The RPB7 Orthologue E′ Is Required for Transcriptional Activity of a Reconstituted Archaeal Core Enzyme at Low Temperatures and Stimulates Open Complex Formation*

RNA polymerases from Archaea and Eukaryotes consist of a core enzyme associated with a dimeric E′F (Rpb7/Rpb4) subcomplex but the functional contribution of the two subunit subcomplexes to the transcription process is poorly understood. Here we report the reconstitution of the 11-subunit RNA polymerase and of the core enzyme from the hyperthermophilic Archaeon Pyrococcus furiosus. The core enzyme showed significant activity between 70 and 80 °C but was almost inactive at 60 °C. E′ stimulated the activity of the core enzyme at 60 °C, dramatically suggesting an important role of this subunit at low growth temperatures. Subunit F did not contribute significantly to catalytic activity. Permanganate footprinting at low temperatures dissected the contributions of the core enzyme, subunit E′, and of archaeal TFE to open complex formation. Opening in the –2 and –4 region could be achieved by the core enzyme, subunit E′ stimulated bubble formation in general and opening at the upstream end of the transcription bubble was preferably stimulated by TFE. Analyses of the kinetic stabilities of open complexes revealed an unexpected E′-independent role of TFE in the stabilization of open complexes.

Transcription in cells of Archaea and Bacteria is catalyzed by a single RNA polymerase (RNAP), 2 whereas eukaryotic cells contain three different types of RNAPs (I, II, and III) that carry out specialized functions. Despite their morphological similarity to Bacteria, Archaea have a transcriptional machinery that is more akin to the eukaryotic machinery (1)(2)(3).
Like eukaryotes, Archaea use extrinsic transcription factors for initiation. The process of transcription initiation in Archaea can be dissected in three steps. The general transcription factor TATA-binding protein (TBP) interacts with the archaeal TATA box, transcription factor B (TFB) stabilizes the binding of TBP to the promoter. The TBP-TFB promoter complex mediates recruitment of RNAP (4,5). These extrinsic factors show the major structural features of eukaryotic TBP and TFIIB and interact with DNA and RNAP in a similar fashion as their eukaryotic counterparts (6,7). This exceptional degree of similarity between the archaeal and eukaryotic transcriptional machineries extends also to the RNAP.
Archaeal RNAP consist of 11 or 12 different subunits (8 -10) that display a high level of primary sequence similarity to the subunits present in eukaryotic RNAPII. With the exception of subunits RPB8 and RPB9, orthologs of other RNAPII subunits have been identified in all archaeal genomes studied so far. A recent in silico study revealed a high degree of conservation of subunits shared by pol II and Archaea in particular in the regions of RNAP comprising the catalytic center and proteinprotein binding studies showed a similar pattern of subunit interactions between the subunits of pol II and of Pyrococcus RNAP (11). Although very similar to eukaryotic RNAP in subunit composition and transcription initiation factor requirement the archaeal machinery is less complex than the eukaryotic machinery. Equivalents of the eukaryotic factors TFIIH and TFIIF are missing in archaeal genomes. Eukaryotic TFIIE is a heterodimer consisting of subunits ␣ and ␤ (12). The archaeal homolog of TFIIE, TFE, is monomeric and corresponds to the essential N-terminal half of TFIIE␣, which is required for basal and activated transcription in vitro and interacts with TBP and RNAPII (13,14). Archaeal TFE was reported to stimulate transcription on some weak promoters in vitro, in particular at suboptimal concentrations of TBP (15,16). The crystal structure of eukaryotic RNAPII has been solved (17)(18)(19) but many key questions concerning structure-function relationships in eukaryotic RNAPs cannot be addressed because an active eukaryotic RNAP could not yet be reconstituted from single subunits or subcomplexes. Recently, the reconstitution of the archaeal RNAP from the methanogen Methanocaldococcus jannaschii has been reported (20) and was used as a tool to define the minimal set of subunits for an active pol II-like enzyme. The macromolecular complex consisting of subunits AЈAЉBЈBЉDLNP was the minimal active assembly in nonspecific assays and the RNAP reconstituted in the absence of subunit K or subunits F/EЈ was still capable of specific transcription. The activity of the recombinant Methanocaldococcus enzyme was stimulated at weak promoters by archaeal TFE and this activation was dependent upon the presence of the EЈ/F subcomplex (Ref. 21; the orthologs of RpB7/4) that is only loosely associated with the archaeal and/or the eukaryotic core enzyme (12,22).
We are exploring the mechanism and regulation of transcription in the hyperthermophilic Archaea Pyrococcus furiosus (23)(24)(25)(26)(27). In this contribution we report the reconstitution of a highly active RNAP from 11 bacterially expressed subunits. The reconstituted core enzyme lacking subunits EЈ and F (⌬EЈF) was used to investigate the effects of subunits EЈ, F, and TFE on RNAP activity. We found that promoter-dependent activity of the core enzyme was at low temperatures strictly dependent upon the presence of EЈ. Furthermore, we provide a model dissecting the roles of the core enzyme, EЈ, and TFE in open complex formation.

EXPERIMENTAL PROCEDURES
Cloning and Expression Procedures-The open reading frames encoding the P. furiosus RNAP subunits were amplified by PCR from genomic DNA using oligonucleotides corresponding to the 5Ј and 3Ј end of the open reading frames and introducing restriction sites. For the RNAP reconstitution experiments the PCR products were cloned into pET-30a-c(ϩ) (Novagen) or pET151/D-TOPO (Invitrogen), and verified by sequencing (Geneart).
The resultant expression plasmids were transformed into BL21Star(DE3)Codon Plus (Stratagene) and expression of the subunits was induced during logarithmic growth of the cells by addition of isopropyl ␤-D-thiogalactopyranoside to 0.5 mM. The cultures were then incubated overnight at 20°C.
Recombinant Subunits Purification-The His 6 -tagged subunits B, AЈ, AЉ, and K were highly insoluble and expressed in inclusion bodies (IB) in Escherichia coli cells. It was necessary to purify these subunits under denaturing conditions. The cell pastes were resuspended in lysis buffer A (20 mM Tris, pH 8, 1 mM PMSF, 5 mM ␤-mercaptoethanol, 0.3 mg/ml lysozyme) and sonicated. After centrifugation the pellets containing the IBs were extensively washed in IB buffer (20 mM Tris, pH 8, 0.5 M NaCl, 0.1% Tween 20 (Pierce), 1 mM PMSF, 5 mM ␤-mercaptoethanol). The IBs were solubilized in binding buffer A (20 mM Tris, pH 8, 0.5 M NaCl, 5 mM imidazole, 6 M guanidine hydrochloride, 1 mM PMSF, 5 mM ␤-mercaptoethanol) for 1 h at room temperature. After centrifugation the supernatants were loaded onto a Ni-NTA column (HisTrapFF, GE Healthcare) and further washed with washing buffer (20 mM Tris, pH 8, 0.5 M NaCl, 5 mM imidazole, 6 M urea, 1 mM PMSF, 5 mM ␤-mercaptoethanol). The refolding of the column-bound proteins was achieved by washing the column with decreasing linear urea gradient ranging from 6 to 0 M in refolding buffer (20 mM Tris, pH 8, 0.5 M NaCl, 10 mM imidazole, 1 mM PMSF, 5 mM ␤-mercaptoethanol). The refolded proteins were eluted with imidazole gradients ranging from 10 to 300 mM imidazole in refolding buffer. No further purification was required. The solubilized subunits were stored at Ϫ80°C until they were used for reconstitution of wild type and core enzyme.
In Vitro Assembly of the Recombinant RNAP-500 g of all 11 subunits were combined in a final volume of 0.5 ml. The mixture was transferred to a dialysis frame (Slide-A-Lyzer 3.5k, Pierce) and denatured in transcription buffer (TB) (40 mM NaHEPES, pH 7.3, 250 mM NaCl, 2.5 mM MgCl 2 , 10% (v/v) glycerol, 0.1 mM EDTA, 100 M ZnSO 4 , 1 mM PMSF, 5 mM ␤-mercaptoethanol) containing 6 M urea for 20 min. The dialysis frame with the denatured subunits was then transferred into TB with 3 M urea for another 20 min. Renaturation was achieved by dialysis in TB for 1 h. The renatured RNAP assemblies were heated for 10 min at 70°C to remove misfolded aggregates, and loaded on an analytic Superdex 200 or onto a Superose 6 column (200 10/300 GL, GE Healthcare).
Promotor-directed in Vitro Transcription Assay-In vitro transcription assays were performed as described (27). Standard transcription reactions contained 300 ng of gdh promoter, 42 nM TBP, 55 nM TFB, and 10 nM purified or recombinant RNAP in a total volume of 25 l of transcription buffer (40 mM NaHEPES, pH 7.3, 250 mM NaCl, 2.5 mM MgCl 2 , 5% (v/v) glycerol, 0.1 mM EDTA, 5 mM ␤-mercaptoethanol, and 0.1 mg/ml bovine serum albumin). The amounts of DNA template, TBP, TFB, and purified RNAP, reconstituted RNAP, or ⌬EЈF RNAP used for the experiments described in Fig. 5 are indicated in the legend. NTPs were added to a concentration of 40 M (ATP, GTP, and CTP) and 2 M UTP and [␣-32 P]UTP at 0.15 MBq (110 TBq/mmol). The components were added at 4°C and then incubated at 70°C for 30 min. Labeled transcripts were analyzed by electrophoresis in 6% (w/v) polyacrylamide urea gels as described (27). The transcription products were visualized using an Image Plate and Image Analyzer (FLA-500, Fuji, Japan).
Immobilized in Vitro Transcription Assays and Isolation of Stalled Ternary Complexes-Biotinylated C-minus cassettes of the gdh promoter attached to streptavidin-coated magnetic beads were used as DNA templates for stalling of transcription complexes at position ϩ20 (23). Stalling of complexes was achieved by omitting CTP from the nucleotide mixture. A standard reaction contained 168 fmol of immobilized DNA template, 70 nM ⌬EЈF RNAP, 285 nM TBP, 60 nM TFB, 40 M ATP and GTP, 2 M UTP, and 0.15 MBq of [␣-32 P]UTP (110 TBq/mmol) in 25 l of transcription buffer not containing glycerol. Ternary complexes were isolated as reported previously (23) and all washing and resuspending steps were carried out with preheated (60 -70°C) transcription buffer. Standard immobilized transcription reactions were incubated for 30 min at 60 or 70°C and stopped by the addition of loading dye as described (23) followed by denaturation for 3 min at 95°C.
KMnO 4 Footprinting of Initiation Complexes-Footprinting experiments were performed using immobilized templates labeled with [␥-32 P]ATP on the free 5Ј-end of the nontemplate DNA strand. DNA templates were attached to magnetic beads via the biotin-labeled 5Ј-end of the coding DNA strand as described previously (23). A footprinting reaction contained 168 fmol of DNA template, 70 nM purified RNAP or 70 nM RNAP⌬EЈF, 285 nM TBP, and 60 nM TFB in 25 l of transcription buffer not containing ␤-mercaptoethanol. Complexes were incubated for 5 min at 60 or 70°C, isolated, washed with transcription buffer (60 or 70°C), and resuspended in transcription buffer preheated to 60 or 70°C. KMnO 4 was added to a final concentration of 23 mM. The samples were incubated for 5 min at 60 or 70°C as indicated. The reaction was stopped and exposed to piperidin treatment as described previously (23).

Determination of Heparin Stability of Open Complexes-
The stability of open complexes was measured using a modification of a heparin challenge assay (28). 26 nM RNAP or 58 nM ⌬EЈF RNAP were preincubated 5 min at 60 or 70°C as indicated in Fig. 5C with 286 nM TBP, 60 nM TFB, and 200 ng of gdh promoter in transcription buffer in 50-l reactions. Heparin was added to the reaction to a final concentration of 800 g/ml. At this concentration of heparin single round conditions were established (data not shown). At various times 10-l aliquots were transferred to tubes containing 5 l of NTP mixture (40 M ATP, GTP, CTP, 2 M UTP, 0.09 MBq of [␣-32 P]UTP (110 TBq/mmol)) in transcription buffer and transcribed 10 min at 60 or 70°C as indicated. Reactions were stopped by the addition of loading dye following denaturation for 3 min at 95°C. For the 100% value, an aliquot was taken prior to heparin addition and incubated with NTPs as described above.

RESULTS
Purification and in Vitro Assembly of the P. furiosus RNAP Subunits-All P. furiosus RNAP subunits were expressed and purified in recombinant form in E. coli. The largest subunits B, AЈ, and AЉ and the small subunit K were insoluble and were purified under denaturing conditions. The other subunits were soluble. The purified subunits analyzed by SDS-PAGE and Coomassie staining are shown in Fig. 1.
For the in vitro assembly assay all 11 subunits were combined in equimolar amounts. When this fraction was assayed in specific transcription assays in the presence of TBP and TFB using the homologous glutamate dehydrogenase (gdh) promoter as template a run-off transcript was detected ( Fig. 2A, lane 1). This result provides evidence that an active Pyrococcus RNAP can be reconstituted simply by combining all purified subunits. This fraction was subjected to stepwise dialysis against transcription buffers containing decreasing urea concentrations (6, 3, and 0 M). After this step the specific activity of this fraction was greatly increased (Fig. 2A, lane 2). This finding indicates that the denaturation and renaturation procedure enhances the formation of specific protein-protein contact between RNAP subunits. When this fraction was heated for 10 min at 70°C to remove misfolded aggregates as described (20) the specific RNAP was slightly reduced ( Fig. 2A, lane 3) indicating that the heat treatment and centrifugation step leads also in part to denaturation of active RNAP molecules. The assembled complexes after the heat treatment step were purified by Superdex 200 column chromatography (Fig. 2B).
Superdex 200 size-exclusion chromatography showed that the RNAP eluted as a single homogenous peak. This RNAP eluted in the same fractions as endogenous RNAP and the marker protein ferritin (molecular mass 440,000). When the activity of RNAP was assayed in specific run-off transcription assays performed at 70°C, fractions 18 -24 of the column were capable of specific transcription (Fig. 2B). When the activity in the various fractions was analyzed the highest activity was found in later eluting fractions 22-24, which did not contain the highest amount of RNAP. The subunit composition of the various fractions did not differ significantly but the reconstituted RNAP contained substoichiometric amounts of subunits EЈ and F (Fig. 3, lane 1). Subunits EЈ and F eluted as a distinct nearly stoichiometric complex in fractions 33 and 34 (data not shown). When subunit EЈ was added to the various fractions and the synthesis of run-off transcripts was studied at 60°C, a clear EЈ-induced activation of transcription was observed in all fractions (Fig. 2B, lower panel). The EЈ-dependent stimulation of transcription was increased in the later eluting fractions rep-FIGURE 1. Analysis of purified subunits used for the reconstitution of the RNAP by SDS-PAGE and Coomassie staining. Note that the electrophoretic mobility of some small subunits with His 6 tag and in particular of subunit F with the His 6 tag differed considerably from that of the corresponding subunits of the purified enzyme. The gene encoding subunit EЈ overlaps in most Archaea with a gene encoding a polypeptide EЉ containing a zinc-finger motif (11). EЉ was cloned and purified in addition but was not contained in purified Pyrococcus RNAP (data not shown).
resenting the enzyme fractions with higher specific activity. The mechanism of EЈ-induced stimulation of transcription will be addressed in the following sections of this paper in more detail but these experiments indicate that two fractions of reconstituted RNAP exist, one fraction with lower specific activity (ϳ2.2 nmol of UMP/mg) that was only slightly activated by EЈ and a second fraction of high specific activity (ϳ6.9 nmol of UMP/mg) that was strongly activated by EЈ. The active fraction could be also separated from the less active fraction by Superose 6 gel filtration chromatography (data not shown). The reason for the different activities of these fractions is presently unclear. Probably, the first eluting and less active RNAP fraction contained still some aggregated subunits and subunits containing misfolded domains or helices that were inactive or less active.
To investigate the interaction of subunits EЈ and F with the active core of RNAP in more detail, the RNAP was reconsti-tuted in the absence of these subunits. This ⌬EЈF RNAP showed a similar elution profile like the enzyme reconstituted from 11 subunits (Fig. 2C). An active RNAP was eluted in fractions 19 -26. This finding indicates that the enzyme lacking these subunits was capable of synthesizing specific run-off transcripts. The ⌬EЈF RNAP is therefore also designated as core enzyme here. The most active fractions of the core enzyme were eluting later (fractions [23][24][25][26] indicating that the lower activity of fractions 19 -21 containing higher amounts of RNAP (see SDS-PAGE of RNAP fractions in Fig. 2B) was not caused by a lower content of subunits EЈ and F in these fractions. Although both fractions appear to be suitable for characterization of the reconstituted ⌬EЈF enzyme we used the RNAP with higher specific activity for the more detailed analyses of the EЈ-core enzyme interaction described later in this paper. When the activities of the reconstituted and the core enzyme were compared under standard conditions (70°C) with that of the endogenous RNAP, the less active fractions of reconstituted RNAP showed ϳ20%, the highly active fractions 50% of the activity of the endogenous RNAP (Fig. 2D). Fraction 25 of the core enzyme displayed ϳ40% of the activity of the endogenous RNAP. At 60°C, the activity of the endogenous RNAP was reduced by a factor of ϳ2, that of the more active reconstituted RNAP by a factor of ϳ4, and that of the core enzyme by a factor of ϳ8 (Fig. 2D). Synthesis of the run-off transcripts was strictly dependent upon the presence of TBP and TFB. This finding indicates that the run-off products were synthesized by an Archaea RNAP dependent upon the presence of Archaea transcription factors. This result and our finding that no RNA synthesis was observed at temperatures Ͻ55°C (data not shown) exclude the possibility that the activity observed is due to E. coli RNAP.
Recombinant RNAP Activity in Promoter-directed in Vitro Transcription-To characterize the enzymatic catalytic properties of the recombinant enzyme, we performed specific in vitro transcription assays using a variety of archaeal promoters as templates. Beside the gdh promoter analyzed in Fig. 2, the reconstituted enzyme synthesized run-off transcript from linearized templates containing the aaa ϩ atpase, hsp20, phr, and malE promoters from P. furiosus and the tRNA Val promoter from Methancoccus vannielii (data not shown). The recombinant enzyme was able to transcribe the gdh promoter in a temperature range from 60 to 80°C specifically (data not shown).
These results indicated that the reconstituted enzyme is able to initiate transcription at canonical archaeal promoters containing a TATA box and a BRE element. We conclude that the recombinant P. furiosus RNAP can perform all the steps   APRIL 13, 2007 • VOLUME 282 • NUMBER 15 JOURNAL OF BIOLOGICAL CHEMISTRY 11051 required for specific in vitro transcription, from RNAP promoter recruitment via TBP and TFB to promoter escape and elongation. The quantification of the reconstituted RNAP activity show that this enzyme has about 40 -50% ( Fig. 2D and Fig. 3) of the specific activity of the endogenous enzyme purified from Pyrococcus cells (Fig. 3, lanes 1 and 3). The ⌬EЈF RNAP showed a specific activity of 5.3 nmol of UMP/mg of protein.

DNA Opening by the Archaeal Rpb7 Homolog
To test the functional role of individual subunits in promoterdependent transcription, RNAP was reconstituted in the absence of one of the small subunits, K, N, H, and K. Very similar results were obtained as reported by Werner and Weinzierl (20) for the M. jannaschii RNAP. The ⌬K showed almost the same level of activity like the complete enzyme, the ⌬N showed low activity and leaving out subunit H or P from reconstitution reactions almost completely abolished activity (data not shown).
Subunit EЈ Stimulates Transcription at Low Temperatures-Pyrococcus enzyme reconstituted in the absence of EЈ and F showed reduced but still significant activity in standard transcription assays at the gdh promoter (Fig. 2D). Therefore, the EЈF subcomplex is not absolutely required for promoter directed in vitro transcription assays conducted at 70°C. Recent proteomic results have shown that cellular levels of subunit EЈ in the methanogen Methanococcoides burtonii are higher at low growth temperature (29) suggesting a specific role of this subunit in transcription at low temperatures. This finding had prompted us to investigate the effect of subunit EЈ on transcription of endogenous and reconstituted Pyrococcus RNAP at low temperatures (see also Fig. 2B and Fig. 4). At 55°C, the activity of the endogenous RNAP was very low and only slightly stimulated by EЈ (Fig. 4A). At 60°C, the weak basal activity levels were strongly stimulated upon addition of EЈ. The activity was increased with the amount of EЈ added and was maximal in the presence of 300 ng (553 nM) of EЈ per reaction. At 70°C, the activity of RNAP was high and only moderately stimulated by EЈ (Fig. 4A). As reported for some other archaeal promoters a ϳ1.5 stimulation of specific transcription from the gdh promoter was observed after addition of TFE and this activation was further increased slightly when both EЈ and TFE were added to transcription reactions (Fig. 4A, right panel). These findings suggest a specific effect of subunit EЈ on transcription at low temperatures and therefore transcription at 60°C was analyzed in more detail.
At 60°C the activities of reconstituted and the core enzyme were stimulated significantly by the addition of increasing amounts of EЈ (Fig. 4B). In the presence of 553 nM EЈ, the activity of the reconstituted enzyme was stimulated 3-fold, that of the core enzyme 7-fold (Fig. 4B). Compared with the endogenous RNAP the reconstituted RNAP was more susceptible to stimulation by EЈ. This can be explained by our finding that the reconstituted RNAP contains, in contrast to the endogenous enzyme, subunit EЈ in substoichiometric amounts (see Fig. 3). When EЈ was added to isolated stalled complexes, the extent of resumption of the stalled RNAP was not affected by the addition of EЈ at neither 70 nor 60°C (Fig. 4C). This finding suggests that EЈ does not stimulate already initiated complexes but seems to act on the level of initiation of transcription. Because EЈ can form a specific subcomplex with F also, the effect of subunit F on the stimulation of the core enzyme by subunit EЈ was studied. The stimulatory effect of EЈ on the core enzyme was not significantly increased by the addition of subunit F (Fig. 4D). This finding indicates that subunit F is not essential for the EЈ-mediated activation of core RNAP at 60°C.
To investigate the effects of TFE on the activity of the core RNAP, TFE was added to reactions conducted at 60 and 70°C. At 60°C, TFE elicited a ϳ2-fold effect on the core enzyme. This TFE-dependent stimulation was optimal in the presence of 250 ng of TFE (500 nM) in the reaction and was not increased by the addition of higher amounts of TFE (Fig. 4E). At 70°C only a very weak stimulatory effect of TFE was observed (Fig. 4E).
To investigate the effect of TFE on the EЈ-mediated activation of transcription at 60°C, TFE was added to reactions containing core enzyme, EЈ, or F or a combination of EЈ and F (Fig.  4, F and G). When TFE was added at optimal concentrations (500 nM) to reactions containing EЈ transcription was increased by a factor of 14 (Fig. 4F, lane 3). The activity of core enzyme was increased by a factor of 25 when F, EЈ, and TFE were added in combination (Fig. 4F, lanes 2 and 9). This 25-fold stimulation was observed in the presence of 100 and 250 ng of TFE and was reduced to a 20-fold effect when more (500 ng) TFE was added (Fig. 4G). This finding indicates that TFE was contained in optimal concentrations in the reactions shown in Fig. 4F. The ϳ2-fold stimulatory effect of TFE was not increased by the addition of subunit F (Fig. 4F, lanes 7 and 4). Subunit F when added separately did not activate transcription (Fig. 4F, lanes 5  and 6). These findings indicate that the stimulatory effect of TFE is primarily mediated by subunit EЈ but further increased Increasing amounts of EЈ were added to transcription reactions containing the gdh promoter and run-off products synthesized at 55, 60, and 70°C were analyzed. The reaction mixture contained template DNA (2 pM), endogenous RNAP (10 nM), TBP (285 nM), and TFB (60 nM). EЈ was added to 184, 368, and 553 nM. The amount (ng) of EЈ added to one reaction is indicated on top of the autoradiogram. B, EЈ-activated transcription of the reconstituted RNAP and ⌬EЈF RNAP. Endogenous RNAP was replaced by recombinant RNP or ⌬EЈF RNAP (65 nM). The effect of the addition of increasing amounts of EЈ was analyzed (92, 184, 368, and 553 nM, respectively). The standard deviation calculated from three independent experiments is shown. C, EЈ had no effect on transcription elongation. Immobilized transcription was stalled at position ϩ20. The starting reaction mixture contained 65 nM ⌬EЈF RNAP, 285 nM TBP, and 60 nM TFB. Stalled ternary complexes were purified, complexes in lanes 1 and 4 were promptly denatured, and complexes analyzed in lanes 2 and 5 were chased. The effect of the addition of 184 nM EЈ to chase reactions was analyzed. The temperature for the chase reaction is indicated on top of the gel. Electrophoresis was performed in a 28% polyacrylamide gel (D). Subunit F shows no significant effect on the EЈ-induced activation. Increasing amounts of F, 284 nM and 1.42 M in reactions analyzed in lanes 3 and 4, respectively, were added to transcription reactions containing 35 nM ⌬EЈF RNAP and 368 nM EЈ (lanes 2-4). Run-off RNA products were analyzed on a 6% polyacrylamide gel. E, a 2-fold stimulation of transcription by TFE at 60°C. Transcription experiments were performed at 60°C as described in A but with 65 nM ⌬EЈF RNAP. The reactions contained in addition TFE as indicated. F, subunits EЈ, F, and TFE interact synergistically in activation of transcription. The effect of EЈ, F, TFE, and combinations of these components on transcription was analyzed at 60°C. G, the EЈF subcomplex increases the stimulatory effect of TFE dramatically. Varying amounts of TFE as indicated were added to reactions of the core enzyme supplemented with EЈ and F and the effect on activity was analyzed. H, 2-fold activation of the core enzyme by TFE and EЈ at 70°C. The effects of EЈ and TFE on synthesis of run-off transcripts by the core enzyme were analyzed at 70°C.
in the presence of the heterodimeric EЈF complex. Subunit F has only a minor effect on the stimulatory activity of EЈ but the core enzyme combined with EЈ and F is much more susceptible to stimulation by TFE than the core enzyme-EЈ combination. This finding indicates an important role of subunit F in mediating the stimulatory activity of TFE.
At 70°C, the effects of TFE on the EЈ-mediated activation of the core enzyme were moderate and similar as described for the M. jannaschii enzyme (21). The ϳ1.5-fold stimulation of the core enzyme by EЈ (Fig. 4H, lanes 8 -10)

was slightly increased by the addition of TFE (lane 3) or both TFE and F (lane 2).
Subunit EЈ and TFE Stimulate Formation of the Open Complex-The findings that EЈ has a strong effect on transcription at low temperatures, only a moderate effect on transcription at 70°C, and no effect on the elongation of stalled complexes suggest that EЈ acts at the level of promoter opening. To investigate this, open complex formation by the endogenous and ⌬EЈF RNAP was studied at 70 and 60°C in the presence and absence of EЈ by permanganate footprinting. At 70°C, the T residues at positions Ϫ2, Ϫ4, and Ϫ6 of the nontemplate DNA strand of the gdh promoter showed high and the T residues at positions ϩ2 and ϩ3 showed somewhat weaker reactivity to KMnO 4 (23) (Fig. 5A, lane 2). The addition of EЈ affected promoter opening by the endogenous RNAP marginally (Fig. 5A,  lane 3). In the presence of both EЈ and TFE in the permanganate footprinting reactions, the sensitivity of all T residues in the bubble was clearly enhanced, particularly at position Ϫ6 (Fig.  5A, lane 4; see also a quantitative analysis of the data below the figure). This finding demonstrates that TFE stimulates open complex formation in the presence of EЈ. When the endogenous RNAP was incubated with the gdh promoter at 60°C the T residue at Ϫ2 displayed significant reactivity toward KMnO 4 , the T residues at positions Ϫ6, Ϫ4 and ϩ2 and ϩ3 were only weakly modified (Fig. 5B, lane 2). Addition of isolated subunit EЈ to the endogenous RNAP increased the KMnO 4 sensitivity of the T residues between Ϫ6 and ϩ3 considerably, indicating that EЈ is involved in open complex formation too. The reactivity of all T residues was further increased upon addition of TFE to the endogenous RNAP.
The endogenous RNAP contains EЈ and therefore the specific role of EЈ in open complex formation can be best addressed in experiments with the core enzyme lacking this subunit. When the ⌬EЈF RNAP was incubated with this template at 60°C, only a weak modification of the T residues at Ϫ2 and Ϫ4 was found (Fig. 5B, lane 5), but no modifications at positions ϩ3, ϩ2, or Ϫ6 could be detected. Upon addition of subunit EЈ, a stimulation of the reactivity of all T residues was observed. The T residues at ϩ2 and ϩ3 showed the highest reactivity, but the T residue at position Ϫ6 was still not modified (Fig. 5B, lane 6). This finding suggests an EЈ induced opening of the bubble mainly at the transcription start site. When TFE was added in addition to EЈ the sensitivity of T residues at positions Ϫ4, Ϫ2, ϩ2, and ϩ3 was further increased and, in addition, also the T residue at Ϫ6 was modified (Fig. 5B, lane 7). The quantitative analysis of the data below the figure corroborates that TFE further stimulates EЈ-induced promoter opening and in particular contributes to bubble opening at the 5Ј end of the open complex. Taken together the results presented here support the conclusion that subunit EЈ stimulates transcription at 60°C by catalyzing open complex formation and that TFE stimulates bubble opening further at the upstream end of the bubble. Then, open complexes were challenged by the addition of heparin (the experimental design is outlined in Fig. 6). The fraction of remaining open complexes at various times after heparin addition was quantified by transcription assays. In these assays At 70°C open complexes formed by the endogenous and core RNAP were significantly destabilized by heparin. The residual activities of the endogenous RNAP and the core enzyme after 1 h of exposure to heparin were ϳ37% (Fig. 6). After a shorter heparin challenge (5-15 min) the residual activity of endogenous RNAP was ϳ60 and that of the core enzyme was ϳ30% (Fig. 6). Open complexes formed by both enzymes were significantly stabilized by TFE when exposed to heparin for 5-15 min. In the presence of TFE the residual activities of the endogenous enzyme and the core enzyme were ϳ75 and ϳ40%, respectively. Open complexes of the core enzyme containing EЈ and TFE displayed the same sensitivity against heparin challenge as complexes containing only the core enzyme and TFE (Fig. 6). When the core enzyme was subjected to heparin challenge in the presence of EЈ, no stabilization was observed (Fig. 6). These findings indicate that open complexes are stabilized by TFE and not by EЈ.
At 60°C, open complexes formed by the ⌬EЈF enzyme displayed a higher stability than at 70°C. The remaining activity of the core enzyme after 60 min exposure to heparin was ϳ80%. When TFE was contained in addition in binding reactions at 60°C no significant inactivation of open complexes was observed (Fig. 6). The addition of subunit EЈ to the core enzyme had no stabilizing effect (data not shown

DISCUSSION
Here we describe the reconstitution of a pol II-like RNA polymerase from individually expressed subunits. Surprisingly, simple incubation of all purified subunits lead to the formation of an RNAP capable of promoter-specific initiation and elongation (Fig. 2), but the efficiency of RNAP reconstitution was greatly increased by denaturation of the 11 subunits in the presence of 6 M urea and stepwise renaturation at 3 M urea and finally, in the absence of urea as described for the reconstitution of the M. jannaschii enzyme (20). The reconstituted subunits BЈAЈAЉDHLPKN eluted as a distinct macromolecular complex after size exclusion chromatography (Fig. 2). This complex contained all RNAP subunits with the exception of EЈ and F in nearly stoichiometric amounts and showed a specific activity in promoter-dependent transcription assays that was comparable with that of the purified enzyme (Fig. 3). Functional studies revealed that the Rpb4/Rpb7 and C17/C25 subassemblies are required for promoter-directed initiation of eukaryotic transcription but dispensable for elongation (30 -32). In contrast to the eukaryotic core enzyme the reconstituted M. jannaschii (20) and P. furiosusus 9 subunit assemblies (Fig. 2B) showed significant catalytic activity in promoter-dependent standard assays conducted at 70°C. Our finding that the Pyrococcus core enzyme is almost inactive at 60°C and can be dramatically stimulated by subunit EЈ provides a functional definition of an archaeal core enzyme. In addition, the property of EЈ in concert with TFE and (to lower extent) with subunit F to stimulate transcription at low temperatures is a useful tool to investigate the interplay of the core enzyme, EЈ, TFE, and F to catalytic activity in various functional assays.
The Rpb7 Ortholog EЈ Stimulates Transcription at Low Temperatures by Catalyzing Open Complex Formation-The EЈ-F complex and its eukaryotic orthologs Rpb7/Rpb4 (RNAPII), Rpc25/Rpc17 (RNAPIII), and Rpa43/Rpa14 (RNAPI) form heterodimers that are a landmark of all non-bacterial RNA polymerases (33)(34)(35)(36)(37). The structure of the Rpb7/Rpb4 complex is very similar to the archaeal EЈF dimer structure (22,17) and to the RNAPIII counterpart of these subunits, C17/C25 (37,38). Their similar structure strongly argues for their functional equivalence. EЈ had no effect on the elongation of stalled complexes (Fig. 4C) indicating that it works on the level of initiation. The strong stimulation of initiation observed was mediated by EЈ alone (Fig. 4, A and B). This finding demonstrates that complex formation of EЈ with F is not a prerequisite for its activating properties. This is a further similarity to the eukaryotic system. In Saccharomyces cerevisiae Rpb7 is an essential protein and Rpb4 is dispensable under optimal growth conditions (reviewed in Ref. 39). The major interaction of Rpb4/Rpb7 with the core enzyme occurs via the tip loops of Rpb7 and this interaction does not involve Rpb4 (39,40). Our finding that EЈ stimulates transcription in the absence of F suggests that EЈ, like Rpb7, can interact as monomer with the core enzyme. The slight stimulation of EЈ-dependent activation of the core enzyme by the addition of F (Fig. 4D) suggests that subunit F may facilitate the association of EЈ with the core enzyme. Hence, weak not yet identified contacts of F with the core enzyme may stabilize the interaction of EЈ with the core enzyme. The finding that the presence of F further stimulates transcription of the core enzyme in reactions containing EЈ and TFE (Fig. 4E, lane 2) suggests that F facilitates TFE recruitment.
A Dual Role of TFE in Initiation-First studies suggested that archaeal TFE stabilizes interactions between TBP and the TATA box (5,16). Recent work showed that TFE increases RNAP recruitment in electrophoretic mobility shift assays (41) and functionally interacts with the EЈF subcomplex (21). Our KMnO 4 footprints at 70°C (Fig. 5A) demonstrated that TFE leads to an increased complex stability by promoting DNA melting similar to the properties reported for eukaryotic TFIIE␣ (12,24,42). The roles of EЈ and TFE in open complex formation were better resolved when initiation by the Pyrococcus core enzyme was analyzed at 60°C. The 7-fold activation of initiation by EЈ was further increased to 14-fold by TFE and the stimulation of initiation was 25-fold in the presence of both EЈF and TFE (Fig. 4, D and G). This finding demonstrates that EЈ and TFE work synergistically during activation. Analyses of kinetic stabilities of open complexes (Fig. 6) show a clear EЈ-independent stabilizing effect of TFE on the core enzyme. This finding indicates that the presence of EЈ is not required for TFE recruitment to the preinitiation complex. Although TFE stabilizes open complexes by the core enzyme significantly in the absence of EЈ (Fig. 6), the major stimulatory effect of TFE on transcription at 60°C strictly requires the presence of subunit EЈ (Fig. 4F, lane 3) 7; and 6). Our data also identify subunit EЈ as the major target for the TFE-induced stimulation of transcription.
The Interplay of Core Enzyme, EЈ, and TFE during Initiation-Structural analyses revealed the presence of a deep cleft in pol II comprising the DNA-binding site and active center (17). One side of the cleft is formed by a mobile clamp that is open in the structure of the 10-subunit core enzyme and closes the cleft in elongation complexes that traps the template DNA inside. Although the role of Rpb4/7 in closure of the clamp during elongation is not known, Rpb7 forms a wedge between the mobile clamp and the linker to the C-terminal repeat and restricts the clamp to the closed position even in the absence of DNA (18,19). Hence, an interaction of Rpb7 with the clamp during initiation is very likely 3 but the role of this interaction and the exact function of Rpb7 in initiation are unclear. The observation that the intracellular levels of EЈ increase during growth at low temperatures (29) suggest that this EЈ facilitates transcription at low temperatures and our present data show a role of EЈ in open-complex formation. It is unclear whether EЈ is essential for growth of archaeal cells but cell-free transcription data demonstrate that EЈ is dispensable for cell-free transcription of the gdh promoter at temperatures between 70 (Fig. 4A) and 80°C, but the presence of EЈ is crucial at 60°C (Fig. 4). Analyses of open complex formation at the gdh promoter provide an explanation of the stimulatory roles of EЈ and TFE in initiation and identify a major function of EЈ during initiation of transcription. EЈ also facilitates open complex formation by the purified RNAP containing EЈ/F but the distinct roles of EЈ and TFE during bubble opening were elucidated by permanganate footprinting experiments with the core enzyme at 60°C. The presence of the core enzyme leads to a weak modification of T residues at positions Ϫ2 and Ϫ4. This melting in this small region is not sufficient to support initiation but shows that the core enzyme interacts with promoter-bound transcription factors and promoter DNA. Therefore, EЈ is not required for stable recruitment of the archaeal RNAP to the promoter, like Rpb4/7 is not required for recruitment of pol II (31). Addition of EЈ stimulates promoter opening in general and leads to an extension of the open region beyond the transcription start site. Most likely bubble opening in this region is crucial for promoter activation, because addition of EЈ to the core enzyme stimulates initiation dramatically (see Fig. 4, B and F). The addition of TFE further increased bubble opening particularly in the upstream region at position Ϫ6 (Fig. 5, A, lane 4, and B, lane  7) and this leads to a further dramatic stimulation of the initiation rate (compare in Fig. 4F, lanes 3 and 10). Recently, cross-linking experiments have revealed that TFIIE␣ approaches promoter DNA at position Ϫ10 and that artificial premelting of the DNA in the Ϫ11/Ϫ1 and in particularly in the Ϫ7/Ϫ8 region increased the stimulating effect of TFII␣ on abortive transcription (42).
Although the EЈ catalyzed promoter opening may not be required under normal growth conditions of the hyperthermophilic Archaea Pyrococcus, the findings reported here have important implications for the mechanism of transcription by a pol II-like polymerase. The pol II core enzyme can be recruited to the TBP-TFIIB-promoter complex and the presence of Rpb4/7 restricts the mobile clamp of RNA polymerase to the closed position. The findings that association of EЈ with the promoter-bound archaeal core enzyme induces further opening of the region slightly melted at Ϫ2 by the core enzyme beyond the transcription start site suggest that binding of EЈ to the core enzyme induces clamp closure. The presence of TFE seems to stabilize clamp closure further and EЈ-mediated contacts of TFE with the core enzyme catalyze further opening of the bubble at the upstream end. Considering the structural similarities between EЈF and Rpb7/4 the novel role of subunit EЈ elucidated in the present work is likely to be also of general significance for the mechanism of initiation by eukaryotic RNAPs.