2 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 Met hanococcus and Pyrococcus depends upon two archaeal transcription fact ors, 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, Antibo dies raised against archaeal TBP and TFIIB bind to polypeptides of ide ntical molecular mass in the aTFB and aTFA fraction. These data identi fy aTFB as archaeal TBP and aTFA as the archaeal homologue of TFIIB, A t the Pyrococcus glutamate dehydrogenase (gdh) promoter these two bact erially produced transcription factors and endogenous RNA polymerase a re sufficient to direct accurate and active initiation of transcriptio n, 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 TAT A box but bound cooperatively with Pyrococcus-TBP generating an extend ed DNase I footprinting pattern ranging from position -19 to -42, Thes e data suggest that the Pyrococcus homologue of TFIIB associates with the TBP-promoter binary complex as its eucaryal counterpart, but in co ntrast to eucaryal TFIIB, it causes an extension of the protection to the region upstream of the TATA box.