 10 volumes of buffer D containing 40 mM imidazole. Columns were eluted with buffer D containing 500 mM imidazole, and four drop fractions (0.2 ml) were collected. Eluted fractions were analyzed in 10% SDS gels (30) or by gel filtration chromatography. Gel filtration chromatography was carried out by applying 200 µl of Ni-NTA eluted fractions to a 24-ml Superose 12 column (Pharmacia Biotech Inc.) that was equilibrated in buffer C containing 10% glycerol instead of 20% glycerol. Columns were run at a flow rate of 0.25 ml/min, and 0.5-ml fractions were collected and analyzed in 10% SDS gels. Molecular mass was calculated as recommended by Stellwagen (31).


Fig. 2. Interaction of Arabidopsis RNA polymerase II alpha -like subunits and yeast RNA polymerase II alpha -like subunits. Recombinant subunits within crude inclusion body fractions were denatured, renatured, and purified on Ni-NTA-agarose columns as described under "Experimental Procedures." Five µl each of the uncleared dialysates, load fractions (dialysates cleared by centrifugation and applied to a Ni-NTA column), flow-through fractions (material that was not retained on the Ni-NTA column), and bound fractions (material that bound to and eluted from the Ni-NTA column) were analyzed by SDS-PAGE. Samples analyzed contained Arabidopsis B13.6 (A), Arabidopsis His-tagged B36a (B), B13.6 and His-tagged B36a (C), B13.6 and Arabidopsis His-tagged B36b (D), yeast B12.5 (E), and B12.5 and yeast His-tagged B44 (F). Subunits were resolved by SDS-PAGE and stained with Coomassie Blue. Molecular weight markers are shown to the left of each gel. Positions of Arabidopsis (At) and yeast (Sc) alpha -like subunits are indicated to the right of each gel.
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Fig. 3. Interactions between Arabidopsis and yeast RNA polymerase II alpha -like subunits. Recombinant subunits were treated as described in Fig. 2. Samples analyzed contained yeast B12.5 and His-tagged Arabidopsis B36a (A), yeast B12.5 and His-tagged Arabidopsis B36b (B), and Arabidopsis B13.6 and yeast His-tagged B44 (C). Samples were subjected to SDS-PAGE and stained with Coomassie Blue. Molecular weight markers are indicated to the left of each gel, and positions of Arabidopsis (At) and yeast (Sc) alpha -like subunits are indicated to the right of each gel.
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Fig. 4. Interactions between RNA polymerase II (B) and RNA polymerase I and III (AC) alpha -like subunits. Recombinant subunits were treated as described in Fig. 2. All alpha -like subunits analyzed were from Arabidopsis except for those indicated as yeast subunits. Samples analyzed contained B13.6 and His-tagged AC42 (A), B13.6 and His-tagged AC43 (B), yeast B12.5 and His-tagged AC42 (C), and yeast B12.5 and His-tagged AC43 (D). Samples were subjected to SDS-PAGE and stained with Coomassie Blue. Molecular weight markers are indicated to the left of each gel. Positions of Arabidopsis (At) and yeast (Sc) RNA polymerase II (B) or RNA polymerase I and III (AC) alpha -like subunits are indicated to the right of each gel.
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Fig. 5. Interactions between Arabidopsis RNA polymerase I and III (AC) alpha -like subunits. Recombinant subunits were treated as described in Fig. 2. Samples analyzed contained AC14 (A), AC14 and His-tagged AC42 (B), and AC14 and His-tagged AC43 (C). Samples were subjected to SDS-PAGE and stained with Coomassie Blue. Molecular weight markers are indicated to the left of each gel. Positions of Arabidopsis (At) and yeast (Sc) RNA polymerase II (B) or RNA polymerase I and III (AC) alpha -like subunits are indicated to the right of each gel.
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Fig. 7. Affinity purification of RNA polymerase II alpha -like subunit heterodimers. Single untagged or His-tagged recombinant subunits, which migrate differently in SDS-PAGE, are shown in lanes 1 and 2 of each panel. Mixed recombinant subunits were denatured, renatured, clarified, and affinity-purified on Ni-NTA-agarose columns as described under "Experimental Procedures." In each panel, lane 3 contains the renatured unclarified dialysate of the mixed subunits, lane 4 contains the clarified dialysate that was applied to the Ni-NTA column, lane 5 contains the flow-through fraction from the column, and lane 6 contains the bound and eluted fraction from the Ni-NTA column. A, Arabidopsis alpha -like subunits: 2.5 µg of B36a (lane 1); 2.5 µg of His-tagged B36a (lane 2); 20 µl of B13.6, B36a, and His-tagged B36a dialysate (lane 3); loaded fraction (lane 4); flow-through fraction (lane 5); and 10 µl of affinity-purified subunits (lane 6). B, yeast alpha -like subunits: 2.5 µg of B44 (lane 1); 2.5 µg of His-tagged B44 (lane 2); 20 µl of B12.5, B44, and His-tagged B44 of dialysate (lane 3), loaded fraction (lane 4); flow-through (lane 5); and 10 µl of affinity-purified subunits (lane 6). C, yeast alpha -like subunits: 3 µg of B12.5 (lane 1); 1 µg of His-tagged B12.5 (lane 2); 40 µl of B12.5, His-tagged B12.5, and B44 of dialysate (lane 3); loaded fraction (lane 4); flow-through (lane 5); and 10 µl of affinity-purified subunits (lane 6). Samples were analyzed by SDS-PAGE and stained with Coomassie Blue. Molecular weight markers are indicated to the left of each gel, and positions of Arabidopsis (At) and yeast (Sc) alpha -like subunits are indicated to the right of each gel.
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RESULTS

Affinity Purification of Renatured Arabidopsis RNA Polymerase II alpha -like Subunits and Yeast RNA Polymerase II alpha -like Subunits

Both the large and small alpha -like subunits in eukaryotic RNA polymerases contain two motifs related to the motifs in the E. coli alpha  subunit (2-12). These motifs consist of a 25-residue amino-terminal block of similarity and a 40-residue block of similarity that are separated by about 130 amino acids in the E. coli alpha  subunit and by 140-200 amino acids in the eukaryotic large alpha -like subunits, but are juxtaposed in the eukaryotic small alpha -like subunits (Fig. 1). To determine if large and small alpha -like subunits interacted with themselves or with one another, we carried out renaturation experiments with recombinant large alpha -like subunits (36-44 kDa) and small alpha -like subunits (13-14 kDa) from Arabidopsis and yeast RNA polymerases using the methods described by Tang et al. (29). The large RNA polymerase II alpha -like subunits that were tested included Arabidopsis B36a and B36b and yeast B44. The Arabidopsis B36a and B36b subunits are encoded by two different genes, and the B36a subunit is the predominant form of the large alpha -like subunit associated with purified RNA polymerase II (6). The small RNA polymerase II alpha -like subunits that were analyzed consisted of Arabidopsis B13.6 and yeast B12.5. Recombinant alpha -like subunits expressed in E. coli were recovered as insoluble polypeptides in inclusion body fractions and solubilized in a denaturing buffer containing guanidine hydrochloride. Various combinations of denatured large and small alpha -like subunits were mixed together and renatured by dialysis against buffers that lacked guanidine hydrochloride. In these experiments, the large alpha -like subunit contained a His tag on the amino terminus, which facilitated purification of soluble, renatured subunits on Ni-NTA-agarose columns. The small alpha -like subunits lacked His tags and were retained on the Ni-NTA columns only if they formed stable complexes with the His-tagged large alpha -like subunits.


Fig. 1. alpha motifs shared by E. coli, yeast, and Arabidopsis RNA polymerases. Schematic diagrams are shown for the E. coli alpha subunit and for yeast and Arabidopsis RNA polymerase I, II, and III alpha -like subunits. Checkered boxes represent the more NH2-terminal alpha -motif, and stippled boxes represent the more COOH-terminal alpha -motif in each subunit.
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When the Arabidopsis B13.6 subunit was subjected to the renaturation protocol, the subunit precipitated and little if any B13.6 subunit was retained in a soluble form (Fig. 2A). In contrast, the bulk of the His-tagged Arabidopsis B36a (or B36b; data not shown) subunit remained soluble during the renaturation protocol and could be purified by Ni-NTA affinity chromatography (Fig. 2B). Renaturation of the B13.6 subunit in the presence of either His-tagged B36a or B36b subunit resulted in a soluble form of B13.6 that bound to the Ni-NTA column along with the His-tagged B36a or B36b subunit (Fig. 2, C and D), suggesting that the B13.6 subunit formed a soluble, stable complex with the B36a or B36b subunit. Similar experiments were carried out with the yeast B44 and B12.5 subunits. In this case, however, the majority of the B12.5 subunit was recovered in the soluble fraction whether renatured in the absence or presence of the His-tagged B44 subunit (Fig. 2, E and F). The yeast His-tagged B44 subunit, like the Arabidopsis His-tagged B36a or B36b subunit, remained soluble during the renaturation protocol and could be recovered by chromatography on the Ni-NTA resin (data not shown). The B12.5 subunit was not retained on the Ni-NTA column if renatured in the absence of the His-tagged B44 subunit (Fig. 2E), but was retained on the nickel resin if renatured in the presence of the His-tagged B44 subunit (Fig. 2F). The results with yeast alpha -like subunits are consistent with the results obtained with the Arabidopsis alpha -like subunits and indicate a small alpha -like RNA polymerase II subunit forms a stable complex with a large alpha -like RNA polymerase II subunit in vitro.

Affinity Purification of Renatured Heterologous RNA Polymerase alpha -like Subunits

Another set of renaturation experiments was carried out to determine if the yeast small alpha -like subunit could stably associate with the Arabidopsis large alpha -like subunit and if the Arabidopsis small alpha -like subunit could stably associate with the yeast large alpha -like subunit. Results based upon the retention of small alpha -like subunits and His-tagged large alpha -like subunits on Ni-NTA columns presented in Fig. 3 indicate that yeast B12.5 forms stable complexes with Arabidopsis B36a (Fig. 3A) and B36b (Fig. 3B) and that Arabidopsis B13.6 forms stable complexes with yeast B44 (Fig. 3C). The renaturation of soluble Arabidopsis B13.6 appears to be less effective when carried out in the presence of the yeast B44 subunit, however, than renaturation in the presence of Arabidopsis B36a or B36b (compare Fig. 3C to Fig. 2, C and D). This less effective renaturation with the Arabidopsis B13.6 and yeast B44 subunit appears to result from precipitation of these two subunits when renatured together, while the yeast B44 subunit remains soluble when renatured alone or in combination with the yeast B12.5 subunit (compare Fig. 3C to Fig. 2F). Because only small amounts of B13.6 and B44 complexes remained soluble, little of this complex was applied and bound to the affinity resin (Fig. 3C).

To determine if the large and small alpha -like subunit interactions are specific for RNA polymerase II alpha -like subunits, renaturation experiments were conducted with mixtures of small alpha -like RNA polymerase II subunits and large alpha -like RNA polymerase I and III subunits. The large RNA polymerase I and III alpha -like subunits subjected to analysis were Arabidopsis AC42 (42 kDa) and AC43 (43 kDa). The Arabidopsis AC42 and AC43 subunits are encoded by two different genes, and the AC42 subunit is the predominant form associated with RNA polymerase III, but the form associated with RNA polymerase I has not been determined (5). Arabidopsis B13.6 or yeast B12.5 subunits were subjected to the renaturationation protocol as mixtures with His-tagged Arabidopsis AC42 or AC43. When His-tagged AC42 or AC43 subunits were renatured alone, they behaved similar to the Arabidopsis B13.6 subunit in that the bulk of these subunits precipitated during the renaturation protocol (data not shown). Only small amounts of soluble His-tagged AC42 or AC43 could be recovered on the Ni-NTA resin. Likewise, when His-tagged AC42 or AC43 were renatured with Arabidopsis B13.6, most of the AC42, AC43, and B13.6 precipitated, and only small amounts of His-tagged AC42 or AC43 could be recovered on Ni-NTA columns (Fig. 4, A and B). No B13.6 subunit was retained on the nickel column. Similar results were obtained when renaturation experiments between yeast B12.5 and Arabidopsis AC42 or AC43 were conducted (Fig. 4, C and D). In this case, however, the yeast B12.5 subunit remained soluble during the renaturation protocol, but failed to bind to the Ni-NTA column even though small amounts of His-tagged AC42 or AC43 were retained on the nickel resin.

Affinity Purification of Renatured Arabidopsis RNA Polymerase I and III alpha -like Subunits

To assess whether Arabidopsis AC42 and AC43 subunits could form stable complexes with a RNA polymerase I and III small alpha -like subunit, the Arabidopsis small alpha -like subunit, AC14 (7), was renatured in the absence or presence of a His-tagged AC42 or AC43 subunit. Most of the AC14 subunit remained soluble whether or not an AC42 or AC43 subunit was present during the renaturation protocol, but the AC14 subunit was only retained on the Ni-NTA column if renatured in the presence of a His-tagged AC42 or AC43 subunit (Fig. 5, A-C). Most of the AC42 and AC43 subunits precipitated out of solution if renatured in the absence of AC14, but both subunits remained soluble if renatured in the presence of AC14. The AC42 and AC43 subunits failed to stay in solution if renatured in the presence of small alpha -like Arabidopsis B13.6 or yeast B12.5 subunit (Fig. 4, A-D), indicating that the large RNA polymerase I/III alpha -like subunits only remain soluble if they form a stable complex with a small alpha -like subunit. These results suggest that Arabidopsis RNA polymerase I and III large alpha -like subunits, AC42 and AC43, form soluble stable complexes with the Arabidopsis RNA polymerase I and III small alpha -like subunit, AC14. On the other hand, RNA polymerase I and III large alpha -like subunits do not form stable complexes with RNA polymerase II small alpha -like subunits.

Gel Filtration of Renatured Arabidopsis RNA Polymerase II alpha -like Subunits and Yeast RNA Polymerase II alpha -like Subunits

To further characterize alpha -like subunit complexes, renatured alpha -like subunits were fractionated on gel filtration columns. When renatured (but not affinity-purified) Arabidopsis His-tagged B36a was fractionated on Superose 12, a major portion of this subunit eluted as a symmetrical peak with an estimated size of 54 kDa (Fig. 6A). A minor fraction eluted as a broad peak with an average size of about 400 kDa, suggesting that some aggregation of B36a subunits occurs during the renaturation or fractionation process. The Arabidopsis B13.6 subunit could not be analyzed by gel filtration because this subunit precipitated during the renaturation protocol. When the His-tagged B36a subunit was renatured (but not affinity-purified) with a molar excess of B13.6 subunit and fractionated on Superose 12, most of the B36a and B13.6 subunits eluted as a symmetrical peak with an estimated size of 67 kDa (Fig. 6B). The peak fraction containing the B36a and B13.6 subunits eluted one fraction earlier than the peak fraction containing only the B36a subunit. A minor fraction of the B36a and B13.6 subunits eluted in fractions with an estimated size of 200-500 kDa, suggesting that some aggregation occurs similar to that found with the B36a subunit alone. His-tagged B36A and B13.6 complexes that had been renatured together and purified by affinity chromatography on an Ni-NTA column were also analyzed by gel filtration on Superose 12. This purified complex displayed a fractionation profile similar to that observed with the unpurified B36a·B13.6 mixture (Fig. 6C); however, high molecular weight aggregates were not observed with the affinity-purified B36·B13.6 complex. Taken together, the gel fractionation results are consistent with B36a and B13.6 associating together as a stable heterodimer.


Fig. 6. Analysis of Arabidopsis and yeast alpha -like subunit interactions by gel filtration. Recombinant subunits were from inclusion body fractions that had been subjected to denaturation, renaturation, and clarification (A and B) or subjected to denaturation, renaturation, clarification, and affinity purification on Ni-NTA columns (C-E). Recombinant subunits within crude inclusion body fractions were denatured, renatured, and purified on Ni-NTA-agarose columns as described under "Experimental Procedures." Samples were fractionated on a Superose 12 column, and aliquots from fractions were subjected to SDS-PAGE and stained with Coomassie Blue. Samples analyzed contained Arabidopsis B36a (A), Arabidopsis B36a and B13.6 (B), affinity-purified Arabidopsis B36a and B13.6 (C), affinity-purified yeast B44 and B12.5 (D), and affinity-purified Arabidopsis B36a and yeast B12.5 (E). Fraction numbers are shown above each panel and molecular mass markers are shown above panel A.
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Affinity-purified complexes containing yeast His-tagged B44·yeast B12.5 and Arabidopsis B36a·yeast B12.5 were also analyzed by gel filtration on Superose 12 (Fig. 6, D and E). In each of these cases, the large alpha -like subunit and small alpha -like subunit fractionated as a stable complex similar to that observed with affinity-purified Arabidopsis His-tagged B36a·B13.6 complexes. The yeast His-tagged B44·B12.5 complex eluted in fractions with an average molecular mass of greater than 67 kDa, however. The yeast His-tagged B44 subunit shows this same fractionation pattern (i.e. eluting in fractions with an estimated molecular mass in excess of 67 kDa) when renatured by itself and analyzed by Superose 12 gel filtration (data not shown). Since the large alpha -like subunit may not form a globular structure, it is possible that some fractionation abnormalities are observed on Superose 12. Support for the fractionation abnormalities of the yeast large alpha -like subunit as opposed to association between these subunits was obtained from the next set of experiments.

Affinity Purification of His-tagged and Untagged alpha -like Subunit Mixtures

Gel filtration experiments did not allow us to definitively determine if large and small alpha -like subunits formed heterodimers or some type of higher order multimers. For example, complexes observed in affinity-purified fractions or gel filtration fractions might consist of two large alpha -like subunits associated with one small alpha -like subunit, one large subunit associated with two small subunits, or two large subunits associated with two small subunits. To further verify that one large and one small alpha -like subunit associate with one another to form heterodimers and not higher order multimers, renaturation experiments were carried out with one His-tagged large alpha -like subunit, a second untagged large alpha -like subunit, and an untagged small alpha -like subunit. Complexes were purified by affinity chromatography on Ni-NTA columns, and purified complexes were analyzed by SDS-gel electrophoresis. Because His-tagged and untagged large alpha -like subunits displayed different mobilities on SDS gels, these subunits could be distinguished from one another. Fig. 7A shows that with affinity-purified renatured mixtures of Arabidopsis His-tagged B36a, untagged B36a, and untagged B13.6, only the His-tagged B36a and B13.6 subunits bound to the affinity resin. No untagged B36a subunit was found in the affinity-purified complex. Likewise, Fig. 7B shows that with affinity-purified mixtures of yeast His-tagged B44, untagged B44, and untagged B12.5, only the His-tagged B44 and B12.5 subunits bound to the affinity resin. These results indicate that only one molecule of the large alpha -like subunit associates in a complex with the small alpha -like subunit.

To determine if only one molecule of the small alpha -like subunit is associated with this complex, renaturation experiments were carried out with mixtures of yeast His-tagged B12.5, untagged B12.5, and untagged B44. Complexes were purified by affinity chromatography on Ni-NTA columns, and purified complexes were analyzed by SDS gel electrophoresis. His-tagged and untagged B12.5 subunits displayed different mobilities on SDS gels and could be distinguished from one another. Fig. 7C shows that affinity-purified complexes contained the His-tagged B12.5 subunit and B44 subunit, but no untagged B12.5 subunit. These results indicate that only one molecule of the small alpha -like subunit associates in a complex with the large alpha -like subunit. Taken together, our results indicate that large and small alpha -like subunits from Arabidopsis and yeast form stable heterodimers in vitro.


DISCUSSION

Previous results obtained by using the yeast two-hybrid system have indicated that a large alpha -like subunit and small alpha -like subunit from yeast RNA polymerase I and III and Arabidopsis RNA polymerase II could interact stably with one another in vivo (6, 15). These studies also indicated that large alpha -like subunits do not interact with themselves and small alpha -like subunits do not interact with themselves. Extragenic suppression studies in yeast support the interaction between a large and small alpha -like subunit in yeast RNA polymerases I and III (15). Using an antibody pull-down experimental approach, Ulmasov et al. (6) further demonstrated that an in vitro translated epitope-tagged Arabidopsis RNA polymerase II large alpha -like subunit associated with an untagged RNA polymerase II small alpha -like subunit. While these results are consistent with one large alpha -like subunit and one small alpha -like subunit associating as a heterodimer in RNA polymerases I, II, and III, the experimental approaches described above do not rule out additional interactions with other molecules that might be required for stable association of alpha -like subunits and do not establish stoichiometries for these alpha -like subunit interactions. For example, previous studies do not rule out the possibility that B36(B13.6)2 or (B36)2B13.6 complexes were formed. We have taken an alternative approach using in vitro association studies with recombinant alpha -like subunits from yeast and Arabidopsis RNA polymerases that allows us to rule out accessory protein requirements for alpha -like subunit interactions, to establish stoichiometries for alpha -like subunits in purified stable complexes, and to provide a framework for the reconstitution of eukaryotic RNA polymerases. Our results indicate that one large alpha -like subunit associates with one small alpha -like subunit to form stable heterodimers in vitro and that heterodimerization of alpha -like subunits in vitro is not species-specific but is specific for the class of RNA polymerase (i.e. RNA polymerase II versus RNA polymerases I and III).

E. coli and other prokaryotic RNA polymerases contain an alpha 2 homodimer that is associated with beta ' and beta  in core enzymes. The alpha 2 homodimer is formed as the initial event in the E. coli RNA polymerase assembly pathway (32). The assembly pathway for eukaryotic nuclear RNA polymerases has not been established, but studies in yeast have suggested that the third largest subunit in yeast RNA polymerase II, which is related to the E. coli alpha  subunit, may homodimerize and subsequently interact with the second largest and largest subunits in enzyme assembly (33). Our results suggest an alternative to the alpha 2 homodimer enzyme assembly pathway in eukaryotic RNA polymerases might involve the initial heterodimerization of a large alpha -like subunit and a small alpha -like subunit. Future experiments will be required to determine if alpha 2 heterodimers can stably associate with second largest and/or the largest subunit of eukaryotic RNA polymerases to form a core enzyme analogous to beta 'beta (alpha )2.

Mutation studies with the E. coli alpha  subunit have shown that the two motifs conserved in eukaryotic alpha -like subunits are important in enzyme assembly of prokaryotic RNA polymerase (34-38). Mutations in the NH2-terminal alpha -like motif in yeast AC40 subunit can be suppressed by elevated expression of the AC19 subunit (15), and mutations in the yeast B44 subunit alpha -like motifs prevent assembly of the B44 subunit with the second largest and largest subunits of yeast RNA polymerase II (33). NH2-terminal and COOH-terminal mutations in the Arabidopsis B36b subunit that disrupt either alpha -like motif prevent interaction between the B36 and B13.6 subunits in in vitro pull-down experiments (6). Taken together, preliminary results with the eukaryotic alpha -like subunits suggest that alpha -motifs conserved in prokaryotic alpha  subunits and eukaryotic alpha -like subunits may be important for subunit association and enzyme assembly.

There is evidence that the alpha  subunits in E. coli RNA polymerase are not structurally (i.e. tertiary structure within the core enzyme) or functionally equivalent (39-45). Based on the nonequivalence of the alpha  subunits in prokaryotic RNA polymerases, we propose that eukaryotes may have evolved alpha -like subunits that are nonequivalent in primary and tertiary structure as well as in function. alpha -Like subunits in eukaryotes are clearly not equivalent in function to prokaryotic alpha  subunits because eukaryotic subunits lack the COOH-terminal motif of prokaryotic alpha  subunits that plays a role in transcriptional activation and repression and in binding to promoter DNA elements (45-47). Eukaryotic enzymes may have evolved distinct alpha -like subunits that serve nonequivalent roles in enzyme assembly, in contacting other subunits, and possibly in enzymatic function.

Recent immunoelectron microscopy results with antibodies directed against the largest (beta '-like), second largest (beta -like), and alpha -like subunits of yeast RNA polymerase I have provided information about the location of these subunits within the enzyme and have indicated that the large and small alpha -like subunits are located near each other within the apical region of the enzyme (48). These results have not, however, provided definitive information about the stoichiometries of the alpha -like subunits within the yeast enzyme. Our results, based upon in vitro subunit interaction studies, are consistent with there being one large alpha -like subunit and one small alpha -like subunit in eukaryotic RNA polymerases. Nonetheless, there remains the possibility that additional alpha -like subunits might associate with eukaryotic RNA polymerases by interacting with a higher order subunit complex (i.e. after additional subunits have associated with the alpha -like subunit heterodimer).


FOOTNOTES

*   This work was supported by National Research Initiative Competitive Grants Program Grant USDA CSRS 94-37301-0300. This is Journal Series contribution 12,582 from the Missouri Agricultural Experimental Station.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Dagger    To whom correspondence should be addressed: Dept. of Biochemistry, 117 Schweitzer Hall, University of Missouri, Columbia, MO 65211. Tel.: 573-882-7648; Fax: 573-882-5635; E-mail: bctguilf{at}muccmail.missouri.edu.
1   The abbreviations used are: ORF, open reading frame; EST, expressed sequence tag; PCR, polymerase chain reaction; NTA, nitrilotriacetic acid.
2   R. Larkin, unpublished results.

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