Protein-Protein Interactions in the Archaeal Transcriptional Machinery

Protein-Protein Interactions in the ArchaealTranscriptional MachineryBINDING STUDIES OF ISOLATED RNA POLYMERASE SUBUNITSAND TRANSCRIPTION FACTORS*□S

Received for publication, May 31, 2006, and in revised form, July 26, 2006 Published, JBC Papers in Press, August 1, 2006, DOI 10.1074/jbc.M605209200

Bernd Goede‡, Souad Naji§, Oliver von Kampen‡, Karin Ilg§, and Michael Thomm§1

From the ‡Lehrstuhl fur Allgemeine Mikrobiologie, Universitat Kiel, am Botanischen Garten 1-9, 24107 Kieland the §Lehrstuhl fur Mikrobiologie und Archaeenzentrum, Universitaet Regensburg, 93053 Regensburg, Germany

Transcription in Archaea is directed by a pol II-like RNA po-lymerase and homologues of TBP and TFIIB (TFB) but the crys-tal structure of the archaeal enzyme and the subunits involved inrecruitment of RNA polymerase to the promoter-TBP-TFB-complex are unknown. We described here the cloning expres-sion and purification of 11 bacterially expressed subunits of thePyrococcus furiosus RNAP. Protein interactions of subunitswith each other and of archaeal transcription factors TFB andTFB with RNAP subunits were studied by Far-Western blottingand reconstitution of subcomplexes from single subunits insolution. In silico comparison of a consensus sequence ofarchaeal RNAP subunits with the sequence of yeast pol II sub-units revealed a high degree of conservation of domains of theenzymes forming the cleft and catalytic center of the enzyme.Interaction studies with the large subunits were complicated bythe low solubility of isolated subunits B, A�, andA�, but an inter-action network of the smaller subunits of the enzymewas estab-lished. Far-Western analyses identified subunit D as structur-ally important keypolypeptide ofRNAP involved in interactionswith subunits B, L, N, and P and revealed also a strong interac-tion of subunits E� and F. Stable complexes consisting of sub-units E� andF, ofDandL and aBDLNP-subcomplexwere recon-stituted and purified. Gel shift analyses revealed an associationof the BDLNP subcomplex with promoter-bound TBP-TFB.These results suggest a major role of subunit B (Rpb2) in RNAPrecruitment to the TBP-TFB promoter complex.

Archaeal RNA polymerases (RNAP)2 are multisubunitenzymes that resemble in sequence subunit composition andfunctional aspects eukaryotic RNAP. Fig. 1 shows the subunitstructure of eukaryotic RNA polymerase II (pol II), of the Pyro-coccus RNAP and of the RNAP from Escherichia coli. Homolo-

gous subunits are indicated by the same colors. Archaeal RNAPdisplay greater similarities with all eukaryotic RNAP than withthe four subunits of the bacterial core enzyme. We refer heremainly to pol II as the subunit interactions within this enzymeare known from the crystal structure (1, 2). Archaeal RNAPshave clear homologues to Rpb4 and Rpb7 of pol II, which werefirst called F and E (3). In the genomes of most Archaea, thegene encoding E overlaps at its 3�-end in a different readingframe with a second gene containing a zinc finger motif. Todiscriminate between the first gene that is homologous to rpb7and the second gene that has no homolog in yeast but is highlyconserved inArchaea, the first one is designated in data bases asrpoE� and the second one as rpoE�, and the corresponding pro-teins as E� andE�. Only E�has been detected in purified archaealRNAP. Two subunits shared by all three eukaryotic RNAP,Rpb12, and Rpb10 have the subunits P andN as archaeal homo-logues but no bacterial homologues. In addition, subunit H ofthe archaeal enzyme has a homologue in pol II (and also in polI and pol III) but not in the bacterial enzyme.The gene encoding the largest subunit in eukaryotic RNAP,

Rpo1 and �� of the E. coli enzyme is split into two genes encod-ing subunits A� and A� in all Archaea. The RNAP of Pyrococcusand of Crenarchaeota show the subunit composition BA�A�DE�FLHNKP (4, 5). In methanogens and extreme halophilicArchaea subunit B is split into the subunits B� and B� (6). This Bsplit defines the second major type of archaeal RNAP with thesubunit composition A�B�B�A�DE�FLHNPK.The archaeal RNAP is recruited to the preinitiation complex

by association to promoter-bound transcription factors TBPand TFB (7, 8), which are interacting with the TATA-box andBRE element of archaeal promoters (reviewed in Ref. 9). BothTBP and TFB consist of two imperfect direct repeats. TFB hasin addition an N-terminal domain forming a zinc ribbon and aB-finger (see Figs. 6C, 9, and 10). A third archaeal transcriptionfactor, TFE, is homologous to the N-terminal part of subunit �of eukaryotic TFIIE (11, 12). TFE is not required for promoter-directed transcription but can stimulate the activity of somepromoters by a factor of 3–4. TFE can also complement somemutants of TFB indicating that these proteins interact synergis-tically and contribute to catalytic core functions of RNAP (10).The path of the DNA in the Pyrococcus RNAP has been stud-

ied by photochemical cross-linking (13, 14, 15). These studiesrevealed that subunit B of Pyrococcus RNAP cross-links theRNAP between the TATA-box and the transcription start site

* This work was supported by the Deutsche Forschungsgemeinschaft. Thecosts of publication of this article were defrayed in part by the payment ofpage charges. This article must therefore be hereby marked “advertise-ment” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

□S The on-line version of this article (available at http://www.jbc.org) containssupplemental models.

1 To whom correspondence should be addressed: Lehrstuhl fur Mikrobiolo-gie, Universitaet Regensburg, Universitaetsstrasse 31, 93053 Regensburg.Tel.: 0049-941-943-3160; Fax: 0049-941-943-2403; E-mail: [email protected].

2 The abbreviations used are: RNAP, Archaeal RNA polymerases; PMSF, phen-ylmethylsulfonyl fluoride; TBP, TATA-binding protein; pol, polymerase;PDB, Protein Data Bank; TFB, transcription factor B.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 281, NO. 41, pp. 30581–30592, October 13, 2006© 2006 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

OCTOBER 13, 2006 • VOLUME 281 • NUMBER 41 JOURNAL OF BIOLOGICAL CHEMISTRY 30581

by guest on April 13, 2018

http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

and that subunits A�, A�, and H contact the DNA downstreamof the start site. In vivo and in vitro binding assays were used toinvestigate the interactions of subunits of the RNAP fromMethanocaldococcus jannaschii. The eukaryotic subunits Rpb4and Rbp7 form a heterodimer that reversibly associated withthe pol II core. As predicted from the similarity to the eukary-otic system the archaeal homologues of these polypeptides, Eand F, forma complex (3) and archaeal F interactedwith humanRpb7 to form an archaeal-human F-Rpb7 hybrid (16). SubunitsD, L, N, and P were shown to associate to a tetrameric D-L-N-Pcomplex (16). The eukaryotic homologues of these subunits,Rbp3, Rpb10, Rpb11, and Rpb12 are in close interaction andclustered together in the pol II structure (1). This assembly ofthe archaeal subunits D-L-N-P was able to recruit the largestsubunit B in vitro and used as a frame for the reconstitution ofactiveM. jannaschii RNAP from individual subunits (17).We are exploring the mechanism and regulation of tran-

scription in Pyrococcus using a cell-free transcription system (7,18, 20). We report here cloning and expression of all RNAPsubunits from Pyrococcus. The interaction of these polypep-tides with each other and with the archaeal transcription fac-tors TBP and TFB were studied by far-Western analyses, col-umn chromatography and gel electrophoresis. Our resultsreveal many interactions predicted from the structural similar-ities to the pol II system, the existence of various subcomplexesand an interaction of the BDLNP subcomplex with promoter-bound TBP and TFB.

EXPERIMENTAL PROCEDURES

Cloning of RNAPSubunits—The coding region of RNAP sub-units B (PF1564), A�(PF1563), A� (PF1562), D (PF1647), E�(PF02569, F (PF1036), H (PF1565), K (PF1642), L (PF0050), N(PF16439 and P, (PF2009) from Pyrococcus furiosus DSMZ3638, were PCR-amplified using genomic DNA as template.The oligonucleotides were complementary to the 5�- and3�-ends of the genes and contained the restrictions sites NdeI atthe 5�-end and BamHI at the 3�-end. The PCR fragmentsencoding subunits B,D, E�, F, L,H,K, andPwere cloned into thecorresponding restriction sites of the expression vector pET-33b (Novagen). The expressed proteins carry a His6 tag and arecognition site for heart muscle kinase (HMK) at the N termi-nus. The PCR products encoding subunits F and H were alsocloned in a modified version of pET-33b resulting in proteinscontaining the His6 tag and the HMK site at the C terminus.Details of the modified vector and of the oligonucleotidesequences are available from the authors on request. The PCRfragments encoding subunitsA� andA�were cloned in theNdeIsite and BamHI site of pET-14b. The expressed subunits A� andA� contained only aHis6 tag at theN terminus andwere used astargets in Far-Western experiments. To obtain subunits A� andA� with a HMK site in addition, the PCR products containingthe HMK recognition site at the 5�-end were cloned intopET151/D-TOPO (Invitrogen).Identification of a Consensus Sequence for Subunits of

Archaeal RNAP and Bioinformatic Work—Multiple se-quence alignments of the genes encoding RNAP subunits ofup to 18 Archaea and of the subunits of pol II from S. cerevi-siae revealed an amino acid consensus sequence for each

subunit with the exception of Rbp8 and Rpb9, which have nohomologues in Archaea. Most of the genes encodingarchaeal RNAP subunits were extracted from whole genomefiles available at NCBI or at SRS. BLAST search and othercommon bioinformatic resources were used to identifyunannotated entries. A number of missing genes becameavailable by local BLAST search in Bioedit on the basis of thewhole genome file in raw format. Multiple sequence align-ments were carried out usingMalign, an algorithm especiallysuitable when genes are compared that show low sequencesimilarities and different lengths (scoring matrix: PAM250).Because subunit B is split in two parts in several Archaea(rpoB�and rpoB�) two single alignment steps were carriedout and combined in a subsequent step to obtain betterresults. MAlign2Msf was used to convert the data into theMSF file type. After import into Bioedit the data were for-matted, a consensus sequence was generated and shadingwas applied. As final step export as RTF file and import intoMS Word was performed. The consensus sequence of eachalignment was also used to generate two-dimensional simi-larity diagrams (Fig. 3) and to visualize the distribution ofidentities and similarities in the three-dimensional model ofS. cerevisiae polII (PDB: 1NT9; Fig. 4). A small Delphi pro-gram was written to draw the two-dimensional diagrams andas a helper tool for sequence analysis and various conversionsteps. In the diagrams a vertical line represents identitybetween the archaeal consensus and the amino acidsequence of pol II. Lines of half-length indicate similarities.To visualize the homologous regions of archaeal RNAPs andof pol II in the three-dimensional structure of pol II, theconsensus sequence for each subunit was converted in aProSite search pattern and applied to the S. cerevisiae pol IImodel (PDB: 1NT9) using the Cn3D 4.1 annotate function.Expression and Purification of Proteins—For the expres-

sion of the proteins the plasmids were transformed in theexpression strains BL21(DE3)Star-CP (subunits B, A�, D, E�,F, H, K, L, N, and P) and in BL21(DE3)pLysS (subunit A�).The proteins were expressed by inducing exponentially cul-tures with 1 mM isopropyl-1-thio-�-D-galactopyranoside for3 h. For Far-Western dot blot experiments (Fig. 5A, firstthree panels), subunit B was purified after SDS-polyacrylam-ide gel electrophoresis. Gel slices containing this subunitwere incubated in a solution containing 0.1% (w/v) SDS.Then, SDS was precipitated and the protein refolded by dilu-tion and dialysis as described by Ref. 21. Subunit B used asprobe (Fig. 5A, lower panel) for dot blots and B used for gelblots and for reconstitution of the DLNPB subcomplex andsubunits A�and A� were renatured from inclusion bodies.First, cells were suspended in lysis buffer (20 mM Tris-HCl,pH 8, 1 mM PMSF, 5 mM 2-mercaptoethanol, 0.3 mg/mllysozyme) and sonicated. After centrifugation the pelletscontaining the inclusion bodies were extensively washed inpurification buffer (20 mM Tris-HCl, pH 8, 0.5 M NaCl, 0.1%Tween 20, 1 mM PMSF, and 5 mM 2-mercaptoethanol) Theinclusion bodies were solubilized in binding buffer (20 mMTris-HCl, pH 8, 0.5 M NaCl, 5 mM imidazole, 6 M guanidineHCl, 1 mM PMSF, 5 mM 2-mercaptoethanol) for 1 h at 20 °C.After centrifugation the supernatant was loaded onto a

Interactions of RNAP Subunits

30582 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 281 • NUMBER 41 • OCTOBER 13, 2006


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

Ni2�-NTA column (HisTrapFF, GE healthcare) and washedwith binding buffer containing 6 M urea and no guanidineHCl. The refolding of the bound protein was performed oncolumn using a decreasing linear urea gradient (10 columnvolumes) ranging from 6 M to 0. The refolded proteins wereeluted with an imidazole gradient ranging from 5 to 300 mMimidazole. For the purification of subunits D, L, N, E�, F, K,H, and P cells were resuspended in a buffer containing 50mMNaPO4, pH 8, 10 mM imidazole, 300 mM NaCl, and 10% v/vglycerol. Cells were disrupted by passage through a Frenchpressure cell. The lysate was clarified by centrifugation at100,000 � g for 20 min at 4 °C. The supernatant was directlyapplied to a Ni2�-NTA column (subunits D, E�, K) or afterheating for 20 min at 80 °C (subunits F, H, L, N, and P) andseparation of precipitated E. coli proteins by centrifugation.Bound proteins were stepwise eluted with 300mM imidazole.Some subunits were further purified by MonoQ- orSuperdex75-chromatography.Far-Western Blotting—The whole procedure was a variation

of the protocols described by Arthur and Burgess (22) and Bur-gess et al. (23). Dot blot-solubilized proteins were spotted ontoa nitrocellulose membrane (Optitran BA-S 85 Reinforced NC,Schleicher and Schull, order number 439196). The affinity ofRNAP subunits to bind to themembrane and/or to detach fromthe membrane during the following incubations steps differedconsiderably. In particular subunit B and H showed a tendencyto detach from the membrane during the following incubation.This detachment was inhibited by drying the membranes afterspotting of the proteins briefly at 50 °C. To control the amountof proteins used for the protein-protein interactions studiesproteins were spotted in parallel on two membranes for eachexperiments and one membrane was stained with Ponceau Sand the second used for the Dot Blot. 0.5 to 3 �g of protein wasspotted for each individual subunit onto the membrane toobtain similar signals with Ponceau S staining. In particular theamount of subunits A�, A�, F, and H added to the membranewas higher, but also somewhat higher amounts of subunits B, P,and purified RNAP were spotted onto the membrane to obtainsimilar staining signals with Ponceau S. After drying the mem-brane used for the dot blot was blocked and subunit B refoldedby incubation overnight in probing buffer (20 mM HEPES, pH7.2; 200 mM KCl, 2 mMMgCl2, 0.1 mM ZnCl2, 1 mM dithiothre-itol; 0.5% (v/v) Tween 20, 1% (w/v) nonfat-dried milk and 10%(v/v) glycerol (23). After probing and autoradiography theamount of protein on themembrane was controlled by stainingwith Ponceau S.Gel Blot—Cloned RNAP subunits or purified RNAP were

electrophoretically transferred (Semidry system Bio-Rad) afterSDS-polyacrylamide electrophoresis to nitrocellulose mem-branes (PROTRANBA750.05�m;Schleicher and Schull, ordernumber 10402196). Proteins on the membrane were refoldedby incubation overnight in probing buffer. The transfer of pro-teins onto the membranes was verified after probing and auto-radiography by staining with Ponceau S.Labeling of Probes—70 pmol RNAP subunit was incubated

with 20 �Ci of [�-32P]ATP (6000 Ci/mmol) and 10 units ofHMK (Novagen) in a total volumeof 10�l of the buffer suppliedwith the enzyme for 70 min at 30 °C.

Probing—The blocked nitrocellulose membrane was incu-bated in 10 ml of probing buffer containing 10 �Ci of 32P-la-beled RNAP subunits for 2 h at 4–8 °C. The blot was washedtwo times in probing buffer dried and exposed to a Phosphoim-ager (FLA-5000, Fuji).Electrophoresis under Non-denaturating Conditions—Inter-

actions of subunits D and L and E� and F were investigatedby electrophoresis in native 8–15% polyacrylamide gels asdescribed in Ref. 24.Reconstitution of BDLNP RNAP Subcomplexes—Equimolar

amounts (2.5 nmol) of RNAP subunits were incubated in tran-scription buffer (40 mM HEPES, pH 7.3, 250 mM NaCl, 2.5 mMMgCl2, 10% (v/v) glycerol, 1 mM EDTA, 1 mM PMSF, 5 mM2-mercaptoethanol) for 1 h at 20 °C. The complex formationwas analyzed by Superdex 200 (GE Healthcare) column chro-matography. Alternatively, the BDLNP complex was reconsti-tuted by denaturation and renaturation of recombinant sub-units. 2.5 nmol of subunits B andD and 5 nmol of subunits N, L,and P were combined in a final volume of 500 �l of transcrip-tion buffer containing in addition 6 M urea. The mixture wastransferred to a dialysis frame (Slide-A-Lyzer 3.5k, Pierce) andincubated for 20 min at 20 °C. Then, the mixture was dialyzedagainst transcription buffer containing 3 M urea for 20 min at20 °C. Renaturation was achieved by dialysis in transcriptionbuffer for 1 h. The renaturated subcomplexes were heated for10 min at 70 °C to remove misfolded aggregates. The BDLNPsubcomplex was purified by Superdex 200 chromatography(Superdex 200 10/300 GL, GE Healthcare).Electrophoretic Mobility Shift Assay—The DNA sequence of

the P. furiosus gdh promoter was amplified from genomic DNAby PCR. 90 bp of 32P-labeled DNA fragments encoding the pro-moter region from position�60 to� 30 were end-labeled withT4 polynucleotide kinase (MBI Fermentas) according to theinstructions of the manufacturer. The labeled DNA was puri-fied using mini Quick Spin Columns (Qiagen). DNA bindingreactions were conducted for 30 min at 70 °C in a 12.5-�l vol-ume of transcription buffer containing in addition 0.1 mg/mlbovine serum albumin, 240 nM TBP, 60 nM TFB, and 8.6 nMRNAP, or 100 nM BDLNP subcomplex. The reactions wereloaded onto a native 4% polyacrylamide gel (buffer containing25 mM Tris-HCl, pH 8.5, 10% glycerol, and 0.5 mM 2-mercap-toethanol), electrophoresed at room temperature and analyzedby phosphorimaging.

RESULTS AND DISCUSSION

The Archaeal RNAP Displays High Sequence Similarity withthe Catalytic Core of Pol II—To investigate themolecular archi-tecture of an archaeal RNAP we cloned and expressed 11 sub-units of the Pyrococcus RNAP (Figs. 1 and 2) ranging in molec-ular mass from 127 003 (subunit B) to 5757 (subunit P). Thesequences of the genes encoding these subunits were alignedwith the sequences of subunits of 17 archaeal RNAP and fromthis an archaeal consensus sequence for each individual subunitwas derived (“Experimental Procedures” and supplementarydata). This consensus sequence was aligned with the pol IIsequence and identical amino acids and amino acids highlyconserved between archaeal RNAP and S. cerevisiae pol II wereindicated by horizontal bars in the schematic representation of




http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

the gene (Fig. 3) and highlighted in yellow and green color in adepiction of the three-dimensional structure of individual sub-units of pol II and in the crystal structure (2) of the 12 subunitenzyme (Fig. 4; see also three-dimensionalmodel in the supple-mentary data).Rbp1 corresponding to subunits A� andA� harbors the active

center and the Mg2� ion in the active center. The C-terminaldomain of subunit Rpb1 is not present in Archaea and theregions corresponding to the clamp head, the foot and the jawshowed a low degree of sequence conservation. In particularconserved in the archaeal subunits A� and A� (Figs. 3 and 4)were regions homologous to parts of the clamp core (positions1–95 and 235–346 of this subunit), to parts of the active site andof the metal A site of the catalytic center (346–375 and 436–500) and of the cleft (809–871 and 1059–1141).Rpb2 of S. cerevisiae and subunit B of Pyrococcus RNAP are

similar in size. Highly conserved in the archaeal subunit B werethe sequences homologous to parts of the fork (position 465–547), two sequences corresponding to regions of Rpb2 involvedin hybrid binding (from position 750–852 and from 952–1127)

and a sequence homologous to a part of the wall (from 852–952) and of the anchor (1127–1151) of pol II (Figs. 3 and 4).Homologous parts of the smaller subunits of archaeal RNAPand of pol II are also depicted in Figs. 3 and 4. It is obviousthat the highest degree of sequence conservation was foundin the region encoding the major cleft of RNAP harboringthe active center which is formed mainly by the two largestsubunits of pol II.Subunits Rbp7 and E�, Rpb11 and L and RpB12 and P are

similar in size. The other smaller subunits of pol II are larger inmolecular mass (see Fig. 3) mainly because of extensions at theN terminus (Rbp4 and Rpb6) or at the C terminus (RpB3).These extensions showed no sequence similarity to archaealRNAP subunits. No homologues of subunits Rpb8 and Rpb9were detected in the genome of Pyrococcus and of otherArchaea. These subunits are indicated in Fig. 4C in light blue. Athree-dimensional model showing the amino acids conservedbetween archaeal RNAP and yeast pol II is available in the sup-plementary materials.Far-Western Analyses of Interactions of RNAP Subunits—

The 11 cloned archaeal RNAP subunits were used to investigateprotein-protein interactions of all components of the basalarchaeal transcription machinery. A HMK recognition site wascloned at the N or C terminus of genes encoding RNAP sub-units to allow specific labeling of the proteins with 32P. TheN-terminally labeled subunits B, D, E�, F, L, N, K, and P andC-terminally labeled subunits F and H were used as probes forthe detection of interactions of subunits immobilized on nitro-cellulose membranes. In one set of experiments the native pro-tein was simply immobilized on nitrocellulose membranes(Far-Western dot blot). In a second approach the RNAP sub-

FIGURE 1. Homologous subunits of RNAP from the three domains of life.The subunit pattern of the RNAP from P. furiosus, pol II of S. cerevisiae and ofthe E. coli enzyme are shown. Identical colors indicate homology. Note thatsubunit Rpb1 of eukaryotes is split in Archaea into the two polypeptides, A�and A�. Subunit � of the E. coli RNAP is homologous to a part of subunit D ofArchaea and of Rpb3 of yeast and to subunits L and Rpb11. The molecularmass of the subunits of Pyrococcus RNAP is indicated to the left.

FIGURE 2. Cloned and bacterially expressed subunits of P. furiosus RNAPused as probes and targets for protein-protein interaction studies. Puri-fied single subunits of P. furiosus RNAP expressed in cells of E. coli and RNAPpurified from Pyrococcus cells were separated on an 8 –15% polyacrylamidegradient gel under denaturing conditions and stained with Coomassie Blue.All recombinant proteins had a 25-amino acid extension at the N terminus(subunits B, A�, A�, D, E�, L, N, K, and P) or at the C terminus (subunits F and H).This extension contained as His6 tag, a PKA recognition site and a thrombincleavage site. Therefore, the molecular mass of cloned subunits is higher thanthe molecular mass of subunits of the purified enzyme. This extension makesa difference in particular in the case of the smaller subunits. Lanes 1–9, sub-units A�, D, E�, F, L, H, N, K, and P; lanes 13 and 14, purified RNAP; lanes 15 and16, subunits B and A�. The molecular mass of standard proteins is given to theleft.




http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

units were transferred to themembrane after electrophoresis indenaturing polyacrylamide gels (Far-Western gel blot). Detec-tion of interactions was performed after treatment of the trans-ferred proteins by a procedure allowing refolding of proteindomains at the membrane (23).Subunits A� and A� precipitated during the labeling and

binding assays and were therefore only used as immobilizedtargets of binding reactions but not as labeled probes. A typicaldot blot experiment is shown in Fig. 5. Subunit D interactedwith subunits P and B, subunit L exclusively with D. This resultestablished a strong and specific interaction of the subunits Dand L. Subunit E� bound strongly to F and weakly to TBP andTFB. Subunit F bound strongly and exclusively to E�. This resultconfirmed the specific interaction of these proteins which areknown to form a specific subcomplex in theMethanocaldococ-cus RNAP (25). Subunit N interacted with D and P (Fig. 5).Subunit P bound toD. These result showed that the subunits D,L, N, and P which were used as platform for the reconstitutionofMethanocaldococcus RNAP (26, 17) formed also an interac-

tive network in the Pyrococcus RNAP (see Fig. 9). Interactionsof labeled D with L and of labeled L with D were confirmed byfar-Western gel blots (Fig. 5, B and C). D as probe interactedwith B and L (Fig. 5B). B as probe bound in dot blots to D, F, L,andN,weakly to P and in addition toTFB (Fig. 5A, lower panel).Our results established in addition protein-protein contacts ofsubunit B with D both when D and B were used as probes (Fig.5, B and A, lower panel). In addition, contacts of B with N wereshown both in dot blots (Fig. 5A) and gel blots (data not shown)with B as probe.These data establish an interactive network between the

subunits BDLNP and between subunits E� and F which werepreviously shown to form subcomplexes in the Methanocal-dococcus system (3, 16, 17). The high sequence similarity ofessential parts of the two largest subunits of archaeal RNAPand pol II (see Figs. 3 and 4) suggest that the archaeal sub-units B and A�, A� form the catalytic center and have manycontact sites similar as in pol II. Strong interactions of sub-units B with A� and A� could not be shown by Far-Western

FIGURE 3. The consensus of archaeal RNAP subunits shows extensive homology to domains of the active core of pol II. The consensus sequence for thegenes encoding archaeal RNAP subunits was aligned with the homologous eukaryotic subunits. The genes encoding the subunits of yeast pol II are shown inthe top lane of each panel. DNA regions conserved in the consensus of the archaeal RNAP subunit are indicated in the pol II gene by full-length black bars(identical with archaeal consensus) and half-length bars (similar to archaeal consensus). A, top lanes, comparison of rpoA encoding A� and of rpoA2 encodingA� with the gene encoding the largest subunit Rpb1 of yeast pol II. The extent of the genes encoding subunits A� and A� is indicated in the pol II gene by blueboxes. The C-terminal domain of pol II that is missing in the archaeal enzyme is boxed in red. Second lane, domains or helices identified in the crystal structureof pol II (1); 1, clamp core; 2, clamp head, 3, clamp core; 4, active site; 5, dock; 6, active site (metal A); 7, pore; 8, funnel; 9, cleft; 10, foot; 11, cleft; 12, jaw, 13, cleft;14, clamp core; 15, linker; 16, CTD. Third lane, amino acids containing hydroxyl groups as potential phosphorylation sites are indicated by bars. B, comparisonof Rpb 2 and Rpb5 with archaeal subunits B and H. The labeling and symbols are in all the following panels like in the first panel. Domains and helices of subunitRpb2 in the crystal structure of pol II: 1, external; 2, protrusion; 3, lobe; 4, protrusion; 5, fork; 6, external; 7, external; 8, hybrid binding; 9, wall; 10, hybrid binding;11, anchor; 12, clamp. Domains and helices in the crystal structure of subunit Rpb5: 1, Jaw; 2, assembly. C, comparison of Rpb 3, Rpb11, Rpb10, and Rpb12 withsubunits D, L, N, and P of the archaeal enzyme. Domains and helices in the crystal structure of subunit Rpb3: 1, dimerization; 2, domain; 3, zinc loop; 4, domain2; 5, dimerization; 6, loop; 7, dimerization; 8, tail. Domains and helices in the crystal structure of subunit Rpb11: 1, tail; 2, dimerization; 3, tail; Domains and helicesin the crystal structure of subunit Rpb10: 1, zinc bundle; 2, tail. Domains and helices in the crystal structure of Rpb12: 1, zinc ribbon; 2, tail. D, comparison ofsubunits Rpb6, Rpb4, and Rpb7 with subunits K, F, and E�. Domains and helices in the crystal structure of subunit Rpb6: 1, tail (not contained in the three-dimensional model); 2, assembly.




http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

and dot blot analyses probably due to the low solubility of theisolated large subunits and because of the tendency of theisolated proteins to precipitate. However, at least weak inter-actions of subunits B and A� could be detected (Fig. 5A) and

in the light of the high sequence similarity of the large sub-units of pol II and of the archaeal RNAP (Figs. 3 and 4) thesewere considered as being significant. Additional contactswere found between H and A� (Fig. 5A). The homologous

FIGURE 4. Visualization of regions conserved between pol II and the archaeal enzyme in the crystal structure of pol II. A, upper panel, structure of singlesubunits of pol II and lower panel, crystal structure of pol II holoenzyme as described in Ref. 2. The color of the subunits is like in Fig. 1. B, the regions of pol IIidentical in sequence to the corresponding archaeal subunit are shown in yellow, regions showing high similarity are shown in green, sequences with nosequence similarity in blue, and sequences missing in the archaeal enzyme are shown in light-blue. C, left, backbone model for the 12 subunits of pol II shownas ribbon diagram; the color code for each subunit is as indicated in A. Middle, space filling model of pol II using the same color code for subunits as in A. Right,backbone model of pol II indicating regions with high and low similarity of pol II to the archaeal enzyme, the some colors as in B were used to indicate identity,similarity, no similarity and regions missing in the archaeal enzymes. D, backbone model of pol II viewed from top showing the deep cleft and the position ofsubunit H at the end of the lower jaw. Left, complete subunit H is indicated in gray; right, the N-terminal domain of subunit H, not conserved in the archaealenzyme was removed from the model.




http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

proteins of pol II, Rbp5, and Rpb1, are in close contact in thecrystals of pol II (Fig. 9) and therefore also this H-A� inter-action observed here was as predicted. Additional contacts,e.g. those of B with N were also as predicted from the crystalstructure of pol II.Fig. 9 summarizes the protein-protein interactions of sub-

units ofPyrococcusRNAPestablished in this study in relation tothe interactions found in the crystal structure of pol II. Thesimilarities of the interaction patterns of subunits within thearchaeal and eukaryotic enzyme are evident. The interfacesbetween the large subunits B and A�, A� are well conserved

between the eukaryotic and archaeal polymerase (Fig. 3).3Therefore, the strong interactions and extensive interfacesbetween the three large archaeal subunits corresponding toRpb1 and Rpb2 are expected to be very similar in the archaealand eukaryotic enzymes although we could not show strongprotein-protein interactions between these subunits in thepresent work. The reason for that is probably the low solubilityof the isolated large subunits. The subunits B�, A�, and K pre-

3 P. Cramer, personal communication.

FIGURE 5. Interactions of isolated RNAP subunits analyzed by Far-Western blotting. A, dot blot, recombinant RNAP subunits and TBP And TFB werespotted on NC membranes and probed with 32P-labeled subunits D, E�, F, H, K, L, N, P, and B as indicated; binding of probes to immobilized subunits wasdetected by autoradiography. B and C, Far-Western gel blot of purified RNAP and isolated subunits after separation on an 8 –15% denaturing polyacrylamidegel.




http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

cipitated during expression in E. coli and had to be renaturedfrom inclusion bodies. We assume that the tendency of theseproteins to precipitate and to denature is the major reason forthe lack of detected interactions between isolated large sub-units. It is interesting to note that subunit H interacted with theA� subunit corresponding to amino acids 100–1430 of Rpb1 aspredicted from the crystal structure. The subunit H and Rpb5are in proximity of theDNAregion from�4 to�15 in initiationcomplexes (27, 13).The general architecture of pol II is characterized by a deep

cleft between the two large subunits, which are anchored at oneend to the subassembly of subunits Rpb3,10, 11, and 12 (theD-L-N-P orthologs; 28; see also Fig. 4D). Rpb5 and regions ofRpb1 form a pair of jaws that appear to guide the DNA to theactive center. The cross-linking data of Bartlett et al. (13) andour finding that H interacts with A� as predicted support theconclusion that the archaeal subunit H is located in the three-dimensional structure of the enzyme on a similar position asRpb5 in the eukaryotic enzyme. Rpb5 forms the end of thelower “jaw” of polII that is in contact with downstream DNA.Interestingly, the highly conserved proline residues 86 and 118of Rpb 5, which are facing the DNA (28) are not conserved inthe archaeal subunit H (Fig. 4D and supplemental materials),which lacks the N-terminal domain of Rpb5. DownstreamDNA is not involved in sequence-specific contacts with RNAPand therefore subunit H seems to function collectively with A�and A� in guiding DNA toward the active center during elon-gation of transcription.In contrast to the larger insoluble subunits, the interaction

patterns of the soluble subunits D, L, N, and P described herecould be clearly established and the results are as predictedfrom the crystal structure of pol II. This finding suggests thatthe same conserved sites in pol II and the archaeal enzyme areinvolved in interactions of these subunits. It is unclear whetherthe same amino acids are responsible for theD-L interactions inthe archaeal enzyme as in the Rpb3-Rpb11 interactions. Theconservation of regions 1 and 3 of Rpb3 and of region 2 ofRpb11, which are involved in dimerization between these sub-units (see Fig. 3C) with the corresponding regions of subunits Dand L is not in particular high. But subunits DL from a stablesubcomplex that can be easily identified by gel electrophoresis(Fig. 7). Therefore, these subunits can serve as an excellentmodel to identify the motifs and principles involved in interac-tions of a multisubunit enzyme by mutational studies.The interactions of the 10 subunit core enzyme of RNAP

which contains the mobile clamp of RNAP in the open confor-mation (1) with the subunits Rbp7�Rpb4 which maintains theconformation of a transcribing complex (2, 29) are of particularinterest. Rpb7 interacts with a pocket of pol II formed by sub-units Rpb1, Rpb2 and Rpb 6 (reviewed in Ref. 30). The highstructural similarity of Rpb7/4 and of E�/F (3, 25, 31) suggeststhat the interactions sites are conserved between the archaealand eukaryotic enzyme, although we could only detect interac-tions of E� and Fwith subunit B. Our recent data suggest that E�stimulates transcription of thePyrococcus core enzyme and thatF is not required for this activation but stabilizes the E�-core

interaction.4 The N-terminal region of Rpb4 makes a contactwith Rpb1 (29), but a different interaction site must stabilizebinding of E� to the archaeal core, because the larger N-termi-nal domain of Rpb4 is not conserved in the archaeal subunit F,which ismuch smaller than Rpb4 (see Fig. 3 and supplementarydata). In fact, we show an interaction of F with B (Fig. 5) indi-cating that a different subunit is involved in binding of F to thecore enzyme in the archaeal system. Analyses of the interac-tions of E� and F with core RNAP are of key interest for anunderstanding of conformational changes in the RNAP duringthe first steps of transcription. The transcriptional activation ofPyrococcus RNAP by E� and the known crystal structure of theinteracting tip domain of E� (3) are excellent tools to unravel themechanism of E� induced RNAP activation by mutational andfurther structural studies.In general, the archaeal subunits are smaller than the corre-

sponding pol II subunits. RpB2 and B have approximately thesame size, themajor difference betweenRpb1 andA�, A� are thesplitting of the archael protein into two subunits and the lack ofthe tandem repeats at the C terminus of the archaeal enzyme.Only subunits N and Rpb10 and E� and Rpb7 have similar size.All other archaeal subunits are significantly smaller than theireukaryotic counterparts, in particular H, K, P, and F (Fig. 3).All these smaller subunits with the exception of subunit K,

forwhichwe could not identify a binding partner, seem to inter-act with each other in a similar manner like their orthologs inpol II. Therefore, the present work suggests that the basic inter-action sites between RNAP are conserved between these twodomains of life. Hence, the archaeal polymerase seems to rep-resent the ancient version of pol II containing all the structuralelements required for formation of a stabile structure and cat-alytic activity. It is most likely that the subunits Rpb8 and Rpb9and the additional domains found in the eukaryotic polymeraseevolved later to cope with the complex regulatory patternsencountered in higher cells. An example for this is the N-ter-minal domain of Rpb5, which is involved in activation of tran-scription (33).Far-Western Analyses of RNAP Transcription Factor Inter-

actions—The archaeal RNAP is recruited to the preinitiationcomplex by association with promoter-bound TBP and TFB.The nature of this interaction and the subunits involved inRNAP transcription factor contacts are unknown. Therefore,the RNAP transcription factor interactions are of special inter-est. The structure of the eukaryotic preinitiation complex hasnot yet been solved and RNAP-transcription factor-interac-tions elucidated in the archaeal system are also of potentialsignificance for the eukaryoticmachinery. Inspection of Fig. 5Areveals that labeled subunits D, E�, N and P interacted clearlywith immobilized TBP and TFB. In dot blot experiments usinglabeled TBP and/or TFB as probe, both factors bound to TBPandTFB but only very weak binding signals were detected (TFBbound very weakly to P, D, and K, TBP very weakly to D and P;data not shown). Probably, the dimerization of TBP in solution,the low stability of TFB in solution and its tendency to bindnon-specifically to nitrocellulose membranes hampered the

4 S. Naji, S. Gruenberg, and M. Thomm, manuscript in preparation.




http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

detection of interactions of TBP and TFB with RNAP subunitsby this approach. When TFB and TBP were used as probes forgel blots the binding signals were still weak, but binding of TFBto A� and E� and of TBP to A� and E� was detected (data notshown).To investigate the interaction of TBP and TFB with individ-

ual subunit of RNAP in a way allowing to estimate the strengthof protein-protein interactions, binding of the seven smallerRNAP subunits to serial dilutions of TBP and TFB immobilizedon a membrane was analyzed by the far-Western dot blot pro-cedure. When binding of labeled RNAP subunits to immobi-lized TBP was analyzed, the intensity of binding signalsdecreased in the order P, N, TFB, E�, K, D, H, F, and L. WhenTFB was bound to nitrocellulose membranes, the bindingintensity of labeled probes decreased in the order P, N, D, E�, K,TBP, H, F, and L (data not shown).Considering the crystal structure of pol II and the great

similarities of the general architecture of the archaeal andeukaryotic enzyme3 it is highly unlikely that all these smallsubunits are in the preinitiation complex in contact withTBP and TFB. It seemsmore likely that the ability of TBP andTFB to interact with most of the small subunits is restrictedto sites exposed only in isolated subunits. Subunit K of Sul-folobus was recently shown to interact with TFB (34) in twohybrid assays. In the light of our findings that many isolatedsmall subunits show a tendency to bind strongly to both TFBand TBP it is advisable to see interactions of isolated smallsubunits with TBP and TFB with some caution. In pol II, thetwo larger subunits flanking the deep cleft and forming theactive center of the enzyme have been identified as majorinteraction sites of TBP and TFIIB by photocross-linking.We have also identified subunit A� as binding target of bothTBP and TFB (not shown) and detected also an interaction oflabeled subunit B with TFB (Fig. 5A, lower panel). Analysis ofa subcomplex containing subunits BDLNP showed that sub-units A� and A� are not required for recruitment of RNAP tothe TBP-TFB promoter complex (see below). Structuralanalyses of the archaeal preinitiation complex are requiredto resolve the transcription factor RNAP interactions sites inmore detail.Sequences at the N and C Terminus of TFB Inhibit Binding of

RNAP Subunits—Intramolecular interactions of the N termi-nus with the C terminus of TFB molecules has been observedwith purified eukaryotic TFIIB (35). The conformation of iso-lated TFIIB is known to block biologically significant interac-tions like those with the acidic activator VP16, and with pol IIand TFIIF (35). These intramolecular interactions of purifiedTFIIB molecules can be counteracted by deletion of the N ter-minus of TFIIB, which is binding to the second direct repeat ofthe C-terminal domain of TFIIB.To investigate whether deletion of parts of the archaeal

TFB molecule leads also to improved binding to of TFB toRNAP subunits a series of N-terminal and C-terminal dele-tion mutants of Pyrococcus TFB were constructed (Fig. 6C).Subunits K and D were used as probes in Far-Western gelblots with truncated versions of TFB (Fig. 6). Subunit Kshowed a higher binding affinity to the N- and C-terminaltruncated mutant �39, 283–300 of TFB than to wild-type

TFB (Fig. 6B, compare lanes 1 and 7). This result indicatesthat deletions of TFB can lead to a conformational change inTFB structure supporting interactions with an RNAP sub-unit. A similar result was obtained when binding of subunitD to truncated TFB mutants was studied. Here, the N-termi-nal mutant �2–162 (Fig. 6A, lane 4) and the C-terminalmutant �201–300 (Fig. 6A, lane 6) showed stronger bindingsignals than wild-type TFB (Fig. 6A, lane 1). Deletionmutants of the zinc ribbon at the N terminus of the TFBmolecule, �1–39 (Fig. 6A, lane 2), and of both the zinc rib-bon and of the B-finger, �2–117 (Fig. 6A, lane 3), showed alower binding affinity to subunit D than WT-TFB (Fig. 6A,lane 1). The TFBmutant �2–199 lacking in addition the firstdirect repeat of TFB, displayed an even lower affinity to sub-unit D (Fig. 6A, lane 5). However, the zinc ribbon regionseems not to be necessary for interactions with subunits Dand K because�2–199 interacts like wild-type TFB with sub-unit K (Fig. 6B, lane 5) and �2–162 shows even a higherbinding activity to subunit D (Fig. 6A, lane 4) than the wild-type protein.The major conclusions derived from analysis of truncated

TFBmolecules are that deletions can change the conformationof the protein dramatically and can also enhance the bindingcapacity of themutant formof TFB at least in the case of TFB-Dinteractions by an unknown mechanism. Analysis of the dele-tionmutant�1–39,283–300 lacking parts of the N and of the Cterminus suggested that intramolecular interaction of the Cterminus and N terminus of TFB molecules are possiblyinvolved in inhibition of the TFB-K interaction, but do notinterfere strongly with TFB-D binding.Analyses of RNAP Subcomplexes—The interactions of some

components of the RNAPwere analyzed in addition by electro-phoresis in native polyacrylamide gels and by Co-immobiliza-tion assays on Ni2�-NTA columns to confirm and extend the

FIGURE 6. Interactions of subunits D and K with N- and C-terminally trun-cated versions of TFB. A and B, Far-Western gel blots of TFB (lane 1) andtruncated versions of TFB (lanes 2–7) probed with labeled subunit D (A) or K(B). C, schematic representation of various N- and C-terminal deletionmutants of TFB. Hatched region, C-terminal tag; Zn, zinc ribbon, the arrowsindicate the two direct repeats of TFB. Thin lanes indicate deleted regions; theboxes indicate the wild-type sequences. The labeling code (1–7) used formutants in C is also used in A and B.




http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

FIGURE 7. Identification of stable subcomplexes of subunits DL, E�F, co-immobilization of D and P, and identification of a stable BDLNP complex.A and B, native gel electrophoresis. The labeled subunit L (70 ng) was incubated for 2 h at 30 °C with increasing amounts of subunit D in TEN buffercontaining 10% (v/v) glycerol and 0.5% (v/v) Tween 20. Complex formation was then analyzed on a 8 –15% native PA gel. Lane 1, no D, lane 2, 9 ng of D;the reactions analyzed in lanes 3–10 contained increasing amounts of subunit D (5 ng for each reaction); B, the labeled subunit F (70 ng) was incubatedfor 90 min at 37 °C in TEN buffer containing 10% glycerol with increasing amounts of subunit E�. Lane 1, no E�, lane 2, 62 ng E�, reactions analyzed in lanes3–10 contained increasing amounts (62 ng per reaction) of subunit E�. C, co-immobilization of subunits D and P. His6-tagged subunit D (40 �g) wasimmobilized on a Ni2�-NTA resin. After extensive washing of the column, 40 �g of subunit P were added, the column was washed again and immobilizedprotein were eluted by a linear gradient ranging from 50 mM to 1000 mM imidazole. The proteins in the flow-through fraction and in the fractions elutingfrom the column were analyzed on an 8 –15% PA gel and stained with Coomassie Blue. Lane 1, standard proteins; lane 2, subunit D (fraction applied tothe column); lane 3, subunit P (fraction applied to the column); lane 4, flow-through fraction after addition of D; lane 5, flow-through fraction afteraddition of P; lanes 6 –11, fractions eluted by the imidazole gradient. D, identification of a BDLNP subcomplex. Isolated RNAP subunits were reconsti-tuted by denaturation and renaturation and complex formation was identified by Superdex 200 chromatography. The peak elution fractions of standardproteins are indicated by arrows. The fractions eluting from the column were analyzed on an 8 –20% PA gel and silver-stained. The fractions containingthe BDLNP subcomplex are indicated in the figure.




http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

results that had been produced by Far-Western blotting.Whenlabeled subunit L was incubated with subunit D the formationof a D-L complex was observed after gel electrophoresis (Fig.7A). A complex formation between labeled subunit F with sub-unit E� could be shown as well (Fig. 7B). These results confirmthat strong interactions between subunits D and L and E� and Fexist as observed by Far-Western analyses (see Figs. 5 and 6). Aninteraction of subunit Dwith subunit P could be shownwhenDwas immobilized by its His6-tag on a Ni2�-NTA column andsubunit P without His tag was added to the column (Fig. 7C).The co-elution of these proteins by a linear imidazole gradi-ent indicated that these polypeptides associate on the col-umn. A complex formation of subunit B with L was alsoshown by this co-immobilization assay (data not shown).These results are consistent with the Far-Western blottingexperiments in respect to binding of subunits P and D (Fig.5A). Binding of B to L and of B to P was only found when Bwas used as probe but not in the inverse experiment using Pand L as probe. Therefore, the B-L and B-P interactions areless certain and are indicated in Fig. 9 as unidirectionalbinding.Gel filtration experiments revealed that only a few soluble

subcomplexes are stable during chromatography. Complex for-mation between subunits D and L and between subunits E� andF was clearly demonstrated by Superdex 75 chromatography(data not shown). When the subunits B, D, L, N, and P wereincubated and applied to a Superdex 200 column a stableBDLNP complex could be identified (data not shown). Thesame complex was formed after denaturation of recombinantsubunits in 6 M urea and stepwise renaturation by dilution in

buffers containing 3M andnourea (“Experimental Procedures,”Fig. 7D). The same procedure was successfully used to recon-stitute a highly active RNAP from 11 isolated subunits5 but theDLNP complex observed during reconstitution of the Meth-anocaldococcus enzyme (17) could not be identified in recon-stitution experimentswith recombinantPyrococcusRNAP sub-units (Fig. 7D; data not shown). Most combinations of twosubunits were unstable and precipitated during size exclusionchromatography, e.g. when P was incubated with D, B, with A�and B with A� (data not shown).The BDLNP Subcomplex Associates with Promoter-bound

TBP-TFB—Analyses in the pol II system indicated that the twolargest subunits Rpb1, homologous to A� and A�, and Rpb2,homologous to subunits B of the archaeal enzyme (Figs. 1, 3,and 4), are mainly involved in binding of transcription factors(19, 36). The archaeal RNAP forms a large complex of low elec-trophoretic mobility in mobility shift assays with promoterbound TBP-TFB (13; Fig. 8, lane 6). To investigate whether thisisolated BDLNP complex can associate with the promoter-bound TBP-TFB platform, the subcomplex was added to DNAbinding reactions containing TBP and TFB and DNA alone.The BDLNP subcomplex produced a smear with a probe har-boring the Pyrococcus gdh promoter (Fig. 8, lane 3) suggestingthat it interactedweakly and in a nonspecificmannerwithDNA(compare lanes 1 and 3). TBP-TFB formadistinct complexwiththe gdh promoter (Ref. 32; Fig. 8, lanes 4 and 5). When thepurified subcomplex was added to reactions containing TBPand TFB a third complex was observed that showed lower elec-trophoretic mobility than the DNA-TBP-TFB complex (com-pare lanes 4 and 5 with lane 6). The purified RNAP forms aslower migrating complex indicating the difference in molecu-lar mass between the subcomplex and the complete enzyme(Fig. 8, lane 2). This finding demonstrated that the BDLNPsubcomplex was able to form a stable complex with promoter-bound transcription factors TBP and TFB.

5 S. Naji, manuscript in preparation.

FIGURE 8. The BDLNP subcomplex is recruited by promoter-bound TBP-TFB. The renatured BDLNP complex purified by Superdex 200 chromatogra-phy was incubated in DNA binding reactions with a DNA probe containingthe Pyrococcus gdh promoter in the presence and absence of TBP and TFB.Binding of the subcomplex occurs only to TBP-TFB-DNA complexes. The pres-ence of individual components of the transcriptional machinery in bindingreactions is indicated on top of each lane. In lanes 4 and 5, the Superdexfractions 18 and 20 from Fig. 7D were analyzed.

FIGURE 9. Schematic representation of interaction of subunits in pol IIand the archaeal enzyme. Right panel, modified interaction diagram of pol IIsubunits based on the crystal structure of pol II according to Ref. 1 and con-sidering the interactions of Rpb3 and Rpb7 according to Ref. 2. The color codeis the same as in Figs. 1 and 4. Left panel, interaction diagram of Pyrococcussubunits based on Far-Western analyses. The homology of subunits to pol II isindicated by the color code. The thickness of the lines connecting the sub-units gives an estimation of the strength of interactions. Connecting lines inblue color indicate an interaction established by one labeled probe, connect-ing lines in red color indicate interactions established by both interactingpartners as probes.




http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

Our results show that subunits A�, A�, H, K, E�, and F are notrequired for RNAP recruitment. Rpb1 represent the majormass of pol II in the region below the cleft. It forms an essentialpart of the clamp, of the active center and of the upper andlower jaw. More importantly, the “dock” domain formed byRpb1 has been suggested as binding site of TFIIB in the preini-tiation complex (1, 2). Rpb1 andRpb2 of pol II were found in theeukaryotic preinitiation complex in close proximity of the threeTFIIB domains (19, 36). Hence, our finding that stable recruit-ment to promoter-bound factors requires only subunit B (theRpb2 ortholog) bound to the D-L-N-P anchor is highly unex-pected. Our novel findings suggests that the dock domain is notessential for RNAP binding and that the important interactionssites with promoter-bound transcription factors reside in theouter surface of the B-D-L-N-P (Rpb 2-3-10-11-12) subcom-plex (Fig. 7). The gel-shift assay established for binding of theB-D-L-N-P subcomplex to promoter-bound TBP-TFB (Fig. 8)might be a useful tool to identify the sites involved in recruit-ment of a pol II-like RNAP by mutational analyses.

REFERENCES1. Cramer, P., Bushnell, D. A., and Kornberg, R. D. (2001) Science 292,

1863–18762. Armache, K. J., Kettenberger, H., and Cramer, P. (2003) Proc. Natl. Acad.

Sci. U. S. A. 100, 6964–69683. Todone, F., Brick, P., Werner, F., Weinzierl, R. O. J., and Onesti, S. (2001)

Mol. Cell 8, 1137–11434. Lanzendorfer, M., Langer, D., Hain, J., Klenk, H. P., Holz, I., Arnold-

Ammer, I., and Zillig, W. (1994) System. Appl. Microbiol. 16, 654–6645. Puhler, G., Leffers, H., Gropp, F., Palm, P., Klenk, H. P., Lottspeich, F.,

Garrett, R. A., and Zillig, W. (1989) Proc. Natl. Acad. Sci. U. S. A. 86,4569–4573

6. Schnabel, R., Thomm, M., Gerardy-Schahn, R., Zillig, W., Stetter, K. O.,and Huet, J. (1983) EMBO J. 2, 751–755

7. Hausner, W., Wettach, J., Hethke, C., and Thomm, M. (1996) J. Biol.Chem. 271, 30144–30148

8. Bell, S. D., and Jackson, S. P. (2000) J. Biol. Chem. 275, 12934–129409. Geiduschek, E. P., and Ouhammouch, M. (2005) Mol. Microbiol. 56,

1397–140710. Werner, F., and Weinzierl, R. O. J. (2005)Mol. Cell. Biol. 25, 8344–835511. Bell, S. D., Brinkman, A. B., van der Oost, J., and Jackson, S. P. (2001)

EMBO Rep. 2, 133–13812. Hanzelka, B. L., Darcy, T. J., and Reeve, J. N. (2001) J. Bacteriol. 183,

1813–181813. Bartlett, M. S., Thomm, M., and Geiduschek, E. P. (2004) J. Biol. Chem.

279, 5894–590314. Renfrow, M. B., Naryshkin, N., Lewis, L. M., Chen, H. T., Ebright, R. M.,

and Scott, R. A. (2004) J. Biol. Chem. 279, 2825–283115. Bartlett, M. S. (2005) Curr. Opin. Microbiol. 8, 1–816. Werner, F., Eloranta, J. J., andWeinzierl, R. O. J. (2000)Nucleic Acids Res.

28, 4299–430517. Werner, F., and Weinzierl, R. O. J. (2002)Mol. Cell 10, 635–64618. Hethke, C., Geerling, A. C. M., Hausner, W., de Vos, W., and Thomm,M.

(1996) Nucleic Acids Res. 24, 2369–237619. Chen, H.-T., and Hahn, S. (2004) Cell 119, 169–18020. Spitalny, P., and Thomm, M. (2003) J. Biol. Chem. 278, 30497–3050521. Burgess, R. R. (1996)Methods Enzymol. 273, 14522. Arthur, T. M., and Burgess, R. R. (1998) J. Biol. Chem. 273, 31381–3138723. Burgess, R. R., Arthur, T. M., and Pietz, B. C. (2000) Methods Enzymol.

328, 14124. Ehlers, C., Grabbe, R., Veit, K., and Schmitz, R. A. (2002) J. Bacteriol. 184,

1028–104025. Meka, H.,Werner, F., Cordell, S. C., Onesti, S., and Brick, P. (2005)Nucleic

Acids Res. 33, 6435–644426. Eloranta, J. J., Kato, A., Teng, M. S., and Weinzierl, R. O. J. (1998) Nucleic

Acids Res. 26, 5562–556727. Kim, T.-K., Ebright, R. H., and Reinberg, D. (2000) Science 288,

1418–142228. Cramer, P., Bushnell, D. A., Fu, J., Gnatt, A. L., Maier-Davis, B., Thomp-

son, N. E., Burgess, R. R., Edwards, A.M., David, P. R., and Kornberg, R. D.(2000) Science 288, 640–649

29. Bushnell, D. A., and Kornberg, R. D. (2003) Proc. Natl. Acad. Sci. U. S. A.100, 6969–6973

30. Choder, M. (2004) Trends Biochem. Sci. 29, 674–68131. Jasiak, A. J., Armache, K. J., Martens, B., Jansen, R. P., and Cramer, P.

(2006)Mol. Cell 23, 1–1132. Vierke, G., Engelmann, A., Hebbeln, C., and Thomm, M. (2003) J. Biol.

Chem. 278, 18–2633. Miyao, T., and Woychik, N. A. (1998) Proc. Natl. Acad. Sci. U. S. A. 95,

15281–1528634. Magill, C. P., Jackson, S. P., and Bell, S. D. (2001) J. Biol. Chem. 276,

46693–4669635. Roberts, S. G. E., and Green, M. R. (1994) Nature 371, 717–72036. Chen, H.-T., and Hahn, S. (2003)Mol. Cell 12, 437–447




http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

Bernd Goede, Souad Naji, Oliver von Kampen, Karin Ilg and Michael ThommTRANSCRIPTION FACTORS

BINDING STUDIES OF ISOLATED RNA POLYMERASE SUBUNITS AND Protein-Protein Interactions in the Archaeal Transcriptional Machinery:

doi: 10.1074/jbc.M605209200 originally published online August 1, 20062006, 281:30581-30592.J. Biol. Chem.

10.1074/jbc.M605209200Access the most updated version of this article at doi:

Alerts:

When a correction for this article is posted•

When this article is cited•

to choose from all of JBC's e-mail alertsClick here

Supplemental material:

http://www.jbc.org/content/suppl/2006/08/09/M605209200.DC1

http://www.jbc.org/content/281/41/30581.full.html#ref-list-1

This article cites 36 references, 19 of which can be accessed free at


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/lookup/doi/10.1074/jbc.M605209200

http://www.jbc.org/cgi/alerts?alertType=citedby&addAlert=cited_by&cited_by_criteria_resid=jbc;281/41/30581&saveAlert=no&return-type=article&return_url=http://www.jbc.org/content/281/41/30581

http://www.jbc.org/cgi/alerts?alertType=correction&addAlert=correction&correction_criteria_value=281/41/30581&saveAlert=no&return-type=article&return_url=http://www.jbc.org/content/281/41/30581

http://www.jbc.org/cgi/alerts/etoc

http://www.jbc.org/content/suppl/2006/08/09/M605209200.DC1

http://www.jbc.org/content/281/41/30581.full.html#ref-list-1

http://www.jbc.org/

Protein-Protein Interactions in the Archaeal Transcriptional Machinery

Documents

Transcript of Protein-Protein Interactions in the Archaeal Transcriptional Machinery