A Cell-Free Transcription System for the Hyperthermophilic Archaeon Pyrococcus Furiosus

Hethke, Carina; Geerling, A.C.M.; Hausner, Winfried; Vos, Willem M. de; Thomm, Michael

doi:10.1093/nar/24.12.2369

Cited by 73 publications

(85 citation statements)

References 36 publications

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“…Promotor-directed in Vitro Transcription Assay-In vitro transcription assays were performed as described (27). Standard transcription reactions contained 300 ng of gdh promoter, 42 nM TBP, 55 nM TFB, and 10 nM purified or recombinant RNAP in a total volume of 25 l of transcription buffer (40 mM NaHEPES, pH 7.3, 250 mM NaCl, 2.5 mM MgCl 2 , 5% (v/v) glycerol, 0.1 mM EDTA, 5 mM ␤-mercaptoethanol, and 0.1 mg/ml bovine serum albumin).…”

Section: Methodsmentioning

confidence: 99%

“…The components were added at 4°C and then incubated at 70°C for 30 min. Labeled transcripts were analyzed by electrophoresis in 6% (w/v) polyacrylamide urea gels as described (27). The transcription products were visualized using an Image Plate and Image Analyzer (FLA-500, Fuji, Japan).…”

Section: Methodsmentioning

confidence: 99%

“…We are exploring the mechanism and regulation of transcription in the hyperthermophilic Archaea Pyrococcus furiosus (23)(24)(25)(26)(27). In this contribution we report the reconstitution of a highly active RNAP from 11 bacterially expressed subunits.…”

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confidence: 99%

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

Naji

Grünberg

Thomm

2007

Journal of Biological Chemistry

120

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RNA polymerases from Archaea and Eukaryotes consist of a core enzyme associated with a dimeric E F (Rpb7/Rpb4) subcomplex but the functional contribution of the two subunit subcomplexes to the transcription process is poorly understood. Here we report the reconstitution of the 11-subunit RNA polymerase and of the core enzyme from the hyperthermophilic Archaeon Pyrococcus furiosus. The core enzyme showed significant activity between 70 and 80°C but was almost inactive at 60°C. E stimulated the activity of the core enzyme at 60°C, dramatically suggesting an important role of this subunit at low growth temperatures. Subunit F did not contribute significantly to catalytic activity. Permanganate footprinting at low temperatures dissected the contributions of the core enzyme, subunit E , and of archaeal TFE to open complex formation. Opening in the ؊2 and ؊4 region could be achieved by the core enzyme, subunit E stimulated bubble formation in general and opening at the upstream end of the transcription bubble was preferably stimulated by TFE. Analyses of the kinetic stabilities of open complexes revealed an unexpected E -independent role of TFE in the stabilization of open complexes. Transcription in cells of Archaea andBacteria is catalyzed by a single RNA polymerase (RNAP), 2 whereas eukaryotic cells contain three different types of RNAPs (I, II, and III) that carry out specialized functions. Despite their morphological similarity to Bacteria, Archaea have a transcriptional machinery that is more akin to the eukaryotic machinery (1-3).Like eukaryotes, Archaea use extrinsic transcription factors for initiation. The process of transcription initiation in Archaea can be dissected in three steps. The general transcription factor TATA-binding protein (TBP) interacts with the archaeal TATA box, transcription factor B (TFB) stabilizes the binding of TBP to the promoter. The TBP-TFB promoter complex mediates recruitment of RNAP (4, 5). These extrinsic factors show the major structural features of eukaryotic TBP and TFIIB and interact with DNA and RNAP in a similar fashion as their eukaryotic counterparts (6, 7). This exceptional degree of similarity between the archaeal and eukaryotic transcriptional machineries extends also to the RNAP.Archaeal RNAP consist of 11 or 12 different subunits (8 -10) that display a high level of primary sequence similarity to the subunits present in eukaryotic RNAPII. With the exception of subunits RPB8 and RPB9, orthologs of other RNAPII subunits have been identified in all archaeal genomes studied so far. A recent in silico study revealed a high degree of conservation of subunits shared by pol II and Archaea in particular in the regions of RNAP comprising the catalytic center and proteinprotein binding studies showed a similar pattern of subunit interactions between the subunits of pol II and of Pyrococcus RNAP (11). Although very similar to eukaryotic RNAP in subunit composition and transcription initiation factor requirement the archaeal machinery is less complex than the eukaryotic machine...

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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

Naji

Grünberg

Thomm

2007

Journal of Biological Chemistry

120

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“…The plasmid was digested with EcoRV or HincII ( Fig. 1) for in vitro generation of run-off transcripts (20). For primer extension, a [␥-…”

Section: Methodsmentioning

confidence: 99%

The Ferredoxin-dependent Conversion of Glyceraldehyde-3-phosphate in the Hyperthermophilic ArchaeonPyrococcus furiosus Represents a Novel Site of Glycolytic Regulation

Oost¹,

Schut²,

Kengen³

et al. 1998

Journal of Biological Chemistry

101

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The fermentative conversion of glucose in anaerobic hyperthermophilic Archaea is a variant of the classical Embden-Meyerhof pathway found in Bacteria and Eukarya. A major difference of the archaeal glycolytic pathway concerns the conversion of glyceraldehyde-3-phosphate. In the hyperthermophilic archaeon Pyrococcus furiosus, this reaction is catalyzed by an unique enzyme, glyceraldehyde-3-phosphate ferredoxin oxidoreductase (GAPOR). Here, we report the isolation, characterization, and transcriptional analysis of the GAPOR-encoding gene. GAPOR is related to a family of ferredoxin-dependent tungsten enzymes in (hyper)thermophilic Archaea and, in addition, to a hypothetical protein in Escherichia coli. Electron paramagnetic resonance analysis of the purified P. furiosus GAPOR protein confirms the anticipated involvement of tungsten in catalysis. During glycolysis in P. furiosus, GAPOR gene expression is induced, whereas the activity of glyceraldehyde-3-phosphate dehydrogenase is repressed. It is discussed that this unprecedented unidirectional reaction couple in the pyrococcal glycolysis and gluconeogenesis gives rise to a novel site of glycolytic regulation that might be widespread among Archaea.

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“…Instead, all densities we obtained with the deposited structure factors were consistent with the B-reader helix observed by us. P. furiosus protein preparations Endogenous P. furiosus (Pfu) RNA polymerase and recombinant Pfu TBP were purified as described 17 . Pfu TFB was cloned with an N-terminal His 6 -Tag in pET14b (Novagen) in E. coli BL21(DE3)pLysS (Novagen).…”

Section: Modeling the Closed And Open Complexesmentioning

confidence: 99%

RNA polymerase II–TFIIB structure and mechanism of transcription initiation

Kostrewa

Zeller

Armache

et al. 2009

Nature

273

430

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Preparation of yeast proteinsEndogenous S. cerevisiae ten-subunit Pol II core enzyme was prepared as described 1 . An E. coli expression vector was derived from pET21b (Novagen) for the coexpression of the translational fusion of S. cerevisiae Rpb4:20 glycine linker:TFIIB and Rpb7:His 6 under the control of separate T7 promoters. Details of the vector design are available on request. Following expression in E. coli, cells were lysed by sonication in buffer A (50 mM Tris, 150 mM NaCl; pH 7.5, 0.3 mg/L leupeptin, 1.4 mg/L pepstatin A, 0.17 g/L PMSF, 0.33 g/L benzamidine and 10 mM β -mercaptoethanol). The lysate was cleared by centrifugation and applied to a Ni-NTA agarose column (Qiagen). The column was washed with buffer A containing 2 M NaCl, and the protein was eluted with a gradient of 10 mM to 200 mM imidazole in buffer A containing 150 mM NaCl. Peak fractions were diluted twofold and loaded onto a Mono-S cation exchange column (Amersham) equilibrated with buffer A containing 100 mM NaCl. The fusion protein was eluted over a total of 15 column volumes with a gradient of 0.1-1 M NaCl in buffer A. Peak fractions were concentrated and applied to a Superose 6 gel filtration column (Amersham) equilibrated with buffer B (5 mM HEPES pH 7.25, 40 mM ammonium sulfate, 10 μM ZnCl 2 , 10 mM DTT). Peak fractions were concentrated, shock-frozen in liquid nitrogen, and stored at −80°C. The TBP core domain (S. cerevisiae residues 61-240) expression vector was a generous gift from Dr. Sean Juo. Expression and purification of the yeast TBP core domain was as described 2 except that Superose 12 size exclusion chromatography was performed with buffer B. Peak fractions were concentrated, shock-frozen in liquid nitrogen, and stored at −80°C. 10-subunit Pol II was incubated with two molar equivalents of nucleic acid scaffold (Template, 5'-cgacacagcatcaaatgcacgatgtaacttttataggcgcccaacc;Nontemplate, 5'-ggttgggcgcctataaaagttacatcgtgcaaaatcgttatgagaa; RNA, 5'-gctgtgtcg) as described 3 and 2.5 molar equivalents of TBP. After incubation for 20 minutes at 20°C 3-5 molar equivalents of TFIIB-Rpb4/7 fusion protein were added. After incubation for 20 min. at 20°C, the complex was purified on a Superose 6 size exclusion column (Amersham). Fractions corresponding to the complex were pooled and concentrated to 4 mg/ml.Crystallization, data collection, and structure determination Crystals were grown at 20 °C using the hanging drop vapor diffusion method by mixing 1.5 µl of sample solution with 1.5 µl of reservoir solution (800 mM sodium ammonium tartrate, 100 mM HEPES pH 7.5, 5 mM DTT). Crystals were transferred stepwise to mother solution containing additionally 0-22% glycerol over 8 h, slowly cooled down to 8 °C, incubated for another 24 h, and plunged into liquid nitrogen. Diffraction data were collected in 0.75° increments at the protein crystallography beamline ID 29 at ESRF. Diffraction data were processed with XDS and scaled with XSCALE 4 . The structure was solved by molecular replacement with PHASER 5 using the first 12-subunit Pol II ...

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A Cell-Free Transcription System for the Hyperthermophilic Archaeon Pyrococcus Furiosus

Cited by 73 publications

References 36 publications

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

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

The Ferredoxin-dependent Conversion of Glyceraldehyde-3-phosphate in the Hyperthermophilic ArchaeonPyrococcus furiosus Represents a Novel Site of Glycolytic Regulation

RNA polymerase II–TFIIB structure and mechanism of transcription initiation

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