Interactions between the Human RNA Polymerase II Subunits*

(Received for publication, January 24, 1997, and in revised form, April 16, 1997)

Joël Acker Dagger , Michael de Graaff §, Isabelle Cheynel , Vladimir Khazak , Claude Kedinger par and Marc Vigneron

From the Institut de Génétique et de Biologie Moléculaire et Cellulaire (CNRS/INSERM/ULP), F-67404 Illkirch Cedex C.U. de Strasbourg, France

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

As an initial approach to characterizing the molecular structure of the human RNA polymerase II (hRPB), we systematically investigated the protein-protein contacts that the subunits of this enzyme may establish with each other. To this end, we applied a glutathione S-transferase-pulldown assay to extracts from Sf9 insect cells, which were coinfected with all possible combinations of recombinant baculoviruses expressing hRPB subunits, either as untagged polypeptides or as glutathione S-transferase fusion proteins. This is the first comprehensive study of interactions between eukaryotic RNA polymerase subunits; among the 116 combinations of hRPB subunits tested, 56 showed significant to strong interactions, whereas 60 were negative. Within the intricate network of interactions, subunits hRPB3 and hRPB5 play a central role in polymerase organization. These subunits, which are able to homodimerize and to interact, may constitute the nucleation center for polymerase assembly, by providing a large interface to most of the other subunits.


INTRODUCTION

In eukaryotic cells transcription of protein coding genes is carried out by RNA polymerase II (RPB),1 in combination with a number of cofactors that control the selectivity and/or efficiency of transcription initiation, elongation and termination (for recent reviews, see Refs. 1-3). The initiation process involves the formation of a multi-protein complex, referred to as the preinitiation complex (PIC), on the template DNA. The PIC may be assembled stepwise on the promoter by sequential binding of RPB and general transcription factors (3, 4), as suggested by in vitro DNA-binding experiments (3, 4). Recent observations also suggest that formation of the PIC may result from the direct promoter association of a large preformed complex (referred to as the holoenzyme) comprising, in addition to the above mentioned components, several polypeptides, some of which are involved in the chromatin clearing of the target promoter region (5). The description, at the molecular level, of the spatial organization of this complex edifice necessitates a detailed knowledge of the structure of its central component, RPB.

Depending on its origin, RPB consists of 10-14 polypeptides ranging from 220 to 10 kDa (6-8). To date, most studies have focused on the yeast polymerases; much less is known about the human (h) RPB, although the sequences encoding the subunits homologous to the yeast RPB have now been identified (see Table I).

Table I. Simplified nomenclature for the human RNA polymerase II subunits

A systematic nomenclature for the human (h) RNA polymerase II (RPB) subunits used in the present work (leftmost column) is compared to the biochemical nomenclatures of the human (20) (second column from the left) and yeast (6) (third column from the left) subunits, and to the yeast genetic nomenclature (7, 35, 43, 44) (rightmost column).

Human
Yeast
Protein (this study) Protein Protein Gene

hRPB1 hRPB220 B220 RPB1
hRPB2 hRPB140 B150 RPB2
hRPB3 hRPB33 B44 RPB3
hRPB4 B32 RPB4
hRPB5 hRPB25 ABC27 RPB5
hRPB6 hRPB14.4 ABC23 RPB6
hRPB7 hRPB19 B16 RPB7
hRPB8 hRPB17 ABC14.5 RPB8
hRPB9 hRPB14.5 B12.6 RPB9
hRPB10alpha hRPB7.0 ABC10alpha RPC10
hRPB10beta hRPB7.6 ABC10beta RPB10
hRPB11 hRPB14 B12.5 RPB11

Electron microscopy image processing and two-dimensional crystal analyses have been applied to yeast RNA polymerase I (RPA) and RPB (9, 10) and have suggested a rather complex structure. Several subunit-specific epitopes have recently been localized within the yeast RPA, using immunoelectron microscopy (11). Potential interactions between the various subunits of yeast RNA polymerase III (RPC) have been tested by the two-hybrid system. However, few contacts could be detected by this method and comprised only RPC-specific subunits (12).

Functional domains could be assigned to specific subunits, on the basis of primary sequence conservations between subunits of DNA-dependent RNA polymerases from all living organisms and experimental approaches such as affinity labeling, protein and DNA blotting, cross-linking, protease-susceptibility measurements, and functional inhibition by subunit-specific antibodies (11). Both largest subunits (so-called beta ' and beta -like subunits, respectively) have been found to interact with the DNA template and RNA, while only the second largest binds the initiator nucleotide. Together, these observations strongly suggest that these large subunits contain most of the catalytic activities. On the other hand, the phenotypes associated with the deletion of six yeast subunit genes could be independently compensated for, at least to some extent, by their human counterparts (13-16). These results further emphasize the role of conserved sequence elements, but also indicate that the polymerases can accommodate, at least to some extent, variations in primary structure between subunits.

In the present study, we have undertaken a systematic analysis of the interactions that may exist between the various hRPB subunits. Using a baculovirus expression system, we were able to overexpress all the hRPB subunits (except hRPB4, which was originally not available) in a soluble form, either as untagged subunits or as glutathione S-transferase (GST) fusions. The interactions between pairs of subunits were then tested in a GST-pulldown assay for the available subunits. While not all subunits interact under these conditions, the results reveal a number of reciprocal bindings whose relevance is discussed in the context of the complete enzyme. Detailed knowledge of the interaction network between the subunits will ultimately help in generating functional complexes from recombinant polypeptides.


MATERIALS AND METHODS

Nomenclature

For the sake of simplicity, the subunits of hRPB (initially based on their molecular mass in kDa) have been renamed, following the genetic nomenclature of yeast RPB subunits (7, 17). Accordingly, each hRPB subunit was attributed a number (n = 1-9, 10alpha , 10beta , and 11), as indicated in Table I.

Cloning

Cloning of sequences encoding hRPB1, 2, 3, 5, 6, 7, 8, 9, 10alpha , and 10beta subunits has been described previously (in Refs. 18, 19, 20, 21, 22, 15, 13, 23, 13, and 13, respectively). Recently, the sequence of a subunit named hRPB14 was described (24). Our attempts to reclone this cDNA from our HeLa cell cDNA library failed. Instead, upon screening data bases with the hRPB14 sequence, we identified a human expressed sequence tag (EST) (HUMGS00602; GenBankTM accession no. [GenBank]) exhibiting a strong homology with the 5', but not the 3' portion of hRPB14. Using primers derived from the 3' portion of the EST sequence and from the 5' portion of the hRPB14 sequence, we PCR-amplified a fragment of the expected size (420 bp) from our HeLa cDNA library. The resulting fragment was cloned and its sequence established (accession no. [GenBank]). This cDNA, which represents the major detectable species in our library, will be referred to henceforth as hRPB11. Independently, a human cDNA was recently published (25) (accession no. [GenBank]), which encodes a polypeptide identical to hRPB11. The original hRPB14 (24) may therefore represent either a rare related protein or a cloning artifact consisting of a chimeric cDNA.

Subcloning of the hRPB Open Reading Frames into Baculovirus

PCR-mediated mutagenesis was used to insert unique restriction sites (NheI, SpeI, or XbaI, depending on the target cDNA) at the boundaries of each hRPB open reading frame. These sites have been chosen because they generate cleavage products which are compatible with NheI, SpeI, XbaI, and AvrII sites, and may be inserted into any one of them.

The DNA fragments containing the open reading frames of all subunits except hRPB1 and hRPB2 were inserted into the XbaI site of the pVL1393 baculovirus transfer vector (PharMingen) (Fig. 1, left). The hRPB1 and hRPB2 sequences were subcloned together into the XbaI and SpeI cloning sites of the pAcAB4 transfer vector (PharMingen), respectively. The hRPB1 NheI fragment lacking its proper termination codon was inserted, after filling-in of the ends, into the repaired XbaI site, creating an artificial stop codon (underlined) at the resulting junction: 5'-GCT AGC TAG A-3'. The corresponding hRPB1 peptide consisted of 1972 amino acids including the C-terminal extra Ala-Ser residues. The XbaI fragment spanning the hRPB2 coding sequence was inserted into the SpeI cloning site of the same vector. The resulting plasmid enabled the creation of a baculovirus that simultaneously expressed hRPB1 and hRPB2 (Fig. 1, middle).


Fig. 1. Structure of the recombinant hRPB baculovirus transfer vectors. The main features of the vectors used to transfer the hRPB sequences into the baculovirus genome are schematically represented. The listed hRPB sequences (open boxes), with their natural translation initiation and termination sites (closed boxes), and PCR-generated boundaries, were inserted individually into the XbaI and NheI sites of the pVL1393 and pVLGST plasmids, respectively (see "Materials and Methods"). The hRPB1 and hRPB2 sequences were also inserted together into the XbaI and SpeI sites of the pAcAB4 vector, respectively. Arrows below the hRPB sequences indicate to the orientation of insertion. Open and shaded arrowheads on the plasmids refer to the polyhedrin (PH) and p10 (P10) promoters, as indicated. The ampicillin resistance gene (AmpR) and prokaryotic origin of replication (Ori) are shown.
[View Larger Version of this Image (36K GIF file)]

In another series of constructions, the pVL1393 vector was modified by insertion of an EcoRI fragment spanning the GST coding sequence (excised from the pGEX-3X plasmid; Pharmacia Biotech Inc.) into the unique EcoRI site of pVL1393. In addition, the polylinker was altered so as to include unique NheI and NdeI cloning sites that allowed insertion of the subunit sequences and subsequent expression of the corresponding GST fusion proteins, with the GST at the N-terminal position (Fig. 1, right). In the case of the GST-hRPB1 construct, six His residues were added to the natural hRPB1 before reaching a translation termination site. In all other cases, translation terminated at the natural position.

The resulting transfer vectors were then recombined with linearized baculovirus DNA (BaculoGold DNA, PharMingen) in Sf9 cells, using procedures recommended by the manufacturer. The Sf9 cells were grown in the TNM-FH medium as described (26) at 28 °C. The recombinant viruses were plaque-purified, and correct expression of the corresponding hRPB subunits was verified by Western blot analysis. Viral stocks were prepared by a three-step growth amplification.

Baculovirus Infection and Metabolic Labeling

About 2 × 105 Sf9 cells, grown in 24-well dishes (Falcon), were coinfected with pairs of the appropriate baculoviruses. Multiplicities of infection (between 2 and 10 plaque-forming units/cell) were adjusted, so as to balance the amount of recombinant proteins simultaneously expressed from each virus. At 44 h postinfection, the medium was replaced by a Met- or Met/Cys-free medium and 35S-labeled Met (hRPB1, 2, 6, 7, and 8) or a mixture of Met and Cys (hRPB5, 9, 10alpha , 10beta , and 11) (25 µCi each in 500 µl of medium) were added. The cells were collected 4 h later and disrupted by three cycles of freeze-thawing in 500 µl of buffer A (20 mM Tris-HCl, pH 7.5, 20% glycerol, 0.1% Nonidet P-40, 150 mM NaCl, 5 mM beta -mercaptoethanol) containing 2.5 ng/ml each of leupeptin, pepstatin, aprotinin, antipain, and chymostatin (Sigma). The cell lysate was centrifuged (10 min at 14,000 × g), and the cleared supernatant was used for labeled protein analysis.

GST-Pulldown Assay

The 35S-labeled cell extracts (400 µl) were first incubated with 15 µl of protein A-Sepharose (Sigma) to adsorb nonspecific binding proteins. The treated extracts were then incubated with 15 µl of glutathione (GSH)-Sepharose beads (Pharmacia) that were preblocked in phosphate-buffered saline containing 0.3% bovine serum albumin. After 2 h at 4 °C, the beads were washed with buffer A. The final concentrations of salt and detergent were adjusted to minimize nonspecific interactions; NaCl was 300 (hRPB9), 500 (hRPB1, 2, 3, 6, 7, 8, 10alpha , 10beta , and 11) or 1000 mM (hRPB5), and Nonidet P-40 was 0.1 (hRPB9) or 1% (all the other subunits). After washing, the beads were boiled in the electrophoresis sample buffer (20 µl) and the retained proteins were analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) (27). After soaking the gel in a 10% solution of diphenyloxazole in acetic acid, the labeled proteins were revealed by autoradiography.

Antibodies and Immunoblotting

A monoclonal antibody (anti-hRPB6) against the purified bacterially synthesized recombinant hRPB6 subunit was produced, as described previously for the anti-hRPB5 antibody (28). Polyclonal antibodies against hRPB3 were raised in rabbits by injecting a mixture of ovalbumin-coupled synthetic peptides spanning residues 131-141 and 207-217. Proteins that were separated by SDS-PAGE and electroblotted onto nitrocellulose filters were reacted with specific antibodies and revealed by a chemiluminescence detection system (NEN Life Science Products), as described (29).


RESULTS

The GST-Pulldown Assay

The capacity of two proteins to interact with each other may be examined by a number of different techniques, including the yeast two-hybrid system (30), the co-immunoprecipitation test (31), far Western detection (31), or the GST-pulldown assay (32). The two-hybrid assay has been quite informative in many instances, but, for reasons that are not clear, did not give satisfactory results in the present case; no positive results were obtained, even with combinations of recombinant hRPB subunits that, according to genetic evidence in yeast, were expected to interact. Co-immunoprecipitation experiments have also been used with success to identify interacting protein(s) with the help of antibodies directed against one of the partners (31). Here, an extensive study would require a complete set of antibodies capable of precipitating each hRPB subunit. Furthermore, some antibodies may destabilize or even disrupt the interaction that one is looking for, especially in the case of the smallest subunits. Far Western assays, which involve the probing of a protein blot with a protein of interest, are critically dependent on the efficiency of SDS removal and protein renaturation after SDS-PAGE; less stable interactions may be missed in such a system. For these reasons, we used the GST-pulldown assay in which any interacting subunit would be retained on GSH-agarose beads, together with the GST-tagged partner subunit.

Why Baculovirus as an Expression System?

We first tried to overexpress each of these subunits, as untagged polypeptides or GST fusions, in bacterial isopropyl-1-thio-beta -D-galactopyranoside-inducible expression vectors. However, most of the proteins were either produced as insoluble inclusion bodies or found partially degraded in the bacterial extracts (data not shown). Therefore, we opted for the baculovirus expression system; each hRPB cDNA, with the exception of hRPB4, was cloned into appropriate recombinogenic vectors (under the control of the baculovirus P10 or polyhedrin promoter) and introduced into the baculovirus genome by in vivo recombination.

The resulting viruses, upon infection of Sf9 insect cells, allowed efficient synthesis of all subunits, whether free or fused to GST, as both soluble and intact proteins. The only subunit that remained poorly soluble and accumulated in reduced amounts was hRPB2, despite care being taken to coexpress it with hRPB1 (or hRPB3; data not shown), from the same vector. Coexpression was indeed found to improve the yields of proteins otherwise weakly soluble.2 The baculovirus expression system had the additional advantage of providing an eukaryotic environment for protein synthesis and assembly, as opposed to bacterial or in vitro systems.

To facilitate detection of the virally expressed proteins, the infected cells were radiolabeled by growing them in the presence of 35S-labeled methionine or methionine/cysteine, at a late time (44 h) postinfection. Since only a limited number of viral or cellular proteins continue to be synthesized at this period after infection, the labeling pattern is reasonably clean and allows easy visualization of the extraneous polypeptides. Efficient incorporation occurred in all recombinant subunits, allowing their easy identification by SDS-PAGE analysis of a crude infected Sf9 cell extract, even in the case of hRPB11, which contains only one of each of these residues. The overall content of each subunit in Met and Cys residues, whether fused to GST or not (Table II), was taken into account for a rough estimation of the relative amounts of proteins visualized.

Table II. Methionine and cysteine content of each hRPB subunit and of the GST moiety

The number of methionine (M) and methionine + cysteine (M+C) residues present in each polypeptide was derived from the corresponding nucleotidic and deduced peptidic sequences.

Protein M M+C

hRPB
  1 56 80
  2 41 66
  3 1 7
  5 7 9
  6 5 6
  7 5 8
  8 4 5
  9 3 12
  10alpha 3 7
  10beta 2 6
  11 1 2
GST 10 14

Interactions between RPB Subunits Were Examined Pairwise

Sf9 cells were coinfected with pairs of baculovirus recombinants expressing the various RPB polypeptides, one as a GST fusion and the other as an untagged protein. Potential interactions between each combination of tested subunits were scored by the coretention of the untagged subunit onto the GSH-agarose beads, as revealed by autoradiography after SDS-PAGE (Fig. 2). In case of ambiguous identification of a particular band, due to overlapping electrophoretic mobilities of the tagged and untagged molecules, the proteins were blotted onto nitrocellulose and probed with specific antibodies (Fig. 2, B and D, and data not shown). The strength of the interactions between the subunits was deduced from the relative intensities of the bands corresponding to the GST-fused subunits and their non-fused counterparts (see legend to Table III).


Fig. 2. Interactions between hRPB subunits as revealed by the GST-pulldown assay. A representative set of experiments showing the interactions between GST-fused and free hRPB subunits is presented. Sf9 plates were coinfected each with a different combination of two recombinant baculoviruses, one expressing a GST-fused subunit or the GST alone (GST-hRPB series and GST, as indicated on the top of each panel), the other expressing one (panels B-I) or two (panel A) non-fused subunits (names and positions indicated on the right of each panel). After cell labeling, extracts were prepared and processed as described (see "Materials and Methods"). Aliquots of the crude extracts were analyzed directly (Extracts; arrowheads point to the bands corresponding to the GST-hRPB or GST polypeptides) or after incubation in the presence of the GSH-agarose beads (GST-pulldown; arrows point to coretained non-fused hRPB). In panels B and D (bottom), the presence of the coretained free subunit was verified by Western blot analysis (W), by probing the blot with polyclonal antibodies directed against hRPB3 and monoclonal antibodies against hRPB6, respectively.
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Table III. Summary of the hRPB interactions as detected by the GST-pulldown assay

The strengths of interactions between pairs of hRPB subunits (non-fused and GST-fused) were deduced from sets of experiments including and similar to those shown in Fig. 2. Subunits hRPB1 and hRPB2 were coexpressed, because of the weak hRPB2 solubility. Results were based on the relative intensities of the bands corresponding to the GST-fused subunits (GST-hRPB columns) and each non-fused subunit (leftmost column). Considering the relative Met/Cys content of each subunit (see Table II), the interactions were qualified as "strong" (+++), "significant" (++), or "weak" (+), from a comparison of at least two independent GST-pulldown experiments; a lack of interaction (-) was reflected by the weak and irregular detection of the untagged subunit, under the experimental conditions used. ND, not determined.

hRPB GST-hRPB
1 2 3 5 6 7 8 9 10alpha 10beta 11

1 ND ND + +  -  - +++  - + +  -
2 ND ND + +  -  - +  - + +  -
3 +++ +++ +++ +++ + +++ ++  - +++ +++ +++
5 +++ + +++ ++ +++ + ++  - ++ ++  -
6 +  - ++  -  - +  -  - +  -  -
7 +++  - ++ +++  - ++ +  - + +  -
8 ++ + + ++  - + +  - ++ +  -
9  -  -  -  -  -  -  -  -  -  -  -
10alpha  - ND +++  -  -  -  -  - +  -  -
10beta +++ + +++ +++  -  -  -  -  -  -  -
11  -  - +++  -  -  -  -  - +  -  -

Each of the GST-hRPB fusion protein was coexpressed pairwise with every available non-fused hRPB subunit, using this baculovirus system. This allowed the examination of the reciprocal interactions between any tagged subunit with any non-tagged one and thereby allowed us to take into account possible influences of the GST tag on subunit interactions. The results of GST-pulldown experiments such as those shown in Fig. 2 and others (data not shown) are summarized in Table III.

In each series of GST-pulldown experiments, the specificity of the binding reactions was systematically controlled by coexpressing the non-fused GST protein and verifying the lack of interaction with any of the hRPB proteins. In cases where some nonspecific interaction was detected with the GST protein alone (as in Fig. 2A, for example), the amount of nonspecifically bound protein was subtracted from the GST-hRPB-pulldown quantitations.

hRPB3 and hRPB5 Are the Major Anchoring Sites for the Other hRPB Subunits

Three hRPB subunits were repeatedly found in this assay to interact with themselves (hRPB3, hRPB5, and hRPB7), suggesting that they may be present at two (or more) copies within the whole enzyme molecule. Strikingly, together hRPB3 and hRPB5 were able to contact all of the other hRPB subunits, with perhaps the only exception being hRPB9.

We did not analyze separately the interaction pattern of the non-fused hRPB1 and hRPB2 subunits with the GST fusions. To improve the solubility of the untagged hRPB2 subunit, it was coexpressed with hRPB1, from the same recombinant baculovirus. The behavior of both proteins together was examined in the GST-pulldown assay (Fig. 2A). Because of the likelihood of direct interactions between these two large subunits, unambiguous interpretation of the observed results was difficult. However, combining them with those obtained with the GST-hRPB1 and hRPB2 fusion proteins (panels B-I), suggested that both hRPB1 and hRPB2 efficiently interact with hRPB3, whereas only hRPB1 strongly binds to hRPB5. This conclusion was further corroborated by immunoprecipitation experiments in which antibodies against hRPB1 effectively coprecipitated hRPB3 or hRPB5, from extracts of cells coinfected with the corresponding recombinant baculoviruses (data not shown). The hRPB1 subunit also contacts hRPB7, hRPB10beta , and, more weakly, hRPB8. By contrast, it is noteworthy that GST-hRPB8 is the fusion protein that interacts most efficiently with hRPB1 (Fig. 2A).

The reciprocal interactions analyzed in this study were generally not dependent on which of the two subunits was fused to GST, indicating that the GST tag did not critically interfere with the recognition of the partner subunit. However, a few subunits did seem to be affected in their interaction capacities by the GST moiety. hRPB5, when fused to GST, was unable to contact hRPB6 (Fig. 2D) and hRPB10alpha (Fig. 2G); similarly, fusion of GST to hRPB7 impeded interaction with hRPB1 (Fig. 2A and data not shown). These observations suggest that the contacts established by the hRPB5 and hRPB7 subunits may involve their N-terminal portion and that this domain must remain free to allow efficient interactions. Alternatively, the binding sites are localized elsewhere on these proteins, but they may be masked, either directly or allosterically, by the N-terminally fused GST.

Subunits hRPB6 and hRPB11 exhibit only a few contacts with other subunits, essentially with hRPB3 and hRPB5. Subunit hRPB9 did not show any binding capacity in our GST-pulldown assay, whether tested as a GST fusion (Fig. 2) or as an untagged protein (data not shown). This result suggests that hRPB9 may be assembled into the RPB complex through multiple interactions, each one alone being too weak to be detected by our binding assay. If true, hRPB9 binding may only be observed with larger preassembled complexes. Alternatively, the possibility exists that this subunit, which contains a putative zinc-ribbon motif (33, 34), is not properly folded under the present expression conditions.


DISCUSSION

The hRPB subunits have been tested pairwise by the GST-pulldown assay, for their capacity to interact with each other. The observed interactions have been schematically represented on a two-dimensional representation of the hRPB (Fig. 3). Interestingly, interactions that were readily detected in extracts from cells that had been coinfected with two recombinant baculovirus vectors (like GST-hRPB3 and hRPB5) could not be reproduced after mixing extracts from cells infected separately with each virus. Although the mixing assay has not been systematically repeated with each subunit combination, this result suggests that coexpression of the tested polypeptides is required for proper folding and optimal interaction between present subunits. Since subunit coexpression is likely to occur during native polymerase assembly, this result may reflect a greater specificity of the interactions as tested by coinfection of cells compared with mixing of extracts.


Fig. 3. An integrated view of the interactions detected between hRPB subunits. The hRPB complex is schematically represented with each subunit as spheres to approximate scale. Contacts that were identified by the GST-pulldown assay (Table III and Fig. 2) are represented by arrows pointing toward the non-fused subunit retained in this assay. For the sake of simplification, only the most prominent interactions have been outlined: unilateral and reciprocal interactions are represented by single and double arrows, respectively; thickness of the arrows indicates the estimated contact strength. Subunits that interact with themselves are shown as homodimers (hRPB7 is represented at one copy, in accordance with its stoichiometry in the yeast polymerase; Ref. 42). Subunits hRPB4 (not available) and hRPB9 (no contacts detected) are not represented. Potential interactions between the two largest subunits (not directly documented in the present study) are symbolized by the dashed double-headed arrow.
[View Larger Version of this Image (66K GIF file)]

The Pattern of hRPB Subunit Interactions Deduced from the Present Study Finds Confirmation in Other Species

Some of the interactions detected in this system between hRPB subunits have previously been documented with their yeast homologues: these results, which were based either on genetic analyses or two-hybrid assays, further reinforce the present conclusions. A mutation (rpoB1) of the gene for RPB1 has been found that facilitates the release of the RPB4·RPB7 heterodimer from the yeast polymerase (35). Our observation that hRPB1 and hRPB7 strongly interact in the GST-pulldown assay may be related to this finding. On the other hand, RPB4 has also been shown to participate to the recruitment of RPB4·RPB7 on the yeast enzyme (36); it will therefore be worth examining also the ability of hRPB4 to interact with hRPB1. The growth defect of another RPB1 mutant (rpo21-4) has been shown to be compensated by overexpression of the RPB6 subunit (35), a result suggesting that both RPB1 and RPB6 subunits are in close contact. The direct, albeit weak interaction that we see between hRPB1 and hRPB6 further supports this conclusion, particularly if one assumes that such contacts may be stabilized in the context of the complete enzyme.

From the study of mutants altered in each of the three largest subunits of yeast RNA polymerase II (RPB1, RPB2, and RPB3), it has been suggested that RPB2 and RPB3 form a complex that subsequently interacts with RPB1 during RPB assembly (37). In agreement with this conclusion, we detect strong interactions between the hRPB1 and hRPB3 as well as between the hRPB2 and hRPB3 subunits, suggesting that human and yeast RPB assembly may follow similar pathways.

Subunits AC40 and AC19 (both of which are shared by yeast RPA and RPC) are capable of interacting in a two-hybrid assay (12), consistent with the direct and strong interaction between hRPB3 (an AC40 homologue) and hRPB11 (an AC19 homologue) detected here. The homologous subunits from Arabidopsis thaliana (RPB3 and RPB11) have recently also been shown to interact both in double-hybrid and co-immunoprecipitation assays (38). That these interactions occur in vivo is suggested by the observation that the effects of yeast mutations affecting either AC40 or AC19 can be compensated by overexpression of AC19 and AC40, respectively (12). Finally, the reality of these interactions was best illustrated by the colocalization of both subunits on the surface of the yeast RPA, as revealed by immunoelectron microscopy (11).

Overexpression in yeast of ABC10beta , a subunit shared by all three polymerases (RPA, RPB, and RPC), has been shown to efficiently suppress a mutation in AC40 (12). Together with our results showing strong interactions between hRPB10beta and hRPB3 (the human homologues of ABC10beta and AC40, respectively), this effect suggests that these subunits directly contact each other in the natural polymerase molecule. By contrast, overexpression of ABC10beta only partially compensates for a mutation affecting AC19 (12), suggesting that this effect might be indirect. Our results indicating that hRPB10beta and hRPB11 do not contact each other directly, but through their common target hRPB3 (see Fig. 3), are in agreement with such a conclusion.

Toward RNA Polymerase Assembly

Two subunits (hRPB3 and hRPB5) were found to establish nearly 60% (33 over 56) of all contacts detected. Some properties of these subunits are reminiscent of those of the Escherichia coli alpha  subunit, which, as a dimer, recruits the two largest subunits, beta ' and beta , during prokaryotic RNA polymerase assembly. Our observation that each of these subunits may homodimerize further supports the conclusion that they play a central role in the architecture of the native hRPB complex; clearly, a stoichiometry of 2 within the whole enzyme may facilitate multiple contacts with other subunits by providing a larger molecular interface. Interestingly, RPB3 and RPB5, their yeast homologues, are also represented with a stoichiometry of 2 in the yeast enzyme (39). A short stretch of sequence homology, the so called "alpha -motif," has in fact been noticed upon comparison of subunits alpha , RPB3 and AC40 (12). Mutagenesis of this motif within AC40 confirmed that its integrity is essential for yeast survival (12). Finally, the ability of hRPB5 to directly interact with a transcriptional activator, the hepatitis B virus HBx factor (40), is also reminiscent the activator-binding property of the prokaryotic alpha  subunit.

The hRPB7 subunit also revealed the capacity to interact with itself, although its yeast homologue, RPB7, is present in the enzyme of cells with a variable stoichiometry ranging from 0.2 to 1, depending on whether the cells are in exponentially growing or stationary phases (41, 42). It will be of interest to examine if hRPB7 interacts with hRPB4 and whether these subunits, like their yeast counterparts RPB7 and RPB4, can readily dissociate from the polymerase without affecting basal transcriptional activity.3

It is usually, and perhaps naively, accepted that the strongest interactions are the most physiologically relevant. This implicit assumption might have to be revised, particularly in the case of multi-subunit complexes. In such complexes the stability of the whole edifice most likely results from the multiplicity rather than strength of contacts established between each component. Thus, in complement to the present pairwise binding measurements, it will be important to examine the binding potential of hRPB subcomplexes of increasing complexity. Together, both approaches should help elucidating the order of subunit assembly during the polymerase biosynthetic pathway.

Strikingly, the two smallest subunits, hRPB10alpha and hRPB10beta , were found to bind a number of different subunits: given their size (7 and 7.6 kDa, respectively), it is difficult to imagine how these relatively short polypeptides might establish contacts with such an array of subunits, in the native enzyme. It is possible, therefore, that some of the interactions detected with these small subunits are nonspecific. Alternatively, the detected contacts may represent all possible interactions that these small subunits may establish with other ones during polymerase assembly. Some of the interactions occurring at an early step of this complicated process may actually be excluded later on, as higher order complexes are being elaborated. In this respect, it may be relevant that the strong interaction seen between hRPB5 and hRPB8, when coexpressed alone, is disrupted in the presence of a third subunit, hRPB6, which establishes tight contacts with hRPB5, but excludes hRPB8.2

Altogether, these results clearly provide additional confirmation of the interactions observed with the GST-pulldown assay. Furthermore, this opens up avenues toward the reconstitution of an enzymatically active polymerase. The success of such an enterprise will first of all depend on the availability of baculovirus vectors capable of expressing several hRPB subunits, so as to limit as much as possible the number of required recombinant viruses. The construction of such vectors, which has recently been achieved,4 should facilitate coordinate expression of the various subunits. Reconstitution of an entirely recombinant polymerase will allow the detailed functional study of each subunit by use of the powerful in vitro genetics.


FOOTNOTES

*   This work was supported in part by funds from INSERM, CNRS, the Centre Hospitalier Universitaire Régional, the Association pour la Recherche sur le Cancer, the Ligue Nationale contre le Cancer (to J. A.), the Fondation pour la Recherche Médicale, and the Human Frontier Science Program.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.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) X98433[GenBank].


Dagger    Present address: IGR, 94805 Villejuif Cedex, France.
§   Recipient of EMBO Long Term Fellowship ALTF 55-1995. Present address: Dept. of Experimental Therapy, Netherlands Cancer Institute, 1066 CX Amsterdam, Netherlands.
   Present address: The Fox Chase Cancer Center, Philadelphia, PA 19111.
par    To whom correspondence should be addressed. Tel.: 33-3-88-65-34-46; Fax: 33-3-88-65-32-01; E-mail: kedinger{at}igbmc.u-strasbg.fr.
1   The abbreviations used are: RPB, RNA polymerase II; h, human; PIC, preinitiation complex; PCR, polymerase chain reaction; RPA, RNA polymerase I; RPC, RNA polymerase III; GST, glutathione S-transferase; EST, expressed sequence tag; PAGE, polyacrylamide gel electrophoresis.
2   J. Acker, C. Kedinger, and M. Vigneron, unpublished observation.
3   V. Khazak, J. Estojak, J. Majors, G. Sonoda, J. R. Testa, and E. A. Golemis, submitted for publication.
4   J. Acker, I. Cheynel, C. Kedinger, and M. Vigneron, manuscript in preparation.

ACKNOWLEDGEMENTS

We thank Marguerite Wintzerith for her contribution to the initial steps of this work, R. Gopalkrishnan for critical reading of the manuscript, C. Hauss for expert technical assistance, the IGBMC chemistry staff for oligonucleotides, the antibody staff and S. Vicaire for automated DNA sequencing, and H. Boeuf, B. Chatton, R. Gopalkrishnan, and P. Lutz for helpful discussions.


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