Stimulation of transcription in vitro by binding sites for nuclear factor I

Nuclear factor I (NFI) is a site-specific DNA binding protein required for the replication of adenovirus DNA in vitro and in vivo. We have examined the effect of natural and synthetic binding sites for NFI (FIB sites) on RNA synthesis in HeLa whole cell extracts. The natural binding site used is the 26bp FIB-2 site previously isolated from the human genome. When present upstream of the TATA box of the adenovirus major late promoter, the FIB-2 site stimulates RNA synthesis 3 to 5-fold. This stimulation occurs with either orientation of the FIB-2 site. A point mutation in FIB-2 that decreases NFI binding at least 100-fold reduces, but does not completely abolish, the stimulation of transcription. A number of synthetic binding sites for NFI were tested for the ability to increase RNA synthesis. The strongest binding sites stimulated transcription the most, while the weakest sites had the least effect. These studies strongly suggest a role for NFI and cellular FIB sites in the control of RNA synthesis.