Two transcription factors related with the eucaryal transcription factors TATA-binding protein and transcription factor IIB direct promoter recognition by an archaeal RNA polymerase

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.