Steffan J S, Keys D A, Dodd J A, Nomura M
Department of Biological Chemistry, University of California, Irvine 92697-1700, USA.
Genes Dev. 1996 Oct 15;10(20):2551-63. doi: 10.1101/gad.10.20.2551.
Transcription of Saccharomyces cerevisiae rDNA by RNA polymerase I involves at least two transcription factors characterized previously: upstream activation factor (UAF) consisting of Rrn5p, Rrn9p, Rrn10p, and two more uncharacterized proteins; and core factor (CF) consisting of Rrn6p, Rrn7p, and Rrn11p. UAF interacts directly with an upstream element of the promoter and mediates its stimulatory function, and CF subsequently joins a stable preinitiation complex. The TATA-binding protein (TBP) has been known to be involved in transcription by all three nuclear RNA polymerases. We found that TBP interacts specifically with both UAF and CF, the interaction with UAF being stronger than that with CF. Using extracts from a TBP (I143N) mutant, it was shown that TBP is required for stimulation of transcription mediated by the upstream element, but not for basal transcription directed by a template without the upstream element. By template competition experiments, it was shown that TBP is required for UAF-dependent recruitment of CF to the rDNA promoter, explaining the TBP requirement for stimulatory activity of the upstream element. We also studied protein-protein interactions and found specific interactions of TBP with Rrn6p and with Rrn9p both in vitro and in the yeast two-hybrid system in vivo. Thus, these two interactions may be involved in the interactions of TBP with CF and UAF, respectively, contributing to the recruitment of CF to the rDNA promoter. Additionally, we observed an interaction between Rrn9p and Rrn7p both in vitro and in the two-hybrid system; thus, this interaction might also contribute to the recruitment of CF.
酿酒酵母核糖体DNA（rDNA）由RNA聚合酶I转录，这一过程至少涉及两个先前已被鉴定的转录因子：上游激活因子（UAF），由Rrn5p、Rrn9p、Rrn10p以及另外两种未被鉴定的蛋白质组成；核心因子（CF），由Rrn6p、Rrn7p和Rrn11p组成。UAF直接与启动子的上游元件相互作用并介导其刺激功能，随后CF加入稳定的预起始复合物。已知TATA结合蛋白（TBP）参与所有三种核RNA聚合酶的转录过程。我们发现TBP与UAF和CF都有特异性相互作用，且与UAF的相互作用强于与CF的相互作用。使用TBP（I143N）突变体的提取物表明，TBP是上游元件介导的转录刺激所必需的，但对于没有上游元件的模板所指导的基础转录则不是必需的。通过模板竞争实验表明，TBP是UAF依赖的CF募集到rDNA启动子所必需的，这解释了TBP对上游元件刺激活性的需求。我们还研究了蛋白质 - 蛋白质相互作用，发现在体外以及体内酵母双杂交系统中，TBP与Rrn6p和Rrn9p都有特异性相互作用。因此，这两种相互作用可能分别参与了TBP与CF和UAF的相互作用，有助于CF募集到rDNA启动子。此外，我们在体外和双杂交系统中都观察到Rrn9p和Rrn7p之间的相互作用；因此，这种相互作用也可能有助于CF的募集。
