Eisenmann D M, Arndt K M, Ricupero S L, Rooney J W, Winston F
Department of Genetics, Harvard Medical School, Boston, Massachusetts 02115.
Genes Dev. 1992 Jul;6(7):1319-31. doi: 10.1101/gad.6.7.1319.
Mutations in the Saccharomyces cerevisiae gene SPT15, which encodes the TATA-binding protein TFIID, have been shown to cause pleiotropic phenotypes and to lead to changes in transcription in vivo. Here, we report the cloning and analysis of one such mutation, spt15-21, which causes a single-amino-acid substitution in a conserved residue of TFIID. Surprisingly, the spt15-21 mutation does not affect the stability of TFIID, its ability to bind to DNA or to support basal transcription in vitro, or the ability of an upstream activator to function in vivo. To study further the spt15-21 defect, extragenic suppressors of this mutation were isolated and analyzed. All of the extragenic suppressors of spt15-21 are mutations in the previously identified SPT3 gene. Suppression of spt15-21 by these spt3 mutations is allele-specific, suggesting that TFIID and SPT3 interact and that spt15-21 impairs this interaction in some way. Consistent with these genetic data, coimmunoprecipitation experiments demonstrate that the TFIID and SPT3 proteins are physically associated in yeast extracts. Taken together, these results suggest that SPT3 is a TFIID-associated protein, required for TFIID to function at particular promoters in vivo.
酿酒酵母基因SPT15发生突变，该基因编码TATA结合蛋白TFIID，已证明这种突变会导致多效性表型，并在体内引起转录变化。在此，我们报告了其中一种突变spt15 - 21的克隆与分析，该突变在TFIID的一个保守残基处导致单氨基酸替换。令人惊讶的是，spt15 - 21突变并不影响TFIID的稳定性、其与DNA结合或在体外支持基础转录的能力，也不影响上游激活剂在体内发挥功能的能力。为了进一步研究spt15 - 21缺陷，我们分离并分析了该突变的基因外抑制子。spt15 - 21的所有基因外抑制子都是先前鉴定的SPT3基因中的突变。这些spt3突变对spt15 - 21的抑制具有等位基因特异性，这表明TFIID和SPT3相互作用，并且spt15 - 21以某种方式损害了这种相互作用。与这些遗传数据一致，免疫共沉淀实验表明TFIID和SPT3蛋白在酵母提取物中存在物理关联。综上所述，这些结果表明SPT3是一种与TFIID相关的蛋白，是TFIID在体内特定启动子处发挥功能所必需的。
