Two distinct transcription factors bind to the HSV thymidine kinase promoter in vitro.

We have characterized an in vitro transcription system derived from uninfected HeLa cells that accurately initiates RNA synthesis at the herpes virus thymidine kinase (TK) promoter. Analysis of linker-scanning, single-site, and promoter-inversion mutants reveals that the TK upstream elements previously mapped in vivo are accurately recognized in vitro. A protein fraction required for TK transcription in reconstitution experiments was found to contain multiple protein species that bind specifically to the TK promoter. DNAase I footprint experiments with wild-type and mutant promoters reveal that the TK upstream elements contain three distinctive protein binding sites, two of which appear to be recognized by the Sp1 transcription factor and one which interacts with a cellular protein that binds to "CCAAT" sequences. Optimal expression of the thymidine kinase gene appears to require the coordinate interaction of these two types of transcription factors with the three upstream elements of the promoter.