PMC

Analysis of the copy number of pYS4 in P. furiosus (P. f.) by Southern blotting. Identical amounts from 25 ng to 400 ng of EcoRV-digested genomic DNA of P. furiosus wild-type (lanes 2 to 6) and pYS4 transformant (lanes 7 to 11) were used and analyzed with the rpoD probe. Lane 1 contained the EcoRV-digested vector pYS4 as a positive control.

Schematic diagram of the shuttle vectors pYS2, pYS3 and pYS4. To create pYS3, the pyrE marker of pYS2 was replaced with a simvastatin resistance cassette via BamHI restriction sites. This enables the overexpression of the HMG-CoA reductase under the control of the gdh promoter. The plasmid pYS4 contains an additional fragment for the expression of a His₆-tagged version of RNAP subunit D. This further copy is under the control of the fbp promoter and followed by the hpyA1 terminator sequence. The fragment was inserted into the EcoRV restriction site of pYS3. dso, double-stranded origin of replication; sso, single-stranded origin of replication; ori, origin of replication.

Comparison of the expression of wild-type and His₆-tagged subunit D encoded on plasmid pYS4 by a Western blot analysis. To analyze the relative amount of subunit D in the transformant grown with starch or pyruvate and in wild-type cells grown with starch, we used comparable amounts of RNAP in each extract. For titration of equal amounts of RNAP, we used an anti-subunit A" antibody (upper signal). The corresponding amounts of proteins in crude extracts from the transformant grown with starch were 218 ng, 436 ng, and 872 ng (lanes 1 to 3). Lanes 4 to 6 contained the transformant grown with pyruvate with protein amounts of 1,122 ng, 2,244 ng, and 4,488 ng. Wild-type crude extracts were 166 ng, 332 ng, and 664 ng protein (lanes 7 to 9). For identification of subunit D, we used an anti-subunit D antibody (lower signals). Lane 10 contains a prestained size marker (Pager Ruler; Fermentas) and lane 11 contains 50 ng of purified RNAP (with a His tag at subunit D). P. f., P. furiosus.

Purification and functional analysis of the RNAP. (A) Silver-stained gradient SDS gel (4 to 20%) of 2 μg purified RNAP from P. furiosus (P. f.). Lane 2 contains a wild-type RNAP, purified as described previously (). Lane 3 contains the RNAP from the transformant grown with starch and purified with Ni-NTA and gel filtration chromatography. The corresponding fractions (identical volumes) of similar amounts of two-step-purified cell extracts from wild-type cells (lane 4, without RNAP activity) and transformants grown with starch (lane 5) and pyruvate (lane 6) were analyzed on a 10% SDS gel. The band labeled with an asterisk is an unknown protein which was copurified in all three extracts. The corresponding subunits of the RNAP are indicated. Lane 1 contains a molecular size marker. (B) In vitro transcription with the purified RNAP fractions. The RNAP fractions from the transformants grown with starch (lane 2) or pyruvate (lane 3) and from the wild type (lane 1) were used to transcribe the gdh promoter in the presence of the archaeal transcription factors TBP and TFB. Lanes 4 to 6 contain control experiments without transcription factor TBP, transcription factor TFB, or both. The transcription assays were performed as described previously ().