TATA box-mediated polymerase III transcription in vitro

We have defined conditions whereby a functional TATA box can mediate efficient in vitro transcription by RNA polymerase III. A TATA box is absolutely required for this reaction as a single-point mutation in this sequence completely abolishes transcription. Two protein components are also required: a HeLa cell phosphocellulose fraction (fraction B) and at least one other factor that can be supplied by various crude nuclear extracts or by HeLa cell phosphocellulose fractions C and D. The order of addition is critical; fraction B must be preincubated with the template DNA for TATA box-dependent polymerase III transcription to occur. Various TATA sequences are quite similar in their ability to mediate transcription by polymerases II and III. Despite the similarity in sequence requirements, fraction B does not appear to contain any detectable transcription factor (TF) IID activity, and TATA box-mediated polymerase III transcription does not appear to require TFIID in the form contained in phosphocellulose fraction D. It was recently reported that TFIID is required TFIID in the form contained in phosphocellulose fraction D. It was recently reported that TFIID is required for polymerase III transcription of the yeast and human U6 genes (Margottin, F., Dujardin, G., Gerard, M., Egly, J.-M., Huet, J., and Sentenac, A. (1991) Science 251, 424-426; Simmen, K. A., Bernues, J., Parry, H. D., Stunnenberg, H. G., Berkenstam, A., Cavallini, B., Egly, J.-M., and Mattaj, I. W. (1991) EMBO J. 10, 1853-1862). We propose that fraction B may contain TFIID in a modified form that is not functional for polymerase II transcription.