Analysis of protein-DNA interactions within the transcription preinitiation complex of Pyrococcus furiosus
Renfrow, Matthew Blain
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Initiation of transcription requires the assembly of a multi-protein preinitiation complex (PIC) on promoter DNA. The protein-DNA interactions within the transcription PIC of the archaeal hyperthermophile, Pyrococcus furiosus (Pf) were probed by site-specific DNA-to-protein photocrosslinking. This involved the assembly of the PIC including Pf RNA polymerase (Pf RNAP) and general transcription factors Pf TATA-binding protein (Pf TBP) and Pf transcription factor B (Pf TBP) on 41 azido-phenacyl derivatized DNA promoter fragments containing the Pf glutamate dehydrogenase upstream promoter + gene. The region from -40 to +1 (transcription start site is +1) of both the template and nontemplate DNA promoter was probed. Pf RNAP-DNA promoter interactions span the entire probed region. Pf TFB-DNA promoter interactions occur upstream and downstream of the TATA box promoter element. Pf TBP-DNA interactions center around the TATA box promoter element. Results provide evidence of the homology between eukaryal RNA polymerase II gene transcription system and the archaeal gene transcription system. Pf TFB-DNA interactions are more extensive in the archaeal PIC than those seen in the analogous eukaryal system between transcription factor IIB (TFIIB) and promoter DNA. The Pf TFB-DNA interactions reported here provide support for a mechanism by which Pf TFB carries out its roles in PIC assembly of recruiting Pf RNAP to the complex and accurate start site selection.
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