Abstract
The precise binding site in the adenovirus E2 promoter for the E1A-inducible factor E2F was determined. DNase footprinting revealed two distinct regions of protection which spanned sequences from -33 to -49 and from -53 to -71. Chemical modifications of DNA further delineated nucleotides involved in DNA-protein contacts in each binding region. The E2F binding sites are clearly distinct from the binding site for another E2 promoter binding factor, located at -68 to -80, previously described by SivaRaman et al. [(1986) Proc. Natl. Acad. Sci. USA, 83, 5914-5918]. As determined by DNase footprinting using crude nuclear extracts, both factors were present in extracts of Ad5-infected cells and were found to bind simultaneously to their respective sites on the promoter. In contrast, E2F was not evident in extracts of uninfected cells, whereas there was no difference in the -68 to -80 footprint as a function of the extract. Thus, although multiple factors interact with the E2 promoter, only the E2F factor is unique to the infected extract. The implications of the formation of a multi-factor promoter complex as a possible mechanism of transcriptional regulation are discussed.