Abstract
The core promoters for mammalian protein-coding genes often contain a TATA box, an initiator (Inr) element, or both of these control elements. The TFIID complex is essential both for TATA activity and for the activity of a common class of Inr elements characterized by an approximate consensus sequence PyPyA+1NT/APyPy. Although the complete set of proteins required for basal TATA-mediated transcription has been established, the requirements for TFIID-dependent Inr activity remain undefined. In this study we set out to reconstitute Inr activity with purified and recombinant general transcription factors. For this analysis, Inr activity was measured as the ability of an Inr to enhance the strength of a core promoter containing an upstream TATA box. Inr activity was not detected in reactions containing TFIIB, RAP30, RAP74, RNA polymerase II, and either TBP or TFIID, even though these factors were sufficient for TATA-mediated transcription from supercoiled templates. By use of a complementation assay, a factor that imparts Inr activity was identified. This factor, named CIF, stimulated Inr activity in reactions containing the TFIID complex, but activity was not detected with TBP. Further characterization of CIF suggested that it contains multiple components. Functional and immunological experiments demonstrated that one of the CIF components is the mammalian homolog of Drosophila TAF(II)150, which is not tightly associated with mammalian TFIID. These results reveal significant differences in the factor requirements for basal TATA and Inr activity. Further elucidation of these differences is likely to explain the need for the core promoter heterogeneity found within protein-coding genes.
