Abstract
We reported previously that cell-free transcription in the Archaea Methanococcus and Pyrococcus depends upon two archaeal transcription factors, archaeal transcription factor A (aTFA) and archaeal transcription factor B (aTFB). In the genome of Pyrococcus genes encoding putative homologues of eucaryal transcription factors TATA-binding protein (TBP) and TFIIB have been detected. Here, we report that Escherichia coli synthesized Pyrococcus homologues of TBP and TFIIB are able to replace endogenous aTFB and aTFA in cell-free transcription reactions. Antibodies raised against archaeal TBP and TFIIB bind to polypeptides of identical molecular mass in the aTFB and aTFA fraction. These data identify aTFB as archaeal TBP and aTFA as the archaeal homologue of TFIIB. At the Pyrococcus glutamate dehydrogenase (gdh) promoter these two bacterially produced transcription factors and endogenous RNA polymerase are sufficient to direct accurate and active initiation of transcription. DNase I protection experiments revealed Pyrococcus-TBP producing a characteristic footprint between position -20 and -34 centered around the TATA box of gdh promoter. Pyrococcus-TFIIB did not bind to the TATA box but bound cooperatively with Pyrococcus-TBP generating an extended DNase I footprinting pattern ranging from position -19 to -42. These data suggest that the Pyrococcus homologue of TFIIB associates with the TBP-promoter binary complex as its eucaryal counterpart, but in contrast to eucaryal TFIIB, it causes an extension of the protection to the region upstream of the TATA box.
Highlights
Recent work established that cell-free transcription in Archaea is mediated by transcription factors (1–3)
To investigate the relationship of the factors encoded in the genome of Pyrococcus that are related to eucaryal TATA-binding proteins (TBP) and TFIIB with Pyrococcus transcription factors archaeal transcription factor A (aTFA) and archaeal transcription factor B (aTFB), the cloned Pyrococcus genes were overexpressed in E. coli, the polypeptides purified to near homogeneity, and their function analyzed in the Pyrococcus cell-free system
When a recombinant plasmid that is harboring the glutamate dehydrogenase gene of Pc. furiosus linearized with BamHI was used as template, a run-off transcript of 173 nucleotides was synthesized by the reconstituted Pyrococcus cell-free transcription system (Ref. 12; Fig. 1A, lane 7). This system consists of highly purified RNA polymerase, the Superdex fraction of aTFB and aTFA purified from the crude extract by a two-step procedure (12)
Summary
Templates for Cell-free Transcription Reactions—The plasmid pLUW479 containing the promoter and a part of the coding region of the gdh gene of Pyrococcus furiosus was used in standard transcription reactions (12). Expression and Purification of the Pyrococcus Homologue of TFIIB—A DNA fragment encoding the open reading frame of Pyrococcus woesei TFIIB gene was amplified by using the polymerase chain reaction with the following oligonucleotides: 5Ј-GGAATTCCATATGAATAAGCAAAAGGTTTGTC-3Ј and 5Ј-GTCATTCGAATTCATGCTATAGGAACTTTAATC-3Ј. The recombinant Pc-TFIIB remained in the supernatant and was further purified by Mono Q and Superdex 200 chromatography as described previously (11). The resulting clone pTBPPW. was transformed into the E. coli strain BL21(DE3), induction of gene expression was performed for 3 h, and the cell extract was heated for 15 min at 80 °C. Western Blot Analyses—Western blot analyses with antibodies directed against recombinant Thermococcus TBP and Pyrococcus TFIIB were performed as described previously (11). The DNA fragments were purified by phenol treatment, precipitated with ethanol, and analyzed on a 6% sequencing gel (45 watts for 2.5 h)
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