Linker scanning analysis of TBP promoter binding factor DNA binding, activation, and repression domains

The transcription activator TATA box-binding protein promoter-binding facto r (TPBF) is both an activator and repressor of TBP gene expression in Acant hamoeba. TPBF bears little similarity to previously characterized families of factors. In order to identify domains that are involved in DNA binding, activation, and repression, we constructed several alanine linker scanning mutants and tested them for their ability to function in a variety of assay s. The DNA binding domain comprises a large 100-amino acid domain within th e central third of the protein, suggesting that DNA recognition is accompli shed by interactions derived from several structural units within this doma in. Surprisingly, transcription activation and repression are impaired by m utations within either of two discrete amino acid sequences located on eith er side of the DNA binding domain. These data suggest that TPBF activation and repression are accomplished by interactions with the same target. Since TATA elements can function bidirectionally, and in solution TBP can bind t o TATA elements in either orientation, we propose that TPBF functions in pa rt by orienting TBP or TFIID correctly on the TATA box.