LOCALIZATION OF SUBUNITS OF TRANSCRIPTION FACTORS IIE AND IIF IMMEDIATELY UPSTREAM OF THE TRANSCRIPTIONAL INITIATION SITE OF THE ADENOVIRUSMAJOR LATE PROMOTER

The assembly of a preinitiation complex containing RNA polymerase II o n promoter DNA is a complex process that involves several general tran scription factors. Using 5-[N-(p-azidobenzoyl)-3-aminoallyl] photocros s-linking, we previously determined the locations of the two large sub units of transcription factor (TF) IIA (A35 and A21), TATA box-binding protein (TBP), RNA polymerase II-associated protein (RAP) 30, and TFI IB along the Ad2 ML promoter. We have now localized TFIIE34 and RAP74 just upstream of the transcription start site. The two sub-units of TF IIF, RAP74 and RAP30, cross-linked to nucleotides that probed adjacent spaces on the same face of the DNA helix beginning just downstream of TBP at -19 and extending to -5. Specific photocross-linking of TFIIE3 4 required the presence TFIIE56. In addition, TFIIE and RAP74 strongly stimulated cross-linking of RAP30 and the large subunits of RNA polym erase II to position -19. Our topological data support the idea that R AP74 and TFIIE34 may be involved in melting of the promoter DNA upstre am of the initiation site.