Kasten M M, Stillman D J
Department of Oncological Sciences, Huntsman Cancer Institute, University of Utah Health Sciences Center, Salt Lake City 84132, USA.
Mol Gen Genet. 1997 Oct;256(4):376-86. doi: 10.1007/s004380050581.
The yeast SIN3 gene functions as a transcriptional repressor, despite the fact that Sin3p does not bind DNA directly. We have conducted a two-hybrid screen to look for proteins that interact with Sin3p, using the PAH2 domain of Sin3p as bait. Five new genes, STB1-STB5 were identified, as well as the STB6 gene, which is similar to STB2. STB1, STB2, STB3, and STB6 are novel genes, and STB4 and STB5 encode C6 zinc cluster DNA-binding proteins. None of these genes is essential for viability, and several of these genes may encode transcriptional activators. Several special problems were encountered in using a transcriptional repressor in a two-hybrid screen. For example, the STB genes will interact with a LexA-Sin3(PAH2) fusion protein containing a region of Sin3p, but a LexA-Sin3p fusion protein containing full-length Sin3p, along with a STB clone, does not produce two-hybrid activation of a transcriptional reporter. In addition, a sin3 mutation reduces the transcriptional activation by two-hybrid partners, suggesting that a sin3 mutation reduces the transcriptional efficiency of the Gal4p and VP16 activation domains. We have shown previously that Sin3p is part of a large multiprotein complex, and we show here that Stb1p and Stb2p are present in this complex.
酵母SIN3基因起着转录抑制因子的作用，尽管Sin3p并不直接结合DNA。我们利用Sin3p的PAH2结构域作为诱饵，进行了双杂交筛选以寻找与Sin3p相互作用的蛋白质。鉴定出了五个新基因STB1 - STB5，以及与STB2相似的STB6基因。STB1、STB2、STB3和STB6是新基因，STB4和STB5编码C6锌簇DNA结合蛋白。这些基因中没有一个对细胞活力是必需的，并且其中几个基因可能编码转录激活因子。在双杂交筛选中使用转录抑制因子时遇到了几个特殊问题。例如，STB基因会与含有Sin3p区域的LexA - Sin3(PAH2)融合蛋白相互作用，但含有全长Sin3p的LexA - Sin3p融合蛋白与一个STB克隆一起，不会产生转录报告基因的双杂交激活。此外，sin3突变会降低双杂交伙伴的转录激活，这表明sin3突变会降低Gal4p和VP16激活结构域的转录效率。我们之前已经表明Sin3p是一个大型多蛋白复合物的一部分，并且我们在此表明Stb1p和Stb2p存在于该复合物中。
