The orientation of DNA in an archaeal transcription initiation complex

RNA polymerase from the hyperthermophile archaeon Pyrococcus furiosus (Pfu) forms specific and transcriptionally active complexes with its conjugate t ranscription factors TBP (the archaeal TATA binding protein homolog) and TF B (the archaeal homolog of eukaryotic RNA polymerase II and III transcripti on factors TPHB and Brf) at the Pfu glutamate dehydrogenase promoter. A pho tochemical crosslinking method was used to map the vicinity of the catalyti c subunits of Pfu RNA polymerase to DNA locations distributed along the pol ymerase-promoter interface. The largest component of this archaeal polymera se is split into two subunits, A' and A ", whose relatively sharp boundary of DNA crosslinking (probed on the transcribed strand) is centered five to six base pairs downstream of the transcriptional start site. A strong argum ent based on this information, on the well-defined homology between the cor e bacterial, archaeal and eukaryotic RNA polymerase snbunits, and on the re cently determined structure of a bacterial RNA polymerase specifies the dir ectionality of DNA in the archaeal transcription complex;and its trajectory downstream of the transcriptional start site.