Dissociable Rpb4-Rpb7 Subassembly of RNA Polymerase II Binds to Single-strand Nucleic Acid and Mediates a Post-recruitment Step in Transcription Initiation*

Stephen M. OrlickyDagger , Phan T. Tran§, Michael H. Sayre§||, and Aled M. EdwardsDagger **

From the Dagger  Banting and Best Department of Medical Research and Department of Medical Genetics and Microbiology, C. H. Best Institute, University of Toronto, Toronto, Ontario M5G 1L6, Canada and the § Department of Biochemistry, Johns Hopkins University, Baltimore, Maryland 21205

Received for publication, April 13, 2000, and in revised form, November 16, 2000


    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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The Rpb4 and Rpb7 subunits of yeast RNA polymerase II form a heterodimeric complex essential for promoter-directed transcription initiation in a reconstituted system. Results of template competition experiments indicate that the Rpb4-Rpb7 complex is not required for stable recruitment of polymerase to active preinitiation complexes, suggesting that Rpb4-Rpb7 mediates an essential step subsequent to promoter binding. Sequence and structure-based alignments revealed a possible OB-fold single-strand nucleic acid-binding motif in Rpb7. Purified Rpb4-Rpb7 complex exhibited both single-strand DNA- and RNA-binding activities, and a small deletion in the putative OB-fold nucleic acid-binding surface of Rpb7 abolished binding activity without affecting the stability of the Rpb4-Rpb7 complex or its ability to associate with polymerase. The same mutation destroyed the transcription activity of the Rpb4-Rpb7 complex. A separate deletion elsewhere in the OB-fold motif of Rpb7 also blocked transcription but did not affect nucleic acid binding, suggesting that the OB-fold of Rpb7 mediates both DNA-protein and protein-protein interactions required for productive initiation.


    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cellular RNA polymerases contain a core set of subunits exemplified by the alpha , beta , and beta ' subunits of the eubacterial enzyme. Eukaryotic nuclear RNA polymerases have an additional 8-9 accessory subunits that are shared, unique, or similar in the three classes of polymerase (I, II, and III) (1). RNA polymerase II, which is mainly responsible for transcribing protein coding genes, consists of twelve subunits, named Rpb1-12, from largest to smallest (2). The Rpb1, Rpb2, Rpb3, and Rpb11 subunits are the functional and structural homologues of bacterial core subunits. Along with accessory Rpb subunits, RNA polymerase II uses additional proteins called general transcription factors (GTFs)1 to initiate transcription in a promoter-dependent fashion (reviewed in Refs. 3 and 4). The accessory Rpbs and GTFs seem to play a role analogous to that of the sigma  subunits of prokaryotic RNA polymerases, which enable the core catalytic enzyme (alpha 2beta beta ') to bind specifically to start site regions, initiate transcription efficiently, and respond to regulatory inputs.

Several characteristics of a heterodimeric complex of Saccharomyces cerevisiae Rpb4 and Rpb7 bear resemblance to prokaryotic sigma  factors; the Rbp4-Rpb7 complex is absolutely required for accurate initiation but dispensable for RNA chain elongation (5), and the complex can associate reversibly with core (10-subunit) RNA polymerase II (5). Moreover, the Rpb4 and Rpb7 subunits are less abundant than other polymerase subunits in vivo (6) and appear to be critical for cellular adaptations to stress, a hallmark of many sigma  factors (6-9). Finally, overexpression of Rpb7 in wild-type yeast induces cell filamentation, suggesting that Rpb7 may promote gene-specific transcription (10).

Rpb7 is essential for cell viability in yeast (11) but Rpb4 is not (12). Some of the stress-sensitive phenotypes linked to an rpb4 null mutation are suppressed by overproduction of Rpb7 (9). Biochemical evidence suggests that the yeast Rpb4 subunit may facilitate association of Rpb7 with core polymerase (5). Taken together, these findings suggest that the Rpb7 subunit is key to the transcriptional activity of the Rpb4-Rpb7 complex. Here we show that Rpb7 contains a putative OB-fold domain that seems structurally and topologically related to a subfamily of domains typically found in proteins involved in ssRNA and ssDNA binding. Biochemical studies with wild-type Rpb4-Rpb7 complex and variants with targeted mutations in this domain reveal a possible link between nucleic acid-binding activity and an essential step in promoter utilization that occurs after formation of a stable preinitiation complex.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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Baculovirus Expression of Yeast Rpb4 and Rpb7-- Open reading frames (ORFs) were amplified by polymerase chain reaction from yeast genomic DNA using primers containing an NdeI restriction site at the 5' end and a BamHI restriction site at the 3' end of each ORF. The RPB4 gene was subcloned into the pET15b bacterial expression plasmid (Novagen), appending a hexahistidine tag and a thrombin cleavage site to the N terminus of Rpb4. The DNA insert encoding His-tagged Rpb4 was excised with XbaI and BamHI and ligated between the XbaI and BglII sites of the baculovirus transfer vector PacAB 4 (Pharmingen) to create PacAB 4/RPB4. The RPB7 gene was cloned into PacAB 4/RPB4 between the SmaI and BamHI sites to create PacAB 4/RPB4/7. To create recombinant baculoviruses, PacAB 4/RPB4/7 was cotransfected into Sf9 cells using calcium phosphate precipitation with 0.5 µg of baculovirus GoldTM digested with Bsu 36I (New England BioLabs). Recombinant virus containing the RPB4 and RPB7 genes, identified by blue/white plaque screening, was plaque-purified twice and then amplified for large-scale protein expression.

To prepare individual Rpb4 and Rpb7 recombinant baculoviruses, RPB4 and RPB7 genes were cloned into the baculovirus transfer vector PVL 1392 (Pharmingen). The RPB7 ORF was first cloned between the NdeI and BamHI sites in PET11a (Novagen), and RPB4 was cloned into pET15b. Each construct was digested with XbaI and BamHI, and the resulting ORF-containing fragments were subcloned between the XbaI and BamHI sites of PVL 1392.

In-frame deletions in RPB7 in the PVL 1392 construct were prepared with the QuikChange site-directed mutagenesis system (Stratagene). After confirming each deletion by sequencing, RPB7 alleles were subcloned between the EcoRI and BamHI sites of pFasBac 1 (Invitrogen). Clones were transformed into DH10Bac competent cells (Life Technologies, Inc.) to generate recombinant baculoviruses. Rpb7 variant proteins were coexpressed with Rpb4 in Sf9 cells by coinfection with recombinant baculoviruses.

20 plates of Sf9 cells (150 × 20 mm; Nunc), each containing roughly 2 × 107 cells, were infected with 250 µl of high titer virus stock. The optimal multiplicity of infection for protein expression was determined by SDS polyacrylamide gel electrophoresis. Infected cells were scraped from plates and collected by centrifugation at 800 × g for 20 min. Cell pellets were washed in 25 ml of 50 mM Hepes (pH 7.5), 150 mM NaCl to remove any remaining medium and then resuspended in Buffer A (50 mM Hepes (pH 7.5), 750 mM NaCl, 10% glycerol, 5 mM imidazole, 0.1% Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride, 1 mM benzamidine). Resuspended cells were flash frozen and stored at -80 C.

Purification of Rpb4-Rpb7 Complexes-- Infected cells were thawed on ice and broken by sonication (3 times for 1 min). Subsequent steps were performed at 4 °C. The sonicate was clarified by centrifugation at 100,000 × g for 45 min in a Beckman Ti70 rotor. The supernatant was loaded onto a 2-ml His-Bind resin column (Novagen) and washed with Buffer A until the eluate contained less than 10 µg of protein per ml (Bio-Rad protein assay). The His-Bind column was then washed with 10 columns of Buffer A containing 50 mM imidazole but lacking Nonidet P-40 and protease inhibitors. Cleavage to remove the hexahistidine tag was performed on the column by loading 30 µg of thrombin (Parke-Davies) in 2 ml of buffer containing 50 mM Hepes (pH 7.5), 150 mM NaCl, 5% glycerol (cleavage buffer) and incubating the column at 4 °C overnight. Thrombin was next eluted from the column by washing with 2 column volumes of cleavage buffer. The Rpb4-Rpb7 complex lacking the histidine tag was then eluted by raising the NaCl and imidazole concentrations to 500 and 50 mM, respectively (residual uncleaved His-tagged Rpb4-Rpb7 complex remained on the column and was recovered by elution with 500 mM imidazole). The cleaved (untagged) complex was diluted to 100 mM NaCl and purified further on a Mono Q column (5 × 50 mm; Amersham Pharmacia Biotech) developed with a linear gradient (10 ml) of potassium acetate (pH 7.5) from 100 mM to 1 M at 0.5 ml/min. Fractions containing the Rpb4-Rpb7 complex (identified by SDS polyacrylamide gel electrophoresis) were pooled, dialyzed for 12 h against 50 mM Hepes (pH 7.6), 400 mM potassium acetate, 10% glycerol, 1 mM dithiothreitol, and concentrated in a Centricon 30 device (Amicon). Expression and purification of Rpb4-Rpb7 variant complexes were carried out exactly as described for the wild-type complex.

Biochemical Assays-- To test for single-strand nucleic acid-binding activity, purified Rpb4-Rpb7 complex was incubated for 30 min on ice with 25 fmol of a 32P end-labeled oligonucleotide (18-mer) in 20 µl of buffer containing 40 mM Hepes (pH 7.2), 100 mM potassium acetate, 10% glycerol. Bound and free probes were resolved by electrophoresis in a 7.5% polyacrylamide gel containing 2% glycerol and 0.5× TBE. Radiolabeled DNA was visualized by autoradiography and quantified using a phosphorimager (Storm). RNA binding (65-mer) was carried out under the same conditions except for the addition of 0.5 units of RNase inhibitor (Promega).

For transcription assays, wild-type RNA polymerase II, TFIIF, TFIIH, and Mediator were purified from yeast whole-cell extract (13-16). RNA polymerase II lacking Rpb4 and Rpb7 subunits (pol IIDelta 4/7) was purified from a rpb4Delta yeast strain as described (5). Yeast TBP, TFIIB, and TFIIE were purified from recombinant Escherichia coli strains (17, 18). Plasmid DNA templates contained the CYC1, AdML, or TEF1 promoters fused to G-less cassettes (13, 19). Template competition experiments employed the CYC1/G-less cassette plasmids pJJ460 and pJJ470 (20). ATP, CTP, UTP, and [alpha -32P]UTP (3000 Ci/mmol) were from Amersham Pharmacia Biotech. Preinitiation complexes were assembled during a 10-min incubation at 23°C in reaction mixtures (20 µl) containing 20 mM Hepes-KOH (pH 7.6), 7 mM magnesium acetate, 100-110 mM potassium acetate, 250 ng of plasmid DNA, 100 ng of RNA polymerase II, 30 ng of TBP, 30 ng of TFIIB, 15 ng of TFIIE, 50-100 ng each of TFIIF and TFIIH, and (where indicated) 100 ng of Mediator. Purified Rpb4-Rpb7 complex or dilution buffer was added as indicated, and transcription was initiated by adding 5 µl of a solution containing 20 mM Hepes-KOH (pH 7.6), 7 mM magnesium acetate, 2.5 mM ATP, 2.5 mM CTP, 125 µM UTP, 5 µCi of [alpha -32P]UTP. For template competition experiments, this NTP mix (5 µl) also contained 250 ng of competitor template DNA(s) as indicated. Labeling reactions were carried out at 23°C for 15-30 min and then terminated by adding stop mix (0.2 ml) containing 0.3 M NaCl and 2 units of T1 ribonuclease (Calbiochem). Radiolabeled transcripts were processed for denaturing gel electrophoresis and detected by autoradiography as described (21).

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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A Predicted Nucleic Acid-binding Domain in Rpb7-- The sequence of the archaea Rpb7 homologue is similar to that of the ribosomal S1 domain from E. coli. The S1 protein exemplifies the OB-fold (22), a structure found in many other proteins with single-stranded nucleic acid-binding activity (23, 24) (Fig. 1a). The OB-fold motif consists of a 5-stranded beta -sheet that is coiled to form a closed beta -barrel and an alpha -helix that connects the third and fourth strands (Fig. 1, b and c). Some general observations about the interaction of OB-fold proteins with single-stranded nucleic acid can be gleaned from the two known structures of OB-fold/nucleic acid complexes. First, the beta -strands 1-3 (Fig. 1b, green) form a scaffold across which nucleic acid is splayed, making hydrogen bond and stacking contacts. Second, the loop between the first and second strands (L12; Fig. 1b, red) makes key hydrogen bonds with the phosphate backbone. Third, the loop that connects the fourth and fifth beta -strands (L45; Fig. 1b, yellow) clamps down on the nucleic acid strand, making hydrogen bond and stacking interactions.


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Fig. 1.   Panel A, sequence alignment of yeast Rpb7, an S1 domain from E. coli, and the rpoE RNA polymerase subunit from Sulfolobus. Black and gray boxes mark amino acid residues conserved in all three or in two of the three proteins, respectively. Panel B, positions of Rpb7 deletions superimposed on a typical OB-fold structure (residues 180-290 of the largest subunit of human replication protein A). The DNA channel in replication protein A lies between the D1 and D3 deletions when visualized in this orientation. Panel C, boxes enclose missing amino acid residues in Rpb7 deletions D1, D2, and D3.

Our study had the following three goals: to purify a transcriptionally active recombinant Rpb4-Rpb7 complex, to test its nucleic acid-binding activity, and to explore its role in transcription initiation. To generate recombinant Rpb4-Rpb7 complex, subunits were first expressed individually or together in E. coli. Although both subunits accumulated to high levels in cells, they proved to be insoluble. However, a soluble complex was recovered from insect cells coinfected with recombinant baculoviruses expressing Rpb4 and Rpb7 or from cells infected with a single recombinant baculovirus encoding both proteins. Purification of the complex from insect cell lysates was facilitated by adding a hexahistidine tag and thrombin cleavage site to the N terminus of Rpb4. The complex was immobilized on a metal affinity column and subsequently released (after extensive washing to remove unbound contaminants) by digestion of Rpb4 with thrombin. Following subsequent anion exchange chromatography, the purified complex was judged to be at least 95% homogeneous by SDS polyacrylamide gel electrophoresis and Coomassie Blue staining (Fig. 2), in a yield of 200 µg per liter of Sf9 cells.


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Fig. 2.   Purification of wild-type (W.t.) and deletion mutant (D2 and D3) Rpb4-Rpb7 complexes. Purified complexes were resolved by denaturing gel electrophoresis and stained with Coomassie Blue. Bands representing Rpb4 and Rpb7 subunits are indicated.

We designed mutant Rpb4-Rpb7 complexes containing small in-frame deletions in the predicted OB-fold of Rpb7 using information from both sequence alignments and detailed structural knowledge of OB-fold interactions with nucleic acids (Fig. 1b). We constructed three Rpb7 mutants predicted to be compromised for ssDNA-binding (Fig. 1c). The first deletion, Rpb7Delta 91-97 (D1), was predicted to disrupt beta -strand 1 and part of the L12 loop. The second deletion, Rpb7 Delta 108-113 (D2), was predicted to disrupt beta -strand 3, which harbors an aromatic residue (Phe in this case) critical for stacking interactions in all known structures. The third deletion, Rpb7 Delta 151-158 (D3), was predicted to disrupt the L45 loop, which contributes to stacking interactions in one structure but is less well conserved among the known OB-fold proteins.

The three Rpb7 variants were coexpressed with histidine-tagged Rpb4 (Fig. 2). The structural integrity of mutant proteins was tested by monitoring the ability of each mutant to dimerize with Rpb4. The Rpb7D1 mutant was expressed but failed to copurify with Rpb4, suggesting severe mis-folding. This mutant was not analyzed further. By contrast, both the Rpb7D2 and Rpb7D3 variants formed a stable complex with Rpb4, as judged by copurification with histidine-tagged Rpb4. Moreover, the resulting mutant Rpb4-Rpb7 complexes were able to interact normally with pol IIDelta 4/7 (5) as judged by comigration with the polymerase on a size-exclusion column (data not shown) and by competition experiments with wild-type Rpb4-Rpb7 complex (see below).

Nucleic Acid Binding by Rpb4-Rpb7 Complexes-- To test purified wild-type Rpb4-Rpb7 for single-strand nucleic acid-binding activity, we performed gel mobility shift assays with a radiolabeled ssDNA oligonucleotide probe. As predicted from its OB-fold motif, the Rpb4-Rpb7 complex bound to ssDNA in a saturable and reversible manner (Fig. 3) with an apparent dissociation constant of 0.7 µM (determined by half-maximal binding at equilibrium with protein in large molar excess). Gel mobility shift assays with an RNA oligonucleotide probe revealed similar binding to ssRNA with an apparent dissociation constant of 1.2 µM (data not shown; see Fig. 4).


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Fig. 3.   DNA-binding activity of wild-type Rpb4-Rpb7 complex. A radiolabeled DNA probe was incubated with indicated amounts (micrograms) of purified Rpb4-Rpb7 complex as described under "Experimental Procedures." Resulting complexes were resolved from unbound DNA by electrophoresis in a nondenaturing polyacrylamide gel. Bands formed by free DNA and protein-DNA complexes (arrows) were detected by autoradiography.


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Fig. 4.   RNA-binding activity of wild-type and deletion mutant (D2 and D3) Rpb4-Rpb7 complexes. A radiolabeled RNA probe was incubated with indicated amounts (micrograms) of purified Rpb4-Rpb7 complexes as described under "Experimental Procedures." Products were analyzed as for Fig. 3.

We were unable to measure the binding activity of wild-type Rpb7 in isolation, because the protein was insoluble in the absence of Rpb4 (the same held true for N- and C-terminal Rpb7 deletion variants and for several internal deletion variants, as well). We therefore tested the Rpb4-Rpb7D2 and Rpb4-Rpb7D3 complexes on ssRNA and ssDNA to identify region(s) in Rpb7 that mediate nucleic acid binding. The Rpb7D3 mutation deleting the putative L45 loop between the predicted beta -strands had no apparent effect on nucleic acid binding (Fig. 4). By contrast, the affinity of the Rpb4-Rpb7D2 complex for RNA (Fig. 4) and DNA (not shown) was at least 10-fold lower than normal. These results suggest that this part of Rpb7 may be principally responsible for the observed nucleic acid binding, as expected from structure predictions.

Transcription Initiation in Reconstituted System-- Crude cell extract prepared from yeast lacking the RPB4 gene fails to support promoter-directed transcription, but activity can be restored by adding purified Rpb4-Rpb7 complex resolved from polymerase by ion exchange chromatography in the presence of urea (5). To confirm that the Rpb4-Rpb7 complex is required in transcription reactions reconstituted with purified components (and therefore lacking nonspecific inhibitors present in crude extracts; see Ref. 13), we assayed the activity of baculovirus-expressed Rpb4-Rpb7 complex with the minimal set of yeast GTFs and highly purified pol IIDelta 4/7. As expected (5), pol IIDelta 4/7 was completely inactive in promoter-directed transcription in this well defined system. Moreover, addition of purified recombinant Rpb4-Rpb7 complex supported transcription by pol IIDelta 4/7 at levels achieved by native (12-subunit) polymerase (Fig. 5a, lanes 1-4).


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Fig. 5.   Transcription activities of recombinant Rpb4-Rpb7 complexes. Panel A, transcription reactions with a CYC1 promoter template contained pol IIDelta 4/7 (lanes 1-4 and 6-11) or wild-type polymerase (lanes 12-14) and indicated amounts (ng) of wild-type (WT) or mutant (D2 and D3) Rpb4-Rpb7 complexes. Control reactions lacked Rpb4-Rpb7 complex (lane 1) or polymerase (lane 5). The arrow marks bands formed by labeled RNA transcripts in a denaturing polyacrylamide gel. Panel B, order of addition experiments with wild-type and mutant Rpb4-Rpb7 complexes. Transcription reactions contained 100 ng of pol IIDelta 4/7 and either 10 ng of wild-type Rpb4-Rpb7 complex alone (WT 4/7; lane 5), or with 10 ng of mutant (D2 and D3) Rpb4-Rpb7 complex (lanes 1-4 and 6-13) added either sequentially or simultaneously as indicated. Molar ratios of mutant to wild-type complexes are indicated above the lanes. Transcripts are marked as in A. Panel C, supercoiled (sc) or linearized (lin) plasmid templates containing three different promoters (TEF1, AdML, and CYC1) were transcribed with pol IIDelta 4/7 in the absence or presence of wild-type recombinant Rpb4-Rpb7 complex (10 ng) as indicated. Transcripts are marked as in A.

In contrast to wild-type Rpb4-Rpb7 complex, complexes containing the Rpb7D2 or D3 mutants were completely inactive in transcription (Fig. 5a, lanes 6-11). We performed order of addition transcription experiments to test whether the D2 or D3 mutant complexes could compete with wild-type complex for stable binding to polymerase. When pol IIDelta 4/7 was preincubated with wild-type Rpb4-Rpb7 complex, subsequent transcription was only slightly reduced by adding excess mutant complexes (Fig. 5b, lanes 1-5). By contrast, when polymerase was preincubated with the D2 or D3 mutant complex first, subsequent transcription in the presence of wild-type Rpb4-Rpb7 fell sharply (Fig. 5b, lanes 6-9); when wild-type and mutant Rpb4-Rpb7 complexes were copreincubated prior to transcription, the mutant complexes inhibited RNA production in a dose-dependent manner (Fig. 5b, lanes 10-13). Taken together with size-exclusion chromatography behavior (not shown), these results suggest that both of the mutant Rpb4-Rpb7 complexes bind normally to RNA polymerase II and can even displace wild-type Rpb4-Rpb7, as expected for reversible binding to the same site on polymerase.

To test the generality of the requirement for the Rpb4-Rpb7 complex, we tried transcription with several different promoters. The complex was required for initiation on the TEF1, CYC1, and AdML promoters (Fig. 5c) regardless of DNA template topology (linear or supercoiled). As expected (25), addition of Mediator to the reconstituted system boosted basal transcription by pol IIDelta 4/7 in the presence of wild-type Rpb4-Rpb7 complex, but it did not obviate the strict requirement for Rpb4-Rpb7 (not shown).

Rpb4 and Rpb7 Act after the Formation of a Stable Preinitiation Complex-- Transcription initiation can be resolved biochemically into an ordered series of steps beginning with the assembly of a preinitiation complex and recruitment ("commitment") of polymerase to the promoter (see Refs. 26-28; reviewed in Refs. 3 and 4). Previous binding studies comparing the affinity of wild-type (12-subunit) polymerase and pol IIDelta 4/7 for a TBP/TFIIB complex implicated Rpb4 and/or Rpb7 in the assembly of a preinitiation complex (29). To explore this question further in the functional context of transcription, we assessed the ability of pol IIDelta 4/7 to form a stable preinitiation complex in the absence or presence of Rpb4-Rpb7. Reactions contained two otherwise identical CYC1 promoter templates encoding G-less cassette transcripts of different lengths (20), and saturating amounts of TBP, TFIIB, TFIIE, and TFIIF, as determined by factor titrations and by DNase I footprinting assays showing full TATA box occupancy by TBP under identical conditions.2 When pol IIDelta 4/7 and the GTFs were incubated with both templates simultaneously, subsequent transcription reactions yielded equal amounts of both sets of promoter-directed transcripts (Fig. 6, lanes 1 and 2). However, if pol IIDelta 4/7 and GTFs were preincubated with DNA I prior to adding DNA II and NTPs, transcription ensued almost exclusively on DNA I (lane 3). No such template commitment was observed if TBP and TFIIB were omitted from the preincubation and added with DNA II (data not shown). In contrast to TBP and TFIIB, Rpb4-Rpb7 complex was not required for template commitment; the degree of commitment to DNA I was identical regardless of whether the Rpb4-Rpb7 complex was present in the preincubation step (compare lanes 3 and 4). These results indicate that polymerase can join a stable, active preinitiation complex in the absence of Rpb4 and Rpb7 and therefore suggest that the essential function of the Rpb4-Rpb7 complex (Fig. 5a) comes after polymerase binds to the promoter.


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Fig. 6.   Stable recruitment of RNA polymerase II into preinitiation complexes in the absence of Rpb4 and Rpb7. pol IIDelta 4/7 (100 ng) was preincubated with GTFs and Mediator for 30 min in the absence or presence of a short G-less cassette template (DNA I) and Rpb4-Rpb7 complex (10 ng) as indicated above the lanes and in a schematic diagram. Rpb4-Rpb7 complex was then added to reactions in which it was lacking, followed 15 s later by simultaneous addition of DNA I and a long G-less cassette template (DNA II; lanes 1 and 2), or by addition of DNA II alone (lanes 3 and 4), along with NTPs for RNA synthesis. Transcription reactions were stopped after 15 min. Specifically initiated transcripts from each template (RNA I and RNA II) were detected as in Fig. 5.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In this study we focused on the nucleic acid-binding activity of the yeast Rpb4-Rpb7 complex and on its role in transcription initiation. Our results show that the Rpb4-Rpb7 complex binds fairly strongly to ssDNA and RNA in vitro, as predicted from the sequence and structural homology of the OB-fold motif of Rpb7 to the S1 domain (22, 30). The complex binds RNA and DNA with nearly equal affinity, and a mutation in the putative OB-fold abrogates binding to both substrates, indicating a common site for interaction. Because the OB-fold occurs in both RNA- and DNA-binding proteins, and the two types of interaction seem structurally indistinguishable, the physiological substrate remains to be determined.

The Rpb4-Rpb7D2 complex bearing a deletion in beta -strand 3 of the putative OB-fold DNA-binding surface (Fig. 1) was indeed defective in nucleic acid binding and moreover was completely devoid of transcription activity, raising the possibility that nucleic acid binding is key to the function of the Rpb4-Rpb7 complex. However, the Rpb7 D3 deletion disrupting the putative L45 loop (Fig. 1) was also completely inactive in transcription, even though it retained nucleic acid-binding activity. Our sequence alignment may not accurately predict the exact boundaries of the putative OB-fold, and the D3 deletion may lie outside the critical DNA-binding surfaces. Even if the alignment is correct, the L45 loop in Rpb7 may simply be less important for stabilizing nucleic acid interactions compared with other OB-folds. In any case, this portion of Rpb7 evidently has a key role in transcription distinct from DNA binding. Indeed, the ssDNA-binding activity of the Rpb4-Rpb7 complex may be peripheral to transcription activity, and the deleterious effect of the D2 mutation on both activities may be coincidental. Further genetic, biochemical, and structural studies are needed to fully delineate the role of the OB-fold motif of Rpb7 in DNA/RNA-binding and transcription. The C25 subunit of RNA polymerase III appears to be a Rpb7 homologue (31). It will be interesting to see whether this subunit also possesses nucleic acid-binding activity and if so, whether it is important for transcription initiation on class III promoters.

Endogenous pol IIDelta 4/7 was incapable of promoter utilization in a crude extract unless supplied with purified Rpb4-Rpb7 complex or (alternatively) Gal4-VP16, a potent transcription activator (5). The extract provided GTFs and Mediator, as well as pol IIDelta 4/7, and likely contained Rpb7, as well (Rpb7 is essential for yeast viability). Thus, our previous experiments did not rule out a role for Rpb7 in activated transcription. Results reported here confirm that the Rpb4-Rpb7 complex is indeed required for basal transcription in a completely defined reconstituted system. We also find that the complex is required for transcription in the presence of Mediator, indicating an important role for the Rpb4-Rpb7 complex in transcription by RNA polymerase II holoenzyme (16, 25, 32). Further work is needed to explore the role of Rpb7 in activation by enhancer-binding proteins. The defining biological feature of sigma  factors is their capacity to confer promoter class specificity on polymerase and thereby orchestrate multigene transcription programs for cellular development and responses to stress (reviewed in Ref. 33). The real test to see whether yeast Rpb4-Rpb7 complex plays a similar role will come from whole-genome analysis with suitable mutants (e.g. see Ref. 34).

Reconstructions of yeast RNA polymerase II structures from electron micrographs place the Rpb4-Rpb7 complex in the putative DNA-binding cleft of the polymerase and suggest that the complex favors a closed conformation of the polymerase "arm" domain around the cleft (29). These observations led to the hypothesis that Rpb4-Rpb7 may stabilize polymerase binding by contacting DNA in the cleft and somehow coupling DNA entry to closure of the cleft. It is tempting to speculate that the Rpb7D2 mutation may disrupt the DNA "sensor," whereas the D3 mutation may disrupt protein-protein interactions that transduce the DNA signal to the polymerase arm domain. A more detailed structural model of polymerase bound to DNA places Rpb4-Rpb7 downstream of the catalytic site in the cleft (35), and because our biochemical findings indicate that Rpb4-Rpb7 complex mediates a step following template commitment, we speculate that it may stabilize the open promoter complex prior to initiation and/or an early transcribing complex prior to promoter escape, perhaps by binding to ssDNA in the transcript "bubble" (in analogy to bacterial sigma  factors) or to nascent RNA. Sorting this out will require further biochemical studies using cross-linking, permangenate footprinting, and kinetic measurements along the lines used for analyzing mammalian systems (36-40).

    ACKNOWLEDGEMENTS

Purified yeast Mediator was a generous gift from Larry Myers and Roger Kornberg. We thank Matthew Healy, Pilar Tijerina, and Byung Ahn for providing purified transcription factors and Stefan Larsen for helping prepare some of the figures.

    FOOTNOTES

* This work was supported in part by grants from the Medical Research Council of Canada (MRC) (to A. M. E.) and by Grant GM50724 from the National Institutes of Health (NIH) (to M. H. S.).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.

Recipient of Predoctoral Research Fellowship CA73466 from the NIH.

|| Current address: Division of Molecular and Cellular Mechanisms, NIH Center for Scientific Review, 6701 Rockledge Dr., Bethesda, MD 20892. Recipient of a Junior Faculty research award from the American Cancer Society.

** MRC Scientist. To whom correspondence should be addressed: Banting and Best Dept. of Medical Research and Dept. of Medical Genetics and Microbiology, C. H. Best Inst., University of Toronto, 112 College St., Toronto, Ontario M5G 1L6, Canada. Tel.: 416-946-3436; Fax: 416-978-8528; E-mail: aled.edwards@utoronto.ca.

Published, JBC Papers in Press, November 21, 2000, DOI 10.1074/jbc.M003165200

2 M. S. Healy, unpublished data.

    ABBREVIATIONS

The abbreviations used are: GTFs, general transcription factors; ss, single-strand; ORF(s), open reading frame(s); pol IIDelta 4/7, RNA polymerase II lacking Rpb4 and Rpb7 subunits; TF, transcription factor; TBP, TATA-binding protein; D, deletion.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. Archambault, J., and Friesen, J. D. (1993) Microbiol. Rev. 57, 703-724[Abstract]
2. Sawadogo, M., and Sentenac, A. (1990) Annu. Rev. Biochem. 59, 711-754[CrossRef][Medline] [Order article via Infotrieve]
3. Reinberg, D., Orphanides, G., Ebright, R., Akoulitchev, S., Carcamo, J., Cho, H., Cortes, P., Drapkin, R., Flores, O., Ha, I., Inostroza, J., Kim, S., Kim, T., Kumar, P., and Lagrange, T. (1998) Cold Spring Harbor Symp. Quant. Biol. 63, 83-103[Medline] [Order article via Infotrieve]
4. Conaway, R. C., and Conaway, J. W. (1997) Prog. Nucleic Acid Res. Mol. Biol. 56, 327-346[Medline] [Order article via Infotrieve]
5. Edwards, A. M., Kane, C. M., Young, R. A., and Kornberg, R. D. (1991) J. Biol. Chem. 266, 71-75[Abstract/Free Full Text]
6. Choder, M., and Young, R. A. (1993) Mol. Cell. Biol. 13, 6984-6991[Abstract]
7. Maillet, I., Buhler, J. M., Sentenac, A., and Labarre, J. (1999) J. Biol. Chem. 274, 22586-22590[Abstract/Free Full Text]
8. Rosenheck, S., and Choder, M. (1998) J. Bacteriol. 180, 6187-6192[Abstract/Free Full Text]
9. Sheffer, A., Varon, M., and Choder, M. (1999) Mol. Cell. Biol. 19, 2672-2680[Abstract/Free Full Text]
10. Khazak, V., Sadhale, P. P., Woychik, N. A., Brent, R., and Golemis, E. A. (1995) Mol. Biol. Cell 6, 759-775[Abstract]
11. McKune, K., Richards, K. L., Edwards, A. M., Young, R. A., and Woychik, N. A. (1993) Yeast 9, 295-299[Medline] [Order article via Infotrieve]
12. Woychik, N. A., and Young, R. A. (1989) Mol. Cell. Biol. 9, 2854-2859[Medline] [Order article via Infotrieve]
13. Sayre, M. H., Tschochner, H., and Kornberg, R. D. (1992) J. Biol. Chem. 267, 23376-23382[Abstract/Free Full Text]
14. Henry, N. L., Sayre, M. H., and Kornberg, R. D. (1992) J. Biol. Chem. 267, 23388-23392[Abstract/Free Full Text]
15. Svejstrup, J. Q., Feaver, W. J., LaPointe, J., and Kornberg, R. D. (1994) J. Biol. Chem. 269, 28044-28048[Abstract/Free Full Text]
16. Myers, L. C., Gustafsson, C. M., Bushnell, D. A., Lui, M., Erdjument-Bromage, H., Tempst, P., and Kornberg, R. D. (1998) Genes Dev. 12, 45-54[Abstract/Free Full Text]
17. Tijerina, P., and Sayre, M. H. (1998) J. Biol. Chem. 273, 1107-1113[Abstract/Free Full Text]
18. Feaver, W. J., Henry, N. L., Bushnell, D. A., Sayre, M. H., Brickner, J. H., Gileadi, O., and Kornberg, R. D. (1994) J. Biol. Chem. 269, 27549-27553[Abstract/Free Full Text]
19. Gebara, M. M., Sayre, M. H., and Corden, J. L. (1997) J. Cell. Biochem. 64, 390-402[CrossRef][Medline] [Order article via Infotrieve]
20. Woontner, M., A., W. P., Bonner, J., and Jaehning, J. A. (1991) Mol. Cell. Biol. 11, 4555-4560[Medline] [Order article via Infotrieve]
21. Flanagan, P. M., Kelleher, R. J. D., Sayre, M. H., Tschochner, H., and Kornberg, R. D. (1991) Nature 350, 436-438[CrossRef][Medline] [Order article via Infotrieve]
22. Bycroft, M., Hubbard, T. J. P., Proctor, M., Freund, S. M. V., and Murzin, A. G. (1997) Cell 88, 235-242[Medline] [Order article via Infotrieve]
23. Bochkarev, A., Pfuetzner, R. A., Edwards, A. M., and Frappier, L. (1997) Nature 385, 176-181[CrossRef][Medline] [Order article via Infotrieve]
24. Murzin, A. G. (1993) EMBO J. 12, 861-867[Abstract]
25. Kim, Y. J., Bjorklund, S., Li, Y., Sayre, M. H., and Kornberg, R. D. (1994) Cell 77, 599-608[Medline] [Order article via Infotrieve]
26. Buratowski, S., Hahn, S., Guarente, L., and Sharp, P. A. (1989) Cell 56, 549-561[Medline] [Order article via Infotrieve]
27. Davison, B. L., Mulvihill, E. R., Egly, J. M., and Chambon, P. (1983) Cold Spring Harbor Symp. Quant. Biol. 47, 965-975[Medline] [Order article via Infotrieve]
28. Conaway, R. C., Bradsher, J. N., and Conaway, J. W. (1992) J. Biol. Chem. 267, 8464-8467[Abstract/Free Full Text]
29. Jensen, G. J., Meredith, G., Bushnell, D. A., and Kornberg, R. D. (1998) EMBO J. 17, 2353-2358[Abstract/Free Full Text]
30. Langer, D., Lottspeich, F., and Zillig, W. (1994) Nucleic Acids Res. 22, 694[Medline] [Order article via Infotrieve]
31. Sadhale, P. P., and Woychik, N. A. (1994) Mol. Cell. Biol. 14, 6164-6170[Abstract]
32. Koleske, A. J., and Young, R. A. (1994) Nature 368, 466-469[CrossRef][Medline] [Order article via Infotrieve]
33. Gross, C. A., Chan, C., Dombroski, A., Gruber, T., Sharp, M., Tupy, J., and Young, B. (1998) Cold Spring Harb. Symp. Quant. Biol. 63, 141-155[Medline] [Order article via Infotrieve]
34. Holstege, F. C., Jennings, E. G., Wyrick, J. J., Lee, T. I., Hengartner, C. J., Green, M. R., Golub, T. R., Lander, E. S., and Young, R. A. (1998) Cell 95, 717-729[Medline] [Order article via Infotrieve]
35. Cramer, P., Bushnell, D. A., Fu, J., Gnatt, A. L., Maier-Davis, B., Thompson, N. E., Burgess, R. R., Edwards, A. M., David, P. R., and Kornberg, R. D. (2000) Science 288, 640-649[Abstract/Free Full Text]
36. Lagrange, T., Kim, T. K., Orphanides, G., Ebright, Y. W., Ebright, R. H., and Reinberg, D. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 10620-10625[Abstract/Free Full Text]
37. Kugel, J. F., and Goodrich, J. A. (2000) J. Biol. Chem. 275, 40483-40491[Abstract/Free Full Text]
38. Holstege, F. C., Fiedler, U., and Timmers, H. T. (1997) EMBO J. 16, 7468-7480[Abstract/Free Full Text]
39. Dvir, A., Conaway, R. C., and Conaway, J. W. (1996) J. Biol. Chem. 271, 23352-23356[Abstract/Free Full Text]
40. Dvir, A., Garrett, K. P., Chalut, C., Egly, J. M., Conaway, J. W., and Conaway, R. C. (1996) J. Biol. Chem. 271, 7245-7248[Abstract/Free Full Text]


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