Mitchell M T, Hobson G M, Benfield P A
E. I. du Pont de Nemours Co., Inc., Wilmington, Delaware 19880-0328.
J Biol Chem. 1992 Jan 25;267(3):1995-2005.
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.
我们已经确定了一些条件，在这些条件下，一个功能性的TATA框能够介导RNA聚合酶III进行高效的体外转录。对于此反应，TATA框是绝对必需的，因为该序列中的单点突变会完全消除转录。还需要两种蛋白质成分：一种来自HeLa细胞的磷酸纤维素组分（组分B）和至少一种其他因子，该因子可由各种粗核提取物或HeLa细胞磷酸纤维素组分C和D提供。添加顺序至关重要；组分B必须与模板DNA预孵育，才能发生依赖TATA框的聚合酶III转录。各种TATA序列在介导聚合酶II和III转录的能力方面非常相似。尽管在序列要求上有相似性，但组分B似乎不包含任何可检测到的转录因子（TF）IID活性，并且TATA框介导的聚合酶III转录似乎不需要磷酸纤维素组分D中所含形式的TFIID。最近有报道称，酵母和人类U6基因的聚合酶III转录需要TFIID（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）。我们提出，组分B可能含有一种修饰形式的TFIID，这种形式对聚合酶II转录无功能。
