FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2069174810> ?p ?o ?g. }

• W2069174810 endingPage "30592" @default.
• W2069174810 startingPage "30581" @default.
• W2069174810 abstract "Transcription in Archaea is directed by a pol II-like RNA polymerase and homologues of TBP and TFIIB (TFB) but the crystal structure of the archaeal enzyme and the subunits involved in recruitment of RNA polymerase to the promoter-TBP-TFB-complex are unknown. We described here the cloning expression and purification of 11 bacterially expressed subunits of the Pyrococcus furiosus RNAP. Protein interactions of subunits with each other and of archaeal transcription factors TFB and TFB with RNAP subunits were studied by Far-Western blotting and reconstitution of subcomplexes from single subunits in solution. In silico comparison of a consensus sequence of archaeal RNAP subunits with the sequence of yeast pol II subunits revealed a high degree of conservation of domains of the enzymes forming the cleft and catalytic center of the enzyme. Interaction studies with the large subunits were complicated by the low solubility of isolated subunits B, A′, and A″, but an interaction network of the smaller subunits of the enzyme was established. Far-Western analyses identified subunit D as structurally important key polypeptide of RNAP involved in interactions with subunits B, L, N, and P and revealed also a strong interaction of subunits E′ and F. Stable complexes consisting of subunits E′ and F, of D and L and a BDLNP-subcomplex were reconstituted and purified. Gel shift analyses revealed an association of the BDLNP subcomplex with promoter-bound TBP-TFB. These results suggest a major role of subunit B (Rpb2) in RNAP recruitment to the TBP-TFB promoter complex. Transcription in Archaea is directed by a pol II-like RNA polymerase and homologues of TBP and TFIIB (TFB) but the crystal structure of the archaeal enzyme and the subunits involved in recruitment of RNA polymerase to the promoter-TBP-TFB-complex are unknown. We described here the cloning expression and purification of 11 bacterially expressed subunits of the Pyrococcus furiosus RNAP. Protein interactions of subunits with each other and of archaeal transcription factors TFB and TFB with RNAP subunits were studied by Far-Western blotting and reconstitution of subcomplexes from single subunits in solution. In silico comparison of a consensus sequence of archaeal RNAP subunits with the sequence of yeast pol II subunits revealed a high degree of conservation of domains of the enzymes forming the cleft and catalytic center of the enzyme. Interaction studies with the large subunits were complicated by the low solubility of isolated subunits B, A′, and A″, but an interaction network of the smaller subunits of the enzyme was established. Far-Western analyses identified subunit D as structurally important key polypeptide of RNAP involved in interactions with subunits B, L, N, and P and revealed also a strong interaction of subunits E′ and F. Stable complexes consisting of subunits E′ and F, of D and L and a BDLNP-subcomplex were reconstituted and purified. Gel shift analyses revealed an association of the BDLNP subcomplex with promoter-bound TBP-TFB. These results suggest a major role of subunit B (Rpb2) in RNAP recruitment to the TBP-TFB promoter complex. Archaeal RNA polymerases (RNAP) 2The abbreviations used are: RNAP, Archaeal RNA polymerases; PMSF, phenylmethylsulfonyl fluoride; TBP, TATA-binding protein; pol, polymerase; PDB, Protein Data Bank; TFB, transcription factor B. 2The abbreviations used are: RNAP, Archaeal RNA polymerases; PMSF, phenylmethylsulfonyl fluoride; TBP, TATA-binding protein; pol, polymerase; PDB, Protein Data Bank; TFB, transcription factor B. are multisubunit enzymes that resemble in sequence subunit composition and functional aspects eukaryotic RNAP. Fig. 1 shows the subunit structure of eukaryotic RNA polymerase II (pol II), of the Pyrococcus RNAP and of the RNAP from Escherichia coli. Homologous subunits are indicated by the same colors. Archaeal RNAP display greater similarities with all eukaryotic RNAP than with the four subunits of the bacterial core enzyme. We refer here mainly to pol II as the subunit interactions within this enzyme are known from the crystal structure (1Cramer P. Bushnell D.A. Kornberg R.D. Science. 2001; 292: 1863-1876Crossref PubMed Scopus (954) Google Scholar, 2Armache K.J. Kettenberger H. Cramer P. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 6964-6968Crossref PubMed Scopus (194) Google Scholar). Archaeal RNAPs have clear homologues to Rpb4 and Rpb7 of pol II, which were first called F and E (3Todone F. Brick P. Werner F. Weinzierl R.O.J. Onesti S. Mol. Cell. 2001; 8: 1137-1143Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). In the genomes of most Archaea, the gene encoding E overlaps at its 3′-end in a different reading frame with a second gene containing a zinc finger motif. To discriminate between the first gene that is homologous to rpb7 and the second gene that has no homolog in yeast but is highly conserved in Archaea, the first one is designated in data bases as rpoE′ and the second one as rpoE″, and the corresponding proteins as E′ and E″. Only E′ has been detected in purified archaeal RNAP. Two subunits shared by all three eukaryotic RNAP, Rpb12, and Rpb10 have the subunits P and N as archaeal homologues but no bacterial homologues. In addition, subunit H of the archaeal enzyme has a homologue in pol II (and also in pol I and pol III) but not in the bacterial enzyme.The gene encoding the largest subunit in eukaryotic RNAP, Rpo1 and β′ of the E. coli enzyme is split into two genes encoding subunits A′ and A″ in all Archaea. The RNAP of Pyrococcus and of Crenarchaeota show the subunit composition BA′A″ DE′FLHNKP (4Lanzendörfer M. Langer D. Hain J. Klenk H.P. Holz I. Arnold-Ammer I. Zillig W. System. Appl. Microbiol. 1994; 16: 654-664Google Scholar, 5Pühler G. Leffers H. Gropp F. Palm P. Klenk H.P. Lottspeich F. Garrett R.A. Zillig W. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 4569-4573Crossref PubMed Scopus (187) Google Scholar). In methanogens and extreme halophilic Archaea subunit B is split into the subunits B′ and B″ (6Schnabel R. Thomm M. Gerardy-Schahn R. Zillig W. Stetter K.O. Huet J. EMBO J. 1983; 2: 751-755Crossref PubMed Google Scholar). This B split defines the second major type of archaeal RNAP with the subunit composition A′B′B″A″DE′FLHNPK.The archaeal RNAP is recruited to the preinitiation complex by association to promoter-bound transcription factors TBP and TFB (7Hausner W. Wettach J. Hethke C. Thomm M. J. Biol. Chem. 1996; 271: 30144-30148Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 8Bell S.D. Jackson S.P. J. Biol. Chem. 2000; 275: 12934-12940Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar), which are interacting with the TATA-box and BRE element of archaeal promoters (reviewed in Ref. 9Geiduschek E.P. Ouhammouch M. Mol. Microbiol. 2005; 56: 1397-1407Crossref PubMed Scopus (120) Google Scholar). Both TBP and TFB consist of two imperfect direct repeats. TFB has in addition an N-terminal domain forming a zinc ribbon and a B-finger (see Figs. 6C, 9, and 10). A third archaeal transcription factor, TFE, is homologous to the N-terminal part of subunit α of eukaryotic TFIIE (11Bell S.D. Brinkman A.B. van der Oost J. Jackson S.P. EMBO Rep. 2001; 2: 133-138Crossref PubMed Scopus (80) Google Scholar, 12Hanzelka B.L. Darcy T.J. Reeve J.N. J. Bacteriol. 2001; 183: 1813-1818Crossref PubMed Scopus (66) Google Scholar). TFE is not required for promoter-directed transcription but can stimulate the activity of some promoters by a factor of 3–4. TFE can also complement some mutants of TFB indicating that these proteins interact synergistically and contribute to catalytic core functions of RNAP (10Werner F. Weinzierl R.O.J. Mol. Cell. Biol. 2005; 25: 8344-8355Crossref PubMed Scopus (89) Google Scholar).FIGURE 6Interactions of subunits D and K with N- and C-terminally truncated versions of TFB. A and B, Far-Western gel blots of TFB (lane 1) and truncated versions of TFB (lanes 2–7) probed with labeled subunit D (A) or K (B). C, schematic representation of various N- and C-terminal deletion mutants of TFB. Hatched region, C-terminal tag; Zn, zinc ribbon, the arrows indicate the two direct repeats of TFB. Thin lanes indicate deleted regions; the boxes indicate the wild-type sequences. The labeling code (1–7) used for mutants in C is also used in A and B.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 9Schematic representation of interaction of subunits in pol II and the archaeal enzyme. Right panel, modified interaction diagram of pol II subunits based on the crystal structure of pol II according to Ref. 1Cramer P. Bushnell D.A. Kornberg R.D. Science. 2001; 292: 1863-1876Crossref PubMed Scopus (954) Google Scholar and considering the interactions of Rpb3 and Rpb7 according to Ref. 2Armache K.J. Kettenberger H. Cramer P. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 6964-6968Crossref PubMed Scopus (194) Google Scholar. The color code is the same as in Figs. 1 and 4. Left panel, interaction diagram of Pyrococcus subunits based on Far-Western analyses. The homology of subunits to pol II is indicated by the color code. The thickness of the lines connecting the subunits gives an estimation of the strength of interactions. Connecting lines in blue color indicate an interaction established by one labeled probe, connecting lines in red color indicate interactions established by both interacting partners as probes.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The path of the DNA in the Pyrococcus RNAP has been studied by photochemical cross-linking (13Bartlett M.S. Thomm M. Geiduschek E.P. J. Biol. Chem. 2004; 279: 5894-5903Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 14Renfrow M.B. Naryshkin N. Lewis L.M. Chen H.T. Ebright R.M. Scott R.A. J. Biol. Chem. 2004; 279: 2825-2831Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, 15Bartlett M.S. Curr. Opin. Microbiol. 2005; 8: 1-8Crossref PubMed Scopus (38) Google Scholar). These studies revealed that subunit B of Pyrococcus RNAP cross-links the RNAP between the TATA-box and the transcription start site and that subunits A′, A″, and H contact the DNA downstream of the start site. In vivo and in vitro binding assays were used to investigate the interactions of subunits of the RNAP from Methanocaldococcus jannaschii. The eukaryotic subunits Rpb4 and Rbp7 form a heterodimer that reversibly associated with the pol II core. As predicted from the similarity to the eukaryotic system the archaeal homologues of these polypeptides, E and F, form a complex (3Todone F. Brick P. Werner F. Weinzierl R.O.J. Onesti S. Mol. Cell. 2001; 8: 1137-1143Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar) and archaeal F interacted with human Rpb7 to form an archaeal-human F-Rpb7 hybrid (16Werner F. Eloranta J.J. Weinzierl R.O.J. Nucleic Acids Res. 2000; 28: 4299-4305Crossref PubMed Scopus (49) Google Scholar). Subunits D, L, N, and P were shown to associate to a tetrameric D-L-N-P complex (16Werner F. Eloranta J.J. Weinzierl R.O.J. Nucleic Acids Res. 2000; 28: 4299-4305Crossref PubMed Scopus (49) Google Scholar). The eukaryotic homologues of these subunits, Rbp3, Rpb10, Rpb11, and Rpb12 are in close interaction and clustered together in the pol II structure (1Cramer P. Bushnell D.A. Kornberg R.D. Science. 2001; 292: 1863-1876Crossref PubMed Scopus (954) Google Scholar). This assembly of the archaeal subunits D-L-N-P was able to recruit the largest subunit B in vitro and used as a frame for the reconstitution of active M. jannaschii RNAP from individual subunits (17Werner F. Weinzierl R.O.J. Mol. Cell. 2002; 10: 635-646Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar).We are exploring the mechanism and regulation of transcription in Pyrococcus using a cell-free transcription system (7Hausner W. Wettach J. Hethke C. Thomm M. J. Biol. Chem. 1996; 271: 30144-30148Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 18Hethke C. Geerling A.C.M. Hausner W. de Vos W. Thomm M. Nucleic Acids Res. 1996; 24: 2369-2376Crossref PubMed Scopus (68) Google Scholar, 20Spitalny P. Thomm M. J. Biol. Chem. 2003; 278: 30497-30505Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). We report here cloning and expression of all RNAP subunits from Pyrococcus. The interaction of these polypeptides with each other and with the archaeal transcription factors TBP and TFB were studied by far-Western analyses, column chromatography and gel electrophoresis. Our results reveal many interactions predicted from the structural similarities to the pol II system, the existence of various subcomplexes and an interaction of the BDLNP subcomplex with promoter-bound TBP and TFB.EXPERIMENTAL PROCEDURESCloning of RNAP Subunits—The coding region of RNAP subunits 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 DSMZ 3638, were PCR-amplified using genomic DNA as template. The oligonucleotides were complementary to the 5′- and 3′-ends of the genes and contained the restrictions sites NdeI at the 5′-end and BamHI at the 3′-end. The PCR fragments encoding subunits B, D, E′, F, L, H, K, and P were cloned into the corresponding restriction sites of the expression vector pET-33b (Novagen). The expressed proteins carry a His6 tag and a recognition site for heart muscle kinase (HMK) at the N terminus. The PCR products encoding subunits F and H were also cloned in a modified version of pET-33b resulting in proteins containing the His6 tag and the HMK site at the C terminus. Details of the modified vector and of the oligonucleotide sequences are available from the authors on request. The PCR fragments encoding subunits A′ and A″ were cloned in the NdeI site and BamHI site of pET-14b. The expressed subunits A′ and A″ contained only a His6 tag at the N terminus and were used as targets in Far-Western experiments. To obtain subunits A′ and A″ with a HMK site in addition, the PCR products containing the HMK recognition site at the 5′-end were cloned into pET151/D-TOPO (Invitrogen).Identification of a Consensus Sequence for Subunits of Archaeal RNAP and Bioinformatic Work—Multiple sequence alignments of the genes encoding RNAP subunits of up to 18 Archaea and of the subunits of pol II from S. cerevisiae revealed an amino acid consensus sequence for each subunit with the exception of Rbp8 and Rpb9, which have no homologues in Archaea. Most of the genes encoding archaeal RNAP subunits were extracted from whole genome files available at NCBI or at SRS. BLAST search and other common bioinformatic resources were used to identify unannotated entries. A number of missing genes became available by local BLAST search in Bioedit on the basis of the whole genome file in raw format. Multiple sequence alignments were carried out using Malign, an algorithm especially suitable when genes are compared that show low sequence similarities 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 carried out and combined in a subsequent step to obtain better results. MAlign2Msf was used to convert the data into the MSF file type. After import into Bioedit the data were formatted, a consensus sequence was generated and shading was applied. As final step export as RTF file and import into MS Word was performed. The consensus sequence of each alignment was also used to generate two-dimensional similarity diagrams (Fig. 3) and to visualize the distribution of identities and similarities in the three-dimensional model of S. cerevisiae polII (PDB: 1NT9; Fig. 4). A small Delphi program was written to draw the two-dimensional diagrams and as a helper tool for sequence analysis and various conversion steps. In the diagrams a vertical line represents identity between the archaeal consensus and the amino acid sequence of pol II. Lines of half-length indicate similarities. To visualize the homologous regions of archaeal RNAPs and of pol II in the three-dimensional structure of pol II, the consensus sequence for each subunit was converted in a ProSite search pattern and applied to the S. cerevisiae pol II model (PDB: 1NT9) using the Cn3D 4.1 annotate function.FIGURE 3The consensus of archaeal RNAP subunits shows extensive homology to domains of the active core of pol II. The consensus sequence for the genes encoding archaeal RNAP subunits was aligned with the homologous eukaryotic subunits. The genes encoding the subunits of yeast pol II are shown in the 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 encoding A″ 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 blue boxes. 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 structure of pol II (1Cramer P. Bushnell D.A. Kornberg R.D. Science. 2001; 292: 1863-1876Crossref PubMed Scopus (954) Google Scholar); 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, comparison of 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 subunit Rpb2 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 with subunits 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, domain 2; 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 helices in 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 of subunits 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.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 4Visualization of regions conserved between pol II and the archaeal enzyme in the crystal structure of pol II. A, upper panel, structure of single subunits of pol II and lower panel, crystal structure of pol II holoenzyme as described in Ref. 2Armache K.J. Kettenberger H. Cramer P. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 6964-6968Crossref PubMed Scopus (194) Google Scholar. The color of the subunits is like in Fig. 1. B, the regions of pol II identical in sequence to the corresponding archaeal subunit are shown in yellow, regions showing high similarity are shown in green, sequences with no sequence 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 shown as 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 of subunit 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 archaeal enzyme was removed from the model.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Expression and Purification of Proteins—For the expression of the proteins the plasmids were transformed in the expression 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 cultures with 1 mm isopropyl-1-thio-β-d-galactopyranoside for 3 h. For Far-Western dot blot experiments (Fig. 5A, first three panels), subunit B was purified after SDS-polyacrylamide gel electrophoresis. Gel slices containing this subunit were incubated in a solution containing 0.1% (w/v) SDS. Then, SDS was precipitated and the protein refolded by dilution and dialysis as described by Ref. 21Burgess R.R. Methods Enzymol. 1996; 273: 145Crossref PubMed Google Scholar. Subunit B used as probe (Fig. 5A, lower panel) for dot blots and B used for gel blots and for reconstitution of the DLNPB subcomplex and subunits 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/ml lysozyme) and sonicated. After centrifugation the pellets containing the inclusion bodies were extensively washed in purification buffer (20 mm Tris-HCl, pH 8, 0.5 m NaCl, 0.1% Tween 20, 1 mm PMSF, and 5 mm 2-mercaptoethanol) The inclusion bodies were solubilized in binding buffer (20 mm Tris-HCl, pH 8, 0.5 m NaCl, 5 mm imidazole, 6 m guanidine HCl, 1 mm PMSF, 5 mm 2-mercaptoethanol) for 1 h at 20 °C. After centrifugation the supernatant was loaded onto a Ni2+-NTA column (HisTrapFF, GE healthcare) and washed with binding buffer containing 6 m urea and no guanidine HCl. The refolding of the bound protein was performed on column using a decreasing linear urea gradient (10 column volumes) ranging from 6 m to 0. The refolded proteins were eluted with an imidazole gradient ranging from 5 to 300 mm imidazole. For the purification of subunits D, L, N, E′, F, K, H, and P cells were resuspended in a buffer containing 50 mm NaPO4, pH 8, 10 mm imidazole, 300 mm NaCl, and 10% v/v glycerol. Cells were disrupted by passage through a French pressure cell. The lysate was clarified by centrifugation at 100,000 × g for 20 min at 4 °C. The supernatant was directly applied to a Ni2+-NTA column (subunits D, E′, K) or after heating for 20 min at 80 °C (subunits F, H, L, N, and P) and separation of precipitated E. coli proteins by centrifugation. Bound proteins were stepwise eluted with 300 mm imidazole. Some subunits were further purified by MonoQ- or Superdex75-chromatography.FIGURE 5Interactions of isolated RNAP subunits analyzed by Far-Western blotting. A, dot blot, recombinant RNAP subunits and TBP And TFB were spotted 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 was detected by autoradiography. B and C, Far-Western gel blot of purified RNAP and isolated subunits after separation on an 8–15% denaturing polyacrylamide gel.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Far-Western Blotting—The whole procedure was a variation of the protocols described by Arthur and Burgess (22Arthur T.M. Burgess R.R. J. Biol. Chem. 1998; 273: 31381-31387Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar) and Burgess et al. (23Burgess R.R. Arthur T.M. Pietz B.C. Methods Enzymol. 2000; 328: 141Crossref PubMed Google Scholar). Dot blot-solubilized proteins were spotted onto a nitrocellulose membrane (Optitran BA-S 85 Reinforced NC, Schleicher and Schüll, order number 439196). The affinity of RNAP subunits to bind to the membrane and/or to detach from the membrane during the following incubations steps differed considerably. In particular subunit B and H showed a tendency to detach from the membrane during the following incubation. This detachment was inhibited by drying the membranes after spotting of the proteins briefly at 50 °C. To control the amount of proteins used for the protein-protein interactions studies proteins were spotted in parallel on two membranes for each experiments and one membrane was stained with Ponceau S and the second used for the Dot Blot. 0.5 to 3 μg of protein was spotted for each individual subunit onto the membrane to obtain similar signals with Ponceau S staining. In particular the amount of subunits A′, A″, F, and H added to the membrane was higher, but also somewhat higher amounts of subunits B, P, and purified RNAP were spotted onto the membrane to obtain similar staining signals with Ponceau S. After drying the membrane used for the dot blot was blocked and subunit B refolded by incubation overnight in probing buffer (20 mm HEPES, pH 7.2; 200 mm KCl, 2 mm MgCl2, 0.1 mm ZnCl2, 1 mm dithiothreitol; 0.5% (v/v) Tween 20, 1% (w/v) nonfat-dried milk and 10% (v/v) glycerol (23Burgess R.R. Arthur T.M. Pietz B.C. Methods Enzymol. 2000; 328: 141Crossref PubMed Google Scholar). After probing and autoradiography the amount of protein on the membrane was controlled by staining with Ponceau S.Gel Blot—Cloned RNAP subunits or purified RNAP were electrophoretically transferred (Semidry system Bio-Rad) after SDS-polyacrylamide electrophoresis to nitrocellulose membranes (PROTRAN BA75 0.05 μm; Schleicher and Schüll, order number 10402196). Proteins on the membrane were refolded by incubation overnight in probing buffer. The transfer of proteins onto the membranes was verified after probing and autoradiography by staining with Ponceau S.Labeling of Probes—70 pmol RNAP subunit was incubated with 20 μCi of [γ-32P]ATP (6000 Ci/mmol) and 10 units of HMK (Novagen) in a total volume of 10 μl of the buffer supplied with the enzyme for 70 min at 30 °C.Probing—The blocked nitrocellulose membrane was incubated in 10 ml of probing buffer containing 10 μCi of 32P-labeled RNAP subunits for 2 h at 4–8°C. The blot was washed two times in probing buffer dried and exposed to a Phosphoimager (FLA-5000, Fuji).Electrophoresis under Non-denaturating Conditions—Interactions of subunits D and L and E′ and F were investigated by electrophoresis in native 8–15% polyacrylamide gels as described in Ref. 24Ehlers C. Grabbe R. Veit K. Schmitz R.A. J. Bacteriol. 2002; 184: 1028-1040Crossref PubMed Scopus (24) Google Scholar.Reconstitution of BDLNP RNAP Subcomplexes—Equimolar amounts (2.5 nmol) of RNAP subunits were incubated in transcription buffer (40 mm HEPES, pH 7.3, 250 mm NaCl, 2.5 mm MgCl2, 10% (v/v) glycerol, 1 mm EDTA, 1 mm PMSF, 5 mm 2-mercaptoethanol) for 1 h at 20°C. The complex formation was analyzed by Superdex 200 (GE Healthcare) column chromatography. Alternatively, the BDLNP complex was reconstituted by denaturation and renaturation of recombinant subunits. 2.5 nmol of subunits B and D and 5 nmol of subunits N, L, and P were combined in a final volume of 500 μl of transcription buffer containing in addition 6 m urea. The mixture was transferred to a dialysis frame (Slide-A-Lyzer 3.5k, Pierce) and incubated for 20 min at 20 °C. Then, the mixture was dialyzed against transcription buffer containing 3 m urea for 20 min at 20 °C. Renaturation was achieved by dialysis in transcription buffer for 1 h. The renaturated subcomplexes were heated for 10 min at 70 °C to remove misfolded aggregates. The BDLNP subcomplex 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 DNA by PCR. 90 bp of 32P-labeled DNA fragments encoding the promoter region from position –60 to + 30 were end-labeled with T4 polynucleotide kinase (MBI Fermentas) according to the instructions of the manufacturer. The labeled DNA was purified using mini Quick Spin Columns (Qiagen). DNA binding reactions were conducted for 30 min at 70 °C in a 12.5-μl volume of transcription buffer containing in addition 0.1 mg/ml bovine serum albumin, 240 nm TBP, 60 nm TFB, and 8.6 nm RNAP, or 100 nm BDLNP subcomplex. The reactions were loaded onto a native 4% polyacrylamide gel (buffer containing 25 mm Tris-HCl, pH 8.5, 10% glycerol, and 0.5 mm 2-mercaptoethanol), electrophoresed at room temperature and analyzed by phosphorimaging.RESULTS AND DISCUSSIONThe Archaeal RNAP Displays High Sequence Similarity with the Catalytic Core of Pol II—To investigate the molecular architecture of an archaeal RNAP we cloned and expressed 11 subunits of the Pyrococcus RNAP (Figs. 1 and 2) ranging in molecular mass from 127 003 (subunit B) to 5757 (subunit P). The sequences of the genes encoding these subunits were aligned with the sequences of subuni" @default.
• W2069174810 created "2016-06-24" @default.
• W2069174810 creator A5010525072 @default.
• W2069174810 creator A5018631374 @default.
• W2069174810 creator A5041952807 @default.
• W2069174810 creator A5046738629 @default.
• W2069174810 creator A5063401302 @default.
• W2069174810 date "2006-10-01" @default.
• W2069174810 modified "2023-09-29" @default.
• W2069174810 title "Protein-Protein Interactions in the Archaeal Transcriptional Machinery" @default.
• W2069174810 cites W1519016390 @default.
• W2069174810 cites W1535071457 @default.
• W2069174810 cites W1598239857 @default.
• W2069174810 cites W1967938775 @default.
• W2069174810 cites W1968493087 @default.
• W2069174810 cites W1985315760 @default.
• W2069174810 cites W1992413811 @default.
• W2069174810 cites W1993786968 @default.
• W2069174810 cites W1998239575 @default.
• W2069174810 cites W2000668418 @default.
• W2069174810 cites W2005744558 @default.
• W2069174810 cites W2013759002 @default.
• W2069174810 cites W2039922463 @default.
• W2069174810 cites W2041757320 @default.
• W2069174810 cites W2056416339 @default.
• W2069174810 cites W2059691911 @default.
• W2069174810 cites W2071325427 @default.
• W2069174810 cites W2074206760 @default.
• W2069174810 cites W2081288683 @default.
• W2069174810 cites W2084648126 @default.
• W2069174810 cites W2092238955 @default.
• W2069174810 cites W2092558918 @default.
• W2069174810 cites W2093206038 @default.
• W2069174810 cites W2099374359 @default.
• W2069174810 cites W2099775990 @default.
• W2069174810 cites W2104400758 @default.
• W2069174810 cites W2121534706 @default.
• W2069174810 cites W2122212389 @default.
• W2069174810 cites W2127696480 @default.
• W2069174810 cites W2144449918 @default.
• W2069174810 cites W2144926272 @default.
• W2069174810 cites W2151527406 @default.
• W2069174810 cites W2154379317 @default.
• W2069174810 cites W2157455363 @default.
• W2069174810 cites W960870541 @default.
• W2069174810 doi "https://doi.org/10.1074/jbc.m605209200" @default.
• W2069174810 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/16885163" @default.
• W2069174810 hasPublicationYear "2006" @default.
• W2069174810 type Work @default.
• W2069174810 sameAs 2069174810 @default.
• W2069174810 citedByCount "37" @default.
• W2069174810 countsByYear W20691748102012 @default.
• W2069174810 countsByYear W20691748102013 @default.
• W2069174810 countsByYear W20691748102014 @default.
• W2069174810 countsByYear W20691748102015 @default.
• W2069174810 countsByYear W20691748102016 @default.
• W2069174810 countsByYear W20691748102017 @default.
• W2069174810 countsByYear W20691748102018 @default.
• W2069174810 countsByYear W20691748102020 @default.
• W2069174810 countsByYear W20691748102022 @default.
• W2069174810 crossrefType "journal-article" @default.
• W2069174810 hasAuthorship W2069174810A5010525072 @default.
• W2069174810 hasAuthorship W2069174810A5018631374 @default.
• W2069174810 hasAuthorship W2069174810A5041952807 @default.
• W2069174810 hasAuthorship W2069174810A5046738629 @default.
• W2069174810 hasAuthorship W2069174810A5063401302 @default.
• W2069174810 hasBestOaLocation W20691748101 @default.
• W2069174810 hasConcept C11804247 @default.
• W2069174810 hasConcept C185592680 @default.
• W2069174810 hasConcept C55493867 @default.
• W2069174810 hasConcept C70721500 @default.
• W2069174810 hasConcept C86803240 @default.
• W2069174810 hasConcept C95444343 @default.
• W2069174810 hasConceptScore W2069174810C11804247 @default.
• W2069174810 hasConceptScore W2069174810C185592680 @default.
• W2069174810 hasConceptScore W2069174810C55493867 @default.
• W2069174810 hasConceptScore W2069174810C70721500 @default.
• W2069174810 hasConceptScore W2069174810C86803240 @default.
• W2069174810 hasConceptScore W2069174810C95444343 @default.
• W2069174810 hasIssue "41" @default.
• W2069174810 hasLocation W20691748101 @default.
• W2069174810 hasOpenAccess W2069174810 @default.
• W2069174810 hasPrimaryLocation W20691748101 @default.
• W2069174810 hasRelatedWork W1902144740 @default.
• W2069174810 hasRelatedWork W1982038138 @default.
• W2069174810 hasRelatedWork W2051123135 @default.
• W2069174810 hasRelatedWork W2099982459 @default.
• W2069174810 hasRelatedWork W2109298101 @default.
• W2069174810 hasRelatedWork W2146108849 @default.
• W2069174810 hasRelatedWork W2152546257 @default.
• W2069174810 hasRelatedWork W2421485185 @default.
• W2069174810 hasRelatedWork W2742319651 @default.
• W2069174810 hasRelatedWork W2517860465 @default.
• W2069174810 hasVolume "281" @default.
• W2069174810 isParatext "false" @default.
• W2069174810 isRetracted "false" @default.
• W2069174810 magId "2069174810" @default.
• W2069174810 workType "article" @default.