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

Abstract

The transcription activator TATA box-binding protein promoter-binding factor (TPBF) is both an activator and repressor of TBP gene expression in Acanthamoeba. 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 assays. The DNA binding domain comprises a large 100-amino acid domain within the central third of the protein, suggesting that DNA recognition is accomplished by interactions derived from several structural units within this domain. Surprisingly, transcription activation and repression are impaired by mutations within either of two discrete amino acid sequences located on either 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 to TATA elements in either orientation, we propose that TPBF functions in part by orienting TBP or TFIID correctly on the TATA box.